eln: a component of a larger informatics puzzle · eln: a component of a larger informatics puzzle...

www.europeanpharmaceuticalreview.com Issue 4 2013

ELN: a component of a largerinformatics puzzle

Michael H. Elliott, CEO, Atrium Research

Cell based label-freeassays in GPCR drug discoveryNiklas Larsson, Linda Sundström, Erik Ryberg and Lovisa Frostne, AstraZeneca

The rapidmicrobiological

methods revolutionEmanuele Selvaggio, QA Batch Disposition &

Investigation Supervisor, Pfizer

1.877.CRIVER.1www.criver.com/emd

As keepers of the industry’s largest microbial database, our scientists handpick

Learn more at www.criver.com/emd

Charles River supports the pharmaceutical,

medical device, nutraceutical, personal care and

cosmetics industries with its Accugenix® microbial

identification services. Having identified more than

a million samples of unknown microorganisms

over the past decade, our methods have been

shown to increase the efficacy of microbial

identification platforms for environmental

monitoring applications.

While a number of microbial identification

systems are available to the pharmaceutical

market, these platforms tend to underperform

because they do not support the broad range of

microorganisms encountered in environmental

monitoring programs. The associated reference

libraries are often biased toward clinical, rather

than environmental, organisms.

In 2010, we began offering AccuPRO-ID®

microbial identification services, a polyphasic

approach utilising MALDI-TOF and AccuGENX-ID®

16S rDNA sequencing, assuring the highest

accuracy and reportable rate. Since then, we have

processed over 80,000 samples using the Bruker

Daltonics MALDI Biotyper system and continue

to optimise the platform to better accommodate

environmental isolates.

A key activity for achieving better

performance is the continuous expansion of our

reference library. The number of microbial species

frequently encountered in the environmental

arena is tremendous and much higher than

realised by most. The inclusion of these species

in any microbial identification platform’s reference

library is critical to improving reporting and/or

accuracy for nonclinical applications. Therefore,

we developed a systematic approach for adding

relevant microorganisms to the MALDI-TOF

reference library.

The species that most frequently fail to

generate an identification using the MALDI

Biotyper system are frequently analysed by

16S sequencing, enabling the addition of these

fall-through species to the Accugenix® MALDI-TOF

reference library.

To date, these library entries generate

identifications more than twice as frequently as the

entries provided by the manufacturer, confirming

that the most frequent fall-throughs are added to

the Accugenix® reference library first.

The additional library entries generate nearly

37 per cent of all identifications, increase the

number of identifications by more than 10 per cent

and effectively reduce the nonreportable rate by

39.4 per cent.

With continued updates to the Accugenix®

MALDI-TOF reference library, the

percentage of samples generating

identifications will continue to rise.

While low cost and rapid turnaround time

remain strong advantages for MALDI-TOF

microbial identification platforms, our data

demonstrate that building a reference database

inclusive of environmental organisms is essential

to its suitability for nonclinical applications.

Methods

All data presented in this study were generated

from unknown samples received for microbial

identification. Samples submitted for AccuPRO-ID®

MALDI-TOF identification were processed using a

direct formic acid lysis method and/or the extract

method (per Charles River SOPs and the

manufacturer’s instructions) and analysed using

the Bruker Daltonics MALDI Biotyper software.

Samples with a ‘match factor’ greater than 1.75

and separated by > .1 were ascribed probable

species-level identifications. All samples that failed

to produce an identification using the MALDI

Biotyper system were submitted for AccuGENX-ID®

16S rDNA sequencing, where sequence data were

manually assembled and compared against the

validated and proprietary Accugenix® 16S reference

library. Qualified data analysts determined

identifications and confidence levels. 16S-based

sequence identification is used as the reference

method for this study.

The impact of a customised MALDI-TOF library forenvironmental microbial identifications: reducing the

nonreportable rate by 39.4 per cent

Figure 1: Diversity of species routinely identified

Figure 2: Systematic approach for the inclusion of newMALDI-TOF library entries

Figure 3: New entries target the most frequentlyoccurring species

Figure 4: Number of library entries and frequency of additions

Figure 5: Impact of a customised database onreportable identifications

Figure 6: Source of library entries leading to MALDI-TOF identification

For more information about

Accugenix® microbial identification services,

please visit www.criver.com/emd

or contact us at [email protected]

With recent revisions to USP General Chapters 41 and 1251, the pharmaceutical industry

needs to update their SOPs for testing of weighing equipment.

Good Weighing Practice™ (GWP®) from METTLER TOLEDO helps you get in the fast lane to

ensure compliance with USP while saving on testing costs.

Contact the weighing experts and learn more about how USP General Chapters 41 and 1251

affect your weighing processes.

Choose the Fast Lane to USP Compliance with METTLER TOLEDO

www.mt.com/lab-usp

email: [email protected]

When it comes to large, global, multi-site companies, rolling out a new

informatics system can take up to three years from start to finish to implement

across the entire business. While this may seem archaic in today’s age and with

the technology available, pharmaceutical companies have the added

challenge of dealing with highly sensitive data, usually in very large quantities.

Making the transition to a paperless laboratory is a long-term investment and

strategy for a business, usually involving a large investment cost that may not

see immediate gains for the company’s bottom line. With the mergers

and acquisitions that have become a staple of the pharmaceutical industry

over recent years, the added problem of integrating two, three or more different

systems used by different companies then complicates matters further. Is it time for

Pharma to fully embrace cloud computing, which would enable companies to

integrate data much faster? While it seems that the industry recognises the need

for cloud computing, in reality it could take years for cloud platforms to be embraced

by an industry that manages to be both forward thinking and yet reluctant to leave

tried-and-tested methods behind. The end result though will drive productivity and

decision-making as the data becomes easier to handle, analyse and ultimately produce

results for the company.

In this issue of European Pharmaceutical Review, we look at two areas of the

pharmaceutical industry using informatics – biobanking and biologics development

– in the informatics in-depth focus, which begins on page 23. Michael H. Elliott, CEO of

Atrium Research and Editorial Board Member of European Pharmaceutical Review,

looks at the components that make up the larger informatics puzzle and how ELN is

just one piece of the puzzle, while Eva Bürén, Head of IT at the Karolinska Institutet

Biobank looks at how LIMS supports researchers using biobank samples.

If you would like to contribute to European Pharmaceutical Review with the

latest news, editorial contribution or general interest, please send me an email at

[email protected] with your request.

European Pharmaceutical Review

www.europeanpharmaceuticalreview.com 3 Volume 18 | Issue 4 | 2013

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EDITORIAL BOARDAnthony Davies

Director Irish National Centre for High Content Screeningand Analysis Department of Clinical Medicine,

Trinity College Dublin

Sheraz GulVice President and Head of Biology,

European ScreeningPort GmbH

Matthew MoranDirector, PharmaChemical Ireland

Don ClarkPfizer Global Research & Development

Michael J. MillerPresident, Microbiology Consultants

Michael H. ElliottCEO, Atrium Research & Consulting

RUSSELL PUBLISHING LTD Founder Ian Russell

Managing Director Vivien Cotterill-Lee

Editor Helen Bahia

Deputy Editor Annie McKenna

Senior Publications Assistant Karen Hutchinson

Group Sales Director Tim Dean

Sales Director Freddy White

Senior Sales Executive Andrew Johnson

Production Manager Brian Cloke

Front Cover Artwork Steve Crisp

European Pharmaceutical Review (ISSN No: 1360-8606, USPS No: 023-422) is published bi-monthly (6 times per annum) byRussell Publishing Ltd, and distributed in the USA by SP/Asendia,17B S Middlesex Ave, Monroe NJ 08831. Periodicals postage paidat New Brunswick, NJ. POSTMASTER: send address changes toEuropean Pharmaceutical Review, 17B S Middlesex Avenue,Monroe NJ 08831.

European Pharmaceutical Review is published bi-monthly (six times per annum) and is available by subscription at £90.00 for a year, which includes on-line membership access.Back issue copies can be requested at £15.00 per copy.

European Pharmaceutical Review: Published by Russell Publishing Ltd, Court Lodge, Hogtrough Hill, Brasted, Kent, TN16 1NU, UK Tel: +44 (0) 1959 563311 Fax: +44 (0) 1959 563123 Email: [email protected]

ISSN 1360 - 8606Copyright rests with the publishers.All rights reserved©2013 Russell Publishing Limited

Helen Bahia, Editor

Investing in informatics

Welcome...

European Pharmaceutical Review is proud to be affiliated

with JPAG (Joint Pharmaceutical Analysis Group), a not-for-profit

organisation for pharmaceutical analysts. Free subscriptions are

available for members of JPAG. Please contact Karen Hutchinson

at [email protected] for further details.

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Biotechnica

2013

8–10 October, Hall 9 Booth D34

03 INTRODUCTIONInvesting in informaticsHelen Bahia, Editor

07 FOREWORDICHQ2(R1) Validation of Analytical Procedures: Challenges and OpportunitiesDave Elder, Joint Pharmaceutical Analysis Group and GSK

08 NEWS

09 INDUSTRY EXPERT PANEL

10 EVENTS

13 GPCRSCell based label-free assays in GPCR drug discoveryNiklas Larsson, Linda Sundström, Erik Ryberg and Lovisa Frostne, AstraZeneca

18 ION CHANNELSChloride ion channels andtransporters: from curiosities of nature and source of humandisease to drug targetsJonathan D. Lippiat, School of Biomedical Sciences,University of Leeds

35 SHOW PREVIEWMIPTEC

36 RAMAN SPECTROSCOPYRaman spectroscopy: an enabling tool for accelerating pharmaceuticaldiscovery to developmentChanda R. Yonzon, Atul Karande, Sai P. Chamarthy and Brent A. Donovan, Merck & Co. Inc

54 SIX SIGMAImproving operations andperformance: how Rottapharm isusing Lean Six Sigma principlesRichard Hayes, Continuous Improvement Manager,Rottapharm

58 SHOW PREVIEWDRUG DISCOVERY 2013

59 PROTEASESHow naturally occurring inhibitorscan facilitate small molecule drugdiscovery for cysteine proteasesSheraz Gul, Vice President and Head of Biology, European ScreeningPort GmbH

63 SHOW PREVIEWBIOTECHNICA

64 PRODUCT HUBWith Robert Mount, Managing Director, BMG Labtech



41 IN-DEPTH FOCUS:RMMS &ENVIRONMENTALMONITORING

Featuring articles from EmanueleSelvaggio, Pfizer on the RMMrevolution and Chris Delaney, NoonanServices Group, discussing how tocontrol contamination in cleanrooms

Contents

23 IN-DEPTH FOCUS:INFORMATICS

Eva Bürén, Head of IT, KarolinskaInstitutet Biobank looks at using LIMS for biobanking and Michael H. Elliott, CEO, Atrium Research looks at whether ELNs are a good match forbiologics development

Research is still your favorite?MAKE GREAT THINGS HAPPENOpportunities for natural scientists: Do you want to explore, research, change things? Welcome to Merck. With leading research in pharma-ceuticals, through analysis of microorganisms, all the way to liquid crystals for LCDs, Merck continues developing innovative products. We offer excellent development perspectives in challeng ing research

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The guidance covers typical criteria for re-

validation, including changes in API synthesis,

changes in the composition of drug product

or changes in the analytical procedure. However,

the guidance is silent with respect to the

requirements for method validation during

clinical trial development, the expectations for

analytical method technology transfer (TT) and

with respect to validation of extremely sensitive

and sophisticated methods, e.g. trace analysis of

genotoxic impurities. Consequently, there is

often regulatory confusion with respect to

applicability of the ICHQ2(R1) guidelines in

these areas.

There are surprisingly few publications on

the attributable causes for non-robust analytical

methodologies and yet this is a commonly

reported deficiency by regulatory agencies

across the globe. The EMA1 recently indicated

that, ‘During inspections, GMP inspectors

have noted that the root causes for out-of-

specification (OOS) results are sometimes

attributed to a lack of test method validation in

the context of transfer of analytical methods.

Such situations also occur frequently at contract

Quality Control laboratories.’ Therefore, despite

these methods being fully validated according to

ICHQ2(R1), they are still found to be non-robust

during routine QC testing; i.e. not fit for purpose.

Molnár2 attributed these issues to that fact

that ICHQ2(R1) was a statistically based guideline

with limited focus on the resultant chromato -

graphy, particularly critical resolutions. He

indicated that most methods are critically

dependent on a number of key parameters, e.g.

pH, stationary phase (type and batch), temp -

erature, mobile phase, buffer concentration,

etc and in a global economy, it is often difficult to

try and address all of this potential variability

within the existing robustness assessment

contained within ICHQ2(R1). He articulated

that, ‘Therefore, today, we need HPLC methods,

which are adjustable from location to location

to perform the analytical goal of correct

quantitation of the product components.’ This is

obviously only possible if the methods are

developed allowing adjustments between

certain clearly defined limits (termed ‘maps of

critical resolution’). his is the forerunner to apply -

ing Quality by Design (QbD) in the development

of methods and as long as the parameters reside

within pre-defined ‘proven acceptable ranges

(PARs)’, these parameters can be modified to get

the optimal resolution of key impurities (which

again are pre-defined).

A recent example of this QbD approach

was reported by Schmidt and Molnár3 for

the development and validation of a UPLC

method for a second generation anti-histamine

(ebastine) in API and drug product. The authors

utilised chromatography modelling software

(DryLab 4) to develop a Design Space, as per ICH

Q8, for the method, which they described as

‘a region in which changes to method param -

eters will not significantly affect the results.’

They then verified that the Design Space was

accurate with a relative error of prediction (REP)

of only 0.06 per cent. The method was fully

validated as per ICHQ2(R1). Specificity, including

baseline resolution for all impurity peaks could

be achieved in four minutes. This represents

a significant enhancement in productivity

(40-fold), versus the established Ph.Eur. method,

allowing an increased sample throughput of

360 samples/day.

The robustness of the method was assessed

by varying six critical parameters (+1, 0, -1); temp -

erature, ternary composition of the mobile phase,

flow rate, gradient time and initial and final

concentration of the mobile phase composition.

The resultant 729 experiments were statistically

modelled from the established Design Space and

demonstrated that the critical resolution factor of

2.0 can be achieved in all cases.

Additionally, in order to address the

robustness issue, some practioners across

the industry have also advocated the use of

pre-defined rugged stationary phases (rather

than random choice), and by first intent only

developing methods using these columns.



Foreword

Dave ElderJoint Pharmaceutical Analysis Group and GSK

ICHQ2(R1) Validation of Analytical

Procedures: Challengesand Opportunities

The International Conference on Harmonisation (ICH) guideline for the Validation of Analytical Procedures (ICHQ2(R1)) currently

covers validation procedures for the four most common analytical tests: identification tests, quantitative tests for impurities, limit

tests for the control of impurities and quantitative tests for the active moiety(ies) in APIs (active pharmaceutical ingredients) or

drug products. The key underlying concepts and strategies are equally applicable to other analytical methodologies; e.g. particle

size analysis, dissolution, etc. Typical validation parameters covered in the guideline include accuracy, precision, specificity,

detection limits (DL / LOD) and quantitation limits (QL / LOQ), linearity, range and robustness.

1. Concept paper on the revision of chapter 6 of the

EU GMP Guide Quality Control, 12th October 2010,

EMA/INS/GMP/632654/2010 http://www.ema.

europa.eu/docs/en_GB/document_library/

Regulatory_and_procedural_guideline/2010/11/

WC500099260.pdf Assessed on 07th May 2013

2. I.Molnár. Searching for robust HPLC methods –

Csaba Horvarth and the Solvophobic theory,

Chromatographia, 62 (2005) S7-S17

3. A.H.Scmidt, I.Molnár. Using an innovative Quality-

by-Design approach for the development of a

stability indicating UPLC method for ebastine in the

API and pharmaceutical formulations, J. Pharm.

Biomed. Anal., 78-79 (2013) 65-74

Reference


Volume 18 | Issue 4 | 2013 8

Sanofi appoints Carsten Hellmann and David Meeker tothe Executive CommitteeSanofi has announced the appointment of two

new members to the Executive Committee,

effective 1 September 2013. Carsten Hellmann

joins Sanofi from Chr. Hansen Holding A/S and

will take the position of Executive Vice President

Merial, our Animal Health Division, following

the decision of Jose Barella to leave the company

to pursue other opportunities. He will be based in

Lyon and will sit on the Executive Committee.

David Meeker, MD, currently CEO Genzyme

and member of the Global Leadership Team, will

join the Executive Committee as Executive Vice

President, Genzyme.

In his previous role, Carsten was Executive

Vice President, Global Sales, Chr. Hansen

Holding A/S, a position he took upon joining

the company in 2006. Chr. Hansen Holding A/S

is a global biopharmaceutical company that

specialises in natural ingredient solutions for

the food, nutritional, pharmaceutical and agri -

cultural industries.

Merial, the Animal Health Division of

Sanofi, is the world’s third-largest animal

health company and the leader in companion

animal products, rabies vaccines and foot-and-

mouth vaccines with sales of more than

two billion Euros. A fully-fledged member of

the Sanofi family since 2011, there are a number

of significant opportunities for Merial that

remain untapped, particularly in production

animals and emerging markets. As Head of

Merial, Carsten will be tasked with bringing the

opportunities to fruition.

David Meeker was appointed CEO,

Genzyme, in November 2011 following

the acquisition of Genzyme by the Sanofi

Group in February 2011. Under his leader-

ship, a new organisation was put in place

maximising Genzyme’s unique expertise and

know-how, while leveraging Sanofi’s scale

and capabilities. The new organisation incorp-

orates the Rare Disease business and the

Multiple Sclerosis franchise as well as associated

R&D, manufacturing and support functions.

With two new multiple sclerosis medicines,

Aubagio® and LemtradaTM, recently receiving

positive opinions from the European Medicines

Agency (EMA), the business is set on a trajectory

of growth.

www.sanofi.com

AstraZeneca and FibroGen collaborate to develop andcommercialise FG-4592, a treatment for anaemia inchronic kidney disease and end-stage renal diseaseAstraZeneca and FibroGen have announced that they have entered into a

strategic collaboration to develop and commercialise FG-4592, a first-in-

class oral compound in late stage development for the treatment of

anaemia associated with chronic kidney disease (CKD) and end-stage

renal disease (ESRD).

This broad collaboration focuses on the US, China and all

major markets excluding Japan, Europe, the Commonwealth of

Independent States, the Middle East and South Africa, which are covered

by an existing agreement between FibroGen and Astellas Pharma Inc.

The AstraZeneca-FibroGen joint effort will be focused on the

development of FG-4592 to treat anaemia in CKD and ESRD, and may be

extended to other anaemia indications.

FG-4592 is a small molecule inhibitor of hypoxia-inducible factor

(HIF), a protein that responds to oxygen changes in the cellular

environment and meets the body’s demands for oxygen by inducing

erythropoiesis; the process by which red blood cells are produced.

FG-4592 has the potential to address the considerable unmet medical need

for an effective treatment for anaemia that offers the convenience of oral

administration and an improved safety profile as compared to current

standards of care. At present, treatment options involve a combination

of injectable erythropoiesis-stimulating agents (ESAs) and iron

supplements. FG-4592 works through the body’s natural oxygen-sensing

and response system to help produce red blood cells. This can be

compared to the body’s natural response to conditions at high altitude,

where oxygen levels are low, which is to produce more red blood cells.

In Phase II clinical studies, FG 4592 met its primary objective of

demonstrating anaemia correction in treatment-naïve CKD patients not on

dialysis as well as maintenance of haemoglobin levels and anaemia

correction in patients on dialysis. FG 4592 has demonstrated this efficacy

combined with an acceptable safety profile in clinical trials, and has

been shown to achieve anaemia correction in the absence of intravenous

iron supplementation.

The companies plan to undertake an extensive FG-4592 Phase III

development programme for the US, and to initiate Phase III trials in

China, with anticipated regulatory filings in China in 2015 and in the

US in 2017. www.astrazeneca.com

Albiglutide USPDUFA dateextended bythree monthsGlaxoSmithKline plc has announced

that the US Prescription Drug User Fee

Act (PDUFA) goal date for albiglutide,

an investigational once-weekly treat-

ment for adult patients with Type 2

diabetes, has been extended by three

months to 15 April 2014 to provide time for

a full review of information submitted by

GSK in response to the Food and Drug

Administration’s requests.

GSK announced the submission of a

Biologics Licence Application to the US

FDA for albiglutide on 14 January 2013

and this was followed by the submission of

a Marketing Authorisation Application to

the European Medicines Agency (EMA) on

7 March 2013. The EMA filing is pro -

gressing to schedule. Albiglutide is not

approved for use anywhere in the world.

www.gsk.com

“Physical and chemical properties of materials are generally determinedin laboratories. How can these measurements be best employed topredict material performance at manufacturing scale? What are the gapsbetween academic knowledge and manufacturing practice?”John Comer, Chief Scientific Officer, Sirius Analytical Instruments Ltd: Among the

steps involved in pharmaceutical manufacturing, the more important include

sourcing raw materials, synthesising APIs, preparing formulations, manufacturing

tablets and packaging. Measured physical and chemical properties of materials play

a part in all these steps. Properties of raw materials must be checked to confirm their

quality. Industrial-scale synthesis must be optimised by chemical engineering

calculations involving measured values for physical and chemical properties of the materials.

As for formulation, once the formulation approach has been selected, the process must be scaled

up and again, calculations involving measured properties are of great value. Tablet manufacture

and packaging are highly automated but property measurement is still important, not least for

quality control and investigating the stability of a drug under storage conditions. There are

many gaps between academic knowledge and manufacturing practice, so I’ll focus on just one area,

the behaviour of weak acids and bases in solvents. This is an issue during process development

if solvents are to be used. Many APIs and molecular building blocks are ionisable molecules, and

gain or lose protons in response to changing pH. The pKa value expresses the pH at which half

the molecules in solution are charged and half are neutral. Scientists measure the aqueous pKa

value of APIs because it influences ADMET and pharmacokinetics. However, values change

considerably when drugs are dissolved in solvents. For example, the pKa of Ketoprofen, an

anti-inflammatory drug is 4.12 in water, but rises to 11.45 in pure isopropyl alcohol. Such

huge changes mean that aqueous pKa values cannot be used to optimise synthesis procedures.

Academics have demonstrated ways to measure pKa in solvents but the characterisation of each new

solvent involves a lot of experimental work. Industry could profit from better-measured values, but

only after more research is done.

“How are rapid and alternative microbiological methods going toreshape the industry's strategies for microbiology testing?”David Jones, Technical Services Director, Rapid Micro Biosystems: Three trends

seem to be appearing in the QC microbiology area. Test frequency and time to result

is changing; testing is becoming decentralised from the QC laboratory; and the tests

are becoming less technical to perform. Risk analysis is focusing test frequency on

the critical areas of the process while lower risk areas are tested less frequently.

Traditional methods tended to be a single time point taken during the production

cycle. New technologies and modified methods change this. Active air samplers can now take small

samples spread over the full production day to improve the data from the traditional method;

however, the traditional incubation is still required. New technologies improve on that to allow

continuous sampling of air throughout the production day and give real time data. Similar technology

can be used for water testing to continuously monitor the bioburden. Technologies to identify

contaminants are also becoming much faster with real time identification on single cells in under

15 minutes being possible.

PAT initiatives have driven testing closer to the production line. An automated active air sampler

for viable organisms can be attached to the filling line or cleanroom structure to test and generate a

result at the point of use. Small portable endotoxin test machines are available that allow a test to be

performed at site and give a result in 15 minutes. Automated testing equipment can now be placed in

the manufacturing area, to take the samples and perform the analysis without the need to transfer

samples to the QC lab. With the move to the line and the introduction of more automation,

test methods are becoming simpler to perform; machines interpret and report the data. As a result,

the QC staff doesn’t need a microbiologist to perform daily tests; rather, microbiologists can

concentrate on the critical tasks of interpreting the data and problem solving.

This edition of European Pharmaceutical Review’s Industry Expert Panel delves into

the gap between academic research and manufacturing practice, and reshaping

microbial testing strategies.



Industry Expert Panel

Paul WituschekVice President of Sales, Developmentand Clinical ServicesCatalent

Katherine BakeevDirector of Applications Support

B&W Tek, Inc

Tim FreemanManaging Director

Freeman Technology

Fredrik SundbergGlobal Director

GE Healthcare

Ian LewisMarketing ManagerKaiser Optical Systems

Joe GecseyLife Science Application Manager,HACH Particle Counter UnitHACH LANG

Harald StahlSenior Pharmaceutical TechnologistGEA Pharma Systems

Laurent LeblancPharmaceutical and CosmeticsCulture Media R&DbioMérieux Industry

Allen L. BurgensonManager, Regulatory Affairs

Lonza Walkersville, Inc.

