CaptureSelect™ and POROS CaptureSelect™ Affinity Resins for … · 2017-07-21 · Life...

1 Life Technologies™ |

CaptureSelect™ and POROS® CaptureSelect™ Affinity Resins

for Bioprocessing and Analytical Applications

BioProcess International Conference

September 19th, 2013

Bruce Dawson, VP Commercial / Business Development

BAC BV, The Affinity Experts, now part of Life Technologies

[email protected]

+1 (910) 256-5756

mailto:[email protected]


Most selective chromatographic principle:

− Specific recognition Affinity chromatography (AC)

- protein A (IgG purification)

- affinity tags

- target specific antibodies..

Protein Purification

Basic chromatographic purification principles:

− Size Gel filration (GF)

− Charge Ion exchange chromatography (IEX)

− Hydrophobicity Hydrophobic interaction chromatography (HIC)

Reverse phase chromatography (RPC)

− Metal ion binding Immobilized metal ion affinity chromatography (IMAC)

− Mixed Mode combination of e.g. IEC / HIC / RPC

Purification challenge depending on target & feedstock:

− impurity level

− target concentration & stability

− contamination profile (pI, hydrophobicity, MW of HCPs)

− target related impurities: isoforms, ∆ PTMs


Affinity Ligands based on Camelid single Domain Antibodies

Advantages of VHH based affinity ligands (CaptureSelect™Technology):

− Small size:12-15 kDa fragment

> ~1/10th mAb

− Tunable specificity/affinity

− Stable polypeptides

− Easy / flexible formatting

> multimers (bi-heads)

> labeling

− No glycosylation

− Safe and well tolerated by human

− High level production in yeast

> Free of animal derived components

Perfect match with affinity chromatography applications

− Antibody based selectivity + process robustness

Confidential © BAC BV 2010


CaptureSelectTM Affinity Ligands

Advantages of CaptureSelectTM ligands or VHHs:

− Small size:12-15 kDa fragment: ~1/10th mAb

− Tunable specificity/affinity

− Stable polypeptides

− No glycosylation

− Safe and well tolerated by humans

− High level production in yeast (S. cerevisiae): Animal origin free

12-15kDa


CaptureSelectTM Affinity Products

Single-domain [VHH]

antibody fragments

BEAD

COUPLING + Coupling to solid support

PRINCIPLE

Purified sample Complex Impurities Immobilized Ligand

+

Purification target

Impurities

Binding Elution

BENEFITS CaptureSelectTM products help enable: • More efficient purification

• Increased purity and yield

• Lower cost of goods

• Reduced time-to-market for end-users

PRODUCT • Lab-scale purification, >30 products

• Process Development and Biopharmaceutical Manufacturing, 9 commercial

products through partners, 6 in development


One-Step Selectivity Examples

“Non Protein A” Antibody formats

− Human Fab fragments

Recombinant human Proteins

− Biosimilars; FSH, hGH, G-CSF

− FVIII

Viruses, vaccines

− AAV, Influenza

Novel Tag system

− C-tag

From small (G-CSF: ̴20 kDa) to large (FVIII: ̴330 kDa)

From IgG to Fabs


CaptureSelectTM Technology A platform for purification of all biologics

Selectivity

− high purity in single step / feed stock independent

Mild elution conditions

− retaining biological activity of target

Reduction of process steps

− higher yields, reduced costs

Efficient clearance of HCP, DNA, virus

− high selectivity in capture step

Cell Culture Harvest

CaptureSelectTM Resin

Viral Inactivation

Polishing

Viral Filtration

UF/DF


Bioprocess Products Resins launched since 2006

Recombinant and Plasma Proteins

• Suitable for final manufacturing of therapeutic products

• All have Regulatory Support Files

• Leakage ELISAs for all launched bioprocess products

• Over 150 Customers for combined bioprocess products to date

Antibodies and antibody fragments

Vaccines and Viruses

• Human Fc • Human Kappa Light Chain Constant Domain • Human Lambda Light Chain Constant Doimain

