Optimizing downstream purification processes: New ... Capt Sel GP6 053b(1291298174)001:10_UV1_280nm Affi Capt Sel GP6 053b(1291298174)001:10_Cond Affi Capt Sel GP6 053b(1291298174)001:10_pH

The world leader in serving science

Shelly A. Parra Sr. Manager, Global Field Applications

Optimizing downstream purification processes: New approaches and benefits to bioprocess chromatography

2

• Introduction to CaptureSelect™ Affinity Products and Services

• Principles & Advantages

• Introduction to POROS™ Chromatography:

• Principles, Product Attributes & Advantages

• Closing

Overview

3

Leading Capabilities for Every Step of the Bioprocess Workflow

Upstream Downstream

Cell line development and

media optimization

Mixing, cell culture, and fermentation

Harvest and collection Purification Bulk storage

and final fill

Microbial identification

Mycoplasma and viral detection

Sample prep and automation Analytical columns

QC and analysis Gibco™ Freedom™ CHO cell lines

Analytical services

Media optimization services

Gibco™ Media Express™

Cell culture media, feeds, supplements, and bioprocess liquids Single-use mixers, bioreactors, and fermentors

Bioprocess containers (BPCs)

Inflation and integrity test system

Harvest and separation products

Bioprocess containers (BPCs)

Single-use heat exchanger

Single-use aseptic sampling system

POROS™ chromatography resins

CaptureSelect™ affinity ligands and resins

Transfer assemblies

Residual DNA and protein tests

Storage and transport

BPCs, manifolds and containers

Single-use filling system

Acclimate™ freeze/thaw containment system

4

Monoclonal antibodies

• mAb-based therapeutics continue to dominate even with new cell lines emerging

• More Mab Fragments, Fabs, ADCs • Dramatic increases in cell expression levels • Interest in continuous processing

Vaccines • Processes redesigned to be more responsive, more reproducible, with higher yields

Plasma • Interest in modernizing legacy processes

Biosimilars • More drugs coming off patent, need for shorter time to end-user

Gene Therapy • Growing interest in the area and the market expected to grow 15% CAGR

First in human • Speed to clinic is main product development driver

Ease of use • Major shift to disposables in downstream purification

Quality and efficiency

• Continued demand for highest quality resolution, capacity, salt tolerance and operation speed

• Consistent, reproducible material from batch to batch • Supply risk mitigation, cost minimization

Industry Trends Driving Growth in Downstream Purification

5

Purification Overview - Optimizing downstream processes

Throughput Capacity

Resolution/Separation

Yield Balance Required

Development Time

Cost

Many Factors Influence Downstream Process Development

6

BioProduction Solutions for Downstream Processing

POROS™ Ion Exchange • High performance cation and anion exchange

resins • Differentiated surface chemistries provide

unique selectivity

CaptureSelect™ Products and Services • Unique, tunable affinity ligands / resins • High purity in a single step

POROS™ Resins • MabCapture™ A Select resin • High performance Ion Exchange resins