John DubczakGeneral Manager,

Endotoxin and Microbial Detection

Charles River


Volume 18 | Issue 4 | 2013 10

September 2013

ELRIG Drug DiscoveryDate: 3 – 4 September 2013Location: Manchester, UK

e: [email protected]

w: www.elrig.org

EPR Workshop: Label-free assays for screeningDate: 10 – 12 September 2013Location: Hamburg, Germany


w: www.europeanpharmaceutical

review.com/workshops-events/

label-free-assays-september-2013

HUPODate: 14 – 18 September 2013Location: Yokohama, Japan


w: http://hupo.org/2013

Advanced Analytics for Therapeutic Proteins:from Research toManufacturingDate: 18 – 20 September 2013Location: Kloster Irsee, Germany


w: http://events.dechema.de/

analytics2013

3rd Conference Innovation Drug DiscoveryDate: 22 – 25 September 2013Location: Pisa, Italy


w: www.apgi.org/Pise2013

2nd InternationalConference and Exhibition on Biowaivers & BiosimilarsDate: 23 – 25 September 2013Location: Carolina, USA


w: www.omicsgroup.com/

conferences/biowaivers-biosimilars-

2013/index.php

12th Annual BiologicalProduction Forum 2013Date: 23 – 25 September 2013Location: Dusseldorf, Germany


w: www.biological

production.com?mc=EL

Cell Culture Asia Congress 2013Date: 24 – 25 September 2013Location: Singapore


w: www.cellcultureasia-

congress1.com/download-

agenda-marketing

MiptecDate: 24 – 26 September 2013Location: Basel, Switzerland


w: www.miptec.ch

The Future of European PharmaDate: 30 September – 1 October 2013Location: Brussels, Belgium


w: www.ispe.org/2013-flexible-

facilities-eu-conference

October 2013

BiotechnicaDate: 8 – 10 October 2013Location: Hannover, Germany


w: www.biotechnica.de

PDA 8th Annual Global Conference on PharmaceuticalMicrobiologyDate: 21 – 23 October 2013Location: Maryland, USA


w: www.pda.org

CPhL WorldwideDate: 22 – 24 October 2013Location: Frankfurt, Germany


w: www.cphi.com

November 2013

PEGS EuropeDate: 4 – 8 November 2013Location: Lisbon, Portugal


w: www.pegsummiteurope.com

Next GenerationSequencing Congress Date: 18 – 19 November 2013Location: London, UK


w: www.nextgenerationsequencing-

congress.com

World DrugManufacturing SummitDate: 25 – 27 November 2013Location: Dusseldorf, Germany


w: www.wdmsummit.com

December 2013

FT Global Pharmaceuticals andBiotechnology ConferenceDate: 3 – 4 December 2013Location: London, UK


w: www.ft-live.com/pharmabio

January 2014

SLAS 2014Date: 18 – 22 January 2014Location: California, USA


w: www.slas2014.org

PepTalkDate: 13 – 17 January 2014Location: California, USA


w: www.chi-peptalk.com

IFPACDate: 21 – 24 January 2014Location: Washington DC, USA


w: www.ifpac.com

PharmaEVENTS

+

+

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Register now at www.europeanpharmaceuticalreview.com/handheld-raman

Discover the benefits and applications of handheld Raman and how it can facilitatecompliance with PIC/S regulations for raw material identification.

This webinar will discuss the benefits and applications of handheld Raman, including thereasons the industry is interested in the technology, data integrity, method development, howsamples are scanned and the challenges that have been conquered. We’ll also discuss PIC/Sand how it is unifying the regulatory perspective globally, currently including 43 membercountries with applications of Brazil, Japan, South Korea and the United Kingdom under review.We’ll discuss how these regulations are moving towards 100 per cent container testing globallyand how handheld Raman technology can help to achieve it, as well as the importance ofvalidation of instrumentation and methodologies.

Supported by

Organised by:

Advantages of handheld Raman in thepharmaceutical industry

A European Pharmaceutical Review Date:Tuesday, 24 September 2013

Time:15.00 BST

Length:1 Hour

Sulaf AssiAssociate Lecturer in ForensicSciences, Bournemouth University

Presentation: Handheld Ramanspectroscopy for identification ofonline drug products

Katherine BakeevDirector of Analytical Services and Support, B&W Tek

Presentation: Impact of handheld Raman for meeting PIC/S requirements

SPEAKERS:

G protein-coupled receptors (GPCRs) are among

the most important target classes within the

pharmaceutical industry1. Of the currently

marketed small-molecule medicines, approxi -

mately 30 per cent target GPCRs2,3. Historically,

ligand interactions with GPCRs have been

analysed using binding assays providing both

affinity and kinetic data. This technology did not

however take into account functional aspects of

the ligands (for example agonism, inverse

agonism, allosteric effects or signalling path -

way). Many downstream signalling assays like

detection of cAMP, IP3, Ca2+-flux, β-arrestin

recruitment or ERK1/2 phosphorylation have

therefore been applied extensively, expand-

ing the knowledge of functional aspects of

ligands4 (Figure 1, page 14). These assays have

also made it possible to search for new drugs on

orphan receptors. The expanding knowledge

around signalling through different G-proteins,

G-protein independent signalling and signal

bias, have pinpointed the limitations with assays

based on detecting specific intracellular mess -

engers along one single pathway5. However

more recently, label-free biosensor tech-

nologies such as surface plasmon resonance

(SPR), resonant waveguide grating (RWG) and

cell impedance spectroscopy (CIS) have been

developed and applied in drug discovery,

providing an opportunity for the revelation

of pharmacology of greater physiological

and disease relevance than before, including

simultaneous capturing of signalling via

multiple pathways6.

In this review, we focus on the application of

RWG for measurements of DMR in intact cells

upon GPCR activation. DMR is a consequence of

morphological changes and changes in distri -

bution of cellular components after receptor

stimulation. This type of readout is still

somewhat of a black box, but consists of

elements such as protein recruitment, receptor

internalisation, reorganisation of the cyto -

skeleton, as well as altered cell adhesion

(Figure 2, page 15). Activation of all four major

GPCR coupling classes (Gαi/Gαo, Gαs, Gαq/Gα11

and Gα12/Gα13) can be captured with this

technology7, something which is unachievable

with most other platforms. Initial studies

suggested that signalling via different

G-proteins result in class-specific DMR kinetic

profiles8. Subsequent studies have however

revealed that kinetic signatures are cell type

specific and the picture is therefore more

G protein-coupled receptors are one of the major classes of therapeutic targets for a broad range of diseases. The most commonly

used assays in GPCR drug discovery measure production of second messengers such as cAMP or IP3 that are the result of activation

of individual signalling pathways. Such specific assays are unable to provide a holistic view of the cell response after GPCR

activation. This is now changing as label-free technologies and assays on whole cells have been developed that are unbiased

towards the specific downstream pathways and capture the integrated cell response. In this mini-review, we focus on the

application of one of these technologies, namely resonant waveguide grating (RWG) for measurements of dynamic mass

redistribution (DMR) in intact cells upon GPCR activation. Since the technology is sensitive and non-invasive, it is applicable to

most cell types, including primary cells with native receptor expression levels. We discuss how DMR assays have become an

important component of GPCR drug discovery screening cascades and may have the potential to improve the ability to predict if

compounds will be efficacious in vivo.

Cell based label-free assays in GPCR drug discovery

Niklas Larsson, Linda Sundström, Erik Ryberg and Lovisa Frostne

AstraZeneca



GPCRs

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complex than initially suggested. Nonetheless,

DMR provides a G-protein unbiased and path -

way sensitive technology as a powerful

complement to single pathway technologies in

the GPCR assay toolbox.

Examples of applications in the

drug discovery process

We and others have applied cell based label-free

DMR GPCR assays for different purposes in

various phases of early drug discovery (Figure 3,

page 16).

Hit finding

Ligands can be biased with respect to signalling

via different pathways, thereby making the

pharmacology observed highly dependent on

the assay selected9. The potential benefit of

capturing signalling via multiple pathways and

thereby cover compounds with biased

signalling makes DMR an attractive choice for

identification of novel chemical starting points.

However, only a limited number of large DMR

screens have been published so far, likely due to

the relatively recent introduction of the

technology, high plate costs and throughput

challenges including detection of slow

responses using kinetic reading. One published

example is a 100k screen for muscarinic M3

antagonists10. In this screen, a low hit rate

consistent with many other HTS formats was

observed. The output did not suffer from a high

rate of false positives. Most importantly, unique

hits that were not picked up in a corresponding

Ca2+ FLIPR® screen were identified and con -

firmed in a binding assay. Thus, this example

illustrates the potential of DMR to successfully

deliver a relatively clean but unbiased HTS

output including chemistry which is not

captured by single pathway technologies.

Iterative screening

Our own most common application of DMR is as

an integral part of screening cascades within the

lead generation phase, either as the primary

structure-activity-relationship (SAR) driving

assay or as an orthogonal assay. We define an

orthogonal assay as an assay that measures

activity on the same target as the SAR driving

assay but with an independent technology,

which is applied to strengthen confidence in

potency / efficacy measures from the SAR assay.

In this context, DMR assays are of particular

value where other screening friendly assay

alternatives are limited, for example, for GPCRs

such as GPR55 which signals via the Gα12/13

pathway7, for receptors lacking appropriate

radioligands, or for antagonist screens on Gαi

coupled receptors. In addition, DMR assays are

often technically straightforward to establish.

Some concerns about the biological validity

of DMR assays to drive SAR have been raised due

to the undefined / black box nature of the

readout. However, this can be mitigated by

control experiments to confirm target depend -

ence such as demonstrating absence of

compound effects in comparable cell lines

lacking the target. It is also crucial to establish

early correlations between DMR and pathways

specific or binding assay platforms to interro -

gate the chemical series of interest with respect

to potential signal bias. Such a correlation, which

is a prerequisite for application in screening

cascades, has been established both for

overexpressed and endogenously expressed

receptors and is our own experience as

exemplified by the bradykinin B1 receptor

(Figure 3B, page 16).

Translation to biologically relevant

cell models

It is often a key challenge within early drug

discovery projects to establish a link between

assays using recombinant reagents and more

biologically or disease relevant cell models.

For example, overexpression of receptors can

shift both the potency and efficacy of com -

pounds (often overestimate) due to spare

receptors, thus not reflecting the pharmacology

in a native tissue11. Moreover, native tissues also

ensure the right G-protein composition and

expression relative to receptor levels, and this

may even be tissue-specific. Switches in

G-protein coupling have frequently been seen

for receptors expressed in different cell lines,

but are not limited to engineered cells12.

For example, endogenous dopamine D1/D2

receptor responses couples to different

G-proteins in different cell types12 highlighting

the import ance of selecting disease relevant

cells also for in vitro screening.

The high sensitivity of DMR assays enable

measurements of signalling after activation of

endogenously expressed GPCRs. Most import -

antly, since DMR assays do not require genetic

manipulation of cells, even primary cells

from patients can potentially be employed.

An example using non-engineered cells is a


Volume 18 | Issue 4 | 2013 14

GPCRs

Figure 1: GPCR signaling pathways

Simplified illustration of ligand-receptor binding and activation of signaling pathways by different G-proteins.Binding of ligands to the receptor, exchange of GTP to GDP and phosphorylation followed by β-arrestin recruitmentis fundamental and applies to all G protein families whereas other processes are G protein specific. Commonly usedread-outs for studying GPCR activation are indicated by blue, italics; GTPγS, cAMP, Ca2+, IP1, β-arrestin and binding,all of which are based on technologies using labels (e.g engineered cell lines or assay reagents) for detection. DMRis a label-free technology covering all activities that lead to a cell response and can be applied on intact native cells

‘‘The high sensitivity of DMR assaysenable measurements of signalling

after activation of endogenouslyexpressed GPCRs’’

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1280 compound screen using human epider -

moid carcinoma A431 cells endogenously

expressing histamine H1 and β2 adrenergic

receptors13. With respect to cells from native

tissue, it has also been shown that DMR has the

potential to detect prostaglandin E1 (PGE1)

activation of EP2/EP4 prostanoid receptors in

primary human keratinocytes7. In our own lab,

we have shown that activation of Bradykinin B1

receptors (B1R) can be monitored in primary

human preadipocytes and that the DMR

response is target mediated as it can be reduced

by knockdown of B1R using siRNA, demon -

strating specificity of the response (Figure 3C,

page 16). In summary, DMR assays commonly

have sufficient capacity and sensitivity to

provide a relatively high throughput link

between assays on recombinant reagents and

disease models to ensure relevant potency,

efficacy and coupling mechanisms of hit or lead

series early on.

Molecular mechanisms of action studies

Molecular mechanism of action (MMOA) is

defined here as the interaction between a

compound and its target, resulting in a specific

response3. These specific molecular interactions

link structure to function to provide a bio -

logically meaningful response. MMOA studies

are important for selection of functionally

relevant compound series and are generally

performed on small compound sets during late

lead generation and lead optimisation.

A key application in this context is

characterisation of signalling pathways in

physiologically relevant cells. The relative

contribution of different pathways can be

addressed by co-treatment with G-protein /

pathway specific blockers such as pertussis

toxin (Gαi), cholera toxin (Gαs) and YM254890

(Gαq)7 with subsequent measurement of the

remaining signal after agonist stimulation.

When inhibitors are not available, dominant

negative G-protein forms or siRNA can be used

as alternative tools7. Bock e t a l have taken

mechanistic studies one step further by taking

GPCRs

Figure 2: The principle of Dynamic Mass Redistribution (DMR)

Receptor activation results in numerous cellular processes ultimately leading to mass redistribution within the cell.The DMR causes a change in refractive index which can be measured as a change in wavelength (picometer range)using resonant waveguide grating. Available platforms for detection of DMR are the Epic®, Enspire® (with Epicmodule) and SRU Bind® readers. A) Baseline measurement prior to agonist stimulation. B) Measurement afteragonist stimulation, capturing the DMR

advantage of DMR and dualsteric ligands (i.e.

an orthosteric ligand physically linked to an

allosteric modulator) for elucidating the

dependence of promiscuous G-protein activa -

tion upon conformational rearrangements

within the extracellular domain14.

With respect to other MMOA applications,

it is technically possible to assess conforma-

tional mechanisms (e.g. surmountable /

insurmountable, competitive / non-competitive

compounds) but here DMR assays do not offer

any obvious benefits compared to classical

techniques. In contrast, for determination of

efficacy, DMR assays have the potential to

provide relevant measures over the range

from full agonists via partial agonists to

inverse agonists12. This is of particular value in

cases where it is possible to measure on

native(like) cells with endogenous receptor

expression levels.

Concluding remarks

Plate-based label-free technologies like RWG

that can be employed for cell based applications

such as DMR assays have been available for drug

discovery for approximately five years and the

number of applications is still growing. Since

the technology is sensitive, non-invasive and

able to detect signalling via multiple pathways,

it can be applied on disease relevant cell

systems. These properties offer the possibility to

use the same type of readout for hit finding,

iterative screening, (including species and target

selectivity), and disease relevant cell models and

may help to improve the ability to predict if

compounds will be efficacious in vivo.

In the future, we expect label-free cell based

assays with sufficient throughput for early drug

discovery to be commonly applied in com -

pound screening on disease relevant cells,

generating valuable chemical starting points to

be developed into tomorrow’s medicines.


Volume 18 | Issue 4 | 2013 16

GPCRs

Dr. Niklas Larsson obtained his PhD 1999

in Cell and Molecular Biology at Umeå

University, Sweden. Since 2000, he has been

working with early drug discovery projects at

AstraZeneca (Mölndal, Sweden) in different

positions such as Project Leader, Team Leader

and currently as Associate Principle Scientist

within the Discovery Sciences function. His scientific focus is drug

discovery and molecular pharmacology of GPCRs.

Linda Sundström has over 10 years’

experience in GPCR targeted drug

discovery. She received an MSc in Chemical

Engineering from Lund Institute of

Technology, Sweden during which she

completed a thesis work at the Institute of

Biotechnology, University of Cambridge, UK.

She is currently an Associate Principal Scientist at AstraZeneca

Discovery Sciences, Sweden where her primary focus is on GPCR

pharmacology and applications of cellular assays on GPCR targets in

early drug discovery.

Dr. Erik Ryberg obtained his PhD in

Biomedical Science at the University of

Aberdeen, UK in 2009. He has been working

with GPCR related drug discovery at

AstraZeneca for over 10 years in different

positions. He is currently an Associate

Principal Scientist within the Cardiovascular

and Metabolic Diseases function with primary focus on molecular

pharmacology of GPCRs in different phases of drug discovery.

Lovisa Frostne joined AstraZeneca

(Mölndal, Sweden) in 2003 after obtaining

her MSc in Molecular Biology at the

University of Gothenburg, Sweden. She

completed her thesis work at the Institute for

Molecular Bioscience at the University of

Queensland, Australia. During her 10 years in

AstraZeneca, her work has been focused around GPCR pharmacology

in early drug discovery. She now holds a position as Team Leader in

the Bioscience department within the Respiratory, Inflammation and

Autoimmune iMed.

Biographies

1. Ma P, Zemmel R. Value of novelty? Nat Rev Drug

Discov. 2002 Aug;1(8):571-2.

2. Hopkins AL, Groom CR. The druggable genome. Nat

Rev Drug Discov. 2002 Sep;1(9):727-30

3. Swinney DC, Anthony J. How were new medicines

discovered? Nat Rev Drug Discov. 2011 Jun

24;10(7):507-19

4. Zhang R, Xie X. Tools for GPCR drug discovery. Acta

Pharmacol Sin. 2012 Mar;33(3):372-84

5. Whalen EJ, Rajagopal S, Lefkowitz RJ. Therapeutic

potential of beta-arrestin- and G protein-biased

agonists. Trends Mol Med. 2011 Mar;17(3):126-39

6. Cooper MA, Halai R. What is label-free screening and

why use it in drug discovery? European Pharma -

ceutical Review. 2012;17(6):51-3

7. Schroder R, Janssen N, Schmidt J, Kebig A, Merten N,

Hennen S, et al. Deconvolution of complex G protein-

coupled receptor signaling in live cells using dynamic

mass redistribution measurements. Nat Biotechnol.

2010 Sep;28(9):943-9

8. Fang Y, Li G, Ferrie AM. Non-invasive optical biosensor

for assaying endogenous G protein-coupled receptors

in adherent cells. J Pharmacol Toxicol Methods. 2007

May-Jun;55(3):314-22

9. Kenakin TP. Cellular assays as portals to seven-

transmembrane receptor-based drug discovery. Nat

Rev Drug Discov. 2009 Aug;8(8):617-26

10. Dodgson K, Gedge L, Murray DC, Coldwell M. A 100K

well screen for a muscarinic receptor using the epic

label-free system--a reflection on the benefits of the

label-free approach to screening seven-trans -

membrane receptors. J Recept Signal Transduct Res.

2009;29(3-4):163-72

11. Kenakin T, Christopoulos A. Analytical pharmacology:

The impact of numbers on pharmacology. Trends

Pharmacol Sci. 2011 Apr;32(4):189-96

12. Peters MF, Vaillancourt F, Heroux M, Valiquette M, Scott

CW. Comparing label-free biosensors for pharma -

cological screening with cell-based functional assays.

Assay Drug Dev Technol. 2010 Apr;8(2):219-27

13. Tran E, Ye F. Duplexed label-free G protein--coupled

receptor assays for high-throughput screening.

J Biomol Screen. 2008 Dec;13(10):975-85

14. Bock A, Merten N, Schrage R, Dallanoce C, Batz J,

Klockner J, et al. The allosteric vestibule of a seven

transmembrane helical receptor controls G-protein

coupling. Nat Commun. 2012;3:1044

References

Figure 3: Application of DMR GPCR assays in the drug discovery process

A) Phases in the drug discovery process where DMR assays can be applied to GPCR targets and typical number ofcompounds screened / characterised. B) pIC50 correlation between a DMR antagonist and a radioligand bindingassay using HEK293 cells transfected with the bradykinin B1 receptor. DMR was measured with an Epic® reader fromCorning. C) Activation of the bradykinin B1 receptor in primary human preadipocytes. Concentration responsesafter addition of the B1 agonist Des-Arg10Kallidin to preadipocytes. Cells were pretreated with control siRNA orsiRNA to knockdown the bradykinin B1 receptor

‘‘DMR assays have the potential toprovide relevant measures over therange from full agonists via partial

agonists to inverse agonists’’

The first practical steps are the selection

synthesis, management and subsequent

screening of molecular libraries, either small

molecule or biological in origin. The second

critical area is the selection, development

and prosecution of bio-assays for primary

hit identification, validation and profiling.

In the context of drug discovery projects,

hits are the further optimised using multiple

criteria including structure activity relation-

ships, selectivity, physicochemical properties

and liability. Automation is a key enabler to

increase productivity, particularly in structural

based approaches to hit identification and

validation, for example, x-ray crystallography

and NMR spectroscopy.

The Practical workshop: Chemical biology,

drug discovery and screening is designed for

scientists at all levels (undergraduates, post -

graduates and laboratory based scientists within

academic and industrial research organisations)

engaged in early stage drug discovery who have

an interest in the development, validation and

utilisation of cell based assays for screening

against small molecule libraries. The Practical

workshop: Chemical biology, drug discovery

and screening is equally well suited to

technically focused staff from core facilities or

contract research organisations who may wish

to extend their expertise. The evening dinner on

the first day will offer the opportunity for the

participants to network and establish relation -

ships that would be mutually beneficial.

All participants will take part in the practical

sessions and these will involve the development

of screening compatible assays, pharmacology of

standard compounds and proof-of-concept

screen. Participants in this workshop will discuss

and demonstrate practically: (1) the appropriate

steps in selecting suitable assays in light of the

fact that a multitude of assay technologies are

currently available for a given target; (2) how to

select an appropriate technology; which criteria

should be examined during the early stage

chemical biology and drug discovery processes;

(3) whether a generic, flexible set of assay

methodologies or customised solutions should

be applied to the targets being investigated.

The specific aspects of the lectures will

cover general concepts in chemical biology and

drug discovery, the role of biochemical

and cell based assays for drug discovery

purposes, their advantages and disadvant-

ages and how to incorporate them into a drug

discovery workflow.

Workshop topics

The Practical workshop: Chemical biology,

drug discovery and screening will include the

following laboratory sessions:

1. General concepts for biochemical assays

exemplified by kinase and protease targets

2. Pharmacology of standard compounds,

signal stability, choice of liquid handling,

Z′ calculation and proof-of-concept screen

3. Cell viability assays

4. Label-free cell-based assay for GPCR targets

5. Pseudo-label-free kinase assays

The emergence of chemical biology has coincided with increasing numbers of exploratory molecular targets and mechanisms,

both therapeutic and non-therapeutic in origin. Screening using miniaturised microtitre plate format based assays remains the

most widely utilised methodology for identifying novel chemical matter capable of modulating target function in a meaningful,

biologically relevant manner.

Practical workshop: Chemical biology, drugdiscovery and screening



WORKSHOPPREVIEW Date: 23 – 25 October 2013 · Location: European ScreeningPort GmbH, Germany

Organised by: Hosted by:

Sheraz Gul is Head of Biology at the

European ScreeningPort, Hamburg, Germany

where he manages the assay development

and screening of academic targets. Prior to

this, he worked for GlaxoSmithKline for seven

years where he developed biochemical and

cellular assays for high throughput screening

as well as hit characterisation. In addition he has worked in academia

for five years on proteases and kinases. He is the co-author of the

Enzyme Assays: Essential Data Handbook. He is also involved in many

European drug discovery Initiatives involving government, the

pharmaceutical industry and academia (e.g. EU Framework 7 and

IMI). His research interests are directed towards maximising the

impact of HTS for drug discovery.

[email protected]

About the coordinator

The Practical workshop: Chemical

biology, drug discovery and screening

is approved by the Society of Biology for

purposes of Continuing Professional

Development (CPD) and may be counted

as 72 CPD credits if registered on the

Society of Biology CPD Scheme.

SPONSORS

To register your interest in attending, please visit www.europeanpharmaceuticalreview.com/workshop

EARLY BIRD OFFER

www.europeanpharmaceuticalreview.com/workshop

Register by 13 September 2013

to receive 20% DISCOUNT

This satisfies virtually every case from the

classroom to the research lab until we consider

chloride channels. We know that they exist, are

important and might make good drug targets in

several disease areas, but until recently, they

have remained somewhat stigmatised and

unfashionable in the world of therapeutics.

The lack of selective ligands has not helped

at all. The only exception to this has been the

GABA-A receptor that is coupled to an

intrinsic chloride channel that opens upon

binding of the inhibitory neurotransmitter

GABA. Benzodiazepines have been potentiating

this receptor ever since Valium became available

in the 1960s, providing sedative and anti -

convulsant effects. This introduces an important

concept: there are several gene families and

different protein types that can be described as

being chloride channels. This is in contrast to

what we understand of cation-selective ion

channels where there is little flexibility in the

protein structure that can form a pore that is

selective for potassium, sodium or calcium; the

diversity of these ion channels is brought about

by the variations in the protein domains distinct

from the pore that influence the opening-

closing behaviour. To be fair, there do not seem

to be many physiological reasons why an anion

channel should exhibit chloride-selectivity as

there is little physiological role in the membrane

transport of other halides or small anions; any

anion channel will by default be a chloride

channel from a physiological viewpoint. On the

other hand, potassium, sodium and calcium

membrane currents each play different

fundamental roles and selective membrane

Early in their undergraduate education, the student is introduced to various types of integral membrane protein: receptors,

adhesion proteins, ion channels, ion pumps and ion transporters. As they progress through their studies, they find out that

discrete gene families and protein structures are responsible for these different protein classes and there is never any reason to

consider that there might be any ambiguity in assigning any particular protein to its appropriate protein class.

Chloride ion channels and transporters: fromcuriosities of nature and source of human disease to drug targets

Jonathan D. Lippiat

School of Biomedical Sciences, University of Leeds

ION CHANNELS


Volume 18 | Issue 4 | 2013 18

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com

‘‘GABA-A receptors are members of the cys-loop pentameric ligand-gated

ion channel families’’

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permeability to these cations is crucial to cellular

function and indeed life itself.

GABA-A receptors are members of the cys-

loop pentameric ligand-gated ion channel

families, which include several types of

excitatory and inhibitory neurotransmitter-

gated channels. The cystic fibrosis trans -

membrane regulator (CFTR) protein is a

nucleotide-regulated chloride channel. It is

a member of the diverse ATP-binding cassette

(ABC) transporter family and is the only one that

does not seem to transport any substrate across

a membrane, but rather functions as a chloride

channel. The CLC family of voltage-gated

chloride channels arise from another distinct

gene family and have a general structure unique

among membrane proteins. This family of

proteins have brought about many surprises

over the last 30 years and members of this family

will be introduced here and also how they might

be considered drug targets.

