the benefits of high performing chromatography resins...

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Shelly Cote ParraPREP 2014, Boston, MA, USAJuly 23, 2014

The Benefits of High Performing Chromatography Resins Including POROS XQ, A New Strong Anion Exchanger

2

Overview

• Introduction to POROS®

Chromatography:• Principles, Product Attributes &

Advantages

• New approaches and benefits to optimized chromatography• Capture alternatives• Affinity products and services• Rethinking polish applications• Introducing a new AEX resin:

POROS XQ

• Closing

3

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Global Scale • 50,000 employees in 50 countries

• $17 billion in annual revenues

• Unparalleled commercial reach

• 10,000 employees working with customers every day

The World Leader in Serving Science

Unmatched Depth• Innovative technologies

• Applications expertise

• Lab productivity partner

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4

Laser Focus On Your Needs

• Enabling technologies across the workflow

• White glove service and support

• Best-in-class products

• Operational excellence

• Global presence

Services

CellTherapy

Cell Culture

Analytics

Chromatography

Single Use Technology

Technical & Commercial

Expertise

5

Combined Technologies to Drive Solutions– Strong Industry Adoption

• >200 uses of POROS® resins at all stages of development across 24 countries• >60% of the top 25 biopharmaceutical companies use POROS® resins• Proven affinity technology with single domain antibody ligands

• R&D >30 products (>250 customers) • BioProcessing with18 products (>150 customers), 9 products through

partners

6

POROS® Chromatography Resin: Product attributes

• Polystyrene-Divinylbenzene Backbone• Rigid, Incompressible• Easy Handling• Robust Chemical Stability

• Perfusion Chromatography• Pore Structure with Large Throughpores• Unlocks Interior of Bead• Increased Convective Flow, Reduced Diffusional

Limitations• Improved Mass Transfer, More Efficient Purification

• 50 Micron Particle Size• Superior Resolution• Excellent Pressure-Flow Properties• Fully Scalable

7

POROS® Chromatography Resin Product Attributes

Capacity and resolution maintained as linear flow rate increases

Format

Column: 0.46cmDx 20cmL

Capacity: 5 mg/mL hIgG in 20mM MES, 40mM NaCl, pH 5.0

Format


Capacity: 10 mg/mL BSA in 20mM Tris, pH 8.0

Format


Capacity: 5 mg/mL hIgG

Anion Exchange Cation Exchange

Protein A

BSA Capacity at 5% Breakthrough vs Linear Velocity

10

20

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700 800 900 1000

Flowrate (cm/H)

C5

Cap

acity

(mg/

ml)

POROS HQ50 Q Sepharose Fast Flow

hIgG Capacity at 5% BreakThrough versus Linear Velocity

y = -0.0119x + 51.313 y = -0.0433x + 55.2y = -0.0358x + 50.617

20

25

30

35

40

45

50

55

100 200 300 400 500 600 700 800Linear Flow Rate (cm/H)

C5 C

apac

ity (m

g/mL

)

POROS MabCapture A rPA Media 1A rPA Media 1B

IgG Capacity at 5% Breakthrough vs Linear Flowrate

0

10

20

30

40

50

60

70

80

0 100 200 300 400 500 600 700 800

Flowrate (cm/H)

C5

Cap

acity

(mg/

ml)

POROS HS50 SP Sepharose

8

POROS® Chromatography Resin: Principles• Architecture of bead conserves mass transfer at high flows

POROS®

10 cm Bed

Agarose

10 cm Bed

Theoretical Transition

Reprinted from: Hahn, R., Comparison of Protein A Affinity Sorbents, J. of Chromatography B (2003) 790, 35-51. Fig 3 with permission from Elsevier (10)

9

POROS® Chromatography Resin: Elution Efficiency

• POROS® resin improves elution volume versus typical process resin

POROS®

Diffusive

Reprinted from: Hahn, R., Comparison of Protein A Affinity Sorbents, J. of Chromatography B (2003) 790, 35-51. Fig 8 with permission from Elsevier (10)

10

FormatColumn: 0.46cmDx 20cmL, 3.32 ml Gradient: 10 – 100%B, 16 CV Buffer A: 20 mM MES, 25mM NaCl pH 6.2Buffer B: 20 mM MES/ 1.0M NaCl, pH 6.2Sample:

Chymotrypsinogen A 5mg/mL, pI 9.4Cytochrome C 8mg/mL, pI 10.4Lysozyme 4mg/ml, pI 11.3

Total Protein Loaded: 5.175mg, 1.56mg/ml

Resolution is maintained on

POROS as linear flow rate increases

XS005x Separation FR Study001:10_UV1_280nm XS005x Separation FR Study002:10_UV1_280nm XS005x Separation FR Study003:10_UV1_280nm XS005x Separation FR Study004:10_UV1_280nm XS005x Separation FR Study005:10_UV1_280nm

0

500

1000

1500

2000

2500

mAU

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 ml

100 cm/H300 cm/H500 cm/H700 cm/H1000 cm/H

Abs

orba

nce

280n

m (m

AU

)

Volume (ml)

POROS® Chromatography Resin: Product Attributes

11

Implementing POROS® Resins at Scale: Cleanability and scalability

•Robust chemical stability• pH: 1-14

• 5N NaOH• 2M Acetic Acid• 1M HCl

• Ionic Strength: 0-5M salt• Solvents

• Water• 0-100% alcohols• Acetonitrile• Other organic solvents

• Additives• Urea, Guanidine• Ethylene Glycol• Detergents

•Excellent pressure-flow properties

Pressure Flow Curve for POROS 50 HSPacked 3bar in 21cmD Glass Column with SS frits

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 200 400 600 800 1000

Flow Rate (cm/hr)

Pres

sure

(bar

)

19cm Bed Height

29 cm Bed Height

12

Overview



Advantages


POROS XQ

• Closing

13

Capture step alternativesBenefits of high performance chromatography resins

CAPTURE CHROMATOGRAPHY

Removal of WaterConcentration of Target Protein

High ThroughputSpecificity for Target Molecule vs.