Simplifying biomolecule purification and reducing COGs

Cell Culture Clarification

Capture/Affinity Chromatography

Viral Inactivation

Polish/Non-Affinity Chromatography

Viral Filtration

Formulation

7

POROS™ Bulk Resins

• AFFINITY MabCapture™ A Select MabCapture™ A Heparin 50 µm AAV8/9

• ION EXCHANGE XQ XS HS50 HQ50 D50 PI50

POROS XS

>100 mg/mL Capacity

CaptureSelect™ Bulk Resins • ANTIBODY-BASED THERAPEUTICS

KappaXL FcXL IgG-CH1 IgA IgM

• PROTEIN THERAPEUTICS FSH HSA AAV8 & AAV9 Gonadotropin hGH

Services

• Custom Resin Development • CaptureSelect™ Affinity Ligand

Development

Pre-Packed Columns

• GoPure™ 1.2 cm Diameter Column • Atoll columns

Resins for bioprocessing at any scale

Affinity solutions from screening to final

manufacturing

Simplifying purification preparation and enabling custom affinity solutions

CaptureSelect™ Technology

Unique specificity

Bioproduction Purification Products and Services

8





• Closing

Overview

9

Purification Can Be More Efficient

Non-Affinity Capture

Polish 1

Polish 2

Polish 3

Polish 4

Polish 5

Affinity Capture

Polish 1

Polish 2

80% reduction in purification time

Cell culturefeed

Purifiedproduct

CaptureSelect™ solution • Enabling a platform approach

for biomolecules through selectivity /specificity • High purity in single step

• Reduction of process steps • Higher yields, reduced costs

• Mild elution conditions • To retain biological activity of

target

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

Simplify recombinant protein

purification

10

• Technology based on single-domain [VHH] antibody fragments • Combining antibody based selectivity and process robustness

• Unique screening technology for target specificity, mild elution & stability • Animal origin free (AOF) production process in yeast • Safe and well tolerated by humans

• Products in commercial purification processes (US and EU)

CaptureSelect affinity ligands

11

EL

CaptureSelect affinity purification

Load Load Load EL EL EL Load

Purified sample Complex Impurities Immobilized Ligand Purification target Impurities

Binding Mild Elution

Principle

Results

• One-step selectivity to meet customer challenges • Antibody based target specificity → performance independent of feed stock

EL Load Load EL

Proven platform for product specific affinity solutions

rh GH rh G-CSF rh IgG1 Toxin rhFIX pdFIX

12

CaptureSelect™ technology platform

Proven platform for development of product specific affinity solutions

13

Bioprocess Products

Affinity resins for

large scale cGMP

purification

Custom Ligand Design

Tailored solutions on POROS® resin or Agarose

Antibody Toolbox®

Small scale antibody

purification

Protein Purification

Small scale protein

purification

C-tag Small, inert affinity tag

HPLC affinity

columns

Rapid analysis

and purification

- Supporting Bioprocess

resins

Biotin Conjugated

Ligands

Assay dev. or sample

prep

Bioprocess •First approved gene therapy product purified using CaptureSelect™

•First approved FVIII biobetter purified by CaptureSelect™

•Full portfolio of products for antibody formats, biosimilars and biobetters

Services • Customized affinity solutions - target specific - process compatible - scalable • Selective purification of post translational modified protein

Research • Unique TAG system for purification of EPEA tagged proteins

• Complete offering for purification of any antibody format from any source

CaptureSelect product map and formats

14

Broad range of target molecules covered (RUO and cGMP)

• Antibodies • Human IgG, IgA, IgM • Multi-species IgG • Human specific • Mouse and Rat specific • Fab fragment

− CH1 − LC-kappa/lambda

• Fc fragments and Fc-fusions

• Non-Antibody targets (recombinant and plasma derived) • Blood factors: FI, FII, FVII, FVIII, FIX, vWF, • Proteins: HSA, AAT, tPA, ATIII, TF, Fib, ApoA1, ApoH • Hormones; G-CSF, FSH, hGH, hCG, GM-CSF, Insulin • Viruses: AAV serotypes, Adenovirus, Influenza • Affinity Tags: C-Tag (EPEA)

CH3

CH1

CH2

VH

CL

VL

CH3

CH1

CH2

VH

CL

VL

CH4

CH1

CH2

VH

CL

VL

CH3

CH3

CH1

CH2

VH

CL

VL

www.thermofisher.com..captureselect-affinity-products.html

15

Antibody and antibody fragment purification

Fab fragment

Fc fragment

KappaXL - Human CL-kappa (all) - Generic: 100 % Fab-k coverage - Mild elution

FcXL - Human IgG-Fc (CH3) - All IgG subclasses: IVIG - Mild elution

IgG-CH1 / CH1-XL* - Unique selectivity - Generic: all Fab-k/l - No binding to free LCs - All IgG subclasses: IVIG

Caution: For manufacturing, processing, or repacking.