• rhFVIII (B Domain Deleted) • Alpha-1 Antitrypsin • rhFVII • GCSF • rhFIX

• AAV Serotypes 1-8 Product in Approved Process (EMEA) : 2013

Product in various phase II/III

Product in various phase II/III


CaptureSelect™ Bioprocess Resin Product details

Product name Binding

capacity

Primary elution

conditions

Alternative elution

conditions

CaptureSelect™

CH1 Resin

≥ 29 g/L resin* Acidic pH: 3.5 Up to pH 4.5 with addition of

additives like salt, glycols

and/or arginine

CaptureSelect™

IgA Resin

≥ 12 g/L resin*

Acidic pH: 3.0

N/A

POROS®

CaptureSelect™

IgM Resin

≥ 11 g/L resin

Acidic pH: 3.0

Up to pH 5.0 with addition of

additives like salt, glycols

and/or arginine

* Binding capacity measured at 150 cm/hr, 17.5 cm bed height, 7 minutes contact time (Load for CH1 was polyclonal

human IgG; Load for IgA was polyclonal human serum derived IgA)


CaptureSelect™ Bioprocess Resin Product details

Product name Binding capacity* Elution conditions

CaptureSelect™ FSH

Resin

≥ 5 g/L resin Neutral pH:

20 mM Tris, 2.0 M MgCl2 pH 7.0-7.5

Acidic pH: glycine pH 3.0

CaptureSelect™ hGH

Resin

≥ 7 g/L resin 20 mM Citric acid pH 3.0

CaptureSelect™ HSA

Resin

≥ 18 g/L resin Neutral pH elution:

20 mM Tris, pH 7.4, with addition of

- 2.0 M MgCl2 or

- 1 M NaCl, 50%(v/v) propylene glycol or

- 1 M NaCl, 0.5 M L-Arginine pH 7.4

Acidic elution: 0.1 M Glycine pH 3.0

* Binding capacity measured at 150 cm/hr, 2 cm bed height, 0.8 minutes contact time


CaptureSelect™ Lab-Scale Products

Available as slurries from 5 mL to 1 L

High-throughput screening or prepacked column options through partners

Antibody Purification Antibody Toolbox® Products

Products for antibody and

antibody fragment purification

Sample Preparation Proteomics Toolbox® Products

Single-step protein depletion enabling biomarker discovery

and clinical diagnostics sample preparation

Protein Purification

Products for biopharmaceutical purification CaptureSelect™ C-tag purification platform

for C-tagged proteins

Ligand Conjugates

CaptureSelect™ ligands chemically conjugated to biotin for use in

analytical assays


Custom Project Needs

Biosimilars

Complex Proteins

ADC’s

Platforms Scavenging

Increase yield / purity

Reduce the number

of steps in a process

No generic purification

strategy

Enabling technology

Selective removal of unwanted

compounds Offer platform approach

for purification/detection


CaptureSelect™ Technology Platform -Product Development Cycle-

1) Discovery of VHH binding fragments - Immunize - Construct expression libraries

2) Develop Ligand binding and elution conditions - Test for binding and elution Characteristics - Determine stability

3) Ligand testing - Top candidates are cloned into Yeast - Evaluate process conditions in small scale columns

4) Ligand production - Pick lead candidate - Produce in-house at large scale

- Binding

- Specificity

- Ligand Stability

- Elution

5) Product Commercialization - Develop resin or ligand product for research or clinical manufacturing

http://www.gehealthcare.com/


Case Study: Recombinant Factor VIII

Custom Project

Immunize llama with recombinant Factor VIII

− Follow immune response

Construct ligand expression libraries

Llama Immune Response against Target Target reactive VHH Expression Libraries

Step1: Immunization & library construction



Library screening at monoclonal level on application conditions

Based on customer product/process needs

- Binding to Target (no binding to contaminants, HCPs)

- Broad / Narrow Specificity (e.g. species specific)

- Ligand Stability (acidic / caustic cleaning)

- Binding & Elution Conditions (mild)