Other exciting developments from the

chloride channel field have emerged following

the molecular identification of proteins

underlying calcium-activated chloride (ClCa)

channels. Functionally, we have known about

their physiological roles for some time, but

recent molecular identification of the channel

proteins has allowed development of molecular

and pharmacological tools to probe and alter

their activity. This review focuses on the

pharmacological potential of targeting ClCa

channels and CLC proteins; their function and

subcellular localisation are summarised in

Figure 1 (page 20).

An introduction to CLC proteins

The nine members of this protein family

are CLC-1 to 7, CLC-KA and KB. The founder

member, CLC-1, is the voltage-gated chloride

channel of skeletal muscle and will be discussed

below. Many functional studies have been

carried out on a homologue with the honorary

title of CLC-0, isolated from the E l ec t r o pl a x

electric organ, which enables the ray to stun its

aquatic victims. Reconstitution of CLC-0 into

lipid bilayers and electrophysiological recording

of currents flowing through single ion channels

revealed an intriguing property. Usually, when

recording current from a small patch of

membrane containing a single ion channel, one

observes fluctuations between two current

amplitudes: one representing the closed

channel and the other representing the current

flowing through the open channel. The

fluctuations reflect transitions between open

and closed kinetic states. With CLC-0, however, it

was evident that the single ion channel protein

consisted of two equivalent pores that could

open independently1. This channel was

therefore described as being d o u b l e -

bar r el l ed (Figure 2, page 21). It thus came of

no great surprise that the crystal structures of

homologues from enteric bacteria revealed that

the protein complex was a dimer, with each

subunit poss ess ing an ion-conducting pathway2

(Figure 2, page 21).

The second surprise arose when detailed

functional experiments were carried out on

CLC-ec1 from E . c o l i . Rather than functioning

as a chloride channel, like its long-lost relative in

vertebrate skeletal muscle, CLC-ec1 is a 2Cl-/H+

exchange transporter, or antiporter, with strict

2:1 exchange stoichiometry3. This protein is

important for the survival of enteric bacteria in

ION CHANNELS

‘‘The fluctuations reflect transitions between open and

closed kinetic states’’

low pH4 and is likely to exploit a chloride

gradient to keep cellular pH at a tolerable level.

Prokaryotic CLCs might therefore be targets for

inhibitors to treat pathogenic E. coli or

Salmonella infection4. The discovery of ion

exchange behaviour naturally stimulated

further exertions to determine whether any of

this chloride-for-proton exchange existed in

mammalian CLCs, on the assumption that Cl-/H+

exchange might be the true function of

archetypal CLCs and those identified as chloride

channels might just be exceptions to the rule.

This seems to be the case as there is strong

evidence that CLC-3 through to CLC-7 function

as 2Cl-/H+ exchange transporters, primarily

residing in intracellular organelles, whilst CLC-1,

CLC-2, KA and KB are true plasma membrane

chloride-conducting ion channels5-9. Because

the archetypal CLC property is exchange

transport, being found across all forms of cellular

life, the latter subclass of proteins, the bona fide

chloride channels, can therefore be considered

‘broken’ chloride transporters, which have lost

the coupling of chloride transport to the

movement of a second substrate ion.

CLC-1

This founder of the CLC family is the voltage-

gated chloride channel of skeletal muscle and

serves to regulate the membrane potential

and repolarise the membrane following action

potentials to relax the muscle. In most tissues,

we would expect potassium channels to play

this role, which they do in cardiac and smooth

muscle, as well as regulating membrane

excitability in other cell types. The transverse

tubule system is an extension to the plasma

membrane (sarcolemma) and penetrates into

the contractile tissue. It is critical for the rapid

spread of electrical activity throughout the

muscle and orchestrates rapid and controlled

contraction. Potassium efflux from the muscle

cells into the confined space of the t-tubule

would raise the extracellular potassium

concentration and collapse this ion gradient,

which would lead to prolonged membrane

depolarisation. With chloride channels playing

the predominant repolarising role, this is

prevented. Loss of function mutations in CLC-1

lead to myotonia in man, goats, and mice (for a

recent review see10) and is characterised by

impaired muscle relaxation, consistent with the

loss of a repolarising membrane current.

With expression confined to skeletal muscle,

CLC-1 might be an attractive target for drugs

that control muscle contraction by increasing or

decreasing CLC-1 function and thereby reduce

or increase muscle excitability, respectively.

Compounds that increase CLC-1 function might

be able to treat myotonia, particularly in cases

where they could compensate for partial loss of

chloride channel activity. Inhibition of CLC-1

could reduce the threshold for muscle con -

traction and may be useful in cases of muscle

weakness or degenerative diseases such as

muscular dystrophy.

CLC-2

The inwardly-rectifying chloride channel, CLC-2,

has somewhat widespread tissue distribution.

It can be found in central neurones where it

regulates neuronal activity11-13. In astrocytes, its

subcellular targeting to cell junctions is regu -

lated by an interaction with GlialCAM (MLC1),

mutations in which disrupt this targeting and

cause megalencephalic leukoencephalopathy14.

Recent therapeutic interests involve CLC-2

expression in gut mucosa and lung bronchioles

where it plays a role in intestinal and lung

secretions, respectively. Lubiprostone, a drug

used clinically to relieve constipation was

proposed to exert its effect by activating CLC-2,

but this effect is controversial. Whilst this

compound activates CLC-2 channels in some

studies, in others it regulates CLC-2 trafficking

and increases CFTR function via prostaglandin

receptor activation15,16. However, there remains

sufficient evidence that increasing airway CLC-2

function could provide an alternative chloride

pathway in cystic fibrosis17.

CLC-KA/KB

These chloride channels are notably expressed

in renal epithelia and contribute to the permea -

bility of cell membranes to passive chloride flux.

They have a key role in the ascending limb and

distal tubules of the nephron, providing a

basolateral route for chloride reabsorption,

following transport from the primary urine via

the apical membrane. The CLCKB gene, which

encodes CLC-KB, is one of five genes that

underlie Bartter’s syndrome. Loss-of-function

mutations in CLC-KB result in defective chloride

reabsorption and so leads to a salt-wasting

disorder, which is associated with polyuria. One

of the main features of individuals that are

affected by CLC-KB mutations (Bartter’s type III)

is low blood pressure. A more severe form of the

disease is caused by mutations in the BSND gene

(Bartter’s type IV), which encodes Barttin, an

accessory protein important for the trafficking of

both CLC-KA and KB to the plasma membrane18.

This form of the disorder also includes sensory

deafness, which is thought to be brought about

by the loss of trafficking of both CLC-KA and

KB to epithelial membranes in inner ear.

Presumably, loss of either CLC-KA or KB activity,

but not both, can be tolerated by the auditory

system. This suggests that inhibitors selective for

either CLC-KA or CLC-KB, or partial inhibition of

both, may act as a novel loop diuretic with the

potential to lower blood pressure and with few

side-effects. This has led to the study of their

pharmacology and the development of novel

inhibitor derivatives with low micromolar

affinity19, which had diuretic effects when

administered to rats20. On the other hand, drugs

that activate CLC-KB channels may enhance

residual activity of defective channels in Bartter’s

type III patients.

CLC-7

Of the 2Cl-/H+ exchange transporter subclass,


Volume 18 | Issue 4 | 2013 20

ION CHANNELS

Figure 1: Diversity and subcellular localisation of CLC proteins and selected calcium-activatedchloride channels

Of the CLC family CLC-1, 2, KA, and KB are plasmamembrane chloride channels, whilst CLC-3 to 7 residein organelle membranes and function as 2Cl-/H+

exchange transporters. Members of the Bestrophinand TMEM16 families are candidates for plasmamembrane calcium-activated chloride channels. In addition, Best1 also has a regulatory role in theendoplasmic reticulum. Chloride channels areindicated by ‘Cl-’ at either end of the double-headedarrow and 2Cl-/H+ exchange transport indicated bydouble-headed arrows with ‘2Cl-’ at one arrowhead and‘H+’ at the other

‘‘One of the main features of individualsthat are affected by CLC-KB mutations

(Bartter’s type III) is low blood pressure’’

CLC-7 is a promising target for drug action and

its inhibition may be beneficial in osteoporosis.

Once more, this indication takes its origin from

observations of human disease caused by

loss-of-function mutations in CLC-7, which

cause osteropetrosis21. In this disorder, bone

remodelling by osteoclasts is deficient, which is

thought to be caused by defective acid and

enzyme secretion. This leads to dense bone,

which if reproduced by a CLC-7 inhibitor could

reduce the dissolution of bone and therefore

strengthen the skeleton of osteoporosis

patients22,23. In proof-of-concept studies,

pharmacological inhibition of acidification24 or

disruption of CLC-7 function by antibodies25

reduced bone resorption. However, CLC-7

inhibition may not be without complica-

tions because human and mouse studies

suggest that lack of CLC-7 function may also

be associated with neuronal storage and

de generation disorders because of reduced

lysosomal function26.

Loss-of-function human mutations

– lessons from CFTR

It is worth a brief pause at this point to consider

that some of the therapeutic indications for

novel CLC-targeted drugs aim to treat disorders

distinct and at the opposite end of the spectrum

to those that are caused by defective CLC

function. In addition to those described above,

Dent’s I disease is an X-linked kidney disease that

is caused by loss of CLC-5 function (see27 for a

recent review). All are rare inherited disorders

and a major protein defect involves either

reduced protein activity or trafficking to the

target membrane. In fact, many individual

mutations cause ER-retention and a lack of

protein maturation. A parallel could be made

with cystic fibrosis, with most of the affected

individuals possessing the ER-retained ΔF508

mutation. Recent and stratified approaches to

treating cystic fibrosis have been the result of a

dual-pronged attack to correct mutant protein

folding (CF correctors) and/or increase the

activity of plasma membrane CFTR chloride

channels (CF potentiators). Success is emerging

with the potentiator class28, which are effective

in patients with mutations that reduce CFTR

activity without loss of protein biosynthesis or

trafficking (e.g. G551D), however the effective -

ness of folding correctors (which is required in

the majority of cases) remains to be estab -

lished29. Although the diseases associated with

loss of CLC function are all rare genetic disorders,

we may one day be able to treat individuals with

myotonia, Bartter’s syndrome, Dent’s disease

and osteopetrosis, as well as cystic fibrosis

with drugs that correct the actual cause of

their disorder.

Calcium-activated chloride channels

Finally, mention will be made of calcium-

activated chloride (ClCa) channels, which have

clearly-defined physiological roles in a number

of cell types, yet their molecular identification

suffered a few false and stuttered starts. The

story involves four types of protein: members of

CLCA, Bestrophin, Tweety and TMEM16 gene

families. In all cases, their recombinant over -

expression resulted in the generation of

membrane chloride currents that are stimulated

by raising the intracellular calcium concen -

tration and, to varying extents, membrane

depolarisation. The journey for CLCA came to an

end when it was found that this was a protein

that was secreted, but also likely upregulated

the membrane expression of ClCa channels

endogenous to the expression system30. Tweety

and Bestrophin proteins do not exhibit all of the

properties of ClCa channels studied in major

tissues, although Best1 appears responsible for a

component of ClCa in sensory neurons31 and

may also be an important regulator of calcium

release from the endoplasmic reticulum32,33.

Three independent studies proposed TMEM16A

(also called Ano1) as a candidate for a major

component, if not in its entirety, for a calcium-

activated chloride channel34-36. Many subsequent

studies, facilitated by the generation of

molecular tools, have supported this. TMEM16A

is important for regulating membrane

excitability in vascular smooth muscle, is

upregulated in an animal model of pulmonary

hypertension and tone can be reduced by

channel inhibition37,38. In sensory neurones,

TMEM16A couples the presence of inflamm -

atory mediators to membrane hyperexcitability

and TMEM16A inhibition has antinociceptive

effects39. In animal models of asthma, expression

of this particular chloride channel is increased,

and its inhibition may have beneficial effects40.

It is also found in interstitial cells of Cajal of

the intestine and channel function is required

for the rhythmical contraction of smooth muscle

in the intestinal wall41. Furthermore, TMEM16A

activation may provide an alternative pathway

for epithelial chloride secretion in cystic

fibrosis42. Whilst there is still some debate

surrounding the precise function of the rest

of the TMEM16 family, ClCa function has

been also been ascribed to TMEM16B

(Ano2), which is thought to underlie the ClCa

channel in olfactory hair cells43,44. There may

also be roles for this class of ion channel in

cancer cell biology and its inhibition may

prevent cell proliferation45-48.

Concluding remarks

This review has highlighted the diverse roles of

chloride conducting or transporting proteins

and how their dysfunction is coupled to human

disorder or disease-like symptoms in animal

models. There is a severe lack of pharmaco -

ION CHANNELS



Figure 2: Structural and functional properties of CLC proteins

A Representations of CLC structure generated from the CmCLC X-ray data51 using Pymol software; amembrane side-view (left) with intracellularcystathionine-β-synthase (CBS) domains lying belowthe transmembrane helices and an extracellular view(right) looking down onto the transmembranedomains. The individual monomers are coloured redand blue and the location of ions at external andinternal anion binding sites in each monomer areshown in green. B Representations of single CLC ionchannel currents usually recorded by electro -physiology. CLC channel openings are shown to twoequivalent levels, indicating one or two pores open,whilst the typical open-closed behaviour of othertypes of ion channel are shown below. Cartoonsillustrating channel opening and closing transitions areshown next to the simulated currents

‘‘CLC-7 inhibition may not be without complications because

human and mouse studies suggest thatlack of CLC-7 function may also be

associated with neuronal storage andde generation disorders because of

reduced lysosomal function’’

logical reagents that inhibit, activate or improve

membrane trafficking of chloride channels and

transporters. Progress is being made with drugs

that reverse the defective function of CFTR

in cystic fibrosis, which will hopefully result in

medications that are specific to the different

types of inherited mutation. Inhibitors and

activators of TMEM16A calcium-activated

chloride channels42,49 are proving useful

laboratory tools and new compounds might

make effective drugs, particularly if they can

deliver tissue-specific effects. Molecules specific

for particular CLCs are probably the most elusive.

Understanding the structural basis of voltage-

gated CLC activation50 might identify protein

domains that are targetable by rational

structure-based drug design. Such tools will

enable us to put some of the novel therapeutic

ideas that have been reviewed and presented

here to the test.


Volume 18 | Issue 4 | 2013 22

ION CHANNELS

Dr Jon Lippiat completed his PhD on the

structure, function and pharmacology of

potassium channels at the University

of Leicester. He studied pancreatic beta-cell

function and diabetes at the University of

Oxford before his appointment as Lecturer in

Pharmacology at the University of Leeds. His

research involves elucidating the structural and physiological

properties of several different types of ion channel and transporter

and their potential targeting by novel pharmacological reagents.

Biography

1. Miller, C., Open-state substructure of single chloride

channels from Torpedo electroplax. Philos Trans R Soc

Lond B Biol Sci, 1982. 299(1097): p. 401-11

2. Dutzler, R., et al., X-ray structure of a ClC chloride

channel at 3.0 A reveals the molecular basis of anion

selectivity. Nature, 2002. 415(6869): p. 287-94

3. Accardi, A. and C. Miller, Secondary active transport

mediated by a prokaryotic homologue of ClC Cl-

channels. Nature, 2004. 427(6977): p. 803-7

4. Iyer, R., et al., A biological role for prokaryotic ClC

chloride channels. Nature, 2002. 419(6908): p. 715-8

5. Scheel, O., et al., Voltage-dependent electrogenic

chloride/proton exchange by endosomal CLC proteins.

Nature, 2005. 436(7049): p. 424-7

6. Picollo, A. and M. Pusch, Chloride/proton antiporter

activity of mammalian CLC proteins ClC-4 and ClC-5.

Nature, 2005. 436(7049): p. 420-3

7. Leisle, L., et al., ClC-7 is a slowly voltage-gated 2Cl(-

)/1H(+)-exchanger and requires Ostm1 for transport

activity. EMBO J, 2011. 30(11): p. 2140-52

8. Neagoe, I., et al., The late endosomal ClC-6 mediates

proton/chloride countertransport in heterologous

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9. Matsuda, J.J., et al., Overexpression of CLC-3 in HEK293T

cells yields novel currents that are pH dependent. Am J

Physiol Cell Physiol, 2008. 294(1): p. C251-62

10. Tang, C.Y. and T.Y. Chen, Physiology and

pathophysiology of CLC-1: mechanisms of a chloride

channel disease, myotonia. J Biomed Biotechnol, 2011.

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11. Ratte, S. and S.A. Prescott, ClC-2 channels regulate

neuronal excitability, not intracellular chloride levels. J

Neurosci, 2011. 31(44): p. 15838-43

12. Foldy, C., et al., Regulation of fast-spiking basket cell

synapses by the chloride channel ClC-2. Nat Neurosci,

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13. Rinke, I., J. Artmann, and V. Stein, ClC-2 voltage-gated

channels constitute part of the background

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2010. 30(13): p. 4776-86

14. Jeworutzki, E., et al., GlialCAM, a protein defective in a

leukodystrophy, serves as a ClC-2 Cl(-) channel auxiliary

subunit. Neuron, 2012. 73(5): p. 951-61

15. Norimatsu, Y., A.R. Moran, and K.D. MacDonald,

Lubiprostone activates CFTR, but not ClC-2, via the

prostaglandin receptor (EP(4)). Biochem Biophys Res

Commun, 2012. 426(3): p. 374-9

16. Cuppoletti, J., et al., SPI-0211 activates T84 cell chloride

transport and recombinant human ClC-2 chloride

currents. Am J Physiol Cell Physiol, 2004. 287(5): p.

C1173-83

17. Cuppoletti, J., et al., ClC-2 Cl- channels in human lung

epithelia: activation by arachidonic acid, amidation,

and acid-activated omeprazole. Am J Physiol Cell

Physiol, 2001. 281(1): p. C46-54

18. Estevez, R., et al., Barttin is a Cl- channel beta-subunit

crucial for renal Cl- reabsorption and inner ear K+

secretion. Nature, 2001. 414(6863): p. 558-61

19. Liantonio, A., et al., Molecular switch for CLC-K Cl-

channel block/activation: optimal pharmacophoric

requirements towards high-affinity ligands. Proc Natl

Acad Sci U S A, 2008. 105(4): p. 1369-73

20. Liantonio, A., et al., In-vivo administration of CLC-K

kidney chloride channels inhibitors increases water

diuresis in rats: a new drug target for hypertension? J

Hypertens, 2012. 30(1): p. 153-67

21. Kornak, U., et al., Loss of the ClC-7 chloride channel

leads to osteopetrosis in mice and man. Cell, 2001.

104(2): p. 205-15

22. Schaller, S., et al., The role of chloride channels in

osteoclasts: ClC-7 as a target for osteoporosis

treatment. Drug News Perspect, 2005. 18(8): p. 489-95

23. Zhao, Q., et al., CLC-7: a potential therapeutic target for

the treatment of osteoporosis and neurodegeneration.

Biochem Biophys Res Commun, 2009. 384(3): p. 277-9

24. Karsdal, M.A., et al., Acidification of the osteoclastic

resorption compartment provides insight into the

coupling of bone formation to bone resorption. Am J

Pathol, 2005. 166(2): p. 467-76

25. Ohgi, K., et al., Antibodies against ClC7 inhibit

extracellular acidification-induced Cl(-) currents and

bone resorption activity in mouse osteoclasts. Naunyn

Schmiedebergs Arch Pharmacol, 2011. 383(1): p. 79-90

26. Kasper, D., et al., Loss of the chloride channel ClC-7

leads to lysosomal storage disease and

neurodegeneration. EMBO J, 2005. 24(5): p. 1079-91

27. Lippiat, J.D. and A.J. Smith, The CLC-5 2Cl(-)/H(+)

exchange transporter in endosomal function and

Dent's disease. Front Physiol, 2012. 3: p. 449

28. Ramsey, B.W., et al., A CFTR potentiator in patients with

cystic fibrosis and the G551D mutation. N Engl J Med,

2011. 365(18): p. 1663-72

29. Clancy, J.P., et al., Results of a phase IIa study of VX-809,

an investigational CFTR corrector compound, in

subjects with cystic fibrosis homozygous for the

F508del-CFTR mutation. Thorax, 2012. 67(1): p. 12-8

30. Gibson, A., et al., hCLCA1 and mCLCA3 are secreted

non-integral membrane proteins and therefore are not

ion channels. J Biol Chem, 2005. 280(29): p. 27205-12

31. Boudes, M., et al., Best1 is a gene regulated by nerve

injury and required for Ca2+-activated Cl- current

expression in axotomized sensory neurons. J Neurosci,

2009. 29(32): p. 10063-71

32. Kunzelmann, K., et al., Role of the Ca2+ -activated Cl-

channels bestrophin and anoctamin in epithelial cells.

Biol Chem, 2011. 392(1-2): p. 125-34

33. Barro-Soria, R., et al., ER-localized bestrophin 1 activates

Ca2+-dependent ion channels TMEM16A and SK4

possibly by acting as a counterion channel. Pflugers

Arch, 2010. 459(3): p. 485-97

34. Schroeder, B.C., et al., Expression cloning of TMEM16A

as a calcium-activated chloride channel subunit. Cell,

2008. 134(6): p. 1019-29

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References



25 Using LIMS forbiobanking andimplementing LIMS for researchEva Bürén, Head of IT, Karolinska Institutet Biobank

29 Biologicsdevelopment andELN: A good match?Michael H. Elliott, CEO, Atrium Research

33 InformaticsRoundtable

Moderated by John Trigg, Director, phaseFour Informatics

Informatics

SPONSORS

© F

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Ber

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com

LIMS without BoundariesBrowser independence

Database independenceHardware independence

Location independence

The KI Biobank operations have expanded

greatly over the last few years with new

technology for automated high throughput

sample handling in order to increase through -

put and quality of samples.

The IT department

The IT department at KI Biobank has three main

purposes. The first is to develop, maintain and

support systems for the high throughput

processes at the Biobank facility, such as sorting,

aliquoting and storing samples in an automated

way. The second mission is implementing a LIMS

system, SCARAB-LIMS, which support research

groups that store biobank samples in freezers at

different laboratories. The third purpose is to

develop and maintain an infra structure for large

scale prospective studies. The infrastructure is a

highly integrated service consisting of a CRM

system, web survey system, a LIMS system and a

database platform for safe storage of data.

Implementing LIMS for

research: background

The traditional approaches in data management

are lab notebooks, multiple spreadsheet files in

computer folders and standalone databases.

As a result of the Swedish Biobank legislation

from 2003, which stated that all samples stored

for more than two months must be traceable,

managed with quality and to protect the

integrity of the donor, the Karolinska Institutet in

a joint effort with Stockholm County Council

decided to implement a new LIMS system in

2007 as support for clinical researchers in their

daily operations with using biobank samples

within the healthcare system.

LIMS

A laboratory information management system

(LIMS) database application enables a secure

system that fulfils the legal demands for

traceability of a sample. The system has an

audit trail that contains information about

everything being done in the system and

by which operator. The LIMS system also

manages information about the donor and it is

always at the donor’s discretion to withdraw

consent. One of the most important options

for a LIMS system is the consideration of the

value of the sample collections and associated

data. It enables utilisation of sample collections

and easy access of samples and sample informa -

tion using request and withdrawal functions

and it is possible to share data and stimulate

research collaboration.

SCARAB-LIMS

SCARAB, an acronym for Sample Collection

Administration Research and Biobanking – is a

LIMS from LabWare that has been configured

at KI. In the system, a module called the biobank

template is used. The template consists of

standardised functions for biobanking and

Karolinska Institutet Biobank is a core facility at KI established in 2004 to support medical research in short and long-term storage

of valuable human biology samples. Today, the KI Biobank holds four million samples in freezers from approximately 140 different

studies. The studies vary from disease-specific case control studies with the aim to identify biomarkers for complex diseases to large

prospective cohorts with the aim to investigate genetic and lifestyle factors and their interaction on health and disease.

The service provided at the Biobank is focused on study planning, pre-analytical processes, sample storage and retrieval of

specimens. The facility has 20 robots for sorting, aliquoting, DNA extractions and withdrawal of samples. On a daily basis,

approximately 500 blood samples from different research studies are registered and processed into multiple aliquots and stored in

2D barcoded micro tubes in 96 SBS rack format.

Using LIMS for biobankingand implementing LIMS for research

Eva Bürén

Head of IT, Karolinska Institutet Biobank



IN-DEPTH FOCUS: INFORMATICS

© V

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RIN

/ Sh

utte

rsto

ck.c

om

sample collections. The main functionalities are

planning a study, logging of samples manually

or by file, searching for samples or patients by

different criteria, request of samples and storage

location functionality. Back-end functionality for

logging samples integrated to a robot is also

developed in SCARAB-LIMS.

Information security and maintenance

of the system

Due to the large amount of confidential data

that is being collected, the biobank has a great

responsibility in terms of information security

such as confidentiality, availability, integrity and

traceability regarding the collection, storage

and release.

Several laws and regulations, such as the

Data Protection Act (1998:204), Ethical Review

Act (2003:460), Public Access to Information and

Secrecy Act (2009:400), and the Biobank in

Medical Care Act (2002:297) regulate the

security of data management, sample trace -

ability and the option for sample donors to get

access to stored data. Four of the core principles

within information security are;

� Confidentiality – sensitive information

and programs should be protected from

being accessed or disclosed to un -

authorised persons

� Accessibility – access to information and

information systems should be kept at a

level that the business can run efficiently

and smoothly. The level should meet the

needs and goals of the services and system

owners made based on business

requirements. This information will be

restored within a reasonable period of any

loss or interruption

� Accuracy – information processed is

accurate, current, complete and under -

standable, and presented so that it

meets the intended purpose. Information

should not be changed accidentally or

delib erately distorted

� Traceability – in the handling of information,

it should be possible to track who did what

and when.