ImpuritiesHigh Capacity for Target Molecule

UNIT OPERATION FUNCTION REQUIREMENTS

Important resin features: High dynamic binding capacity Salt tolerance Cleanability and column lifetime Robust and reproducible performance Excellent lot to lot consistency Increased flow rate capability Superior resolution a large plus

14

Affinity chromatography perspectives

Advantages: Highly specific separation High fold purification Basis of platform purification (reproducible) Robust with less process optimization

Disadvantages: Low to moderate capacity Chemical stability of affinity ligands can be low Cost of goods, typically 4-5x more expensive than

other resins Leaching Some Mabs can aggregate under typical rPA

operating conditions rPA does not bind IgG3 and some Fc Fusions

“Glove in hand”

15

IEX Chromatography in capture mode perspectives

Advantages:• High capacity• Cost of goods• Flexible- Large “design space” yet platformable;

Direct capture load• Cleanability/ reuse• Reduced cycling• Equivalent impurity clearance and no Protein A

leachate• Less harsh conditions for elution, more consistent

pH

Disadvantages:• Less specific than affinity• Optimization required• Need to know more about target molecule• May require more feed pretreatment (pH

adjustment, dilution, diafiltration)

16

POROS® MabCapture™ A chromatography resinPerformance of as a function of flow rate

y = -0.0119x + 51.313 y = -0.0433x + 55.2y = -0.0358x + 50.617

20

25

30

35

40

45

50

55

100 200 300 400 500 600 700 800

Linear Flow Rate (cm/H)

C5

Cap

acity

(mg/

mL)

POROS MabCaptureA MabSelect MabSelect Xtra

Maintaining high dynamic binding capacity over a large flow rate range is unique resin behavior

17

POROS® MabCapture™ A chromatography resinProcess Modeling of Resin Requirements

Pilot Scale: 1000L Harvest at 1.5g/L ExpressionMedia Usage as a Function of Linear Flow Rate

0

10

20

30

40

50

60

200 300 400 500 600 700

Linear Flow Rate

Med

ia V

olum

e (L

)

rPA Media 1A rPA Media 1B POROS

• Decrease resin requirements by 10% – 40%

• Direct material COGs savings

18

POROS® MabCapture™ A chromatography resinHigh pH Stability

0.1N NaOH Stability of rPA Resins

60.065.070.075.080.085.090.095.0

100.0105.0

0 10 20 30 40 50 60Cycle Number

% R

ecov

ery

of C

5 In

itial

POROS MabCapture A MabSelect

More robust sanitization capability

0%

10%

20%

30%

40%

50%

60%

70%80%

90%

100%

110%

Reco

very

of I

nitia

l C5

Capa

city

1 5 10 20 30 40 50

Cycle Number

Stability of POROS MabCapture A in 0.1M NaOH

FormatColumn: 0.46cmDx 20cmL, 3.324 mlLoad: Polyclonal hIgG, 5mg/mL in 20mM NaPi, 150mM NaCl, pH 7.5Load Ratio: 28 mg IgG/ml resin

Flow Rate: 500 cm/hr

Each cycle included 30min exposure to 0.1M NaOH

Capacity maintained with sanitization

19

• Improved Yield• Process Flexibility• Reduced Number of Unit Operations• Smaller Column Sizes• Linear and Predictable Scalability• Easier Handling and Packing

POROS® XS chromatography resinOverview

Customer Requirements XS Delivers

High capacity>100 mg/mL 5% breakthrough

capacity for monoclonal antibodies and recombinant proteins under a wide range of process conditions

High resolutionOptimized 50 μm particle size for improved impurity clearance and

better yield

High salt-tolerant protein capacity

>100 mg/mL dynamic binding capacity at 15 mS/cm NaCl

concentration

Low back pressure 2.0 bar at 800 cm/H in 20 cm length column

Rigid polymeric bead Incompressible bead with robust physical stability

A high performance CEX resin that

combines all of the above

New industry standard – major improvement on industry proven

chromatography platform that provides:

POROS® XS Just Eclipsed All OtherCation Exchange Resins

• Increased Process Flexibility and Throughput

• Improved Yield• Smaller Column Sizes• Linear and Predictable Scalability• Easier Handling and Packing

20

POROS® XS chromatography resinDesign space for optimal dynamic binding capacity

4.50

Salt

Conc

entr

atio

n (m

M)

5.00 5.50 6.00 6.50

75.00

112.50

150.00

pH

120110

100

90

80

7060

4030

20 0

8090

100

10

IgG Binding Capacity vs. Salt Concentration and pHCapacity at 5% Breakthrough

Format

Column: 0.46 cmD x 20 cmLBuffer: 20 mM MESLoad Conditions: 5 mg/ml IgGFlow Rate: 300 cm/hr