Capture-ELISA Binding kinetics/thermomdynamics

Step 2: Ligand screening operating conditions



Cloning into yeast expression system

Small scale affinity chromatography under process conditions

Column: Tricorn 5/100, 2 cm bed height

Ligand density: 2.5 mg/ml

Flow rate: 150 cm/h

Step 3: Ligand Testing

http://www.gehealthcare.com/



Production of lead candidates at larger scale (10L fermentation) to produce sufficient material for testing

Evaluate ligand candidates

− Rank

− Pick lead candidate

Deliverable

− Research purification resin

> Resin sample for customer evaluation

− Analytical tools

> Measurement techniques

Step 4: Lead selection and scale up



Ligand upscaling

Yeast based production

− No animal derived components

− ISO9001

− Audited by end customers

Resin Development

− Potential for development of analytical techniques and leakage ELISAs

Product Commercialization

− Process and part numbers established as other products

− Documents, validation with regulatory support needed for filing

Step 5: Develop and scale affinity purification product


Purification of recombinant human BDD-FVIII

Effective impurity clearance:

− HCP clearance: ~ 3 logs

− DNA clearance: ~ 5 logs

− Robust viral clearance (model viruses):

enveloped (XMuLV): ~ 4 logs

non-enveloped (MMV): > 5 logs

Molecular Wt

188 kDa

98 kDa

62 kDa

49 kDa

38 kDa

28 kDa

17 kDa

6 kDa

3 kDa

FVIII

REF STDLIGAND

ELUATE

MAb

ELUATE

M Load M

Molecular Wt

188 kDa

98 kDa

62 kDa

49 kDa

38 kDa

28 kDa

17 kDa

6 kDa

3 kDa

Molecular Wt

188 kDa

98 kDa

62 kDa

49 kDa

38 kDa

28 kDa

17 kDa

6 kDa

3 kDa

FVIII

REF STDLIGAND

ELUATE

MAb

ELUATE

M Load M

FVIII

REF STDLIGAND

ELUATE

MAb

ELUATE

M Load M

Resin currently incorporated in rhFVIII

production process

High target selectivity provided by affinity resin specific for Factor VIII


Purification of adeno associated virus (AAV)

control load M elution FT

VP3 VP2

VP1

Affinity ligand technology also applicable for virus particles (AAV)

− Resin specific to certain AAV subtypes

− Reverts a multi-step process into a high yield 2-step purification run

Current development work on viruses focuses on Influenza (HA antigens)


Case Study: Purification of rhFSH

Development of VHH based affinity ligand for purification of rhFSH

− llama immunized with rhFSH

0.0

0.3

0.5

0.8

1.0

1.3

1.5

1.8

2.0

FSH intact alpha-chain beta-chain

Ligand 1

Ligand 2

Ligand 3

Ligand 4

anti intact FSH

anti beta FSH

anti hCG (alpha)

Screening focused on:

– selectivity for intact FSH only

beta chain

alpha chain

2x Elutie frac003:10_UV2_280nm 2x Elutie frac003:10_Cond% 2x Elutie frac003:10_pH 2x Elutie frac003:10_Fractions

-50

0

50

100

150

200

250

mAU

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 ml

2B3 2B5 2B7 2B9 2B11 2C1 2C3 2C5 2C7 2C9 2C11 2D1 2D3 2D5 2D7 2D9 2D11 2E1 2E3 2E5 2E7 Waste 2E9 2E11 2F1 2F3 2F5 2F72F9 Waste

FT E1 pH 2 ST FT E1 pH 2

FSH

VHH Matrix screening:

– Mild elution

• 2 M MgCl2, pH 7


Case Study: Purification of rhFSH

Follicle Stimulating Hormone

- 3 fold yield increase compared to conventional process

- Binds only intact and active form

- Reduction of process steps

Virus filtration

Chromatography III

Chromatography II

Virus inactivation

FSH-Binding Resin

Ultrafiltration

FSH

Affinity process


Case Study: Purification of rhGCSF

Affinity ligand showing true selectivity for rhGCSF

− Custom Project

− High purity after primary capture step

− Feedstock independent

Feed Stock: mammalian “non-mammalian” E.coli (GCSF-fusion)