The system operates via a centralised data-

base. The database servers are maintained by a

central IT department and are set up within the

department firewall.

It is possible to connect to the system via a

web browser but the login must be performed

using a citrix client in order to increase the

security. Backup of the system occurs every

15 minutes. As the database is used by diff-

erent research groups, it is crucial to implement

restrictions that define different levels of

access to data. A useful functionality of the

system is the possibility to allow specific data

to be accessible for a specific group, user or

defined role.

LIMS and biobanking processes

In the initial study planning phase, the customer

relations group at KI Biobank help researchers in

the overall logistic planning and set up of

medical research studies i.e. ethics, logistics,

storage and informatics.

The process starts with the integration of a

study at the Biobank. Information about the

donor ID, study ID and sample types are

collected and imported into the LIMS system.

LIMS then produces sample numbers and

referrals are created and sent to the partici-

pants of the study. The participants use the

healthcare facilities for the withdrawal of

the blood samples. The samples and referrals

arrive at the Biobank, the referral is scanned

as a first step and the information is imported to

the LIMS system. The samples are then sorted

and scanned by a robot. Every sample has

a unique barcode and the barcode is vali-

dated by the LIMS. All samples are loaded

onto a sorting robot that starts by scanning

the unique barcode of the sample, volume

measurements of the sample and tube

type detection by a camera-based system.

The data is sent to LIMS to match the barcode

and tube type against the imported data.

LIMS generates a working list for the robot

based on the data and send it back to the

robot. The tube and sample type determines

further processing of the samples.

LIMS can be configured to communicate

with laboratory equipment, including analytical

instruments and liquid-handling robots.

This not only allows data to flow directly into the

LIMS as it is generated, but also enables

the system to direct the workflow with specific -

ally developed functions. Features like these can

improve efficiency by saving researchers the

task of manually recording and entering data,

and can reduce data transcription errors.

The LIMS enables a powerful functionality

for searching data and for generating reports.

Many LIMS are also equipped with data-mining

and trending tools that can provide unique

insights into the data.

Standards and pre-analytical codes

LIMS enables implementation of standards and

codes to achieve a higher and comparable

quality of collected samples than what is

available today and includes both samples and

information on how samples are collected

and processed. The SPREC code, developed

by ISBER, describes the pre-analytical con-

ditions of a sample. The codes consist of

seven elements describing for example sample

type, storage condition, pre-centrifugation and

container type.

To enable sharing of data and samples

between biobanks more efficiently, BBMRI.se

has developed a standard for biobank sharing:

Minimum Information about Biobank Sharing

(MIABIS). MIABIS includes data describing

biobanks, studies, contact information and

experiment types.

LIMS in large prospective cohorts

LifeGene and EpiHealth are prospective large

population cohorts in which data on health

status, physical measures, lifestyle, exposures

and biological samples are collected. The aim of

these cohorts is to study the influence of genes

and lifestyle factors on health and diseases

across the lifespan on several tens of thousands

of individuals.

Both EpiHealth and LifeGene have collected

data using the same IT infrastructure, which is

built in modules with a number of services /

applications to support research studies.

The services include study administration for

managing participant contact information and

test centres, websites for interaction with

participants and the public, collection of data

through web-based questionnaires, LIMS for

managing physical measurements and registra -

tions of samples at test centres, referrals to

laboratories for analysis and a biobank and

finally the central repository for all information.

At the test centre, a LIMS is used for

sample registration, instrument integration and

registration of physical measurements. The LIMS

system is integrated with the central database

via a citrix solution. The system allows for

different instrumentation dependent on the

study requirements. Implementation of LIMS

based data entry ensures quality of measure -

ment data. When data input is manual, e.g.

measuring waist circumference, reference

values and other logical checks assure

quality. The interface with the LIMS is peda -

gogically designed to ensure that all activities


Volume 18 | Issue 4 | 2013 26


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Validate and Go Live in a Fraction of the TimeLABVANTAGE Pharma is the only pharmaceutical LIMS on the market that reduces traditional implementation cost by up to 85% and implementation time by up to 75% with:

LABVANTAGE 6 Preconfigured for Pharmaceutical-Specific Functionality

Pre-validated Package Executed Based on GAMP 5 Best-Practices Guide

LABVANTAGEPharma PackageImplementation

As Soon as3 Months

Minimum 12 Months

Months

A Typical LIMS Implementation

Approximately a 75% savings in timeApproximately an 85% savings in cost

in the test are performed in the same order regardless of personnel

performing the tests. The system validates if anything is forgotten or

the results are out of the given boundaries. The LIMS records all

activities in the test rooms. This further ensures that time

requirements for handling of blood samples follows study protocol.

LIMS also has functions to log sample transport to either the biobank

or different labs. This gives full control of the time from sampling to

safe storage.

Obstacles when using LIMS

Most LIMS systems offer a wide range of functionality and it is often

difficult for the researcher to define requirements that fulfil their

needs and to make a requirement specification of which functionality

is needed. The user friendliness of the system can also be an issue for

researchers with little or no IT experience.

What information should be stored in LIMS?

Many research groups don’t have any other systems for clinical data

and large analytical data. The LIMS is favourable for sample

management which is the main purpose of LIMS and the strength of

the system.

What is the best choice; to purchase a commercial

system or an in-house development?

There are pros and cons for both alternatives and there is no

simple answer. In-house development put strong demands on IT

experience in programming which might be a favourable solution

for limited workflows. A commercial system provided by different

vendors enables most functionality to be implemented within a

much shorter time frame compared to in-house development.

It’s quite expensive but has a higher security inbuilt in the system.

The conclusion, based on experience at KI Biobank, is that a key

factor for a successful implementation of a new system is to

carefully evaluate performance, integration to other systems or

instruments, user friendliness, functionality to make reports,

support and price offered by vendors and off course the strategies

and long term planning for the business are critical factors for a

successful implementation.


Volume 18 | Issue 4 | 2013


Eva Bürén started working at the KI Biobank in 2009 as a system developer.

She was involved in implementing a laboratory information management

system (LIMS) for research groups with sample collections at KI. In September

2010, she became manager of the LIMS group. In March 2011, as a result of a

merge of three IT-groups into one, Eva became Head of IT at KI Biobank. The IT

group at the Biobank develops, maintains and supports the IT systems within

the organisation. In addition to being head of the group, she is also account

manager of the IT-services in the organisation.

Eva has a background in molecular biology and has mostly been working in research and

development at Uppsala University and in Biotech companies. In Uppsala, she was involved in an EU

project developing methods for gene modification of strawberries. She also has experience from

developing a gene expression method at Global Genomics AB and working as a section manager of a

clinical laboratory. Eva holds an MSc in Medical Science from Uppsala University.

Biography

KI Biobank has for historical reasons two different LIMS systems, LabVantage LIMS

from Software Point and LabWare LIMS from Labware.

Footnote

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[email protected]

www.idbs.com/epr2013

While informatics within the small molecule

community is relatively mature with well-

established tools such as those for structure

activity relationship (SAR) analysis, reaction

planning, modelling and chemical registration,

the rapid increase in biologics investment has

outpaced the deployment of suitable IT systems.

For those systems commercially available,

adoption has been slow. Few on the biologics

side realise the importance of material reg -

istration and inventory management. The

common practices of ad hoc cell line and protein

batch naming, individual methods of data

management through Excel and reporting via

PowerPoint not only lowers efficiency, it results

in an inability to link data entities and easily track

project progression.

Both biologics discovery and development

phases are highly data intensive. Combined with

increasing numbers of projects, attention to

Quality by Design (QbD), and the ever-present

pressure to reduce cycle time to progress into

the clinic, development teams have particularly

acute stress. This combines with the adoption of

new technologies and new methodologies

generating exponential data volume growth.

For example, pre-formulation groups deploying

screening automation for the examination

of drug-like properties, cell culture teams

experimenting with microscale reactors, and

purification departments rolling out automation

for column condition optimisation. As small

molecule teams learned years ago after

investing in chemical library synthesis and high

throughput screening, data management

practices must adapt to avoid causing a

downstream data analysis bottleneck.

Informatics and biologics development

In the past three years, Atrium Research has

examined the workflows of several biologics

organisations, from large multinationals to small

biotech’s. Through a Voice-of-the-Customer

(VoC) analysis, we have found a consistent

pattern to the top three pain points with the

current state of data management amongst

scientists and their managers:

1. Difficulties finding / locating support-

ing data and information. Because data

manage ment is generally left to individuals,

finding where data lives requires tracking

down the person who created it. Therefore,

decisions are made without the necessary

data / information being available

2. Manual processes for data assembly,

storage and reporting. Results in decreased

resource efficiency, longer cycle times and

induction of data errors

3. Lack of standards in entity naming / numb -

ering, data organisation, calculations or

process. Negatively impacts data analysis

and scientific decisions – cannot merge data

or trace lineage of batches. Cannot deter -

mine if changes in upstream processing

impact downstream results.

Study participants were queried on the

capabilities needed to improve job performance

in a future state. The top five responses were:

1. A comprehensive collection of process

development data and information

2. Enable search across experiments and

studies to retrieve both structured data

(e.g., assay results) and documents (e.g.,

study reports)

Sales of monoclonal antibody (mAbs) and antibody-drug conjugates (ADCs) are projected to rise to USD 58 billion by 2016, an

increase of USD 25 billion from 20101

. This contrasts with other sectors of the pharmaceutical market that are declining. IMS

Health, in their latest survey, suggests that the overall US drug market shrunk in 2012 for the first time ever2

. Combined with

longer useful patent life and the complexity and cost of creating biosimilars, it is no wonder pharmaceutical companies are

diverting a larger portion of their R&D expenditures to biologics.

Biologics development and ELN: A good match?

Michael H. Elliott

CEO, Atrium Research




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3. Integrate data, regardless of origin, to

provide traceability and lineage from cell

line development through purification and

formulations. Be able to merge data across

organisational boundaries to increase

process understanding

4. Decrease variability and improve quality.

Enable consistent global data formats,

calculations, and platform components

5. Reduce non-value added activities through

elimination of manual data tasks.

What about ELN?

Many companies ask if Electronic Laboratory

Notebook (ELN) technology is ‘the solution’ to

mitigating the pain points and achieving the

desired capabilities of the future state.

Unfortunately, the answer is ‘no’ when exam -

ining functional requirements across all use

cases. There are too many capability require -

ments to be solved by any one technology

alone. ELN will not provide – without major

compromises – an effective solution to biologics

registration, inventory control, antibody design,

document management or analytical sample

and results tracking.

The correct question is: ‘Can ELN help?’ The

answer to that is ‘maybe.’ ELN is a component

of the larger informatics puzzle and the oper -

ational gains it offers depends on the decisions

made prior to implementation, such as the

project objectives. The system selected also has

an impact on benefits afforded. There are many

flavours of products on the market: systems

designed as generic tools for unstructured

intellectual property protection, products

layered on top of existing LIMS platforms and

systems with an integrated database for

structured data management. What you want to

do, what you select and what you do with it

determines success of failure.

For a generic implementation, one could

decide to take existing Microsoft Office files

and deposit them into the ELN for long-term

retention. The files are stored in a consistent

manner and tagging them with metadata

enables limited search. This does not address

data integration across experiments, let alone

the genealogy of a purified batch nor does

much to decrease inconsistency between

scientists. However, this is the easiest ELN

incarnation to implement and has the least

impact on existing workflows. If other systems

are developed for managing all the structured

data that results from experiments across all

functions, then this manifestation works well.

This approach can also ease the transition to a

comprehensive, workflow-altering deployment.

Start small, get scientists used to the technology

and slowly migrate them to a consistent method

of operation across the organisation is not a bad

way to go. This is a more of a cultural decision

rather than one of technology, assuming you

have selected a system that has the necessary

capabilities to be switched on at a later date.

ELN systems based on a LIMS are less

straightforward to deploy. Laboratory Informa -

tion Management Systems (LIMS) tend not to

adapt well outside of the analytical function.

Not only does the standard sample > test >

result hierarchy not match the workflows very

well, the ability of most LIMS products to easily

adapt to dynamic designs of experiments is not

present. In very late stage development

(i.e., clinical manufacturing), ELN variants that

are either based on a LIMS or have LIMS-

like design (known as ‘laboratory execution

systems’) can succeed as the workflows and

parameters are more fixed. But, this raises the

question of whether a company wants two

systems – one for the clinical manufacturing and

one for early and mid-stage development.

The systems that have had the greatest

success in the market (as measured by benefits)

are those ELN products with an integrated

structured data management component.

Though still not optimal due to their inherent

unstructured data design metaphor (i.e., paper

notebook replacement), these systems enable a

wholesale substitution of ad hoc spreadsheets

through the use of templates. Templates define

the structure for the data entry, calculations and

plots, supported by data dictionaries or

catalogues that establish a common metadata

vocabulary. Templates can be simple forms for

data entry and/or spreadsheets for data

aggregation and analysis. Manual data inte -

gration can be lessened: data can be integrated

directly from instruments such as fermenters

with near real-time analysis and visualisation.


Volume 18 | Issue 4 | 2013 30


Figure 1: S88 – Recipe migration from platform models through clinical manufacturing

Not only do templates ease data entry, but

previous experiments can also be used as a

starting point for a new one where only a few

parameters need to be changed. A limited level

of cross-experiment data integration is possible,

though not nearly at the level of a data ware -

house designed for purpose.

With the level of change management

required to define consistency in terminology,

formats, workflows and calculations, these

types of ELN products are by far the most

difficult to manage and support. An advant-

age is that these systems are not as fixed as a

LIMS and therefore allow greater flexibility to

achieve the desired capabilities. But this is also

a curse, as this openness often means more

internal disagree ments over design con -

siderations. The effort involved to get a group

of scientists – who have their own Excel temp -

lates – to agree on a common format can

be considerable.

A mistake that template builders habitually

make is attempting to put an entire workflow

into a single template, feeling a large monolithic

version will be easier to support. An issue that

often arises is that any changes in the workflow

require a considerable re-architecture, making it

as complex as a LIMS to maintain. There are so

many triggers, extensions and integrations that

modifying one portion routinely will break

another. Our ELN user surveys highlight a trend

of an increasing frustration with vendors over

the complexities and maintenance of templates.

It is not unusual for the vendor support services

team to build the initial versions; they then have

to be called back at considerable expense to

update them later on. In many ways, several

ELN products are starting to look like the very

thing they replaced: big, unwieldy and complex

LIMS products.

The trend of biologics developers to use a

‘platform’ approach (i.e., a standard workflow),

may support a very fixed and limited set of large

templates. But as not all mAbs behave the same

way, changes in workflow and experiment

parameters are inevitable. Therefore, a break -

down of what a platform ‘is’ is necessary to mix

and match what is needed in the ELN workflow.

It is our opinion that the S88 standard can be

applied to ELN and platform biologics develop -

ment to create a modular approach that benefits

both the organisation and the technology.

S88 and ELN

ANSI/ISA-88 (S88) is the international standard

for defining production processes, emphasising

proper practices for the design and operation of

batch manufacturing and control3. It is a stand -

ard framework in use since the first version was

introduced in 1995. Multiple pharmaceutical

companies were involved in the development of

the standard and several are applying it to

biologics development to expedite technology

transfer and support QbD-like initiatives.

S88 provides a common vocabulary between

sites, scientists and engineers, but it is not a hard

and fast standard. It describes a flexible

framework to build models suited to the needs

of the operation.

The key concept throughout S88 is a ‘Recipe’

which is defined as: ‘the necessary set of

information that uniquely identifies the

production requirements for a specific product’.

It is a depiction of the process inclusive of

materials, steps and conditions. Recipes evolve

over time, starting with a set of process models

(i.e., platform components) that eventually turn

into equipment-specific Master Recipes for

clinical manufacturing (see Figure 1, page 30).

The first recipe, which may go through much

iteration during a development cycle, is a

General Recipe. The General Recipe takes the

process models and turns them into a recipe

that is specific to a particular product.

The beauty of S88 and the recipe concept is

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‘‘The systems that have had the greatest success in the market

(as measured by benefits) are those ELNproducts with an integrated structured

data management component’’

that everything is modular, enabling the

common process models to be reused and

mixed across many different recipes. This allows

for component ELN templates that support a

granular design approach. The procedure of

a recipe is built from stages, process operations

and process actions, which can be supported by

the ELN hierarchy.

In the example in Figure 2, a downstream

purification procedure has an ordered set of

stages: preparation, capture, intermediate

purification, polishing and packaging. The

capture stage has two anion exchange chrom -

ato graphy operations that are further broken

down into ‘Process Actions’ or specific pro -

cedural steps and their parameters. This

modularity enables mixing and matching of

process model templates at the action,

operation or stage level, depending on the

needs of the organisation.

An ELN template for the anion exchange

chromatography process action might have

sections for;

� Bill of Items with tables for the entry of:

� Materials (e.g., buffers, MSDS informa -

tion, lots numbers)

� Equipment (e.g., chromatography,

column, pH meters, spectrophotometer

for A280)

� Consumables

� Quality attributes (e.g., ranges for

analytical results)

� References

� Safety information

� An ordered set of steps for executing the

process action with fields for entry of data

such as loaded volume, buffer volume,

temperature, pH, etc. Calculated fields for

parameters such as elution concentration

� A sampling plan describing when samples

are taken and the assay required. There

would be an area for the entry of non-

structured results like a chromatogram.

Once the template is created, it can be

dragged to the ELN page from a library, allow-

ing the user to create experiments by

changing only a few parameters. Once the

parameters are optimised through a series of

experiments, the downstream recipe can

be merged with an upstream recipe to

create an overall process description. This

builds consistently documented institutional

knowledge documented, eases technology

transfer to clinical supplies and assists in the

creation of regulatory documentation.

Individual ad hoc approaches to data

management work for early stage biotech’s

but are not scalable. The increasing invest-

ment in biologics is forcing a change to a

controlled set of practices using common

process models across scientists and loca-

tions. The S88 standard can be applied in

biologics development to build modular

platform components; an ELN can be leveraged

to support the building of recipes though

modular template building. As a component of a

larger informatics puzzle, ELN can eliminate

many of the data management pain points,

helping to realise a future state of data

integration, consistency between scientists, and

improved efficiency.


Volume 18 | Issue 4 | 2013 32


1. BCC Research; ‘Antibody Drugs: Technologies and

Global Markets’; Feb, 2012

2. IMS Institute for Healthcare Informatics; ‘Declining

Medicine Use and Costs: for Better or Worse?’;

May 9, 2013

3. http://www.isa.org

References

Michael H. Elliott is the founder and CEO of Atrium Research, a

scientific informatics market research and consulting firm. He can be

reached at [email protected]

About the author

Figure 2: Example of a Recipe for Downstream Purification

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Externalisation has become a major

business strategy for Life Science

companies. How well aligned is

the laboratory informatics industry

to addressing the demands of

externalisation in terms of collaboration

support, access control and security?

Williams: As Pharma makes itself increasingly

‘virtualised’, there is a critical need for ‘inter-lab

informatics’ to support a network of partners

and IP producers. These are flexible data systems

which control and contextualise disparately

produced data so that it can be used effectively.

Modern, data centric electronic laboratory

notebooks (ELNs) are well aligned to support

this multidiscipline activity, managing data

security and access control. Legacy ‘digital

sticker-book’ ELNs with ‘single-login’ access are

being superseded with scalable systems with

strong ontology and workflow management,

enabling far flung scientists to move beyond

simple data capture and IP protection, towards

real-time collaboration.

Townsend: Over the last decade, the trend

towards centralised systems for laboratory

automation has led enterprise software vendors

to enable their products to support multiple

laboratories, across multiple sites which are

often spread around the world. LabWare

provides features in our LIMS and ELN products

to effectively manage data segregation, access

rights and allocation of functional privileges.

These tools can also be brought into play to

manage collaboration with external organisa -

tions. Furthermore, we also provide customers

with tools to export / import data in a variety of

popular data-interchange formats, which can be

used in cases where online access to systems is

not desirable or impractical.

Bailey: A sophisticated LIMS can work

outside of the organisation by interfacing with

collaborating systems so data flow seamlessly

between them, even within an extensive

workflow among multiple internal and external

users. Its state-of-the-art security will control

access to certain data so while one company can

track delivery of samples, another can access the

results of its tests. Since only certain businesses

implement a LIMS, the onus of this collaboration

is on the LIMS provider to facilitate.

Lab Informatics tools are increasingly

being influenced by consumer

technologies (e.g. mobile devices,

‘social’ tools). Do these technologies

have a place in the lab, and if so,

what role can they play?

Townsend: Mobile devices offer exciting oppor -

tunities for laboratories and adoption of mobile

technology is slowly but surely gathering pace.

For example, in LabWare we have seen how

mobile devices used for LIMS sampling opera -

tions can provide substantial benefits, including

for example use of GPS location tracking and

map technology. As for social tools, LabWare’s

own internet-based customer LIMS/ELN support

forums have delivered enormous value over

many years allowing customers from all corners

of the world to share experiences and knowl -

edge. In the same way, popular social tools are

able to deliver similar benefits as shown by the

growing number of lab informatics forums.

Bailey: Current consumer technology is

almost entirely web-based, which requires

the modern LIMS to run within a mobile

browser without losing the data sophistication



INFORMATICS ROUNDTABLE

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Moderator

John Trigg, Director, phaseFour Informatics

Glyn WilliamsVice President Product Delivery

IDBS

Peter BaileyCEO

LABVANTAGE

Nick TownsendDirector Life Sciences

LabWare

the user requires. Additionally, the smaller and

lighter these devices become, the more

convenient and desirable. For example, rather

than carrying notebooks, pens and traditional

stationery supplies, the user can simply carry a

tablet computer. In regards to social trends,

LABVANTAGE will soon unveil an exciting new

collaboration module that encourages users to

discuss tests and data while still providing

complete auditing and traceability.

Williams: The environment in and around

the laboratory is mobile, as are the people

who capture, review and share the data. But

standalone ‘m-informatics’ platforms do not

solve the problem. It is critical that mobile and

desktop applications access the same data.

Scientists love to think and talk about their

data. Social tools are vital if we are to capture the

full intellectual input of any scientist. Systems

enabling ‘comments’ and ‘tagging’ of data

encourage knowledge sharing and create

context and provenance for the data. This in turn

creates better information flow around the

organisation and increased scientific interaction.

Do you see ‘Cloud’ as a viable

implementation platform for

informatics tools? What do you see

as the benefits and drawbacks?

Bailey: LABVANTAGE understands the value of

the Cloud because we already use it as a daily

tool in our business. It requires no investment

into hardware and additional IT personnel and

there is no long term commitment. We host

distinct configurations for individual training

courses and for custom service projects. These

systems can quickly be created on the Cloud

instead of having to wait for requisitioned

hardware. Additionally, and almost identically

to using a third-party hosting solution, the

Cloud can provide secure, worry-free produc-

tion environ ments for organisations that

prefer to focus on the lab work rather than

the infrastructure.

Williams: ‘Cloud’ offers extensible comput -

ing power and centralised infrastructure for

collaborative working. But putting a desktop

application onto a Cloud does not make it a

Cloud application. The horsepower of the Cloud

can certainly benefit analytics systems and can

add value to truly scalable enterprise ELNs.

However, there remains corporate resistance to

the use of multi-tenanted applications for

storing high-value IP data. This may change with

improved stability of the Cloud and use of

sophisticated security. The best enterprise ELNs

are being developed with the Cloud in mind and

may revolutionise R&D informatics in the same

way as Cloud-CRM systems did for sales forces.

Townsend: The ‘Cloud’ is certainly a viable

platform for informatics but it has not yet been

widely adopted. We find that customers are still

unsure about how to ensure data security.

The benefits of using Cloud technology for IT

applications are well documented and the

opportunities for lab informatics applications

should be no different. Our technology is Cloud-

ready and we are open to work with customers

who wish to adopt the Cloud. However, this will

be driven by our customers as they gradually

take a view on data security and required

customer-vendor contractual arrangements

associated with moving and supporting

applications to the Cloud.

Laboratories often aspire to becoming

‘paperless’; how viable is that

aspiration, and what are the major

hurdles to be overcome?

Williams: The paperless laboratory is possible.

ELNS have already allowed scientists to replace

the paper notebooks in some companies.

Although there is more paper than just

notebooks to be considered, this shift is already

happening. Reducing transcription errors is a

major driver and integrating systems together

helps this process. There are cultural attach -

ments to paper but this is diminishing and must

be overcome. You only have to look at the move

in the newspaper industry toward digital

rather than print, to see where the science sector

is heading.

Bailey: LABVANTAGE has already taken the

paperless initiative. The fundamental of paper

documentation is record keeping, but to do so

‘paperlessly’ requires a method to input, file and

search. We input data using an electronic form

that mimics its paper counterpart, advanced

instrument integration that collects data directly

from instruments, barcodes, etc. All of this data is

stored permanently in the database with a

complete audit trail. Using both standard

reporting and the LABVANTAGE Ad Hoc Query

tool, users can easily retrieve the contents of

these documents. It’s not only paperless, but

also fast and accurate as well.

Townsend: It is feasible to achieve a

paperless state and it is realistic to achieve it with

LabWare LIMS with or without ELN, although

using LIMS with a LabWare ELN will typically

make going paperless easier. The effort to

achieve a truly paperless state should not be

underestimated. It requires carefully planning of

budget and resource to set-up the required

static data elements and implement data

capture templates that are convenient for the

end-user as they go about their daily tasks.

Laboratories usually interact with instruments

and other applications so ‘going paperless’ is also

dependent on the feasibility of providing

seamless and paper free integration.

What do you see as the biggest

challenges for the laboratory

informatics industry over the

next five years?