50

37.50

0.00

High capacity over a broad rangeof process conditions

HighCapacity

HighResolution

HighSalt Tolerance

XS

High resolution maintainedas linear flow rate increases

C5 C

apac

ity (m

g/m

l)

21

Protein A versus CEX captureProcess comparison

•Protein A Process •CEX Process

Harvest• pH 7.0-7.4• Conductivity: 11-15 mS/cm

Column Load

• Direct load

Column Elute

• pH 2-3• Viral inactivation

Harvest• pH 7.0-7.4• Conductivity: 11-15 mS/cm

Column Load

• Titrate to pH 4.5-6.0• Conductivity 8-15 mS/cm

Column Elute

• Gradient or step elution• Viral inactivation pre or post

CEX column

22

CEX provides an effective alternative to Protein A capture

POROS®

MabCapture™ A Process

POROS® XS Process

Loading density (mg IgG/mL Resin) 40 100Yield (%) 89 98% HMW by SEC-HPLC 1.9 2.4CHO HCP (ppm) 39 (340x reduction) 1645 (8x reduction)Leached Protein A (ppm) 47 N/A

CEX capture chromatography with POROS® XS provides noteworthy performance in relation to Protein A affinity chromatography

• 2-3x protein capacity with up to 4.5x reduced resin cost • Effective HCP removal• No generation of leached Protein A

23

Process comparison using CEX or Protein A as capture

Data from Tao, Y., et al (2014), Biotechnol. Bioeng. doi: 10.1002/bit.25192

CEX process

Pro A process

CEX process

Pro A process

CEX process

Pro A process

Post Capture

93 89 1.44 1.13 1733 2849

Post AEX 84 87 1.61 1.05 22 6.5

Post HIC 90 92 0.36 0.25 10 1.5

Post Capture

91 95 0.78 0.91 6008 1347

Post AEX 99 99 0.75 0.87 47 9.5

Post HIC 89 92 0.38 0.49 22 1.4

Yield%

HMW %

HCPppmProcess

mAb A

mAb B

• Both processes provided material that passed specifications

• CEX provides an alternative to a Protein A process with similar yield and quality, but with potential for significant manufacturing and cost benefits

24

CEX provides an cost effective alternative to Protein A capture A CEX capture step using POROS® XS resin can provide at least double the

capacity of a Protein A process using MabSelect Sure with numerous reports of > 70 mg/ml DBC (Kokke, 2014; Tao, 2014, Conley, 2011, unpublished customer data)

CEX based processes provides equivalent product quality (Kokke, 2014, Tao, 2014, Conley, 2011; Lain, 2009: Ferreira, 2007, 2009)

Higher capacity CEX resins allow for economic and throughput benefits with equal recovery, impurity clearance and product quality as compared to affinity based processes (Kokke, 2014, Arunakumari, 2009; Ferreira , 2007)

CEX capture provides a “cheaper and higher capacity generic method for high-titer antibody processes”. (Tao, 2014)

25

Benefits of a high performing capture stepProcess modeling

• Unit Operation Model• 10,000 L Harvest at 5g/L Expression• 1.4mD x 20cmL Protein A Columns, 308 L• 1.8mD x 20cmL POROS® XS CEX Column, 509 L

Decrease processing times by 40 – 80% over traditional soft gel resin processes using POROS® MabCapture™ A or POROS® XS resin

Capture resin Flow rate(cm/hr)

Capacity(mg/ml)

# Cycles required

Cumulative process time

(H)

% Time reduction

GE MabSelect™ 200 43 4 14.1400 35 5 9.6 31%

POROS®

MabCapture™ A

200 48 4 14.1 0%400 47 4 8.0 43%600 44 4 4.0 71%

POROS® XSCEX Resin 300 100 1 3.3 77%

26

Process modeling of a high performing capture step

• Buffer/WFI costs are decreased by 60% using POROS® XS compared to Protein A process

• Cost savings of 15-50% over GE MabSelect™ and 40-64% over GE MabSelect™ SuRe using a high performing capture step

Unit Operation Model− 10, 000 L Harvest at 5g/L Expression− 1.4mD x 20cmL Protein A Columns, 308 L− 1.8mD x 20cmL POROS® XS CEX Column, 509 L

Capture Resin

Flow Rate

(cm/hr)

# Cycles Req’d

Buffer Cost($K)

Labor Costs ($K)

Resin Cost($M)

Total Cost per Batch

($M)

Buffer Cost

Reduction

Total Cost Reduction

from MabSelect

Total Cost Reduction

from MabSelect

Sure

GE MabSelect™200 4 81.3 4.2 3.08 3.17 400 5 101.6 2.9 3.08 3.18 -25% -1%

GE MabSelect™SuRe

200 4 81.3 4.2 4.31 4.40 0% -39%400 5 101.6 2.9 4.31 4.42 -25% -40%

POROS®

MabCapture™ A

200 4 81.3 4.2 2.62 2.70 0% 15% 39%400 4 81.3 2.4 2.62 2.71 0% 15% 39%600 4 81.3 1.2 2.62 2.70 0% 15% 39%

POROS® XSCEX Resin 300 1 33.6 0.98 1.53 1.56 59% 51% 64%

27

Overview



Advantages


POROS XQ

• Closing

28

CaptureSelect™ Affinity Ligands

• Advantages of CaptureSelectTM ligands or VHHs:• Small size:12-15 kDa fragment: ~1/10th mAb• Tunable specificity/affinity• No glycosylation• Safe and well tolerated by humans• High level production in yeast (S. cerevisiae): Animal origin free

