Optim 2An Introduction

Avacta Analytical

• Based in the UK with network of global distributers.

• Leeds University in the North of England.

• Exponential growth over the past 6 years through both natural growth and acquisition.

• Currently around 80 full time employees.

The Challenge

Three points

• The need for speed

• Low sample availability – especially in early development

• The need for more information

Specifically looking at protein stability

Optim 2

Optim 2 High-throughput micro-volume characterisation of protein stability

Quick Overview

• Combines fluorescence and light scattering into one instrument

• Simultaneous investigation of conformational unfolding and aggregation propensity

• Expressed as the thermal midpoint (Tm) and aggregation onset (Tagg)

• All samples temperature controlled allowing for thermal ramping

• Low sample volumes 9µl

• High throughput measurements 96 sample per day

• Proprietary software with powerful data analysis

Application areas

Discovery Research

Lead Drug Identification

Lead Optimisation Clinical Studies GMP

Manufacturing

Preformulation DevelopmentSolubility, stress testing, clone (candidate) selection, early formulation development

Formulation DevelopmentStability, excipient studies. Further product characterisation.

Process DevelopmentExplore reaction space and process variables to optimize yield and stability

Anywhere you need to screen molecules or formulations for stability

Instrumental configuration

Illustrative pre-formulation screen

• Scenario – Pre-formulation study• Objective – investigate physical stability of different formulations.• One candidate under multiple conditions.• Matrix of 3 buffers, 5 pH’s, 3 salt concentrations and 10 excipients

measured all in triplicate• 810 samples in total• Illustrative case of Optim vs. other label free instruments

Illustrative pre-formulation screening

Protein Required (3)

Cost of Protein (1)

Instrument Time (3)

Total Man hours

Labour Cost (2)

Total Cost

Non-Automated - DSC/DLS 4 42 mg $67,000 3650 h (7) 1215 h $39,000 (9) $106,000

Automated - HT-DSC/HT-DLS 5 12.2mg $19,000 2010 h (8) 9.2 h. $288 $19,288

Single instrument - Optim 2 0.73 mg $1,170 74 h 8.4 h $272 $3,369 (6)

Notes:1. 1mg of therapeutic antibody costs ~£1000 2. labour cost calculated at £20/hr3. Sample requirement and instrument usage time data for the DSC and LS measurements were taken from manufacturer’s specifications where available 4. Malvern Zetasizer and Microcal DSC5. Malvern Zetasizer APS and Microcal Capillary DSC6. Additional cost reflects cost of Optim consumables

Optim delivers in two working weeks results that would otherwise take 4 months to complete

7. Total comes from 2030h for DSC & 1620h for DLS8. Total comes from 390 for DSC & 1620 for DLS 9. Total comes from £16,200 DSC & £8,100 DLS

De-risk your development process

Series10

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70260

1890 Non-Automated

Automated

Optim

A three month pre-formulation project (one month data collection a two months data analysis and interpretation).

Here we compare the amount of samples you can screen using classical techniques, automated techniques and Optim

Examples 5.5, 6, 6.5, 7 4Phosphate, Citrate, Acetate,Tris 4NaCl at 0, 50, 150 mM 3Tween 0, 0.01, 0.1 % v/v 3Trehelose 0, 500 mM 2Histidine 0, 50 mg/ml 2Replicates 3 1728

Using intrinsic protein

fluorescence to measure protein conformational

stability

Intrinsic fluorescence to measure protein conformation

• Illuminate protein with UV light

• Aromatic residues fluoresce – mainly tryptophan

• Aromatic residues hydrophobic and buried away from water in folded protein

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nsity

(C

CD

cts

/s)

Wavelength (nm)Tryptophan

TyrosinePhenylalanine

UV light

• Intensity, peak wavelength and shape of spectrum depends on environment around fluorescent residues

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Wavelength (nm)