Bailey: The biggest challenge we see is keeping

pace with the ever-changing technology that

scientists use and the explosion of resulting data

being stored. In the past decade, both software

and hardware were dramatically improved,

which resulted in more data being stored. The

challenge for the LIMS is to discover more

effective ways to make sense of that data. In the

future, managing these global enterprise

databases and delivering business knowledge

will become the essential competence for the

LIMS vendor.

Townsend: The more mature lab informatics

products that are available on the market have a

much greater functional footprint compared to

that of a decade ago, and this footprint continues

to expand. Expectations of what lab informatics

solutions should provide are also growing.

Going forward, a key challenge is to ensure

expectations can be delivered, supp orted and

upgraded in a cost effective manner over the

long-term, especially when projects involve

integration of multiple products. Meeting

expectations will require customers and vendors

to work together and ensure projects are based

on sound product development strategies,

realistic goals and are executed well.

Williams: Informatics is the combination of

people with technology. Critical to success

therefore will be the ability of IT to help people

of many scientific disciplines cope with, and

share digital R&D. Systems must be available

on multiple platforms and bridge across

today’s legacy siloes. Key to this will be to make

enterprise data interoperable by smart data

management and ontology control. The impact

of consumer UI design also sets expectations for

how people interact with software: intuitively

and personally. The smartest companies have

already begun integrating many of these

capabilities. Those who have just started are

behind the curve.


Volume 18 | Issue 4 | 2013 34

INFORMATICS ROUNDTABLE

This autumn, the scientific elite in pharma -

ceutical research will again come together at

MipTec, as they have done annually for over

11 years. The event has grown from its origins as

a specialist conference for laboratory auto -

mation into a leading global conference in the

life sciences sector. By offering a broad, diverse

and highly scientific conference programme,

MipTec plays a vital role in helping Pharma

and Biotech scientists successfully tackle the

significant challenges of today’s drug research

and development work. The exchange of the

latest scientific findings and technological inno -

vations, as well as the improving integration of

science and technology, will be at the centre

of this three day conference. For the organisers,

it is especially important to create an interactive

platform for researchers working in the life

sciences, who would otherwise rarely meet.

The conference

The 2013 conference theme is Personalised

Medicine, covered by internationally renowned

keynote speakers in the drug discovery and

life sciences field. The theme also interlaces in

the jam-packed three day dynamic programme.

BioValley’s Science Day, Strategy Day and

Connect Day explore facets where tech-

nology accelerates life sciences research, and

MipTec’s Nine Science Forums cover cutting-

edge developments that enhance drug

discovery: Medicinal Chemistry, Peptide

Thera peutics, HCS, NGS, Enzymology, Stem

Cells in Biomedicine, Natural Products and

Synthetic Biology as well as Customised

Therapies-Biomarkers and Biobanking and

Industry-Academic Collaboration Models.

The conference provides a platform

for networking and scientific exchange for

academic and industrial scientists. With more

than 100 exhibitors presenting state-of-the-art

equipment and services to advance biomedical

research, more than 100 scientific presentations

and 100 poster exhibitors, delegates will get

expert insights into the most recent develop -

ments and experiences in all dimensions of drug

discovery. Delegates will also have the oppor -

tunity to discuss strategic and business oriented

issues and learn more career options in many of

the exceptional satellite focused sessions.

Industrial Symposiums will

be organised by:

Bucher Biotec, Hamilton, Thermo Fisher,

Cellular Dynamics International, Toolpoint,

SiLA, PerkinElmer, Cellectis Bioresearch, BioTek,

Agilent Technologies and BMG Labtech.

Keynote Presentations

MipTec 2013 and the BioValley Life Sciences

week are proud to welcome the following three

keynote speakers at the conference:

Tuesday 24 September 2013Dr. Christoph Westphal, Founder of the

Longwood Fund (Boston, US). Lecture title:

Drug Innovation in Biotech: Alnylam, Momenta,

Sirtris, Verastem

Wednesday 25 September 2013Dr. Ralf Schumacher, Leader of the Large

Molecule Research Department (LMR) of Roche

Diagnostics (Penzberg, DE). Lecture title:

Strategies and Challenges for the Next

Generation of Therapeutic Proteins

Thursday 26 September 2013Dr. Kári Stefánsson, CEO deCODE Genetics

(Reykjavik, IS), Lecture title: Genetics of

Common Disease

Networking Events

Welcome ReceptionThis year’s conference will offer a Welcome

Reception on Tuesday 24 September 2013 for

exhibitors and delegates in Hall 4.1 in the

Congress Center Basel. Dr. Sylvain Cottens,

Global Head for the Center of Proteomic

(Novartis, NIBR) will welcome guests. Further -

more, the participants of MipTec and the

BioValley Life Sciences Week will have

the opportunity to network with colleagues

and opinion leaders in a relaxed atmosphere.

Poster Session & Apéro The MipTec Poster Session will be held on

Wednesday 25 September 2013. The authors of

the scientific posters will be available for

questions. Posters, recognised for their scientific

quality, will be nominated for poster prizes,

sponsored by Toolpoint and SLAS.

Industrial ExhibitionParallel to the scientific programme, the MipTec

exhibition will take place in Hall 4.1 in the

Congress Center Basel. Parallel to the MipTec

Drug Discovery Conference, over 900

square metres, exhibitors will showcase their

newest developments, products and services in

the field of laboratory and research reagents,

laboratory automation and instruments as

well as hard- and software for the computer-

supported analysis and medicinal chemistry of

industry leaders and decision makers in the

life sciences area.

The largest European conference on drug discovery, MipTec, continues to grow, taking place from 24 – 26 September 2013 and

offering a high calibre programme. Over 3,000 scientists from industry and academia are expected to attend the presentations

and scientific forums at MipTec which will be held jointly with the BioValley Life Sciences Week in the Congress Center Basel.



The detailed pre-programme is available on the

following websites: www.miptec.com;

www.lifesciencesweek.ch

ShowPREVIEW Date: 24-26 September 2013 · Location: Basel, Switzerland

Leading figures from drug discoveryand life sciences to gather in Basel

Within pharmaceutical development, Raman

spectroscopy has been widely used for non-

destructive quantification of actives and

excipients8. Moreover, Raman spectroscopy has

been proven to be a workhorse as a process

analytical technique for process monitoring9,10

(blending, granulation, compression, coating)

and understanding of process induced trans -

formation11. Raman spectroscopy has also been

used to address regulatory concerns over solid

state trans formations within the final dosage

form12. Due to its intrinsic properties such as

rapid analysis time, user-friendly interfaces, non-

destructive nature, Raman spectroscopy has

been used to tackle drug product counterfeit

and intellectual property rights issues13.

This review highlights a few representative

applications of Raman spectroscopy for in-

process characterisation of active pharma -

ceutical ingredients (APIs) and drug products.

Specifically, application of Raman spectro-

scopy is presented as a tool to monitor the

polymorphic state of an API in a slurry,

the content uniformity of a blend in a dry mixing

process, the end point of analyte dispersion in a

suspension and the measurement of a tablet

coating process. In each of these cases, Raman

spectroscopy has provided in-depth insights

into critical quality attributes of the pharma -

ceutical processes assessed, making the

pharmaceutical drug development workflow

monitorable in real-time.

API polymorph evaluation

Raman spectroscopy is a powerful technique for

characterisation of polymorphic forms of an API.

Implementation of in-line Raman probes allows

direct real-time monitoring of solid state

transformation of an API during the preformu -

Raman spectroscopy has emerged as the preeminent analytical tool for a number of applications within drug discovery and

development. Advances in the instrumentation, sensor fabrication and data analysis have enabled the wider acceptance of

Raman spectroscopy1,2

. In discovery, Raman spectroscopy is used to elucidate structural activity relationships3

and to optimise

reaction conditions and associated parameters (such as polymorph and formulation screening)4,5

that impact scale-up required

for the transfer of drug compounds from discovery to development6,7

.

Raman spectroscopy: an enabling tool foraccelerating pharmaceuticaldiscovery to development

Chanda R. Yonzon, Atul Karande, Sai P. Chamarthy and Brent A. Donovan

Merck & Co. Inc

RAMAN SPECTROSCOPY


Volume 18 | Issue 4 | 2013 36

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Table 1 %RSD comparison of Blend I and Blend II for the three components in a DPI

% RSD

Component I Component II Component III

Blend I 5.14 2.31 1.61

Blend II 3.35 2.20 1.02

lation phase of drug development. As shown in Figure 1, a slurry

experiment using 1:1 mixture of Form I and Form II in propanol:water

mixture at room temperature was performed to determine a stable

polymorph of the API. Real-time Raman spectra were collected

every two hours for three days (λex = 785 nm, Power = 400 mW, Tcollection

= 120 seconds). In order to normalise the spectra and monitor

changes over time, first derivative followed by standard normal

variate (SNV) was performed on the Raman spectra. Principal

component analysis (PCA) of the Raman spectra indicates that

Form I (1662 cm-1) converts entirely to Form II (1657 cm-1) within

around 20 hours (data not shown) at room temperature, there-

fore demonstrating that Form II is the more stable form of the API at

room temperature.

Quantitative determination of components in a

dry powder inhaler drug product

The traditional method for determining blend content uniformity of a

mixture is to sample the powder followed by off-line measurements.

A sample thief is used to sample small volumes of powder from the

blender and they are subsequently assayed using liquid chromato -

graphy to analyse the drug product14. This technique is not suitable

for rapidly testing hundreds of samples that are manufactured during

formulation screening and development due to time-consuming

sample preparation and lengthy analysis time. In addition, it is

technically challenging to sample only a few milligrams of powder

using a sample thief, a unit dosage for a dry powder inhaler (DPI),

without disturbing the blend.

In this study, a DPI blending process was used to demonstrate

the use of Raman spectroscopy as a robust and rapid screening

method to measure content uniformity of the powder blends8. The

DPI blend of interest contained three components that have distinct

Raman spectral fingerprints, making the system an excellent

candidate for quantitative analysis of all the three components.

Partial least squares (PLS) calibration models were constructed to

quantitate all three components. DPI blends from two intermediate

blending processes (blend I and II) were evaluated. Raman spectra



RAMAN SPECTROSCOPY

Figure 1: Real-time transformation of an API from Form I to Form II at room temperature

‘‘The traditional method for determining blend contentuniformity of a mixture is to sample the powder followed

by off-line measurements’’

were collected from blend I (n = 35) and blend II

(n = 80). Figure 2 shows the content uniformity

of the batch normalised to 100 per cent for each

component. The relative standard deviation

(RSD) measured for all three components are

five per cent or less for both blend I and II

(Table 1, page 36). As the blending process

continues, the blending %RSD improves for

blend I versus blend II, therefore demon strat-

ing that Raman can be a useful technique

in determining an end point for blending. In

Figure 2 the dotted lines represent the dose

content uniformity (DCU) US specifica tion

(80-120 per cent)15 of a DPI. The data show

that the content uniformity of both blends is

within the suggested DCU specifica tion,

indicating a uniform batch. If a blend would fail

to meet the DCU specification, this method

allows one to verify expediently whether it is the

blending process that is at issue and correct it

prior to making the drug product.

Process monitoring of a nasal spray

drug product

Raman spectroscopy inherently has a minimal

interference due to water, making the method

an excellent candidate to monitor processes

in an aqueous formulation such as an aqueous

nasal spray. In this study, Raman spectroscopy

was implemented to determine the homo -

geneity of an analyte for a nasal spray product

during batch manufacturing16.

The analyte used in this experiment has a

distinct Raman spectral region of interest, which

does not interfere with other ingredients in the

formulation. The PhAT probe was used to collect

the Raman spectra from the top of the vessel so

that at t = 0 minutes, the Raman spectrum of

the drug substance in the mixture was the

most intense. Raman spectra were collected

every 15 seconds with five second accumulation

times. As the analyte is charged into the vessel,

the signal is the highest and as the analyte is

dispersed, the signal decreases to reach a steady

state, implying that the API in aqueous medium

is uniformly dispersed. Figure 3A is the 3D plot

of the real-time Raman spectra of the analyte

signal, which decreases over time. PCA was

performed on the Raman spectra after fitting

them to second order polynomial baseline

correction in order to remove the varying

background (Figure 3B).

To determine the optimal processing

time for analyte dispersion, a running RSD

was calculated on every two consecutive

principal component 1 (PC1) data points.

The steady state of the dispersion process

was considered to be achieved when the RSD

remained below three per cent for fixed number

of minutes.

Tablet coat thickness measurement

Coating processes are important unit operations

associated with the manufacture of many solid

dosage forms. It serves to impart numerous

advantages to a dosage form such as odour /

taste masking, physical / chemical stability and

modified / sustained release. The thickness and

uniformity of coatings provide information on

the quality of the coated tablets. Direct measure -


Volume 18 | Issue 4 | 2013 38

RAMAN SPECTROSCOPY

Figure 3: A) 3D plot of the Raman spectra B) PCA of the Raman spectra

Figure 2: Content uniformity of three components of two intermediate processes – Blend I and II. Unit dose level (~1 mg)

‘‘Raman spectroscopy inherently has a minimal interference due to water,

making the method an excellentcandidate to monitor processes in an

aqueous formulation such as an aqueous nasal spray’’

‘‘Coating processes are important unit operations associated

with the manufacture of many solid dosage forms’’

RamanRxn2tm HybridDual Function Raman - Macro to Micro

Drug Products• Formulation Development• Raw Materials ID• Blending• Granulation• Drying• Tablets / Gelcaps• QA / QC

COMPLIMENTARYRegister for Kaiser’s Customer Presented Raman Webinars, or View a Webinar Archive.

Go to: www.kosi.com/webinars/

Drug Substance / API• API Development• In situ Reaction Monitoring• Reaction Pathway Understanding• Yield Optimization• Crystallization / Form Identification• Bioprocesses• Applicable to Water-Based Chemistries

On-Line or At-Line• API Low Dosage• Amorphous Content• Form Configuration• Coating Quality• Quantitation demonstrated to 0.05%

PAT / QbD Analyzer

ment of coat thickness is seldom carried out;

instead, indirect measurement, such as estima -

tion of coating thickness by weight change,

is practiced.

In this study, Raman spectroscopy was used

to quantify the tablet coating thickness as a

direct measure17. Placebo tablets (normal,

circular, biconvex, six millimetres in diameter)

made by direct compression were used for

coating. Hypromellose contained in the coating

formula has a prominent Raman peak at

approximately 1480 cm−1 where aliphatic ether

deformation of the polymer occurs18. Acquisition

of Raman spectra was carried out at an

excitation wavelength of 785 nanometres.

Each tablet was placed in a sample holder on a

mounting stage at the focal distance of the

probe and scanned on both surfaces using a

beam size of 300 μm. Acquired spectra were

subjected to spectral pre-processing such as

Savitzky–Golay smoothing and SNV algorithm

in order to remove sampling variations.

Raman spectra were correlated against changes

in coating thickness as coating progresses.

As the coating level increased, the intensity due

to the tablet core decreases and the intensity

due to the coating increases (Figure 4A).

Reference thickness measurements of these

tablets were obtained using optical microscopy.

Raman spectra were collected from 25 tablets to

generate PLS calibration model and inde -

pendent Raman spectra were used for

prediction (Figure 4B, page 40). The root-mean-

square error of calibration (RMSEC) and root-

mean-square error of prediction (RMSEP) are

2.16 and 2.73 μm respectively, indicating the

sensitivity and accuracy of Raman technique for

the coating thickness prediction. Measured

RAMAN SPECTROSCOPY

Figure 4A: Raw Raman spectra of blank tablet core and tablets coated to 0.4, 0.8, 1.2, 1.6, 2 and 3% (w/w) coatingweight gain (LP)

‘‘As the coating level increased, the intensity due to the tablet coredecreases and the intensity due to

the coating increases’’

(reference values using optical microscopy) and

Raman-predicted coating thickness values

indicate no statistical significant difference.

Overall, this work demonstrates that Raman

spectroscopy is a viable analytical technique to

measure coating thickness with similar accuracy

compared to that of optical microscopy.

Conclusion

Raman spectroscopy is a powerful technique

that is widely used within drug discovery and

pharmaceutical development. Raman spectro -

scopic methods in conjunction with the

application of in-line probes and/or usage

of chemometric methods are widely used in

a plethora of pharmaceutical applications.

In this review, a few examples of in-process

monitoring, which resulted in better under -

standing of the process and product critical

quality attributes, were discussed. These

examples further demonstrate that applications

of Raman spectroscopy in the pharmaceutical

industry can lead to significant optimisation of

workflows and understanding of in-process

manufacturing steps.


Volume 18 | Issue 4 | 2013 40

RAMAN SPECTROSCOPY

Dr. Chanda Yonzon is an Associate Principal Scientist at Merck

Manufacturing Division. She received her PhD in Chemistry from

Northwestern University, Evanston, IL under the supervision of

Richard P. Van Duyne. She has extensive experience in vibrational

spectroscopic techniques and has applied these techniques to

understand several pharmaceutical drug product dosage forms and

their processes. She has supported numerous projects in preclinical to

commercial and supply stage. She has authored over 25 publications

and presented her research at several conferences.

[email protected]

Dr. Atul Karande is currently a Senior Specialist engineer at Merck’s

Respiratory product development group, where he is part of the drug

product development team. He received his PhD in Pharmaceutics

from National University of Singapore. He has thorough

understanding of quality by design concept in manufacturing of

different pharmaceutical dosage forms. He has supported inhalation

and intranasal projects in preclinical to commercial stage at Merck

Research Laboratory and Merck Manufacturing Division. Dr. Karande

has several original research articles and conference presentations

within pharmaceutical sciences. Before joining Merck, he worked at

Meggle GmbH as a Technical Application Manager focusing on

optimisation of lactose characteristics for improvement of dosage

form performance.

Dr. Sai Chamarthy received an MS degree in Pharmaceutics from

Duquesne University and a PhD in Industrial and Physical Pharmacy

from Purdue University. Dr. Chamarthy is currently an Associate

Director at Merck’s Respiratory product development group, where he

is part of the drug product development team. Dr. Chamarthy is the

current Chair for the Inhalation and Nasal Technology AAPS Focus

Group (INTFG) and an executive committee member for the Process

Modelling and Simulation Focus Group (PMSFG). Dr. Chamarthy has

published over 20 papers in peer-reviewed journals. His research

has also led to over 15 podium and 30 poster presentations at various

national and international conferences.

Dr. Brent Donovan is an Executive Director leading the respiratory

product development department within Merck Research

Laboratories in Summit, NJ. He leads a team of scientists that are

engaged in analytical and formulation development of dry

powder inhalers, metered dose inhalers and nasal spray products.

His group has been working on developing novel methodologies

for the characterisation of inhalation products as well as

supporting numerous projects in all phases of CMC development. He

received his PhD in Physical Chemistry from the University of

Michigan. Dr. Donovan has several published original research

articles within chemistry and pharmaceutical sciences. He previously

worked at Schering-Plough for nine years prior to the merger with

Merck in 2009.

Biographies

1. Corredor, C.C.; Jayawickrama, D.; McGeorge, G.; Both,

D. “Monitoring of blending uniformity: form

conversion and fluid bed drying by near infrared and

Raman spectroscopy” American Pharmaceutical

Review, 2010, 13(1), pp 66-72

2. Widjaja, E.; Kanaujia, P.; Lau, G.; Ng, W. K.; Garland, M.;

Saal, C.; Hanefeld, A.; Fischbach, M.; Maio, M.; Tan, R. B.

H. “Detection of trace crystallinity in an amorphous

system using Raman microscopy and chemometric

analysis” European Journal of Pharmaceutical

Sciences, 2011, 42(1-2), pp 45-54

3. Pivonka, D. “Vibrational analysis of structure activity

relationships (SAR) in molecular binding Applied

spectroscopy” Applied Spectroscopy, 2004, 58(3),

pp 323-331

4. Heinz, A., Strachan, C.J., Gordon, K.C., Rades, T.,

“Analysis of solid-state transformations of

pharmaceutical compounds using vibrational

spectroscopy,” J. Pharm. Pharmaco., 2009, 61(8),

pp 971-988

5. Xie, Y., Cao, W., Krishnan, S., Lin, H., Cauchon,

N.,“Characterization of mannitol polymorphic forms in

lyophilized protein formulations using a multivariate

curve resolution (MCR)-based Raman spectroscopic

method,” Pharm. Res., 2008, 25(10), pp 2292-2301

6. Zhou, G.; Guenard, R.; Ge, Z., “Infrared and Raman

spectroscopy for process development” From

Applications of Vibrational Spectroscopy in

Pharmaceutical Research and Development, 2007,

pp 185-211

7. Yong Z.; “Model for Raman spectroscopic monitoring

of process-induced pseudomorph interconversion”

American pharmaceutical review, 2009, 12(4),

pp 56- 62

8. Yonzon, C.R.; Donovan, B.A., “Raman spectroscopic

method for content uniformity of a dry powder

inhaler,” Amer. Pharm. Rev. 2008, 11(7), pp 70-76.

9. El Hagrasy, A.; Chang, S.; Desai, D.; Kiang, S.

“Application of raman spectroscopy for quantitative

in-line monitoring of tablet coating”, American

Pharmaceutical Review, 2006, 9(1), pp 40-45

10. Wikstroem, H.; Lewis, I.R.; Taylor, L.S. “Comparison of

sampling techniques for in-line monitoring using

Raman spectroscopy”, Applied Spectroscopy 2005,

59(7), pp 934-941

11. Xie, Y.; Cauchon, N. “Raman scattering as a probe for

properties of active pharmaceutical ingredients in

tablet formulations” American Pharmaceutical Review,

2012, 15(2), pp 26, 28-31

12. Gao, Q.; Lew, A.; Takahashi, L.H.; Cassella, J.V. “An

investigation into the morphology of loxapine in a

thermal aerosolization process from crystalline to

amorphous”Journal of Pharmaceutical Sciences, 2011,

100(4), pp 1407-1415

13. Kwok, K.; Taylor, L.S. “Raman spectroscopy for the

analysis of counterfeit tablets”, From Infrared and

Raman Spectroscopy in Forensic Science, 2012, pp

561-572

14. Muzzio, F.J.; Robinson, P.; Wightman, C.; Brone, D.

“Sampling practices in powder blending. International

Journal of Pharmaceutics”, 1997, 155, 153-178

15. U.S. Department of Health and Human Services, Food,

and Drug Administration, Center for Drug Evaluation,

Research, “Guidance for Industry: Metered Dose

Inhaler (MDI) and Dry Powder Inhaler (DPI) Drug

Products” 1998

16. Pu, Y.; Medendorp, J. P.; Yonzon, C.R. “Real-time

monitoring of active ingredient dispersion in a

pharmaceutical aqueous suspension using Raman

spectroscopy” Journal of Raman Spectroscopy, 2011,

42(11), pp 1994-1999

17. Cahyadi, C.; Karande, A.D.; Chan, L.W.; Heng, P.W.S.

“Comparative study of non-destructive methods to

quantify thickness of tablet coatings” International

Journal of Pharmaceutics, 2010, 398(1-2), pp 39-49

18. Romero-Torres, S.; Perez-Ramos, J.D.; Morris, K.R.;

Grant, E.R. “Raman spectroscopic measurement of

tablet-to-tablet coating variability” Journal of

Pharmaceutical and Biomedical Analysis, 2005, 38(2),

pp 270-274

References

Figure 4B: PLS analysis of tablet coating thickness



43 The rapid microbiological methods revolutionEmanuele Selvaggio, QA Batch disposition & Investigation Supervisor, Pfizer

46 Controlling contamination in the pharmaceutical industryChris Delaney, Cleanroom/GMP Specialist, Noonan Services Group

52 RMMs RoundtableModerated by Jeffrey W. Weber, PAT Project Manager, Process Analytical Sciences Group & Chairman, Pfizer Rapid Microbiological Methods Steering Team, Pfizer

RMMs &Environmental

Monitoring

SPONSORS

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For many years after that first experiment

until the second part of the 20th century, all

microbiological methods relied upon growth

methods. The discovery of several species

that were hard to cultivate (for which suitable

in vitro culture conditions have not yet been

defined) as well as the existence of injured and

viable but non-culturable stressed cells (which

cannot be recognised as ‘alive or dead’ by

the cultivable / non-cultivable dichotomy),

became stimulating factors for the research of

alternative techniques.

Although growth-based method provide

the laboratory with critical information about the

amount and the type of organisms, from an

industry perspective the time to results are

usually much longer than desired. In addition, in

the case of critical events such as massive

contaminations, environmental microbial

excursion and test failures, real time / rapid

technologies may have a key role to maintain the

expected quality level to patients and may help

from a quality risk management standpoint.

As per the Eur.Ph. 5.1.6 definition, alternative

methods for the control of microbiological

quality can be divided into three categories:

� Growth-based methods, in which a detect -

able signal is usually achieved through a

period of subculture

� Direct measurement, in which individual

cells are differentiated and visualised

� Cell component analysis, in which the expr -

ess ion of specific cell components offers an

indirect measure of microbial presence.

Usually, the main difficulty is finding the best

technology which fits both the specific

laboratory requirements and project constrains.

Indeed, nowadays there are several tech -

nologies which can be utilised to address any

type of request depending on the application

(sterility testing, bioburden, microbial identifi -

cation, air monitoring etc.). In order to find the

best solution, it must be pointed out that it is

mandatory to consider the particular factors

that make every single project unique.

Points to consider when starting with a

new project are:

� Purpose of the project (cost saving;

lead time reduction, process knowledge

improve ment, quality improvement, etc.)

� Drug product characteristics (Filtera-

bility, Antimicrobial effect, Colour, Matrix

influence, etc.)

� Budget constrains

� Personnel skills

� Laboratory space and ancillary equipment

� Vendor support and experience.