12-15kDa

29

CaptureSelectTM Affinity Ligands and Resins Product Map and Formats

Full portfolio of affinity solutions supporting biopharmaceutical development from screening to final manufacturing

Bioprocess Products

Affinity resins for

large scale process

Custom Ligand Design

Tailored solutions on POROS® resin or Agarose

Antibody Toolbox®

Small scale antibody

purification

Protein Purification

Small scale protein

purification

C-tag

Affinity TAG

HPLC affinity

columns

Rapid analysis

and purification

-Supporting Bioprocess

resins

Biotin Conjugated

Ligands

Assay dev. or sample

prep

Plasma sample

preparation

Sample prep,

albumin, plasma

depletion

30

CaptureSelectTM Bioprocess Resins

Product Target applications Uniqueness

CaptureSelect™ FSH Human Follicle Stimulating Hormone Binds only intact FSH due to alpha/beta chain epitope

CaptureSelect™ KappaXL Human IgG and Fab fragments thereof containing a kappa light chain

Mild elution for fragments and antibodies

CaptureSelect™ FcXL Human IgG antibodies, Fc-fusion proteins

CH3 binding domain, mild elution

CaptureSelect™ IgG-CH1 Human IgG antibodies and Fab fragments thereof

CH1 domain binding, no free light chain binding for fragments

CaptureSelect™ HSA Human serum albumin, albumin fusion proteins

Mild elution conditions

CaptureSelect™ hGH Human growth hormone High purity capture from E. coli

CaptureSelect™ IgA Human IgA, s-IgA Platform for IgA

POROS® CaptureSelect™ AAV9

AAV9 Platform for AAV9

POROS® CaptureSelect™ IgM Human IgM Platform for IgM

Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only.

31

CaptureSelect™ Custom Service Needs

Biosimilars Complex Proteins

Molecule Platforms Scavenging

Increase yield / purity

Reduce the number of purification steps

No generic purification strategy

Enabling technology

Selective removal of impuritiesOffer platform approach

for purification/detection

Innovative processes

33

CaptureSelect™ Purification Platform

• Enabling a platform approach for all biomolecules through Selectivity / Specificity• high purity in single step / feed stock independent

(like protein A for Mabs)

• Reduction of process steps • higher yields, reduced costs

• Mild elution conditions• retaining biological activity of target

• Efficient clearance of HCP, DNA, virus • high selectivity in capture step

34

POROS® CaptureSelect™ Affinity HPLC ColumnsOverview

Needs POROS® column offers

Protein quantitation from complex sample

matrixes

Precise and sensitive measurement of protein titer in cell culture, fermentation harvests and downstream intermediates

Rapid assays POROS® 20 µm HPLC columns allow for quantitation in under 3 minutes

Flexibility POROS® Protein A, G resins and CaptureSelect® ligands allows assay development for most classes of biologic drugs

AutomationHPLC systems provide for automation of chromatography methods, data collection, data analysis and reporting

Sample preparation for subsequent analysis

Post quantitation, eluted product is highly purified and suitable for further analysis including mass spectrometry

Long column lifePOROS® Affinity Chromatography columns can be cycled hundreds of times for cost-effective and reproducible quantitation or purification

For Research Use Only. Not for use in diagnostic procedures.

Protein A, G and CaptureSelect® affinity ligands when combined with POROS® 20

µm resin provides for rapid quantitation and small-scale purification of:

1. Immunoglobulins – IgG and IgM2. Light chains – Human Kappa and Lambda

3. Human serum albumin4. Fusion proteins - Fc or human serum albumin

36

Overview



Advantages


POROS XQ

• Closing

37

Membrane Adsorbers vs. Traditional Chromatography in Flow Through Mode

• Membrane Advantages Faster flow rates 95% less buffer usage Disposability Ease of use

• Membrane Disadvantages Complicated scale-down models Difficult to compare to other

membranes and resin

Chromatography Advantages Reusable at commercial scale Available for all scales

Chromatography Disadvantages Very large diameter columns to ↑ flow

rate to ↓ bottleneck Packed for throughput and not for

binding capacity

So…Where does POROS resin fit in???

38

• Red = 100 cm/hr• Blue = 300 cm/hr• Pink = 700 cm/hr

1272009005:10_UV1_300nm 8232009001:10_UV1_300nm 1272009005:10_Inject 1232009005:10_UV1_300nm 8232009002:10_UV1_300nm 1232009001:10_UV1_300nm 1242009001:10_UV1_300nm

200

400

600

800

1000

1200

mAU

0 20 40 60 80 100 120 ml

POROS ® = Dotted Lines Seph FF = Solid Lines

POROS® Chromatography resin:Impurity Breakthrough Curves

Steep Breakthrough = Efficient Capture

3939

Comparison of AEX Chromatography Columns and Membranes: Scale down approach

AEX Product

Column/ Membrane

Volume (ml)

Column/Membrane

Dimensions

Linear FlowRate

(cm/hr)

Residence Time (min)