CH3

CH2

VL

CH1 CL

CH2

VH VL

CH3

Glycosylation site

Protein A - Human IgG-Fc (CH2-CH3) - Low IgG3 affinity: no IVIG

* CH1-XL currently available for RUO, BioProcess resin expected Sept 2017

16

• Improved version of the KappaSelect ligand • Same binding specificity and epitope • Improved elution characteristics • Improved stability (50 mM NaOH for cleaning)

CaptureSelect KappaXL

Elution

Strip pH 2.0 Elution

Strip pH 2.0

KappaXL KappaSelect

Elution with 100 mM Glycine pH 3.5

Column: 0.4 ml, 2 cm bed height Load: 12 ml 1 mg/ml Human IgG Running buffer: PBS pH 7.4 Flow: 150 cm/h Elution: buffers are indicated Strip buffer: PBS pH 2.0

Elution buffer KappaXL elution

(%)

Kappa Select Elution

(%)

20 mM citric acid pH 3.0 100 74



100 mM glycine pH 3.5 98 48

17

Targeting CH3 as a platform for mAbs and Fc fusions

Binding specificity

Human IgG, Fc fusion proteins from recombinant sources, and plasma IgG/IVIG

Matrix and particle size

Aldehyde-activated agarose, 65 μm

Dynamic binding capacity

30 g IgG per liter of matrix (column: 10 cm bed height, 150 cm/h, polyclonal IgG )

Elution buffer • 20 mM citric acid or acetic acid, pH 3 to 4 • 20 mM sodium acetate, 1.0 M MgCl2, 40% (v/v) propylene glycol, pH 5 to 6

Purification of human IgG1 monoclonal; efficient clearance of product-related impurities using FcXL • Mild elution, making it suitable for Fc fusion

proteins • Human-specific, no binding to bovine

antibodies • Excellent scalability • Non-animal-derived

L FT Wash Elution

FcXL ProtA

Product-related impurities observed in the elution fraction of ProtA purification are mainly present in the flow-through using FcXL


• CaptureSelect FcXL • Combining high specificity and mild elution for the purification of mAbs and Fc-

fusions

18

• CaptureSelect IgG-CH1 (CH1-XL) • Purification of Fabs from CHO cells containing over-expressed light chains and light chain

dimers

Targeting CH1 as a Platform for Antibody Fab Fragments

• Purifies all subclasses of human IgG • Purifies all antibody fragments

(kappa/lambda) • No free light chain binding (only correctly

assembled Fabs)

A true platform for Fab fragments

VH

Affi Capt Sel GP6 053b(1291298174)001:10_UV1_280nm Affi Capt Sel GP6 053b(1291298174)001:10_Cond Affi Capt Sel GP6 053b(1291298174)001:10_pH Affi Capt Sel GP6 053b(1291298174)001:10_Fractions Affi Capt Sel GP6 053b(1291298174)001:10_Logbook

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3500

mAU

3.0

4.0

5.0

6.0

7.0

0 50 100 150 ml

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66 45

30

20

14

High purity of the Fab, no light chain in the eluate

Eluate Marker

CH-1

19

CaptureSelect CH1-XL

0

1000

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3000

mAU

0 20 40 60 80 100 120

Flow through

Elution Strip

0

1000

2000

3000

mAU

0 20 40 60 80

Flow through

Elution

Strip

Polyclonal Fab

IVIG

CaptureSelect CH1-XL - Improved binding capacity - Elution with standard buffers at

pH 4.0 - Available as RUO resin - Resin suitable for cGMP Sept

2017

10

7

18 17

0

5

10

15

20

25

Human Ivig Human Fab

Dyn

amic

bin

ding

cap

acity

(g/l)