The technology of choice is as important as the

vendor of choice. In fact, over the last decade,

the overall number of vendors available has

been exponentially growing. As the majority of

rapid micro methods rely upon either universal

cell biochemical pathways or common macro -

molecular cell components, nowadays both

chemical instrument vendors and consolidated

micro laboratory vendors are competing for

the market.

Microbiology was officially born in 1676 when a Dutch tradesman and scientist from Delft, the Netherlands, observed bacteria

and other microorganisms for the first time using a single-lens microscope of his own design. Almost two centuries later,

a German biologist called Robert Koch founded modern bacteriology and microbiology. In the 1850s at the University of Breslau,

Ferdinand Cohn's main research tool was a large and expensive microscope that his father had bought for him. In the 1850s,

he studied the growth and division of plant cells and he proved that the use of liquid media was disadvantageous for isolating

pure culture. He was determined to find an alternative technique and introduced the gelatine liquid culture media to be poured

on sterilised glass plates to solidify for the first time.

The rapid microbiologicalmethods revolution

Emanuele Selvaggio

QA Batch disposition & Investigation Supervisor, Pfizer



IN-DEPTH FOCUS:RMM’S AND ENVIRONMENTAL MONITORING

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It cannot be denied that vendors play a key

role in the development of new method-

ologies across the site when deciding what

instrument to buy. Vendors are involved at

several stages of the project, e.g. feasibility

studies, design qualification, IQ/OQ, PQ and

sometimes even during filing (if any). There-

fore, lack of experience as well as reliability

issues would inevitably decrease initial expecta -

tions and chances to complete the project on

time and could even affect the possibility for

the project to be successful. Consequently, the

choice of technology is strictly connected to

the vendor of choice.

Whatever the technology is, there are some

common characteristics which should be

addressed by most rapid micro methods; they

are usually better detectors (more sensitive,

requiring fewer microbial cells) and allow the

laboratory to get faster and more accurate

results and to improve precision due to the

automation of most of the critical steps.

Depending on specific cases, RMMs are an

opportunity to reduce the cost/assay and to

improve the overall quality level.

An example of a cost saving project is

related to MALDI-TOF mass spectroscopy.

MALDI TOF technology is about to revolutionise

timing and costs currently related to microbial

identification in pharmaceutical microbiology

laboratories. This technique has been well-

known for years by chemists but only recently

has been successfully developed for microbial

identifications within GMP contexts.

In the pharmaceutical industry, the need for

timely and accurate microbial identifications is

critical for the characterisation of micro -

organisms isolated from the aseptic area as well

as from final product samples.

The ability to explain aberrant results and

provide information about potential sources of

microorganisms is important to maintain

control over the manufacturing process. A wide

range of microbial identification technologies

currently exists and varies in labour intensity,

cycle time and cost.

MALDI-TOF technology combines cost

savings goals with rapidity. In fact, for isolated

colonies (from agar plates), less than five

minutes is required to get a reliable (accurate

and reproducible) result, at the cost of about one

Euro each. This is an embarrassing fact con -

sidering that most common technologies need

hours if not days to get a result, and costs range

from 10 to 20 Euros per identification.

Whatever the process/assay is, the faster

results can be obtained, the more process

knowledge is increased. It must be noted that

compendia assays generate results which

are always delayed, hence most of the time

QA/QC cannot find the real root cause of an out

of limit specification.

Until a process is real-time monitored, it will

never be really under control so any potential

adverse event or unplanned deviation will be

managed too late to be properly addressed.

For these reasons, corrective and preventative

action plans are often ineffective. Further

analysis is usually required even weeks after

an event has occurred and the decision-

making processes are often too conservative

(batch disposition results in rejections due to a

lack of knowledge).

Real time technologies as well as rapid

technologies give those in charge of monitoring

the process quality an incomparable tool to

promptly react to process quality attribute

excursions, managing the present rather than

investigating the past.

Return of Investment calculations represent

a tough challenge for those scientists who

approach a rapid method for the first time.

Although a dedicated financial analysis is


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Mycoplasma DNA-Chip

Your Power for Health

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Germany (Main offi ce): Greiner Bio-One GmbH, [email protected] l Austria: Greiner Bio-One GmbH, offi [email protected]: Greiner Bio-One BVBA/SPRL, [email protected] l Brazil: Greiner Bio-One Brasil, offi [email protected] l China: Greiner Bio-One Suns Co. Ltd., offi [email protected] France: Greiner Bio-One SAS, [email protected] l Japan: Greiner Bio-One Co. Ltd., [email protected] l Netherlands: Greiner Bio-One B.V., [email protected]: Greiner Bio-One Ltd., [email protected] l USA: Greiner Bio-One North America Inc., [email protected]

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required case by case, a rough estimation of the

ROI can be made considering the following

aspects: manpower, disposables, reagents, costs

of goods and any related activity.

The points to consider are:

� Cost / assay

� Where it takes place

� Number of batches / session

� Disposables

� Reagents

� How many samples / assay

Manpower

� How long it lasts

� Number of single operations

� Time for single operations

� Skills required

Others

� Inventory impact (in term of ‘cash’

inventory costs)

� Warehouse (in case of renting)

Whatever the technology to develop is, an ROI

analysis always starts with a process mapping of

the current method to replace. Any single step

must be considered in terms of both manpower

and costs.

When designing the new process flow, ‘lean

laboratory’ principles as well as a DMAIC

approach are strongly recommended to get the

highest advantage from the new technology.

Indeed, rapid microbiology projects are often a

great chance to change the mind-set of

laboratory employees and to improve efficiency

and productivity.

In conclusion, rapid microbiological

technologies along with PAT are nowadays

the pillars upon which companies should

build the future structure required to

address latest BoH expectations (which are

progressively more demanding) and market

challenges which lead companies to an endless

budget dwindle.

IN-DEPTH FOCUS: RMM’S AND ENVIONMENTAL MONITORING

Emanuele Selvaggio received his degree in Organic Chemistry in

2003 from the University of Catania, Italy. During his career, he

worked in several departments such as Engineering Validation,

Quality Assurance and Technology. In 2008, he was certified as Green

Belt in accordance with the Lean Six Sigma Company Program and

has recently completed his Black Belt project. Since 2008, he has been

in charge of leading many OpEx projects related to several areas such

as Quality Assurance, Quality Control and Manufacturing/Pack

Processes. He was also recruited for several global projects such as

‘Validation Standardisation Project’ (Metrics team) and ‘Laboratory

Instruments Qualification Standard Protocol’.

Since 2009, he has been involved with PAT projects, particularly with

Rapid Microbiology projects (Bioburden, Identification and Cell count

based methods) and since 2011 he has been a permanent member

of the rapid micro methods steering team at Pfizer; he has conceived,

developed and successfully leaded a rapid micro project aimed at

reducing the sterility test cycle time from 14 days to less than one day.

He recently joined the Quality Assurance department as QA Batch

disp. & Investigation Spv.

About the author

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Most of the production in the pharmaceutical

industry is carried out in cleanrooms and

although the principle of cleanroom design

goes well back over 150 years in the hospital

setting, it is only since the 1950s that we have

really seen the development of the modern

cleanroom as we know it. This was driven firstly

by NASA’s space program and later by manu -

facturing needs for a clean environment to carry

out its processes. Cleanrooms are used in

practically all industries where particles can

adversely affect the manufacturing process.

These range from the semiconductor industry,

medical devices, pharmaceutical, biotech and

life sciences.

A cleanroom is a room in which the

concentration of airborne particles is controlled

and which is constructed in a manner to

minimise the introduction, generation and

retention of particles inside the room and in

which other relevant parameters such as temp -

erature, humidity and pressure are controlled as

necessary. The main function of a cleanroom is

to protect the manufactured product from

contamination and this is of utmost importance

to both the manufacture and the customer who

is in actual fact the patient. In the pharma -

ceutical industry, the manufacturer’s economic

survival depends on the safety and quality of the

product. The lives of patients and the reputation

of the manufacturer depend on the purity of the

product. Good Manufacturing Practice (GMP) is

widely accepted as the best way to conduct

business in the Pharma sector. GMP ensures

that products are consistently produced and

controlled to the quality standards appro-

priate to their intended use. GMP covers all

aspects of production from materials, premises,

equipment and the training and the personal

hygiene of staff.

Pharmaceutical cleanrooms are classified by

the cleanliness of the air. The method that is

most easily understood and universally applied

is the A to D version of the Federal Standard 209

in which the number of particles equal to and

greater than 0.5 millimetres is measured in one

cubic foot of air and this count is used to classify

the room. The most recent 209E version has

accepted a metric nomenclature. The newer

standard is TC-209 from the international

standards organisation. Both of these standards

classify a cleanroom by the number of particles

found in the cleanrooms air. The cleanroom

classification standards FS209E and ISO 14644-1

require specific particle count measurements

and calculations to classify the cleanliness level

of a cleanroom or clean area. In the UK, British

standard 5295 is used to classify cleanrooms.

Based upon both the US and EU GMP, con -

tinuous particle counting is a requirement for

sterile manufacturing. Other parameters and

controls in sterile manufacturing are temp -

erature, relative humidity, differential pressure

and air velocity and direction.

In the cleanroom, there are several sources

of contamination such as process equipment,

surfaces and of course personnel. Contamina -

tion can be surface or airborne contamination.

The three main types of contamination in a

cleanroom setting may be particulate, microbial

and rogue or cross contamination. Dust,

fibres and hairs are examples of particulate

con tamination, while microbial contamination

may arise in the form of mould, bacteria or yeast.

Rogue or cross contamination comes about if

materials or products are mixed. Operators are

typically the largest source of contamination in

cleanrooms and in order to counteract this

In Ireland and across Europe, the traditional manufacturing sectors have seen major declines in output and there has being a

fall in the numbers employed in this sector. However, one area that has seen growth is the pharmaceutical industry. In Ireland,

the economy is very dependent on the pharmaceutical industry and the industry currently accounts for over 50 per cent

of the country’s exports.

Controlling contamination inthe pharmaceutical industry

Chris Delaney

Cleanroom/GMP Specialist, Noonan Services Group

IN-DEPTH FOCUS:RMMS AND ENVIRONMENTAL MONITORING


Volume 18 | Issue 4 | 2013 46

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Our IMD-A® systems monitor controlled areas in real time, detecting microbes instantaneously so you can act. Our systems utilize advanced optics and signal processing, requiring no staining, no reagents, and little human intervention. Let us help shed light on what may be lurking in your environment.

Our Instantaneous Microbial Detection™ Solutions Can Help Shed Light.

threat, it is important that the design is right and

also that proper controls are put into place.

These controls can cover areas such as gowning,

personnel flow and material flow. In the

remainder of this article, I am going to con -

centrate on the importance of having an

effective environmental monitoring programme

in place and how correct cleaning and sanitisa -

tion is essential in the cleanroom setting. From

my own personal point of view, after working in

some of the leading global pharmaceutical

companies, developing and improving con -

tamination control programmes these are areas

that I have a keen interest in.

Across the pharmaceutical, biotechnology

and medical devices industry, companies

have cleanroom operations of different sizes

and complexities. Manufacturers in these

industries have recognised that there is a

regulatory compliance to demonstrate clean -

room per formance and the control of product

bioburden by monitoring the environment in

their aseptic manufacturing areas. Environ -

mental monitor ing is a programme designed to

demonstrate the control of viable (living

microorganisms) and non-viable particles in

critical areas. Viable monitoring refers to testing

for the detection of bacteria, yeast and mould

and includes the monitoring of personnel,

surfaces and areas for microbial contamination.

A non-viable particle is a particle that does not

contain living micro organisms but acts as

transportation for viable particles. Non-viable

particles are monitored using particle counters

which do not distinguish between viable and

non-viable particles.

Companies must monitor their cleanrooms

to ensure that their desired quality standards

are met. Competition in the pharmaceutical

industry is intense and competitive and

manufacturers cannot afford to have even a

perception of any quality issue associated with

their products.

Results obtained from an EM programme

provide information on the performance of the

HVAC system, the effectiveness of cleaning and

IN-DEPTH FOCUS: RMMS AND ENVIONMENTAL MONITORING

Figure 1: Classification of cleanrooms: comparison of classification

sanitisation procedures and the effectiveness of

hygiene and gowning practices. Therefore, it is

critical that the programme is valid, justified and

compliant. In developing an adequate environ -

mental monitoring programme, there needs to

be a sufficient number of sampling locations

which are representative of the microbial

challenge to critical activities and are based on

the nature of the process being performed

and the impact of personnel and equipment on

microbial levels. The application of risk assess -

ment to environmental monitoring provides

information on the most appropriate locations

to monitor within the cleanroom with regard to

potential impact of activities on product

quality. The use of risk assessment approaches is

an important cGMP tool in microbiological

environmental monitoring. However, each suite

of cleanrooms or isolator will be subtly different.

Every aspect of the environment must be

considered and what level of monitoring best

suits the system decided, the techniques used

and the locations selected must be justified. Risk

assessments should be kept up-to-date and any

modifications to the area should be reflected.

The three areas that are usually monitored

in a cleanroom are personnel, air and surfaces.

Personnel are the biggest source of contamina -

tion in the cleanroom setting and account for

over 90 per cent of contamination in clean -

rooms. More and more companies are seeing

the benefit of providing a good training

awareness programme for their staff. The

environ mental programme must be designed to

highlight that the preventative measures such

as correct gowning, hand washing and personal

flow is being adhered. Personnel monitoring

employs contact plates to assess microbial

contamination of cleanroom personnel. The

contact plates monitor areas of the body that

may interact with the sterile field or exposure

areas. Personnel monitoring is a good indication

of how well personnel are gowning when they

enter the cleanroom. More and more companies

are using this technique, especially in their

aseptic process areas.

Some ways to measure air sampling in the

cleanrooms are air samplers (active air sampling)

and settle plates (passive air sampling). In air

samplers, predetermined volumes of air are

drawn in over a sterile media plate which is later

incubated to reveal the number of viable

organisms. With the settle plates, Petri dishes

containing sterile growth media are exposed

to the environment for a specific period of

time. Once the plates are incubated, viable

microorganisms that landed on the plates will

begin to grow.

The monitoring of surfaces in the clean -

room is another essential part of the EM

programme. This can be carried out by the use of

contact plates or swabs. The contact plates

which contain sterile growth medium are

pressed against the surface and then incu-

bated to see if there is microbial growth.

Likewise, the swabs which are sterile and stored

in a suitable sterile liquid are rubbed over the

test area that needs to be sampled. Swabs are

used for areas that are not flat or areas that

are hard to reach such as machinery. Environ -

mental organisms recovered from the

cleanroom areas should be identified to show

what organisms are present. Knowing this

will provide the organisation with important

information in monitoring and prevent

potential future contamination pitfalls. Alert

and action limits should be implemented

based on the company’s products, the intended

use of the cleanroom and the classification of

the cleanroom.

Prior to the notion of GMP regulations,

cleaning was never considered to be important.

It was the activity that was carried out last thing

in the day or by the newest employee. The

procedures were often brief and very often

limited to one sentence. But things have

changed dramatically over the years and now

regulators must position cleaning very highly on

their agenda when carrying out audits. Cleaning

is an essential element of contamination control

in the cleanroom. In my own opinion, I believe

that the cleaning process must be viewed as

important as the manufacturing process.

There are many decisions that need to be made

about the details of cleanroom maintenance

and cleaning. Applications and procedures

must be agreed upon by cleanroom managers,

quality depart ment and the cleaning team.

For an effective cleaning programme, there

must be a buy-in from all strands of the

organisa tion. More and more pharmaceutical

companies are out sourcing their cleaning and

maintenance programmes and some main -

tenance comp anies provide expertise advice

and support in drawing up such maintenance

and cleaning programs. In the pharmaceutical

industry, more and more emphasis is being

placed on an efficient and effective cleaning

programme. This is being driven by the need to

reduce the cost of maintenance and cleaning,

increasing prod uction time and also by the

importance being place on cleaning and

maintenance by the regulatory bodies. There

are many key elements of an effective cleaning

programme such as:

� What is regarded as clean

� How is clean managed

� What cleaning materials can be used

� When can the cleanroom be cleaned

� The frequency of cleaning that is required.

The disinfectants selected for use must be

appropriate so that the effectiveness of the

disinfectants is assessed and the scientific

rationale for their selection is documented

as required by USP <1072> ‘Selection of a

disinfectant for use in a Pharmaceutical

Manufacturing Environment’. For surface

challenge testing, the test organisms are

enumerated using swabs, surface rinse or

contact plate methods. Neutralisers that

inactivate the disinfectants should be included

in either the diluent or the microbiological


Volume 18 | Issue 4 | 2013 48

IN-DEPTH FOCUS: RMMS AND ENVIONMENTAL MONITORING

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media used for microbial enumeration or both. Like all cleanroom

developments, there have been great advances in the disinfectants

available and their effectiveness. A disinfectant must have a wide

spectrum of activity. It must have the ability to kill different types of

microorganisms. Currently, the majority of the pharmaceutical

industries use phenolics, quats or quat mixes for routine cleaning.

Therefore, rotation of disinfectant and a sporicidal is common in

the industry.

The USP indicates an acceptance criteria as follows, log reduction

(2 for bacterial spores and 3 for vegetative cells), pre-determined

contact time and recommended typical organisms to be selected.

EN 13697:2002 – Quantitative surface test of bactericidal activity –

describes the method to be used to determine the anti-microbial

activity of commercial formulation or active substances on bacteria in

the conditions in which they are used.

It is important that the right equipment and materials are

selected for cleanroom cleaning. Only equipment that has

proven cleanroom performance records should be selected for

use in cleanrooms. There are many products available now

for cleaning in all cleanroom classification. These include cleaning

and disinfecting solutions, cleanroom mops, cleanroom vacuums,

cleanroom wipes and cleanroom mop and bucket systems. From my

own experience, I have seen how an inadequate cleaning and

maintenance programme can lead to various levels of contamination

problems and potential loss in end user product quality. The saying

‘fail to plan and plan to fail’ is very appropriate when it comes to

setting up a cleaning programme in the pharma ceutical industry.

The effectiveness of the cleaning programme affects all sections of

the company and of course the end user, who is most important.

In conclusion, the main purpose of building a cleanroom suite is

to provide a vital element in the assurance quality of the product for

the end user which in the pharmaceutical industry is the patient. The

cleanroom and indeed the pharmaceutical industry has come a long

way over the brief 25 years that I have worked in it and the next

25 years promise to be more challenging and exciting. The develop -

ment of cleanroom technology is likely to continue in the coming

decades. The pharmaceutical industry is also expected to continue to

grow in the coming years and in recent years, cleanroom technology

has been applied to micro and nano systems processes and this also

looks like a high growth rate in the coming years. The development of

cleanroom technology will also be driven forward by the creation and

use of materials of the finest purity, and by the broad based use

of biotechnology. Given the scale of these challenges, cleanroom

technology looks set to remain indispensable to the pharmaceutical

industries in the coming years and it’s an exciting industry to be in.



IN-DEPTH FOCUS

Chris Delaney has spent over 25 years working in cleanrooms. He has

worked in the medical devices, semiconductor and pharmaceutical sectors.

After completing his degree in management, Chris began in the

manufacturing sector and worked in some leading companies in the medical

device and semiconductor industry. In 2004, Chris moved to the service

industry and joined Noonan service group where he now is GMP/Cleanroom

specialist in the Life Science section. Chris has worked in some of the leading

pharmaceutical companies and has developed many contamination control programs. Chris is also an

executive committee member of the Irish Cleanroom Society. He has also developed a number of

training courses on contamination and cleanroom cleaning.

Biography

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The Growth Direct™ System revolutionizes microbial testing.

By providing a single technology to perform all key microbial quality control tests, the Growth Direct™ System automates and accelerates testing with positive results in hours and fi nal CFU counts in about half the time of traditional methods, eliminating error-prone manual steps and saving labor.

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The Growth Direct™ System eliminates many

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The Growth Direct™ System is designed to

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“An environmental monitoring programme

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room complies with microbial count standards

helps verify the effectiveness of facility cleaning

protocols and control the risk of an external

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microbiology lab must perform; automating the

environmental monitoring tests for air, surface

and personnel saves significant time.”

One of the major benefits of The Growth

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Our final results are available in about half the

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The Growth Direct™ System offers several

areas of value to the pharmaceutical industry

– it can save time, reduce errors, reduce the

reliance on paper and allow microbiologists to

perform higher value work for the organisation.

Sperry explains: “First, by reducing the time-to-

result the product can be released faster.

Next, by automating the process, manual errors

associated with enumeration, incubation, and

data entry are eliminated. These types of errors

often generate investigations that can cost the

company tens of thousands of dollars. Finally,

because the Growth Direct™ System replaces

all the incubation and detection typically done

in the lab, highly trained personnel can be

focused on more critical tasks. Finally, the system

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The Growth Direct™ System became

commercially available in 2013 and Rapid Micro

Biosystems has been extremely pleased with

the response thus far. “Pharmaceutical manu -

facturers have seen the value we discussed

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have been eager to visit our facility in the US

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For more information about Rapid Micro

Biosystems and the Growth Direct™ System,

visit www.rapidmicrobio.com



Tham

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Product HUBJulie Sperry, Chief Commercial Officer atRapid Micro Biosystems discusses theirGrowth Direct™ System technology Rapid Micro Biosystems provides a range of products to aid faster detection of

microbial contamination in the manufacture of pharmaceutical, biotechnology and

personal care industries. The company’s new technology, the Growth Direct™

System, uses proprietary digital imaging technology that automatically enumerates

microcolonies days earlier than the traditional visual plate counting methods.

The system captures the native fluorescence (autofluorescence) that is emitted by all

living cells. By detecting microcolonies composed of a small number of cells, the

Growth Direct™ test can automatically report the number of microbes in a sample

days earlier than the current visual colony counting method. It is the first and only

automated system that addresses all key microbial QC applications and fits with the

current regulatory practices; a critical accelerator for adoption.

What are the perceived hurdles for

Rapid Micro Method implementation?

Plummer: The greatest hurdle is the lack of well-

defined guidelines and regulatory expectations.

Existing documents aren’t in step with RMMs

evolution, either not considering RMMs or

not addressing distinctions between them.

Available guidance is open to interpretation and

requires adaptation to ensure test evaluations

and data analyses are applicable to the new

method. Pharma companies express reluctance

in making modifications without knowing if

their validation approach will find regulatory

acceptance. Exacerbating the uncertainty is the

ill-alignment of regulatory bodies in what is

required for validation. This makes imple -

mentation especially difficult for companies

who sell drug products across multiple

regulatory jurisdictions.

Herber: The biggest hurdle for potential

Rapid Micro Method users tends to be the

confusion regarding which new methods

adhere to existing compendia guidelines and

which methods do not. Our Charles River PTS™

cartridge technology for endotoxin testing

helps eliminate this confusion as it follows the

harmonised BET chapters, and thus is used

routinely for endotoxin determination in raw

materials, in-process samples and finished

product. Factor C and various in vitro assays do

not follow current regulatory guidelines and

would require additional validation as an

alternative method.

Stappert: Manufacturers of biologics are

quite reluctant in establishing RMMs due to the

lack of clear validation and/or acceptance

criteria for these methods.

Niccum: TSI’s BioTrak Real-Time Viable

Particle Detector utilises Laser Induced

Fluorescence (LIF) to determine if a particle is

viable in nature. This method does not rely on

culturability of microbial particles. LIF is more

sensitive and ‘sees’ more viable particles than

compendial methods. The highly sensitive, real-

time results offer significant advantages in the

market. To benefit from this technique, the user

community needs to be willing to trial these

innovative products by conducting evaluations

near existing compendial testing locations to

establish correlations. These can then be used as

support for identifying meaningful action and

alert limits.

What is the biggest challenge to

validating RMMs for manufacturing?

Stappert: Although RMMs may be validated by

the manufacturer of the different techniques,

an individual adaptation / testing must be

performed for every matrix / biologics pro-

duced / used by the manufacturer of biologics.

This task can only be achieved by a team play

between the RMM producer and the manu -

facturer of biologics.

Niccum: Existing guidance documentation

available in USP 1223, EP 5.1.6, and PDA TR33

focuses on laboratory based methods where


Volume 18 | Issue 4 | 2013 52

RMMS ROUNDTABLE

© Sergey Nivens / Shutterstock.com

Moderator

Jeffrey W. Weber, PAT Project Manager, Process Analytical Sciences Group & Chairman, Pfizer Rapid Microbiological Methods Steering Team, Pfizer

Carrene PlummerDirector, Quality and

Regulatory AffairsAzbil BioVigilant, Inc.

Ulrich HerberSenior European Product

Manager Endotoxin and MicrobialDetection Service

Charles River

Jörg StappertManager Biochip GroupGreiner Bio-One GmbH

Darrick NiccumSenior Global Product

Manager – BiotechnologyTSI

serial dilutions can be employed to generate

challenges comparing RMMs to traditional

culture plate count results. Aerosol challenges

are more difficult to create and control than

liquid samples. It is known that active samplers

have varying degrees of microbial capture

efficiency so the ‘truth’ data set has a high degree

of uncertainty. Thus, the numbers for the

‘standard’ method are highly variable and add as

much uncertainty as the aerosol instrument

under test.

Plummer: Many RMMs are vastly different in

functionality and performance from the

traditional methods they are intended to

replace. Data generated by these new methods

are oftentimes not directly comparable to data

obtained from traditional methods. Examples

include disparate units of measurement

(e.g., CFU vs. biologic count) and discrete vs.

continuous data streams. Determining what the

new data means and how to distil actionable

manufacturing decisions can seem over -

whelming. Azbil BioVigilant’s applications

engineers have assisted several customers early

in their implementation planning to address and

suggest practical approaches.

Herber: In addition to achieving rapid

turnaround time, one of the key challenges our

manufacturing customers face is maintaining

QA/QC oversight when testing is performed

outside of the central lab. Point-of-use testing

instruments on the production floor, like the

PTS™, must have built-in safeguards such as

password protection and the ability to be

externally controlled by the QA/QC group.