POROS® HQ 50,Life Technologies 0.83 0.46cmD x 5cmL 1000 0.3

POROS ® PI 50,Life Technologies 0.83 0.46cmD x 5cmL 1000 0.3

Fractogel® TMAE (M),EMD Millipore

0.83 0.46cmD x 5cmL 800 0.4

Q Sepharose® Fast Flow, GE Healthcare 0.83 0.46cmD x 5cmL 500 0.6

Sartobind® Q Nano, Sartorius 1 15 membrane

layers250 DNA100 BSA

0.1 Target 10 MV/min

Mustang® Q Coin, Pall 0.35 16 membrane

layers 131 0.1Target 10 MV/min

40

POROS® HQ50 Pressure vs Flow Curve: 5cm vs 20 cm bed height

• A 5 cmL POROS ® column can be operated at 1000 cm/hr with a pressure drop that allows for use with conventional low pressure chromatography columns and systems

POROS HQ50 Pressure vs Flow: 8cm GoPure, 5um Frits, 0.1M NaCl,

system pressure subtracted

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0 200 400 600 800 1000

Flow Rate (cm/hr)

Pres

sure

(bar

)

5cm Bed Height20cm Bed Height

41

DNA Capacity Summary on Membrane Comparison Study

• POROS ® HQ ranked highest for DNA C5 DBC and >6 times higher than QFF

• POROS ® HQ and PI C5/C50 ratios are as efficient as the membranes

• POROS ® HQ and PI had minimal change in performance in the two formats as compared to Fractogel and QFF

AEX Product

C5mg/ml

C50mg/ml

C5/C50Ratio

∆ in C5 from C5 at 300

cm/hr 20 cmL

POROS® HQ 37 52 0.72 5%

Mustang® Q 33 48 0.70 n/a

POROS® PI 24 33 0.73 13%

Sartobind® Q Nano 18 25 0.72 n/a

Fractogel®TMAE 10 26 0.38 53%

Q Sepharose® Fast Flow 6 43 0.13 64%

42





AEX Product

C5mg/ml

C50mg/ml

C5/C50Ratio


cm/hr 20 cmL

POROS® HQ 37 52 0.72 5%


POROS® PI 24 33 0.73 13%


Fractogel® TMAE 10 26 0.38 53%


43





AEX Product

C5mg/ml

C50mg/ml

C5/C50Ratio


cm/hr 20 cmL

POROS® HQ 37 52 0.72 5%


POROS® PI 24 33 0.73 13%


Fractogel® TMAE 10 26 0.38 53%


44

Impact of Conductivity, Flow Rate and Bed Height on Viral Clearance using POROS® HQ Resin

• >4 LRV for MVM up to 50 mM NaCl (8 mS/cm)

• >5 LRV for XMuLV at 150 mM NaCl (18 mS/cm)

• HQ operated with a short bed height at high flow rate achieves good viral clearance

Study ConditionsColumn Format: 0.46 cmD x 5 cm bed heightFlow Rate: 1000 cm/hr Residence Time: 0.3 min

Virus Load NaCl Concentration

Load Conductivity

(mM) (mS/cm) MVM XMuLV25 5 4.98 4.0450 8 4.34 4.95

150 18 0.83 >5.26

Viral Clearance (Log 10)

45

Large Scale Operating Cost Model for POROS® HQ Resin vs Conventional Soft Gel Resin

POROS HQ in Optimized

Format

Conventional Soft Gel

ResinColumn/Membrane Dimensions 80 x 5 cm 80 x 20 cmColumn/Membrane Volume (L) 25.1 100.5Load Capacity (mg of protein/ml of resin) 538 134Linear Flow Rate (cm/hr or MV/min) 1000 150Product Load Process Time (hr) 0.5 3.6Total Process Time (hr)* 1.1 6.7

• POROS HQ Column is ¼ the size of a conventional soft gel resin

• POROS processing time is ~6x faster

* Total process time includes a 30 minute sanitization hold time

Load volume: 2700L

Total protein loaded: 13.5 kg

Load concentration: 5 mg/ml

46

Large Scale Operating Cost Model for POROS® HQ 50 vs Conventional Soft Gel Resin


Format


ResinColumn/Membrane Dimensions 80 x 5 cm 80 x 20 cmColumn/Membrane Volume (L) 25.1 100.5Buffer Volume (L) 602 2412

Pre-Equil:Equil: Wash:Regeneration:Sanitization: Storage:

3 CV5 CV3 CV3 CV3 CV3 CV

3 CV5 CV3 CV3 CV3 CV3 CV

Load volume: 2700L

Total protein loaded: 13.5 kg

Load concentration: 5 mg/ml

POROS uses 4 times less buffer

than conventional soft gel process

47

Large Scale Operating Cost Model for POROS® HQ 50 vs Conventional Soft Gel Resin


Format


ResinColumn/Membrane Dimensions 80 x 5 cm 80 x 20 cmBuffer Cost ($) 1807 7236Column Packing Labor Cost ($) 4500 4500Process Labor Costs ($) 3937 5499Cost of Resin/Membrane ($) 50,200 100,500Total Cost of Processing/ Cycle ($)

1 Cycle 60,444 117,7355 Cycles 16,684 33,735

10 Cycles 11,214 23,23520 Cycles 8,479 17,98550 Cycles 6,838 14,835

• POROS HQ costs almost 2x less than conventional soft gel resin whether cycled or not

48

Overview



Advantages


POROS XQ

• Closing

49

Introducing POROS® XQ Chromatography ResinA new high performance strong anion exchanger

• Design Goals: • Next generation strong AEX resin with fully quaternized amine surface

chemistry • Increase dynamic binding capacity over current AEX product offering,

including POROS® HQ• Retain all the beneficial performance attributes of POROS® HQ