Dynamic binding capacity at 5% breakthrough

IgG-CH1

CH1-XL

- Column volume: 1 ml - Column dimensions: 5/50 mm - Flow: 0.5 ml/min (150 cm/hour) - Residence time: 2 minutes - Equilibration buffer: PBS pH 7.4 - Elution buffer: 50 mM Sodium Acetate pH 4.0 - Strip buffer: 100 mM Glycine pH 2.0

20

CaptureSelect™ Bioprocess Resins: Ab and Ab fragments


Product Target applications Uniqueness

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 and CH1-XL

Human IgG antibodies and Fab fragments thereof

CH1 binding domain, no free light chain binding for fragments

21

Pipeline of affinity products

CaptureSelect™ affinity products for purification of: Therapeutic proteins (non-mAb) & viruses Antibody types

Bioprocess cGMP

FSH, Human Albumin, hCG AAV8, AAV9,

KappaXL FcXL

IgG-CH1

Stage 5: RUO products

hGH, tPA Antithrombin III, Fibrinogen,

Transferrin, ApoH, C1-Inh

IgA, IgM CH1-XL

Stage 4: Lead Selection

Prothrombin, GM-CSF Exotoxin A (PE)

IgG-CH2

Stage 3: Prototype Resins

Insulin, EPO Adenovirus (Adv5), Flu (HA)

IgE, Free LC-kappa

Stage 2: Lead Screening

TSH, IFNa/b, hIL2, Protein C, FV, FX, FXI, FXII, FXIII, FH, vWF,

Lentivirus (VSV-G)

Rabbit IgG, Anti-Idiotypes

Stage 1: Library Construction DNAse Free LC-lambda,

scFv, IgY, Mouse IgG

22

Analytical Products Supporting Bioprocess Resins

Leakage ELISAs

• Reagents for a sandwich ELISA to detect leached ligand in the elution fractions of the CaptureSelect bioprocess affinity resins

• Available for all

CaptureSelect based bioprocess resins

HPLC columns

• Prepacked POROS CaptureSelect Affinity Columns for rapid quantitation of immunoglobulins, fusion proteins, Fabs, and bispecific antibodies

• Applications include titer determination and small scale purification

Conjugated ligands

• Biotinylated ligands for use in a range of analytical assays

• Applications include Capture ELISA, Western blot, Gyros’ Gyrolab®-based immunoassays, and label-free detection platforms

23





• Case Studies • Closing

Overview

24

• 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

POROS™ Chromatography Resin: Product attributes

25

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™ FFFormat

Column: 0.46cmDx 20cmL

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

Maintain high dynamic binding capacity over a large flow rate

range

High Performance Resins Enable Increased Productivity

26

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)

27

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)

28

Superior Resolution Enables Higher Yield and Purity

500cm/H

300cm/H

Conventional resins lose resolution as flow rate increases

POROS resins maintain resolution as flow rate increases

29

POROS™ AEX Separation Compared to Conventional Chromatography Resin

Conventional Resin Loses Resolution as Flow Rate Increases

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

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

Total Protein Loaded: 4.4 mg, 1.3mg/ml

0

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1200

mAU

10.0 15.0 20.0 25.0

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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

30

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

31

Implementing POROS™ Resins at Scale: Improved column lifetime

Cleaning Protocol 20CV: Tris-HCl, pH 8.2

4CV: 0.2N HCl, 1M NaCl

2CV: Tris-HCl

4CV: 0.5N NaOH, 50% Isopropanol

2CV: Tris-HCl

4CV: 5% Acetic Acid, 1M NaCl

POROS HQ50 Cleaning Study

0

20

40

60

80

100

0 30 60 90 130Cycle Number

% R

ecov

ery

Robust stability allows for effective cleaning and strong reuse performance

32

Classical Antibody Purification Ligands Protein A, G, and A/G

• Protein A

• 85% of market–chemical tolerance, easier elution • Binds Fc region at CH2-CH3 sites and binds to VH3 • Does not bind IgG3

• Protein G

• Binds Fc region at CH2-CH3 sites and to the CH1 region

• Poor binding to Ig subtypes (e.g., IgA, IgM, IgE, etc.)