An invalid assay or an expired calibration

must result in the RMM instrument being taken

off-line until cleared for use by the quality

group. Prospective RMM users also should

ensure that their vendor provides support to

overcome these challenges.

What guidance is available for

statistical analysis of RMM compared

to compendial methods?

Niccum: We are eagerly awaiting the revision of

PDA’s TR33. Based on discussions with members

of the panel, there is significant emphasis on

providing appropriate statistical method

guidance for evaluating RMMs to compendial

method results. We anticipate additional

guidance when revisions to USP 1223 and EP

5.1.6 are released. Several courses sponsored by

ECA and PDA provide overviews of statistical

methods. It is imperative that users look beyond

the statistical values presented by instrument

vendors and understand the rational and

assumptions incorporated into the statistical

method utilised.

Stappert: General: ICH Q2B, USP1223, Ph.

Eur. 2.6.7/2.6.21; Specific: CLSI EP 17-A.

Plummer: Published monographs do exist

which can offer some initial, high-level guidance

on statistical approaches that may be suitable

for data analyses (e.g., EP 5.1.6 – Alternative

Methods for Control of Microbiological Quality

and its US counterpart, USP <1223> -Validation

of Alternative Microbiological Methods). These

monographs, however, do not define the

specific statistical methods that are best to apply

depending on the RMM and intended

application. It is highly recommended that RMM

evaluators seek advice from expert statisticians

to help define proper statistical analyses while

keeping the RMM, compendial method and the

Pharma customer’s validation goals in mind.

How can the Pharma industry

accelerate the adoption and

implementation of RMMs?

Plummer: Regulators have publicly expressed

their desire to see more widespread use of

RMMs and have stated their willingness to aid

end users in the implementation process.

The Pharma industry should be proactive and

take advantage of the open, informal dia-

logue that has been offered by the regulators.

Frequent communication with them, including

partici pation from both reviewers and inspec -

tors, is ideal. Working closely with regulators

fosters a smoother and faster transition to

RMM implementation.

Herber: The Pharma industry should

continue meeting with the regulatory bodies,

either during an industry conference or one-on-

one, to discuss RMMs and get agency buy-in

early on. The Pharma industry can speed up

RMM acceptance internally by better under -

standing the true cost of the conventional

microassay. Keep in mind, the conventional test

usually looks more attractive when just the

reagent costs are analysed, but it is only when

you look at hands-on technician time, data entry

and review, production efficiency, retest rate and

subsequent investigations that you can

establish a fair comparison between the

conventional and the rapid method costs.

Niccum: The Pharma industry includes

pharmaceutical manufacturers, instrument

vendors, regulators, and consultants. We must

develop open and honest dialogue where the

core RMM technologies are discussed and not

limit the dialogue to each instrument vendor’s

implementation of that technology. The

compendial methods have known capabilities

and deficiencies. The Holy Grail of a single

RMM suitable for all applications does not

exist. Each RMM has known capabilities and

potential deficiencies. Only through open

discussion and scientific analysis will appropri -

ate applications for each RMM be identified and

promulgated. This is especially true for real-

time methods where both the sampling and

counting functions are incorporated into a

single instrument.

Stappert: Firstly by getting clear guidelines

for validation of RMMs (as already established

since long time for the validation of IVD/clinical

products e.g., FDA_CDRH_HPV guidance) etc.,

followed by a close collaboration between the

RMM manufacturer and the RMM operator for

necessary matrix adaptations.

Are there practical RMMs being

developed to support environmental

and personnel monitoring?

Plummer: Most certainly. The Azbil BioVigilant

IMD-A system was developed with practicality

in mind. It requires no staining, no reagents,

and little operator intervention in order to

monitor the environment and warn users

when a microbial threat may be present.

The system also operates in real-time and

continuously, so immediate information is

available about the sampling environment.

These features allow actionable decisions and

remediation to be made as excursions occur

instead of retrospectively.

Niccum: Real-time viable particle detectors

are an example of a practical environmental

monitoring RMM. The technology is not at the

point where direct replacement of all com -

pendial active sampling and culture based

counting applications exist. This will occur

over time resulting from on-going use and

increased understanding of the technology.

With that said, there are many applications

where real-time viable detectors can be

employed offering significant financial and

quality benefits to pharmaceutical manu -

facturers. As one inspector mentioned, they are

currently another tool in the tool set which

should be used where applicable. TSI refers to

this as the innovation highway resulting in

greater acceptance by the user community.



RMMS ROUNDTABLE

Rottapharm can boast a rich portfolio of original

molecules and drugs, discovered and patented

by its own R&D department. From the original

glucosamine sulphate, an essential molecule

in the treatment of osteoarthritis, to pro-

glumetacina, a non-steroid anti-inflammatory

drug, up to the launching of the first transdermal

active matrix patch with estradiol an innovative

transdermal releasing system for hormone

replacement therapy in the menopause,

research has not stopped but in fact has

continued to grow and develop in a steady

manner. In the pharmaceutical sector,

Rottapharm|Madaus has over 13 projects in

its pipeline.

Rottapharm in Ireland

The Rottapharm manufacturing site in Dublin,

Ireland was officially opened in June 1999 and

commenced manufacturing operations later

that year. The Dublin site employs 150 people

manufacturing a range of capsules, sachets

and tablet formulations in addition to the API

Crystalline Glucosamine Sulphate. The comp -

any’s leading product DONA, the original

Glucosamine Sulphate, is manufactured in

sachet and capsule formats for markets

in Europe, Asia and South America. DONA is the

first drug of choice to be administered as early as

possible in the treatment of osteoarthritis.

Production output has grown steadily over

the years, with the Dublin site becoming one

of the most important manufacturing sites

within the Rottapharm|Madaus group of

companies. Figure 1 (page 55) shows the growth

in terms of percentage output from 2000 to

2012. The increase in growth from 2009 onwards

was as a result of a seven million Euro invest -

ment and facility expansion. The investment

involved the transfer of a number of new pro -

cesses from another facility within the group.

The project expansion resulted in an addition of

four packaging lines, two coating systems and

an encapsulation machine. Volumes of finished

product packaged increased significantly, as

a result and 35 new positions were created.

The project was completed in 2010.

Lean Six Sigma and the Operational

Excellence programme

In order to improve operations and per -

formance, the Senior Management team at

Rottapharm Dublin introduced a Lean Six Sigma

program in 2007. Six Sigma seeks to improve the

quality of process outputs by identifying and

removing the causes of defects and minimising

variability in manufacturing and business

From their headquarters based in Monza, Italy, Rottapharm’s long history of success began in 1961 with the creation of a small

laboratory for independent research. The company from its early beginning has continuously invested in research, innovation

and development of pharmaceutical products for distribution on a worldwide scale. After acquiring the German multinational

Madaus Pharma in 2007, the Group is currently one of the most significant and prominent pharmaceutical companies in Italy and

is present in over 85 countries worldwide. The field of research is above all the undisputed true mission of the company.

Improving operations and performance: howRottapharm is using Lean Six Sigma principles

Richard Hayes

Continuous Improvement Manager, Rottapharm

SIX SIGMA


Volume 18 | Issue 4 | 2013 54

processes. It uses a set of quality management

methods, including statistical methods, and

creates a special infrastructure of people within

an organisation (Black Belts, Green Belts, etc.)

who are experts in these methods. Each Six

Sigma project carried out within an organisation

follows a defined sequence of steps and has

quantified financial targets cost reduction

and/or profit increase. Rottapharm has run over

20 Six Sigma projects over the past five years.

Typically, these projects were completed on

difficult process problems encountered and by

the application of Six Sigma methodologies,

an effective means of problem-solving and

process improvement could be implemented.

These problems were tackled by teams using

the Six Sigma approach of Define the prob-

lem, Measure, Analyse, Improve and Control

(DMAIC problem solving process). The Six

Sigma DMAIC problem-solving approach con -

tinues to be used to date, supporting the overall

Operational Excellence programme within the

plant. The company has also introduced other

lean tools and concepts into the business in

order to remove waste and drive down costs

in the business. Some of these tools and con -

cepts are described below.

Total Productive Manufacturing (TPM)

The lean tool known as Total Production

Maintenance or Total Production Manufacturing

(TPM) is the main driver of the Operational

Excellence programme within the Rottapharm

Dublin facility.

TPM is a way of working in a cellular based

team structure with manufacturing teams,

made up of operators, technician and engineers,

supported by various Pillar Champions, usually

managers of a particular support function such

as materials, maintenance, safety, quality etc.

TPM focuses on equipment, the interaction of

people with that equipment and the waste

associated with the equipment and the various

interactions. TPM doesn’t just focus on equip -

ment alone; all other support departments are

integrated into the overall structure as their

contribution plays a key role in the success of

each project. All the waste associated with both

the actual manufacturing activity and the

support activities are targeted in order to keep

costs down and productivity up.

The TPM project at Rottapharm Dublin

began in May 2010 with 19 people trained on

the nine step TPM improvement plan. Two

separate shift teams focused on a manu -

facturing area while two other shift teams

looked at a sachet filling and packing line, each

team comprised six members. The teams took

20 weeks using one day a week every fortnight

to go through the nine step TPM programme for

their project. Each day that TPM was scheduled,

production activity was suspended and the

teams worked through a programme of activity

designed to improve the equipment uptime and

performance and remove waste associated with

that equipment and the associated interactions.

The success of the projects was immense;

particularly one associated with a packag-

ing line where a dramatic increase in OEE

was obtained rising from an average of

15 per cent to an average of 50 per cent.

Fourteen TPM projects have been rolled out to

date across the plant. The TPM programme is

broken into nine steps:

1. Collecting equipment history and perform -

ance information

2. Defining Overall Equipment Effectiveness

(OEE) measurement and potential and

reviewing progress

3. Assessing the six top losses and setting

improvement priorities

4. Carrying out a critical assessment

of the equipment

5. Carrying out a condition appraisal

of the equipment

6. Planning the refurbishment, spares and

manpower to carry out the work

7. Developing future asset care

8. Developing best practice

9. Problem solving and prevention.

At the end of the nine step TPM activity, the

teams present a close out to Senior Manage -

ment and then progress onto an ongoing

Continuous Improvement phase, the teams

meet every week for two hours reviewing

performance and performing various activities

focusing on improvement and reducing waste.

In order to ensure that progress is maintained

there are eight levels of assessment that each

team must achieve as the team progresses on

their journey. The teams start to become self

empowered and self managing of all activities

within their influence. The TPM tool is very much

a cultural change tool and the benefits of this

rather than as an equipment or maintenance

tool becomes evident after implementation and

using the tool over a period of time.

Total Productive Administration

In September 2012, based on the recognition

that the cellular based structure of TPM was

successful both in terms of achieving better

KPIs and developing a culture of Continuous

Improvement among cross functional depart -

ments, it was decided to introduce the concept

of Total Production Administration (TPA) into the

support functions at Rottapharm Dublin.

Four project areas were chosen, Quality

Assurance (QA), Quality Control (QC), Finance

and Materials. The teams selected were drawn

from members of those departments along with

members from other process and support

departments to provide an external insight to

the team and offer a new perception to the

project in question. The tool used by the teams

was process mapping whereby the teams used

the brown paper method to:

� Review existing process activities

� Remove non-value adding activities

� Develop a new improved process with the

wasteful activities removed.

In one example, the QC team reduced non-test

task activities from a total time of 39 hours to

SIX SIGMA



Figure 1: Production output expressed as percentage for year on year comparison

16.5 hours, a 58 per cent saving of time for a QC

analyst to perform more value adding tasks.

Paperless projects

The lean paperless projects, which began in

2009, have transformed the operations and

maintenance systems within the Dublin site.

Paperless maintenance involved the

installation of a Computerised Maintenance

Management System (CMMS) to support timely

and accurate information on maintenance

activities within the plant. The system was

installed and validated to CFR 21 standards

within six months. Data access and inputs are

performed by Rottapharm maintenance

personnel using military standard laptops.

The CMMS enables maintenance activities

to be logged as they occur. In conjunction with

an Overall Equipment Efficiency (OEE) system

integrated to the equipment, real-time

information on equipment performance can be

generated and a range of KPIs were developed

for performance review. The CMMS and OEE

systems are also linked which allows meter

based maintenance activities with actual

running times determining routine main -

tenance requirements.

The Manufacturing Execution System (MES)

is a software application which manages various

operations on the factory floor. The MES

implemented at Rottapharm includes numerous

interfaces with plant equipment. The project

started in the warehouse in 2009 and has now

been rolled out to over 95 per cent of the

manufacturing and all packaging activities. This

means that batch records are now electronic

rather than paper-based. Over 800,000 wet

signatures have been removed as a result of this

project. This ensures a reduction in processing

errors and allows a faster batch review time

through the use of ‘review by exception’ taking

place. Paper has all but disappeared within the

production and maintenance environment of

the company.

Energy reduction

Rottapharm has taken steps to improve the

energy efficiency of the business over the last

number of years. The primary reasons for this

have been to reduce the emissions of Green

House Gases, Carbon Dioxide (CO2) and also to

reduce the overhead costs of electricity and

natural gas. Green House Gas CO2 emissions

have dropped from a high of 4685.8 tCO2 in 2010

to 3207.3 tCO2 in 2012. Energy consumption

dropped by 44 per cent between 2010 and 2012

with relatively similar production output of

packs of 20.9 million in 2010 and 20.5 million

in 2012.

The energy awareness project team have

also introduced a number of energy reduction

projects over the last number of years, targeting

some of the significant energy users, which has

resulted in reduced consumption of electricity

and natural gas, these are listed as:

� The recirculation of air in the Heating Vent -

ila tion & Air Conditioning (HVAC) system

� The installation of occupancy and daylight

sensitive lighting controls in corridors

� The optimisation of the chilled water plant

by raising operating temperature and

improving control of the chilled water

plant units

� The optimisation of the Building Manage -

ment System (BMS) in order to control

heating requirements in times when areas

are unoccupied

� The phased installation of Light Emitting

Diode (LED) lighting in the facility.

The Green Shoots

Awards Competition

The Green Shoots Awards Competition came

about as a result of the need to promote energy

and waste awareness within the Rottapharm

plant. The competition was launched on World

Environment Day, 5 June 2012.

The competition was successful in engaging

employees in suggesting ideas for both energy

and waste reduction. Over 140 ideas were

submitted to the competition and through a

process of elimination this figure was short-

listed to 12 finalists. Each of the finalists was

assigned a sponsor to aid them in preparing

a more detailed explanation of their idea.

The 12 detailed entries were then judged by

Senior Management and members of the green

shoots awards committee. The overall winner of

the competition was selected along with the

category winners for the best energy and waste

reduction ideas. The winning energy reduction

entry was to examine what equipment etc.

could be switched off during off peak periods for

the whole plant. The waste reduction winning

idea was to install MES screens on the ware -

house turret trucks to speed up stock location.

The overall winning idea was establishing a CRS

standard Spectrum for all raw materials in the

QC laboratory.

The winners were announced on 19

December 2012 and all 12 finalists received a

prize. The ideas suggested, for energy and waste

reduction during the competition, have already

started to be implemented across the plant.


Volume 18 | Issue 4 | 2013 56

SIX SIGMA

Rottapharm designed automation program for automatic enforcement of in process checks and the MESelectronic batch record screen

Figure 2: Rottapharm Dublin energy consumption in kilowatt hours (2009 to 2012)

Visit www.europeanpharmaceuticalreview.comto register now

Register now at www.europeanpharmaceuticalreview.com/nir-spectroscopy

The Process Analytical Technology (PAT) and Quality by Design (QbD) initiatives have been of interest forpharmaceutical manufacturing in the last few years. Implementation of PAT/QbD approaches involves monitoringand controlling critical process parameters that influence the critical quality attributes of the product. One of theprime processes in pharmaceutical solid dosage form is granulation and the drying process. With the PAT and QbDinitiatives, the FDA aims to increase the efficiency of the pharmaceutical production by real-time process analysisand control. Near Infrared spectroscopy (NIRS) is well accepted as a potential PAT analyser due to its rapid andnondestructive technique that additionally requires no sample preparation.

Metrohm is well-established in determining the moisture content by Karl Fischer titration in every conceivable typeof product. To provide advance solutions for pharmaceutical customers, Metrohm now is moving forward, bringingNIRS to help customers to optimise the use of raw materials and to consistently run production closer to targetspecifications with time- and cost-saving analytical techniques.

During this webinar, we will be showing the use of NIR in the process that allows for monitoring low levels ofresidual moisture and other process constituents to yield better process control and endpoint determination.Additionally, we will be focusing on:• Developing a robust and precise method• Minimising implementation efforts • Ensuring calibration model transferability between analysers.

Supported by

Organised by:

Real-time monitoring of themoisture content in differenttype of dryers by NIRspectroscopy as a PAT tool

A European Pharmaceutical Review Date:Tuesday, 8 October 2013

Time:15.00 BST

Length:1 Hour

Dr. Volker J. FrostHead of the CompetenceCenter Spectroscopy,Metrohm NIRSystems

SPEAKERS:

Wim GunsChief Sales Officer,MetrohmNIRSystems

Proposals for the annual ELRIG Drug Discovery

meeting were developed during 2006 and led to

the first meeting being held at the EMCC,

Nottingham. Over the last six years the meeting

has doubled in size, establishing itself as a

leading vendor exhibition in the UK. More

importantly, the Drug Discovery meeting has

become the premier show to attend within the

Life Sciences community, giving fantastic

networking opportunities to scientists from

Pharma, biotech and academia interested in the

challenges of pre-candidate drug discovery. This

year, ELRIG is once again taking the show back to

the Manchester Central Conference Centre

which is fast becoming the hub for the Annual

Drug Discovery event.

Over the two days a superb range of world-

class speakers will be presenting in eight

sessions covering a wide range of topical issues

for Drug Discovery including ‘Advanced Cell

Technologies’, Innovations in Assay Develop -

ment & Screening and also two sessions on

Macromolecules – as Therapeutics and as Tools

for Discovery & Target Validation.

Each meeting will feature an extensive array

of posters, poster taster talk, snapshot talks,

training, competitions and probably the best

exhibition for Drug Discovery products and

services in the UK, which incorporates a thriving

‘Innovation Zone’ for new companies promoting

exciting technologies to the market.

The conference programme will also

include what the organisers hope will be a

challenging and entertaining Dragon’s Den

event in which innovators will present new ideas

to a panel of industry leaders for discussion.

Finally there will be a range of exhibitor

organised meetings and training courses.

At the 2013 meeting, the keynote address

will be by Dr Ruth McKernan, CSO of Pfizer’s

Neusentis Unit, on ‘Practical applications of stem

cell-derived cells within drug discovery’.

In developing the programme for this

meeting, ELRIG has worked with the objective of

bringing together scientists from across the

field, whether they are based in academia,

the pharmaceutical or biotech industries, in

order to establish relationships to advance the

science of drug discovery in the UK and Europe.

As usual, attendance at the show is free –

thanks to the support of the meeting’s sponsors

and exhibitors. For more information, and to

register to attend, please visit www.elrig.org.

In 2013, ELRIG’s Drug Discovery meeting will be in its seventh year. Organised each year by ELRIG (European Laboratory Robotics

Interest Group) and supported by SLAS (Society for Laboratory Automation and Screening), this year’s event will take place from

3 – 4 September in Manchester, UK.


Volume 18 | Issue 4 | 2013 58

ELRIG’s association with SLAS continues andprovides Students, graduates and post-docswith an opportunity to win the SLAS YoungScientist Award at the ELRIG Drug Discovery2013 Annual Conference and Exhibition.

The winner will be invited to present andparticipate in the Student Poster Competitionat the 3rd Annual SLAS Conference andExhibition, 18 – 22 January 2014 at the SanDiego Convention Center in San Diego,California, USA. SLAS Young Scientist Awardwinners receive a USD 500 cash prize,roundtrip coach airfare, shared hotelaccommodations and conference registrationfor SLAS2014.

ShowPREVIEW Date: 3-4 September 2013 · Location: Manchester, UK

© p

etar

g / S

hutt

erst

ock.

com

Details of protease inhibitors in clinical use have

been reviewed and referenced by Abbenante &

Fairlie3 and up-to-date information relating to

clinical trials for a wide range of diseases,

including those that involve protease inhibitors,

can be identified using the National Institutes of

Health clinical trial database (ClinicalTrials.gov)

which currently contains more than 100,000

clinical trials from 180 countries and receives

over 50 million page views per month. Despite

the successes in discovering and developing

orally administered protease inhibitors,

significant challenges still remain with regards

to their safety profiles and demonstrable

efficacy in clinical trials. Nevertheless, the fact

that there are small molecule protease inhibitors

undergoing clinical trials confirms the view that

the protease target class are tractable for drug

discovery4. In this article, the roles of synthetic,

natural products and endogenous cystatin M/E

are discussed, in particular with respect to

facilitating cysteine protease small molecule

drug discovery.

Synthetic and natural product

low molecular mass cysteine

protease inhibitors

Most of the biochemical and structural studies

carried out on proteases have made use of the

model systems such as the serine protease

chymotrypsin and cysteine protease papain and

these have provided valuable insights into their

mechanism of action and specificity character -

istics5-8. The cysteine proteases contain an

essential highly reactive thiol group contributed

by a cysteine residue which is required for

catalytic activity. In addition, they also contain

an imidazole group contributed by a histidine

residue which is largely responsible for con -

ferring the abnormally low pKa of the cysteine

thiol group (3.4 in the case of papain) rather than

the usual pKa >9 associated with the dissociation

of low Mr thiol containing compounds such as

2-mercaptoethanol. The thiol groups in cysteine

proteases have an inherent propensity to react

with reagents such as iodoacetate9 and mercuri -

Cysteine proteases are expressed ubiquitously in the animal and plant kingdom and are thought to play key roles in maintaining

homeostasis. The aberrant function of cysteine proteases in humans are known to lead to a variety of epidermal disease states

such as inflammatory skin disease1

. In marked contrast, the serine proteases have been most widely implicated in disease

states including hypertension, periodontisis, AIDS, thrombosis, respiratory disease, pancreatitis and cancer2

, and a number of

their inhibitors have been approved for clinical use.

How naturally occurringinhibitors can facilitate smallmolecule drug discovery forcysteine proteases

Sheraz Gul

Vice President and Head of Biology, European ScreeningPort GmbH



PROTEASES

‘‘Most of the biochemical and structuralstudies carried out on proteases have

made use of the model systems such asthe serine protease chymotrypsin and

cysteine protease papain’’

benzoate10,11, however they lack specificity

features for cysteine proteases. Specificity for

cysteine protease inhibitors can be introduced

by the incorporation of features that are

complimentary with the binding sites of the

enzymes as exemplified by the irreversible

substrate derived inhibitors based upon

fluoromethyl ketones12, cyanogen bromide13-15

and 2,2'-dipyridyl disulphides16. Many cysteine

protease inhibitors also inhibit serine proteases

due to their similarities in the catalytic mech -

anism of action. However, as the catalytic site

thiol (of cysteine proteases) has a greater

nucleophilicity relative to the hydroxyl of the

catalytic serine (of serine proteases), this can

allow for selectivity towards cysteine proteases.

As protease enzymes have an inherent

propensity to degrade their respective sub -

strates, they are often synthesised in an inactive

form (pro-enzyme) in order to prevent aberrant

activity. This pro-enzyme subsequently under -

goes auto-catalytic processing to release

the pro-domain, which thereby results in the

generation of mature catalytically competent

protease17. The peptide sequences surrounding

the auto-catalytic cleavage sites of proteases are

often used to design protease substrates such

that they contain similar sequences18,19. However,

proteases usually undergo a conformational

change upon autocatalytic processing and the

specificity characteristics of the mature protease

may not directly resemble that of the pro-

enzyme, therefore it is not always the case that a

substrate designed on the basis of the auto-

catalytic cleavage site will be acted upon by the

mature protease.

The mechanisms by which low molecular

mass inhibitors act upon cysteine proteases

include (a) reaction with their catalytic site

thiol group resulting in the formation a

product which cannot undergo any further

reaction, (b) forming a reactive intermediate

that subse quently reacts with the enzyme

via a mechanism that is not part of its usual

catalytic act or (c) reacting with the enzyme

active centre via their usual mech anism and

under going further reaction at such a slow

rate that it is essentially considered to be an

irreversible reaction thereby rendering the

enzyme catalyt ically inactive. An ex -

tensively characterised low molecular

mass cysteine protease inhibitor is the

natural product alkylating agent

L-trans-Epoxy-succinyl-leucylamido

(4-guanidino)butane (E-64) that orig in -

ates from Aspergillus japonicas. This has

been shown to inhibit a variety of plant

cysteine proteases (including papain and

ficin), human cysteine proteases (cathepsin L20,

a protease from human breast-tumour tissue21,

and the calcium-dependent protease calpain

from chicken muscle22, but not to inhibit a

variety of serine proteases (trypsin, chymo -

trypsin, tissue kallikrein, plasmin and pancreatic

elastase) or aspartic proteases (pepsin and

Paecilomyces acid proteases). X-ray crystallo -

graphy studies of papain-E64 complex have

shown that the epoxide residue of E64 interacts

with the papain S1-subsite and the leucyl residue

is bound to the papain S2-subsite. A variety of

E64 derivatives have been synthesised and

tested in vitro against cysteine proteases,

of which CA-074 has been shown to inhibit

cathepsin B with an IC50 in the low nM range with

>1,000 fold selectivity against other related

cysteine proteases cathepsin L and cathepsin

H23,24. Collectively, these studies suggest that

E64 has been a valuable inhibitor for the study

of cysteine proteases.