• Resolution capability, pressure vs flow, and physical stability

• Co-develop with industry based biopharma• Improved salt tolerance• Increased sodium hydroxide stability

• How Accomplished?• Used an optimized base bead and new surface chemistry

• Larger total pore volume• Smaller average pore size• Higher ligand density

50

POROS® Anion Exchange Resin Offerings

POROS®

Resin Surface ChemistryBSA Binding

Capacity (mg/mL)

AEX Applications

D50 Dimethylaminopropyl 90 Bind/Elute:Protein, virus, plasmid

DNA purification

Flow Through: Trace impurity removal by binding impurities (DNA, viruses, HCP,

aggregates, endotoxin)

PI50 Polyethyleneimine(Mixed Amine) 80

HQ50Quaternized

polyethyleneimine(Mixed Amine)

75

XQ Fully quaternizedamine >140

• A full range of weak and strong anion exchange resins with unique surface chemistries, that provide unique selectivity

PHARMACEUTICAL GRADE REAGENT. FOR MANUFACTURING AND LABORATORY USE ONLY.

51

POROS® AEX Chromatography Resins Titration curves comparing XQ and HQ50

____POROS® HQ50

____POROS® XQ

• POROS® HQ50 curve shows the presence of 1°, 2°, and 3° amino functionality in addition to quaternary amine as shown by the gradual titration curve

• POROS® XQ displays a sharp pH transition indicating the presence of quaternary amine groups typical of true strong AEX resins

• Strong AEX resins stay ionized more efficiently over a wider pH range. Each will give unique selectivity

52

pH

NaCl

Con

cent

rati

on (

mM

)

9.08.58.07.57.06.56.0

200

150

100

50

0

> – – – – < 30

30 6060 9090 120

120 150150

C5

BSA Dynamic Binding Capacity at 5% Breakthrough (mg/ml)

POROS® XQ Chromatography ResinDesign space for optimal dynamic binding capacity

Format

Column: 0.46 cmD x 20 cmLBuffer: 20 mM Bis-Tris PropaneLoad Conditions: 10 mg/ml BSAFlow Rate: 300 cm/hr

120-150

>150

90-120

BSA Dynamic Binding Capacity at 5% Breakthrough (mg/ml)

POROS® XQ delivers high

capacity over a broad range of

process conditions

54

0

20

40

60

80

100

120

140

160

0 200 400 600 800 1000

BSA

DB

C, 5

% (m

g/m

l)

Flow Rate (cm/hr)

POROS XQ POROS HQ50 POROS PI50 Q Sepharose™ FF

POROS® XQ PerformanceBSA binding capacity as a function of flow rate

Format


Load Condition: 10 mg/mL BSA in 20mM Tris, pH 8.0

Maintain high dynamic binding

capacity over a large flow rate range

55

POROS® XQ Performance Setting Standards in BSA binding capacity

POROS® XQ provides high

capacity compared to other

commercially available strong

AEX resins

Format


Buffer: 20mM Tris, pH 8.0

Load Condition: 10 mg/ml BSA in Equil


0

20

40

60

80

100

120

140

160

BSA

DB

C, 5

% (m

g/m

l)

56

0

20

40

60

80

100

120

140

160

No Salt, 1.1 mS/cm

50 mM NaCl, 6.6 mS/cm



BSA

DBC,

5%

(mg/

ml)

POROS® XQ

Nuvia™ Q

Capto™ Q

Eshmuno™ Q

Fractogel® TMAE

Capto™ Q Impres

POROS® XQ Performance BSA binding capacity as a function of NaCl concentration

FormatColumn: 0.46cmDx 20cmL

Buffer: 20mM Tris, pH 8.0



57

100

110

120

130

140

150

160

170

180

0 2 4 6 8 10 12 14

BSA

DBC,

5%

(mg/

ml)

Residence Time (min)

DBC vs Flow Rate DBC vs Bed Height

POROS® XQ PerformanceBSA binding capacity vs residence time

Binding capacity is

≥140 mg/ml at a residence

time of 1 minute and

greater

Format

Column Diameter: 0.46cmD

Buffer: 20mM Tris, pH 8.0, 50 mMNaCl


58

0

20

40

60

80

100

120

140

160

1 mg/mL 5 mg/mL 10 mg/mL 20 mg/mL

BSA

DB

C, 5

% (m

g/m

l)POROS® XQ PerformanceBSA binding capacity as a function of load protein concentration

Binding capacity is

consistently >140 mg/ml

over a 20 fold range in load

protein concentration

Format


Buffer: 20mM Tris, pH 8.0, 50 mM NaCl

Load Condition: Varying BSA concentrations in Equil


59

0

20

40

60

80

100

120

0 100 200 300 400 500 600 700 800

DNA

DBC,

5%

(mg/

ml)

Linear Flow Rate (cm/hr)

POROS® XQ POROS® HQ50 Fractogel® TMAE

Q Sepharose™ FF Capto™ Q

POROS® XQ Chromatography Resin DNA binding capacity as a function of linear velocity

POROS® XQ and HQ50 maintain

high DNA dynamic binding capacity over a large flow rate

range

FormatColumn: 0.46 cmD x 20 cmL, 3.2 ml

Load Condition: 2.0 mg/mL DNA from Herring Sperm (Sigma D3159) in 20 mM sodium phosphate, 50 mM NaCl, pH 7.0