• Protein A/G

• Combines five Fc binding domains from Protein A and two from Protein G

• Combined specificity of Protein A and G for a one- resin-fits-all solution

Utilized for multispecies antibody purification from a wide range of sample sources

33

Thermo Scientific™ POROS™ MabCapture™ Select resins Combining speed and selectivity for a more efficient purification

Capacity

Resolution

Productivity

Bead

Protein A: >35 mg/mL Protein G: >12 mg/mL Protein A/G: 20 mg/mL

Optimized 45 µm particle for improved impurity clearance, better yield

Maintains high binding capacity at high flow rates

Incompressible bead with robust physical stability, low backpressure

34

IEX Resin

Mode of Operation Key Applications

Cation Exchange

(CEX)

Bind/elute Polish of many biomolecules (mAb, VLP/viruses, fusion proteins, high pI recombinant proteins)

Overload/ Flow through

Polish for mAb by binding impurities under normal B/E conditions: impurity removal (aggregates, HCP, DNA, viruses)

Anion Exchange

(AEX)

Bind/elute Polish of many biomolecules (low pI recombinant proteins, DNA, viruses, plasmids)

Flow through Polish for mAb: binds impurities (DNA, viruses, HCP, aggregates, endotoxin)

35

0

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POROS XS

SP Sepharose

Volume (mL)

Abs

orba

nce

at 2

80 n

m (m

AU

) POROS XS vs. SP Sepharose FF

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Capto S

Volume (mL)

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80 n

m (m

AU

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POROS XS vs. Capto S

POROS XS is the only CEX resin to successfully combine superior capacity, salt tolerance & resolution

Format

Column: 1cmD x 20cmL; Buffer A: 20mM MES, 25mM NaCl pH 6.2; Buffer B: 20mM MES, 1M NaCl pH 6.2; Gradient: 10% B – 50% B 7.5CV; Flow Rate: 300cm/H; Sample: Chymotrypsinogen; Cytochrome C; Lysozyme

POROS XS Chromatography Resin Setting standards in resolution and capacity

C5

Cap

acity

(mg/

ml)

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140

Capacity of Cation Exchange Resins 100 mM NaCl, pH 4.5, 300 cm/H

0

500

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2500

POROS XS

SP Sepharo

POROS XS vs. GigaCap S

Volume (mL)

Abs

orba

nce

at 2

80 n

m (m

AU

)

POROS XS

Gigacap S

, p ,

0

20

40

60

80

100

120

140

C5

Cap

acit

y (m

g/m

l)

Capacity of Cation Exchange Resins 40 mM NaCl, pH 5.0, 300 cm/H

Global Adoption

36

Run 1 – pH 5.5 Run 2 – pH 5.0 Run 3 – pH 4.5

• Large particle size resin lacks resolution capability • Peak cutting required to separate monomer and aggregate • Decreased product yield

Aggregate Separation on a Non-POROS Resin

37

Run 1 – pH 5.5 Run 2 – pH 5.0 Run 3 – pH 4.5

High resolution POROS Resin provides improved separation

Aggregate Separation on POROS XS

38

• Optimization of baseline separation • Gradient can be optimized to step elution • Improved product yield due to efficient separation of

monomer and aggregate

Optimization of Aggregate Separation on POROS XS

39

POROS Anion Exchange Resin Offerings

POROS®

Resin

Type of AEX

Resin Surface Chemistry

BSA Binding Capacity (mg/mL)

AEX Applications

D50 Weak Dimethylaminopropyl 90 Bind/Elute: Protein, virus, plasmid DNA purification

Flow Through:

Trace impurity removal by binding impurities (DNA,

viruses, HCP, aggregates, endotoxin)