Intrinsic fluorescence to measure protein conformation

Intrinsic fluorescence to measure protein conformation

• When the protein unfolds partially or completely the environment around the fluorescing residues changes

• The fluorescence spectrum changes in response

Change in florescence with temperature

20 30 40 50 60 70 800.750000000000004

0.800000000000004

0.850000000000004

0.900000000000004

0.950000000000004

Temperature (°C)

Intr

insic

Fl I

nten

sity

Ratio

350

:330

nm

We can apply a temperature ramp and observe changes in intrinsic fluorescence

Tm

Vary solution parameters and observe effect on thermal stability

Vary pH to observe effect on thermal stability

Identify observed midpoint of thermal unfolding transition – Tm

Higher Tm – higher conformational stability

20 30 40 50 60 70 800.000

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Temperature (°C)

d Fl

Rati

o/dT

pH 2.5

pH 3.5pH 4.5

pH 6.5

Static Light Scattering to measure protein aggregation propensity

• Illuminate sample with laser - measure scattered intensity at 90°

• For small solute particles scattered intensity proportional to mean solute mass x concentration

• Therefore scattered intensity increases with aggregation

Static Light Scattering (SLS) to monitor protein aggregation

Static Light Scattering (SLS) to monitor protein aggregation

Wavelength dependence of scattering ~ 1/λ4

Optim static light scattering highly sensitive: Dependence of scattering intensity on IgG monomer concentration

266 nm scattering intensity IgG sample

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IgG Concentration (mg/ml)47

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266 nm light scattering for high sensitivity

473 nm light scattering for higher dynamic range

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Dextran Size (kDa)

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nm S

catte

r In

teg

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nsity 0.1 mg/ml

Dependence of scattering intensity on mean solute mass

Optim static light scattering highly sensitive

Apply temperature ramp and observe aggregation

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Using static light scattering to give an indication of the aggregation propensity

pH 2.5 pH 3.5

pH 4.5pH 6.5

Tagg

I. Data rich II. High throughputIII. Low sample Volume

Key features summary

Data rich

• Optim can perform simultaneous measurements

• Measuring intrinsic fluorescence shifts allowing the user to determine a thermal midpoint or Tm

• Light scattering enables the determination of the onset of protein aggregation or Tagg

Provision of multiple stability indicating measurement helps users predict stability profile of their molecules, de-risking your development programme

UK Biopharmaceutical

“The Optim 1000 is a data-rich method of analysis which uses very small amounts of material, for example, less than 320 mg for one entire study. This low sample requirement allowed us to rapidly screen a variety of different formulations, meaning that we could study even more than had previously been possible. Those formulations that were found to be unsuitable were discarded early in the development process, effectively de-risking the programme”

Head of Preformulation, UK

Rapid and high throughput measurements

• Designed for speed

• 96 samples in one day - 48 samples in one run

• High performance imaging spectrograph is able to instantaneously acquire whole spectra measurements, quickly acquiring data

Faster analysis allows you to increase the scope of your investigations

• Sample held in Micro Cuvette Array (MCA)

• Specifically designed to give optimum florescence and light scattering signal from

small sample volumes – 9µl <0.1 mg/ml to 150+ mg/ml (sample dependent) • Sealed for zero evaporation during heating

• Spacing compatible with standard 384 well plate

Low sample volume

Low sample amounts enables more analytics to be completed earlier in the development process when sample availability is low.

University of Kansas

Prof Russell Middaugh co-director, Center for Macromolecule and Vaccine Stabilization, University of Kansas. Published GEN, Sept 1st

“Conventional analytical methods used for preformulation, stability, and formulation studies have previously relied on methods that extrapolate partial data on slow and labour-intensive instrumentation that is incompatible with high-throughput measurements and tight development timelines.

The Optim 1000 microvolume protein analysis and characterization system offers rapid, multimodal analysis of ultra-low sample volumes at high throughputs.”

Summary

Optim helps reduce the risk of your drug development programme

More information, with less sample and in less time than conventional

techniques