60

0

20

40

60

80

100

120

140

0 50 100 150 200 250 300 350 400 450

DNA

DBC,

5%

(mg/

ml)

NaCl Concentration (mM)

POROS® XQ POROS® HQ50

POROS® XQ Chromatography Resin DNA binding capacity as a function of salt concentration

FormatColumn: 0.46 cmD x 20 cmL

Load Conditions: 2 mg/mL DNA from Herring Sperm (Sigma D3159) in 20 mM sodium phosphate, pH 7.0, test salt concentration


High DNA capacity even up to 400 mM NaCl

for both POROS®

XQ and HQ50

61

0

200

400

600

800

1,000

1,200

1,400

4 9 14 19 24 29 34

Abso

rban

ce a

t 280

nm

(mAU

)

Volume (ml)

100 cm/hr 300 cm/hr 700 cm/hr

POROS® XQ Performance Separation versus linear flow rate

FormatColumn: 0.46cmDx 20cmL, 3.32 ml Gradient: 0-50%B, 10 CV Buffer A: 20 mM Tris, pH 8.0Buffer B: 20 mM Tris, 1M NaCl, pH 8.0Sample:

Holo-Transferrin 2mg/mL, pI 5.5Ovalbumin 5.5mg/mL, pI 4.6Soybean Trypsin Inhibitor 8mg/ml, pI 4.5

Total Protein Loaded: 9.3mg, 2.8mg/ml resin

• Resolution is maintained independent of linear flow rate

• POROS® XQ is capable of achieving excellent separation using a very steep gradient

62

0

200

400

600

800

1,000

1,200

1,400

4 9 14 19 24 29 34

Abso

rban

ce a

t 280

nm

(mAU

)

Volume (ml)

POROS XQ POROS HQ

POROS® XQ Performance Separation compared to POROS® HQ50

POROS® HQ and XQ present with

similar resolution properties

FormatColumn: 0.46cmDx 20cmL, 3.32ml Gradient: 0 – 50%B, 10 CV , 300 cm/hrBuffer A: 20 mM Tris, pH 8.0Buffer B: 20 mM Tris/ 1.0M NaCl, pH 8.0Sample:


Total Protein Loaded: 9.3 mg, 2.8mg/ml resin

63

POROS® HQ50 vs. POROS ® PI50 Separation

Separationrun1thru16002:10_UV1_280nm Separationrun1thru16002:10_Cond Separationrun1thru16012:10_UV1_280nm1

0

200

400

600

800

1000

mAU

0.0 5.0 10.0 15.0 20.0 25.0 30.0 ml

FormatColumn: 0.46cmDx 20cmL, 3.32ml Gradient: 0 – 50%B, 10 CV , 300 cm/hrBuffer A: 20 mM Tris, pH 8.0Buffer B: 20 mM Tris/ 1.0M NaCl, pH 8.0Sample:

Transferrin 5mg/mL, pI 5.6Chicken Ovalbumin 10mg/mL, pI 4.6Soybean Trypsin Inhibitor 4mg/ml, pI 4.5

Total Protein Loaded: 4.4 mg, 1.3mg/ml

PI has different selectivity allowing for increased retention and better resolution for this protein sample

____POROS HQ50

____POROS PI50

64

POROS® XQ Performance Separation compared to other AEX resins

0

200

400

600

800

1,000

1,200

5 10 15 20 25 30 35

Abso

rban

ce a

t 280

nm

(mAU

)

Volume (ml)

POROS® XQ Capto™ Q

0

200

400

600

800

1,000

1,200

5 10 15 20 25 30 35

Abso

rban

ce a

t 280

nm

(mAU

)

Volume (ml)

POROS® XQ Capto™ Q Impres

0

200

400

600

800

1,000

1,200

5 10 15 20 25 30 35

Abso

rban

ce a

t 280

nm

(mAU

)

Volume (ml)

POROS® XQ Fractogel® TMAE

0

200

400

600

800

1,000

1,200

5 10 15 20 25 30 35

Abso

rban

ce a

t 280

nm

(mAU

)

Volume (ml)

POROS® XQ Eshmuno™ Q

50 µm POROS®

resin delivers improved scaleable

protein separation

65

POROS® AEX Separation Compared to Conventional Chromatography Resin

Conventional Resin Loses Resolution as Flow Rate Increases

FormatColumn: 0.46cmDx 20cmL, 3.32ml Gradient: 0 – 50%B, 10 CV Buffer A: 20 mM Tris, pH 8.0Buffer B: 20 mM Tris/ 1.0M NaCl, pH 8.0Sample:

Transferrin 5mg/mL, pI 5.6Chicken Ovalbumin 10mg/mL, pI 4.6Soybean Trypsin Inhibitor 4mg/ml, pI 4.5

Total Protein Loaded: 4.4 mg, 1.3mg/ml

0

200

400

600

800

1000

1200

mAU

10.0 15.0 20.0 25.0

0

200

400

600

800

1000

1200

mAU

10.0 15.0 20.0 25.0 ml

POROS HQ 50 Conventional Resin

Abs

orba

nce

280n

m (m

AU

)

Abs

orba

nce

280n

m (m

AU

)Volume (ml) Volume (ml)

____ 100 cm/hr

____ 300 cm/hr

____ 500 cm/hr

____ 100 cm/hr

____ 300 cm/hr

____ 500 cm/hr

66

0

200

400

600

800

1,000

1,200

1,400

0 2 4 6 8 10 12

Abs

orba

nce

at 2

80 n

m (m

AU

)