PI50 Weak Polyethyleneimine (Mixed Amine) 80

HQ50 Strong Quaternized

polyethyleneimine (Mixed Amine)

75

XQ Strong Fully quaternized amine >140

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

40

POROS XQ is a strong AEX resin that successfully combines superior capacity, salt tolerance & resolution

POROS XQ Strong AEX Chromatography Resin Novel solution for high capacity and resolution

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140

160

0 mM NaCl 50 mM NaCl 100 mM NaCl 150 mM NaCl

BSA

DBC,

5%

(mg/

ml)

Capacity of strong AEX resins at various salt conditions

POROS® XQNuvia™ QCapto™ QEshmuno™ QFractogel® TMAECapto™ Q Impres

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1,200

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t 280

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(mAU

)

Volume (ml)

POROS® XQ Capto™ Q

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Abso

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Volume (ml)

POROS® XQ Capto™ Q Impres

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5 10 15 20 25 30 35

Abso

rban

ce a

t 280

nm

(mAU

)Volume (ml)

POROS® XQ Fractogel® TMAE

41

A high performance resin can provide advantages for flow through chromatography

POROS resins allow high linear velocity, so allows high volumetric flow with smaller diameter/volume columns and less buffer (or much faster processing time)

Flow Through with soft gels can require large diameter/large volume columns to permit reasonable volumetric flow.

Resin Type Linear Flow

Rate (cm/hr)

Column Diameter

(cm)

Bed Height (cm)

Column Volume

(L)

Volumetric Flow Rate

(L/min)

Column Volume

Reduction (%)

1000 L Load Time

Reduction (%)

Soft Gel 200 100 10 78.5 26.5

POROS 1000 45 10 15.9 26.5 80%

POROS 1000 100 10 75.8 130.8 80%

42

Yun (Kenneth) Kang, Rajesh Ambat, Troii Hall, Matthew D Sauffer, Stanley Ng,Martha L Healy-Fried, Julia Lee, Josaih C Adaelu, William D Holmes, Warren Emery, Behnam Shanehsaz, Amy Huebner, Bo Qi, Richard Chen, Michael Barry, Dale L Ludwig & Paul Balderes, Pharmaceutical Bioprocessing, Vol. 3, No. 8, Pages 477-487 , DOI 10.4155/pbp.15.28

POROS™ XQ Case Study: Mab polish in 2-step process

http://www.future-science.com/loi/pbp

http://www.future-science.com/loi/pbp

43

Purpose: To design a scalable AEX flow through polish step for acidic/neutral antibodies (pI≤8.0) that had proved challenging using conventional strong AEX resins (QFF). Goal was to maximize impurity clearance (HCP, host DNA, leached ProA, and HMW aggregates) and product yield by utilizing POROS XQ, a salt tolerant strong AEX resin and to optimize to a 2-step purification process: Protein A>AEX FT Process Details:

• 7 Mabs tested • Loading Capacity: 100-300 mg of protein /ml of resin • Study format:

• HTP Screening: 96 well plates, 40 µl resin volume in 270 µl working volume • Development: 1.1cmD x 5.3cmL, 5ml columns • Pilot: 14cmD x 18.5cmL, 2.9L


44


Data from Kang, Yun (Kenneth), et al (Eli Lilly) “Development of an acidic/neutral antibody flow-through polishing step using salt tolerant anion exchange chromatography”, Pharmaceutical Bioprocess (2015), V8 (8), pages 477-487, DOI 10.4155/pbp.15.28

45


• POROS XQ increased flexibility when designing the purification scheme and eliminated the need for load dilution and diafiltration steps allowing for a more efficient and cost effective process


2-8 Fold HMW Clearance

1-9 Fold HCP Clearance

46

Viral Clearance on POROS™ XQ resin with increasing conductivity

• POROS XQ resin provides excellent viral clearance for both model viruses under typical FT/Wash conditions as well as increased conductivity