The cystatins: endogenous

high molecular mass cysteine

protease inhibitors

As described above, proteases are often

expressed in vivo in an inactive form (pro-

enzyme). Upon cleavage of the pro-domain,

it usually dissociates from the mature protease

thereby rendering it catalytically functional.

The activities of mature cysteine protease

enzymes in vivo are regulated by a variety of

endogenous protease inhibitors such as

cystatins. Additional endogenous protease

inhibitors include the serine protease inhibitors

(serpins), a noteworthy example of which is the

myeloid and erythroid nuclear termination

(MENT), a stage-specific protein which has been

shown to inhibit the cysteine proteases

cathepsin L and cathepsin V25 and the tissue

inhibitor of metalloprotease26.

The cystatins are members of a superfamily

of evolutionarily-related proteins (each con -

taining more than 100 amino acid residues) that

can be divided into three major families, namely

Type-1 cystatins A and B (also known as stefins)

which are relatively simple in structure,

containing no disulfide bonds or carbohydrate

and are found intracellular as well as the

cytoplasm of cells as well as body fluids. Type-2

cystatins (C, D, F, G, M/E, S, SN, and SA)

containing two disulfide bonds and no

carbohydrate which are mainly extracellular

secreted polypeptides synthesised with a

significantly shorter (19 to 28) residue signal

peptide and are broadly distributed and found

in most body fluids, and Type-3, also known

as kininogens (L- and H-kininogens) which

are composed of many domains, disulfide

bonds and carbohydrate and these include

H-kininogen (high-molecular-mass, IPR002395)

and L-kininogen (low-molecular-mass) and are

found in a number of species27. The first human

cystatin was identified from the sera of auto -

immune disease patients and was shown to

inhibit the cysteine proteases papain, human

cathepsin H and cathespin B28.

In general, cystatins are competitive,

reversible, tight binding proteins that inhibit

cysteine proteases in a micromolar to pico-

molar range29. They are capable of rendering

their target proteases inactive via a stable

complex and preventing any additional

proteolysis30-33. These inhibitors act upon their

target proteases that have escaped or upon


Volume 18 | Issue 4 | 2013 60

PROTEASES

‘‘The activities of mature cysteineprotease enzymes in vivo are regulated

by a variety of endogenous proteaseinhibitors such as cystatins’’

© lc

ulig

/ Sh

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PROTEASES



Entitled Practical Workshop: Cell based assays for screening, the third

workshop in the on-going series was a very successful event with a total

of 26 attendees across a wide range of the pharmaceutical industry, from

postdoctoral students to industry scientists.

Lectures included an introduction to drug discovery and the design

and development of biochemical and cell based assays for drug

discovery purposes, screening jargon and terms, a selection of assays to

ensure translation of hits between formats and data analysis and

reduction. During practical sessions, attendees were able to trial

equipment supplied by sponsor companies BMG LABTECH, Cellular

Dynamics, PerkinElmer and Promega to enhance their knowledge,

looking at general concepts for cell based assays, screening cell based

assays against a small molecule library and application of cell health,

cardiac hypertrophy and neurite outgrowth assays using human iPS cell-

derived cardiomyocytes and neurons.

As well as providing theoretical and practical knowledge of cell

based assays and screening, the workshop provided an opportunity for

professionals working in drug discovery to network, discuss their current

projects and keep up-to-date with the latest industry developments.

Participants were able to claim 72 Continuing Professional

Development credits if registered on the Society of Biology CPD Scheme.

Our next workshop, ‘Chemical Biology, Drug Discovery & Screening’,

takes place from 23 - 25 October 2013 at the European ScreeningPort

GmbH facility in Hamburg, Germany.

Please visit www.euopeanpharmaceuticalreview.com/workshop for

more information.

Sponsors

In conjunction with European Pharmaceutical Review, the European ScreeningPort GmbH hosted the third in a series of

workshops which examine, by way of practical sessions and lectures, the design and application of assays for screening

applications in drug discovery from 11 – 13 June 2013.

Practical Workshop: Cell based assays for screening

REVIEWHosted by: Organised by:

exogenous proteases of invading micro -

organisms. The absence of these endogenous

inhibitors has been implicated in disease states,

for example, cystatin C has been shown to

pro mote atherosclerosis in apolipoprotein

E deficient mice34. Each cystatin has a single

reactive site and binds to their target cysteine

protease in a non-covalent manner. Although

the cystatins have many common features, the

differences in their structures have a con -

siderable effect upon their abilities to inhibit their

target proteases. The chicken egg white cystatin

has been purified and extensively characterised

with regards to its bio-physical characterisation

and kinetics and mechanism of inhibition of a

variety of proteases35 and has been shown to

be composed of two major forms (Form A and

Form B, composed of 108 and 116 amino acid

residues respectively and containing two

disulfide bonds).

The role of endogenous cystatin M/E as

a cysteine protease inhibitor

There is evidence implicating the role of cysteine

proteases in the maintenance of epidermal

tissues36,37 as well as being down-regulated in

breast cancer38. The most notable example is the

characterisation of wild-type cystatin M/E and its

N64A and W135E variants against cysteine

proteases that led to the identification of key

residues of cystatin M/E that are responsible for

its inhibition profile. Although wild-type cystatin

M/E has been shown to inhibit legumain,

cathepsin V and cathepsin L with Ki with values

<2 nM, the N64A variant results in a significant

decrease in its potency towards legumain

(Ki >100 nM) whilst retaining similar activity

against cathepsin V and cathepsin L39. In the case

of the W135A cystatin M/E mutant, the potency

against legumain and cathepsin L is similar to

that of wild-type cystatin M/E, however, in the

case of cathepsin V, a significant decrease in

potency was observed (Ki >100 nM). The

homology model of cystatin M/E based upon

the crystal structure of cystatin D has led to the

identification of key regions within the protein

that can explain the inhibition profiles cystatin

M/E as well its variants39.

The studies of Grzonka et al40 involved the

characterisation of the potential of various

cystatins to inhibit papain and cathepsins B, H,

L and S and identified the key residues that are

responsible for the inhibition profiles against a

range of plant cysteine proteases (papain, ficin,

actinidin and cathepsin B).

Studies have shown that cystatin A, cystatin

B and cystatin C inhibit the cysteine proteases

cathepsin B, cathepsin H and cathepsin L with

Ki in the double digit nanomolar range.

Many of these enzymes have been implicated in

tissue degradation and excessive proteolytic

activity, leading to diseases such as arthritis,

stroke, Alzheimer’s and cataracts. The structural

basis for the inhibition of the cysteine protease

papain by chicken white cystatin has been

determined and shown to interact with the

S1-S3 subsite of papain and hairpin loops inter -

acting with the S1'-S2' subsite.

The use of cystatins to facilitate

small molecule drug discovery for

cysteine proteases

Considerable progress has been made in

relation to the understanding of the roles

cysteine proteases play in diseases. The

existence of a variety of endogenous protease

‘‘In general, cystatins are competitive,reversible, tight binding proteins that

inhibit cysteine proteases in amicromolar to picomolar range’’

inhibitors, notably the cystatins, have been

relatively under-exploited for the discovery of

inhibitors of proteases despite the extensive

kinetic characterisation of their mechanism of

inhibition of their respective protease target.

As a variety of natural cysteine protease

inhibitors have been identified with a range of

potencies, some which are relatively potent and

elucidation of their mechanisms of action,

identification of key binding interactions and

kinetics of inhibition can be used to facilitate

drug discovery. A comprehensive list of small

molecule cysteine protease inhibitors can be

found in the review of Otto and Schirmeiter41.

Recent examples of proteases against which

inhibitors have been developed, shown to be

efficacious in clinical trials and approved by

the Food and Drug Administration (FDA),

include Sitagliptin which inhibits the serine

protease Dipeptidyl Peptidase 442. Although

the results from these extensive studies can

be exploited in order to identify key inter-

actions for drug discovery purposes, it has

remained a considerable challenge to develop

suitable compounds with appropriate potency

and selectivity.


Volume 18 | Issue 4 | 2013 62

PROTEASES

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proteases and their inhibitors contribute to

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57:345-354

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3. Abbenante G, Fairlie DP (2005) Protease inhibitors in

the clinic. Med Chem 1:71-104

4. Overington, JP, Al-Lazikani, B. Hopkins, AL (2006) Nat

Rev Drug Discov 5:993-996

5. Wharton CW (1997) A Mechanistic Reference. In: Sinnot

ML (ed) Comprehensive Biological Catalysis. vol I.

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6. Brocklehurst K, Willenbrock F, Salih, E (1987) Cysteine

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7. Barrett AJ, Rawlings ND, Woessner JF. (2004). Handbook

of Proteolytic Enzymes, Elsevier, London

8. Brocklehurst K, Gul S, Pickersgill RW (2009) Substrate

recognition. In: Hughes AB (ed) Amino acids, peptides

and proteins in organic chemistry, vol 2. Wiley-VCH

publishers, Germany, pp 473-504

9. Cigic B, Pain RH (1999) Location of the binding site for

chloride ion activation of cathepsin C. Eur J Biochem

264:944-951

10. Satoh M, Yokosawa H, Ishii S (1989) Characterization of

cysteine proteases functioning in degradation of

dynorphin in neuroblastoma cells: evidence for the

presence of a novel enzyme with strict specificity

toward paired basic residues. J Neurochem 52:61-68

11. Ødum L, Yding Andersen C, Jessen TE (2002)

Characterization of the coupling activity for the

binding of inter-a-trypsin inhibitor to hyaluronan in

human and bovine follicular fluid. Reproduction

124:249-257

12. Marzo I, Péréz-Galan P, Giraldo P, Rubio-Félix D, Anel A,

Naval J (2001) Cladribine induces apoptosis in human

leukaemia cells by caspase dependent and -

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Biochem J 359:537-546

13. Robertus JD, Alden RA, Birktoft JJ, Kraut J, Powers JC,

Wilcox PE (1972) An x-ray crystallographic study of the

binding of peptide chloromethyl ketone inhibitors to

subtilisin BPN'. Biochemistry 11:2439-2449

14. Poulos TL, Alden RA, Freer ST, Birktoft JJ, Kraut J (1976)

Polypeptide halomethyl ketones bind to serine

proteases as analogs of the tetrahedral intermediate. X-

ray crystallographic comparison of lysine- and

phenylalanine-polypeptide chloromethyl ketone-

inhibited subtilisin. J Biol Chem 251:1097-1103

15. Machleidt W, Thiele U, Laber B, Assfalg-Machleidt I,

Esterl A, Wiegand G. Kos J, Turk V, Bode W (1989)

Mechanism of inhibition of papain by chicken egg

white cystatin: Inhibition constants of N-terminally

truncated forms and cyanogens bromide fragments of

the inhibitor. FEBS Letters 243:234-238

16. Brocklehurst K, Little G (1973) Reactions of papain and

of low-molecular-weight thiols with some aromatic

disulphides. 2,2'-Dipyridyl disulphide as a convenient

active-site titrant for papain even in the presence of

other thiols. Biochem J 133:67-80

17. Hsu MF, Kuo CJ, Chang KT, Chang HC, Chou CC, Ko TP,

Shr HL, Chang GG, Wang AH, Liang PH (2005)

Mechanism of the maturation process of SARS-CoV 3CL

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18. Harris JL, Backes BJ, Leonetti F, Mahrus S, Ellman JA,

Craik CS (2000) Rapid and general profiling of protease

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19. Thomas DA, Francis P, Smith C, Ratcliffe S, Ede NJ, Kay C,

Wayne G, Martin SL, Moore K, Amour A, Hooper NM

(2006) A broad-spectrum fluorescence-based peptide

library for the rapid identification of protease

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20. Barrett AJ, Kembhavi AA, Brown MA, Kirschke H, Knight

CG, Tamai M, Hanada, K (1982) L-trans-epoxysuccinyl-

leucylamido(4-guanidino)butane (E-64) and its

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21. Mort JS, Recklies AD, Poole AR (1980) Characterization

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22. Sugita H, Ishiura S, Suzuki K, Imahori K (1980) Ca-

activated neutral protease and its inhibitors: in vitro

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23. Murata M, Miyashita S, Yokoo C, Tamai M, Hanada K,

Hatayama K, Towatari T, Nikawa T, Katunuma N (1991)

Novel epoxysuccinyl peptides. Selective inhibitors of

cathepsin B, in vitro. FEBS Lett 280:307-310

24. Steverding D (2011) The cathepsin B-selective

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under reducing conditions. The Open Enzyme

Inhibition Journal 4:11-16

25. Irving JA, Shushanov SS, Pike RN, Popova EY, Brömme

D, Coetzer TH, Bottomley SP, Boulynko IA, Grigoryev SA,

Whisstock JC (2002) Inhibitory activity of a

heterochromatin-associated serpin (MENT) against

papain-like cysteine proteinases affects chromatin

structure and blocks cell proliferation. J Biol Chem

277:13192-13201

26. Murphy G (2011) Tissue inhibitors of metallo -

proteinases. Genome Biol 12:233

27. Ochieng J, Chaudhuri G (2010) Cystatin superfamily.

J Health Care Poor Underserved 21:51-70

28. Brzin J, Popovic T, Turk V, Borchart U, Machleidt W

(1984) Human cystatin, a new protein inhibitor of

cysteine proteinases. Biochem Biophys Res Commun

118:103-109

29. Brömme D, Rinne R, Kirschke H (1991) Tight-binding

inhibition of cathepsin S by cystatins. Biomed Biochim

Acta 50:631-635

30. Barrett AJ (1986) The cystatins: a diverse superfamily of

cysteine peptidase inhibitors. Biomed Biochim Acta

45:1363-1374

31. Bode W, Huber R (2000) Structural basis of the

endoproteinase-protein inhibitor interaction. Biochim

Biophys Acta 1477:241-252

32. Turk V, Bode W (1991) The cystatins: protein inhibitors

of cysteine proteinases. FEBS Lett 285:213-219

33. Turk B, Turk D, Salvesen GS (2002) Regulating cysteine

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guardians and regulators. Curr Pharm Des 8:1623-1637

34. Sukhova GK, Wang B, Libby P, Pan J-H, Zhang Y, Grubb

A, Fang K, Chapman HA, Shi G-P (2005) Cystatin C

deficiency increases elastic lamina degradation and

aortic dilatation in apolipoprotein E-null mice.

Circulation Res 96:368-375

35. Auerswald EA, Nägler DK, Gross S., Assfalg-Machleidt I.,

Stubbs MT, Eckerskorn C, Machleidt W and Fritz H

(1996) Hybrids of chicken cystatin with human

kininogen domain 2 sequences exhibit novel inhibition

of calpain, improved inhibition of actinidin and

impaired inhibition of papain, cathepsin L and

cathepsin B. Eur J Biochem 235:534-542

36. Zeeuwen PL, van Vlijmen-Willems IM, Olthuis D,

Johansen HT, Hitomi K, Hara-Nishimura I, Powers

JC, James KE, op den Camp HJ, Lemmens R,

Schalkwijk J (2004) Evidence that unrestricted

legumain activity is involved in disturbed epidermal

cornification in cystatin M/E deficient mice. Hum Mol

Genet 13:1069-1079

37. Zeeuwen PL, van Vlijmen-Willems IM, Cheng T, Rodijk-

Olthuis D, Hitomi K, Hara-Nishimura I, John S, Smyth N,

Reinheckel T, Hendriks WJ, Schalkwijk J (2010) The

cystatin M/E-cathepsin L balance is essential for tissue

homeostasis in epidermis, hair follicles, and cornea.

FASEB J 10:3744-3755

38. Sotiropoulou G, Anisowicz A, Sager R (1997)

Identification, cloning, and characterization of cystatin

M, a novel cysteine proteinase inhibitor, down-

regulated in breast cancer. J Biol Chem 272:903-910

39. Cheng T, Hitomi K, van Vlijmen-Willems IM, de Jongh

GJ, Yamamoto K, Nishi K, Watts C, Reinheckel T,

Schalkwijk J, Zeeuwen PL (2006) Cystatin M/E is a high

affinity inhibitor of cathepsin V and cathepsin L by a

reactive site that is distinct from the legumain-binding

site. A novel clue for the role of cystatin M/E in

epidermal cornification. J Biol Chem 281:15893-15899

40. Grzonka Z, Jankowska E, Kasprzykowski F,

Kasprzykowska R, Lankiewicz L, Wiczk W, Wieczerzak E,

Ciarkowski J, Drabik P, Janowski R, Kozak M, Jaskólski M,

Grubb A (2001) Structural studies of cysteine proteases

and their inhibitors. Acta Biochim Pol 48:1-20

41. Otto HH, Schirmeiter T (1997) Cysteine proteases and

their inhibitors. Chem Rev 97:133-171

42. Augeri DJ, Robl JA, Betebenner DA, Magnin DR, Khanna

A, Robertson JG, Wang A, Simpkins LM, Taunk P, Huang

Q, Han SP, Abboa-Offei B, Cap M, Xin L, Tao L, Tozzo E,

Welzel GE, Egan DM, Marcinkeviciene J, Chang SY, Biller

SA, Kirby MS, Parker RA, Hamann LG (2005) Discovery

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References

Sheraz Gul is Head of Biology at European

ScreeningPort, Hamburg, Germany where he

manages the assay development and

screening of academic targets. Prior to this, he

worked for GlaxoSmithKline for seven years

where he developed biochemical and cellular

assays for High Throughput Screening as well

as hit characterisation. In addition, he has worked in academia for five

years on proteases and kinases. He is the co-authored of the Enzyme

Assays: Essential Data Handbook. He is involved in many European

Initiatives involving government, the pharmaceutical industry and

academia (e.g. EU Framework 7 and IMI). His research interests are

directed towards maximising the impact of HTS for drug discovery.

[email protected]

Biography

BIOTECHNICA visitors will be treated to a rich

array of displays revolving around the very latest

advances in technology and R&D, as well as the

latest biotech products and their applications in

the fields of medicine, food production, industry

and environmental protection. There will also be

a wide range of services for the biotech and

pharmaceutical sectors. BIOTECHNICA is con -

sidered to be one of the most important

platforms for bringing innovators together

with investors.

Four marketplaces dedicated

to sector trends

The 20th edition of BIOTECHNICA will boast a

new expo concept revolving around dedi-

cated marketplaces, each addressing a

specific theme or focus of interest in the biotech

sector. This new approach will make it easier

than ever for attendees to find what they

are looking for. Each segment will feature its

own forum with scientific talks and company

lectures, as well as networking services and

poster presentations.

The four marketplaces at BIOTECHNICA

are Personalised Medicine Technologies, Innova-

tion in Food, Industrial Biotechnology and

BioServices. The Personalised Medicine

Technologies Marketplace will focus on molec -

ular diagnostics and strategies for personalised

therapies. The forum discussions and talks

will explore next-generation sequencing,

cell-based assays, biomarkers, companion

diagnostics and molecular imaging. Keynote

speakers have been attracted from the top

German clusters in this field: Bio Deutschland,

the association of the German diagnostics

industry (VDGH), the association of pharma -

ceutical research comp anies (vfa) and the bio

section of the vfa.

The advances being made in bio-pharma -

ceuticals through the application of exciting

new technology will be the highlight at the

BioServices Marketplace, which aims to

establish valuable contacts between research

and development on the one hand, and

innovative biotech companies and the pharma -

ceutical industry on the other.

Featuring new topics and innovative marketplaces, BIOTECHNICA 2013 in Hannover will, from 8 – 10 October,

once again serve as the central hub for Europe’s entire biotech sector and its clientele. For the 20th time

BIOTECHNICA will bring exhibitors and trade visitors together to do business. As Europe’s leading trade fair for

biotechnology, the life sciences and laboratory equipment, BIOTECHNICA is the number one event in its field.

One of the keynotes of this year’s trade fair will be bio-economics. Switzerland – the first Partner Country to be

honoured at BIOTECHNICA – will also be a focus of attention.

For more information, please visit:

www.biotechnica.de

ShowPREVIEW Date: 8 – 10 October 2013 · Location: Hannover, Germany

BIOTECHNICA 2013 – Europe’s biotech hub

BMG LABTECH has a number of microplate

readers on the market, including the multi-

mode OMEGA range, which is popular in the life

sciences and the PHERAstar FS which has

become the gold-standard for the high

throughput screening market. “The CLARIOstar

is a mid-range reader that sits between the

OMEGA and PHERAstar FS,” says Mount, “and

because of its flexibility and performance, it can

fit into an individual research lab or a core

facility. It’s ideal for life science researchers, as

well as groups performing medium throughput

screening within pharmaceutical research. It’s an

exciting product with unique technology.”

So what makes the CLARIOstar so unique?

“It’s BMG LABTECH’s first microplate reader to

feature a monochromator and it is the very first

reader to use LVF technology,” says Mount.

“LVF technology has only just come to the

market generally and this is the first time it’s

been employed in a microplate reader.”

There are many benefits of the LVF

mono chromator over a traditional or grating-

based monochromator system and filters.

For example, the LVF monochromator not only

has selectable wavelengths from 320 to 850

nanometres, it also has continually variable

bandwidths selectable from 8 to 100 nano -

metres. This makes LVF monochromators

much more flexible than traditional mono -

chromators with limited or fixed bandwidths.

“This effectively allows the user to design

individual ‘filters’ for different assays, pro-

viding improved assay performance,” Mount

explains. “LVF mono chromators also avoid

stray light which again significantly improves

assay performance.” In addition, the LVF

mono chromator transmits more light than

a traditional monochromator, for various

tech nological reasons, which greatly im-

proves sensitivity.

“A key feature is flexibility; because the

wavelength and bandwidth can be selected in

LVF monochromators, fluorophores can be

scanned in both excitation and emission to

determine the optimal monochromator setting

or filter pairing to be used for the assay,”

Mount discloses.

Assay performance has always been central

to the design of microplate readers at BMG

LABTECH and filters traditionally provided the

best performance for most assays. “We didn’t

want to compromise the performance of our

microplate readers with the convenience of a

monochromator, but we now have the tech -

nology to produce a monochromator with

filter-like sensitivity,” says Mount. “The LVF

monochromator has opened up flexibility and

performance that hasn’t been seen before in a

microplate reader.”

Mount assures that this doesn’t spell the

end of filter-based microplate readers, though.

“The CLARIOstar has two additional detection

technologies, filters and a spectrometer.

Filters still have an important part to play in

micro plate reading; fluorescence polarisation or

time-resolved fluorescence assays, for example,

will always perform better using filters. And

absorbance assays benefit from the ultra-fast

UV-Vis spectrometer,” he says. “Other microplate

readers measure absorbance and fluorescence

assays using the same monochromator. Since

the CLARIOstar uses a dedicated spectrometer

for full spectrum absorbance measurements, it

does not compromise one mode for another by

using the same detection technology. Fast

absorbance spectrometers, which capture an

entire spectrum (220 to 1000 nanometres) in less

than one second per well, is a newer technology

that BMG LABTECH incorporated into their

micro plate readers over five years ago.”

The LVF monochromator is suited to various

applications; for example scanning new or

unusual fluorophores, where the excitation and

emission wavelengths are not known, and using

fluorophores in non-standard buffer conditions

where the pH may have an effect on the peak

maxima. “When multiplexing assays with

fluorophores or lumiphores of similar or

overlapping spectra, the ability to define

wavelengths and select any bandwidth from

8 to 100 nanometres allows the user to employ

‘filters’ that are away from peaks but are

sufficiently wide enough to allow enough

light through to make the assay workable,”

explains Mount. “Wavelength and bandwidth

can be the difference between an okay assay

and a fantastic assay.”

Launched in June 2013, the CLARIOstar is

already receiving positive feedback. “The

product’s tag line is ‘any wavelength, any

bandwidth, any assay’ and that’s literally it,”

Mount reveals. “This LVF monochromator,

together with the way we can use it with the

filters, is just completely novel.”


Volume 18 | Issue 4 | 2013 64

Tham

papo

n / S

hutte

rsto

ck

Established in 1990 in Offenburg, Germany, BMG LABTECH has spent the last 20 years producing and developing microplate

reading technology. The company has pioneered many new technologies in this field, including dual mode ABS/FI readers,

fluorescence polarisation, simultaneous dual emission detection, laser-based excitation, ultra-fast spectrometers and now linear

variable filter (LVF) monochromators. As Dr Robert Mount, Managing Director of BMG LABTECH Ltd., explains: “We could be

considered a niche company in that we only make microplate readers, however we choose to focus our efforts on serving the

market where we have the technology and expertise, and have no plans to dilute it by moving into other markets.”

Product HUBDr Robert Mount, Managing Director, BMG LABTECH discusses theirCLARIOstar microplate reader with LVF monochromator technology

Anything Is Possible.Any Wavelength. Any Bandwidth. Any Assay.

Alpha Technology is a registered trademark of PerkinElmer, Inc. HTRF is a registered trademark of Cisbio International.

Australia · France · Germany · Japan · United Kingdom · United States

Advanced Detection for Fluorescence and Luminescence Assays: Continuously adjustable wavelengths (320 - 850 nm)

and bandwidths (8 - 100 nm) for excitation and emission

Increased sensitivity over conventional monochromators

Integrated fl uorophore library for easy wavelength selection

Use monochromators, fi lters, or a combination of both

Fluorescence and Luminescence spectral scanning

CLARIOstar Additional Features: Full spectral absorbance with ultra-fast UV/Vis spectrometer

Laser-based Alpha Technology

Fluorescence Intensity, FRET, Fluorescence Polarization,

TRF/TR-FRET, Luminescence, and BRET

Reagent injectors for kinetic or cell-based assays

Low volume DNA measurements

www.bmglabtech.com

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The CLARIOstar‘s monochromator has

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CLARIOstar® - High Performance Microplate Reader with

Advanced LVF MonochromatorsTM, Spectrometer, and Filters

eln: a component of a larger informatics puzzle · eln: a component of a larger informatics puzzle...

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