Column Volumes

5 cm L1 min RT

10 cm L2 min RT

20 cm L4 min RT

30 cm L6 min RT

POROS® XQ PerformanceSeparation versus column length/residence time

Format

Column: 0.46cmD, variable heightFlow Rate: 300 cm/hrGradient: 0-50%B, 10 CV Buffer A: 20 mM Tris, pH 8.0Buffer B: 20 mM Tris, 1M NaCl, pH 8.0Sample:


Total Protein Loaded: 2.8mg/ml resin

High resolution across a wide

range of residence times

67

0

200

400

600

800

1,000

1,200

0 10 20 30 40

Abso

rban

ce a

t 280

nm

(mAU

)

Time (min)

20 cmL, 300 cm/hr 5 cmL, 75 cm/hr

POROS® XQ PerformanceSeparation versus bed height at 4 minute residence time

Format

Column: 0.46cmD, 5 and 20 cmLFlow Rate: 75 and 300 cm/hr (4 min residence time)Gradient: 0-50%B, 10 CV Buffer A: 20 mM Tris, pH 8.0Buffer B: 20 mM Tris, 1M NaCl, pH 8.0Sample:


Total Protein Loaded: 2.8mg/ml resin

With controlled residence time,

predictable resolution can be attained at shorter

bed heights

68

0

20

40

60

80

100

120

140

160

180

0 3 6 9 12 15 18 21 24

BSA

DBC

, 5%

(mg/

ml)

Months storage at 25C

100 mM NaOH

20% Ethanol Control

10 mM NaOH/100 mM NaCl

100 mM Acetic acid/ 50 mM Phosphoric Acid

POROS® XQ PerformanceBSA capacity in different storage solutions (accelerated study)

FormatColumn: 0.46cmDx 20cmL

Buffer: 20mM Tris, 50mM NaCl pH 8.0

Load Condition: 10 mg/mL BSA in equilibration buffer


Accelerated stability: Stored for16 days at 60oC

Stability Extrapolation :10 days = 12 months at 25°C16 days = 19.2 months at 25°C

POROS® XQ is stable in

different storage solutions for >18 months at 25°C

Accelerated Stability Degradation Model: Kennon, L., Use of Models in Determining Chemical Pharmaceutical Stability, Journal of Pharmaceutical Sciences, 53 (7):815-818, 1964.

69

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

BSA

DBC,

5%

(mg/

ml)

Exposure Time (hours)

0.5M NaOH 1M NaOH

POROS® XQ PerformanceStability in 0.5M and 1M NaOH: BSA Capacity

POROS® XQ maintains

dynamic binding capacity and

protein separation in 0.5 and 1M

NaOH solutions mimicking >100

sanitization cycles0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

0 20 40 60 80 100 120

Rete

ntio

n Ti

me

(min

)

Exposure Time (hours)

Holo-Transferrin Ovalbumin Soybean Trypsin Inhibitor

70

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 200 400 600 800

Pres

sure

(bar

)

Linear Flow Rate (cm/hr)

6.2 cmD x 20 cmL, 12 um frits, 0.1M NaCl, 3 bar packPOROS XQ POROS XS

POROS® XQ Performance Pressure vs flow curve compared to POROS® XS

• Linear and predictable pressure – flow response

• Ability to operate under high linear flow rates while maintaining < 3b backpressure

71

POROS® XQ Chromatography ResinSetting the standard in chromatography solutions

Combined feature set for effective and flexible bind/elute or flow

through applications

Features Benefits• Unique, fully quaternized amine surface chemistry

• High dynamic binding capacity under a wide range of process conditions

• > 140 mg/mL BSA at 50 mM NaCl• > 100 mg/mL BSA at 100 mM NaCl• > 100 mg/mL DNA at 150 mM NaCl

• High resolution, baseline separation of proteins

• Flow rate independent performance of capacity and resolution

• Incompressible 50 μm PS-DVB bead with robust physical and chemical stability

• Improved yield and efficient impurity clearance

• Increased process flexibility and throughput

• Reduced column sizes, water/buffer requirements

• Effective separation of product and process related impurities

• Reduced number of unit operations

• Linear and predictable scaleablity

• Strong base stability

• Enables higher efficiency and lower cost of goods

HighCapacity

HighResolution

HighSalt Tolerance

XQ

72

Many thanks to our Product Development Team!

• Daniel Fagan• Elliot Haimes• Sarah Horgan• Christine Gebski• Thomas Leete• Lisa Lindsay• Paul Lynch• Norm Mega

• Mark McCoy• Larry Moschini• Shelly Parra• Malcolm Pluskal• Lou Sacco• Andy Tomlinson• Michael Tyman• Mingcheng Xu

HighCapacity

HighResolution

HighSalt Tolerance

XQ

73

Superior performance compared to other resins

Increased flexibility for all parts of chromatographic downstream applications

Established reputation for impurity removal

Enabling high-performance affinity solutions with CaptureSelect

State-of-the-art manufacturing and robust quality

Global support

Thank you!

Providing flexible, high performance solutions for downstream process chromatography

74

PHARMACEUTICAL GRADE REAGENT. FOR MANUFACTURING AND LABORATORY USE ONLY.

© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Fractogel and Eshmuno are trademarks of Merck KGaA. Nuvia is a trademark of Bio-Rad Laboratories, Inc. Capto is a trademark of GE Healthcare Bio-Sciences.

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