• Viral clearance with higher salt concentrations allows for increased flexibility when designing a purification scheme − Minimizes need for load dilution and diafiltration steps − Allows for more efficient and cost effective processes

Load Conductivity

Load Capacity (IgG/ml of

resin)

POROS XQ

XmuLV Log10 Reduction

MMV Log10 Reduction

5 mS/cm 500 >4.31 ± 0.12 >5.10 ± 0.09

10 mS/cm 500 > 4.39 ± 0.14 1.61 ± 0.23

15 mS/cm 500 3.46 ± 0.29 0.19 ± 0.28

Study Conditions Column Format: 0.46cmD x 20 cmL Flow Rate: 300 cm/hr Protein Load: 5 mg/ml Polyclonal IgG Buffer System: 20 mM Bis-Tris Propane, pH 7.0

47

• “POROS XQ is compatible with multivalent buffers which would provide a practical alternative to traditional AEX resins”

• “Unique salt tolerant nature of POROS XQ allows it to be compatible with most Pro A elution buffers eliminating the need for pre-AEX buffer exchange or inline dilution. In addition, wider operating ranges can be defined relative to traditional Q chromatography, which is likely to result in greater process robustness and manufacturing flexibility."

• “Utilization of XQ Chromatography enabled development of a two-column mAb purification platform, able to meet product purity targets for all antibodies evaluated in the study”

POROS™ XQ Provides Value


• Residual HMW ≤1.5% highlighted in Red

• Yield ≥ 90% in Blue • Overlap shading

indicates “sweet spot”

48

SITUATION OUR RESPONSE VALUE DELIVERED

• Large column volumes and long cycle times

• Goal = increase productivity of anion exchange (AEX) flow through step

• POROS HQ • Optimized AEX format • Technical support for

process optimization, column packing and lifetime

10 fold Cycle time reduction

More cost effective Optimized process

2.5X protein load Robust viral clearance & product quality

Case Study: POROS HQ in Short Bed/High Flow Format

Data from Shen, Yuyi et al, (Xoma) “An Optimized Approach for AEX Polish Chromatography to Drive Productivity and Decrease Cost of Goods”, Poster Presentation, BPI, Boston, MA Oct 2014

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POROS™ Quality Testing

• CQA testing designed to model how customers use the material

• Statistical process control to monitor manufacturing processes

• Outsourced testing: bioburden analysis, catalyst and OH polymer SEC testing • All contract labs have been qualified and approved

PROCESS STEP

Size Distribution

Fines Measurement FTIR Extractables

/ Leachates

Pore mode

Protein

Capacity Separation Endotoxin Bioburden

Base Polymer ● ● ●

Product Intermediate ● ●

Final Product ● ● ● ● ● ● ● ●

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• Robust, Defined, Proven Quality System − ISO 9001:2008 & ISO 13485:2003 Certified

> Last Registrar Audit – September 2015

− Process Media Products Backed by a Drug Master File since 2001

− Facility and Quality Systems Routinely Audited by Customers

− Products Used in Multiple FDA/EMEA Approved Therapeutic Processes

− Products being evaluated and incorporated into new processes by existing and new customers

POROS Quality Systems

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Your Partner for Purification

Enabling, purification solutions that simplify processes and reduce COGs

Superior performance compared to other resins

Novel affinity and IEX solutions Established reputation for affinity

purification of recombinant proteins including biosimilars

Established reputation for process and product-related impurity removal

State-of-the-art manufacturing and robust quality

Global support

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A solution that’s fits your needs

PHARMACEUTICAL GRADE REAGENT. FOR MANUFACTURING AND LABORATORY USE ONLY.

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Optimizing downstream purification processes: New ... Capt Sel GP6 053b(1291298174)001:10_UV1_280nm Affi Capt Sel GP6 053b(1291298174)001:10_Cond Affi Capt Sel GP6 053b(1291298174)001:10_pH

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