M St FT EL M St FT EL M St FT EL

GCSF


Naturally-Occurring Affinity Ligands against Antibodies

Protein A and L are commonly used antibody binding proteins originally found in the cell wall of bacteria

Fab (Fragment antigen binding)

Fc region (Fragment crystallizable)

Hinge region

Glycosylation

Fv (Antigen binding site)

CH3

CH2

VL

Prot A wt: Binding to some VH domains of human VH3 class Z domain rProtA: no VH binding

Prot L Binding to human VL-ĸ1, 3 and 4 no binding to VL-ĸ2 or any VL-λ

Prot A CH2-CH3 interface of IgG no binding to human IgG3

CH1 CL

CH2

VH VL

Limited coverage of (new) Ab formats


Expanding Ligand Selectivities for Antibodies

VHH based affinity ligands directed against a broader range of antibody domains

CH3

CH2

VL

CH1 CL

CH2

VH VL

Anti IgG-CH1 - all human IgG subclasses - independent of light chain

- independent of Fv - no binding to free light

chains (FLC)

Anti human light chains (k / λ) - CL domain: similar in all human

isotypes and subclasses: - IgG - IgM - IgA - IgE

- human Fab fragments (all kappa and lambda formats)

Isotype specific - IgG - IgM - IgA

Anti VH - e.g. for scFv / domain Abs

(in development)


CaptureSelect™ Affinity Resins for Bioprocess

CaptureSelect™ CH1 Resin − The platform for human antibodies and fragments

− Eliminates the need to assess binding to rPA/G/L

USP’s: − Purifies all human IgG subclasses and Fab fragments thereof

> Independent of light chain and Fv

− No binding to free light chains or light chain dimers

29 g/l IgG

7 minute residence time

(17.5 cm column, 150 cm/h)

0

5

10

15

20

25

30

35

Cap

acit

y I

gG

g/l

CH1

CaptureSelect™ CH1 Resin

CH3

CH1

CH2

VH

CL

VL


CaptureSelect™ Affinity Resins for Bioprocess

CaptureSelect™ IgA Resin − Binds to all human IgA’s (mono- and

dimeric) and s-IgA (secretory IgA)

USP’s: − No cross binding to IgG’s

− High dynamic binding capacity 12 g/L

− Also suitable for IgA scavenging of plasma derived products

> IgA is the largest contaminant in IVIG preparation

IgA clone CaptureSelect™ IgA Resin

Multi step chromatography

Clone 1 92% 61%

Clone 2 91% 52%

The described method was superior to commonly

applied IgA purification schemes and may be used

as a generic capture step for the isolation of human

IgA irrespective of their heavy and light chain

subtypes. (Reinhart et al., 2012, JIM)


POROS® CaptureSelect™ Affinity Resins

POROS® CaptureSelect™ IgM Resin

− Binds to heavy chain of all human, rat and mouse IgM

− Binds to pentameric and hexameric forms

Features:

− High dynamic binding capacity 11 g/L

− Mild elution (~pH5) possible with additives to maintain IgM stability

− POROS® Resin backbone provides improved binding at lower residence times


CaptureSelectTM Affinity Products for Purification of

Stage Therapeutic proteins

(non-mAb) Antibody Types Vaccines & Viruses

Step 5 (Candidates)

Antithrombin III Fibrinogen Transferrin

Von Willebrand Factor ApoA

Step 4 (Lead Selection)

Prothrombin Exotoxin A (PE)

IgG-CH2

Rotavirus

Step 3 (Matrix Testing)

Insulin ApoH

VH3 Free LC-kappa

Step 2 (Library Screening)

C1-Inhibitor Interferon alpha/beta, hIL2

GM-CSF, hCG, Protein C FV, FX, FXI, FXII, FXIII, FH

Free LC-lambda

Step 1 (Immunization)

tPA

CaptureSelectTM Products Development Pipeline


For Research Use Only. Not intended for any human or animal therapeutic or diagnostic use, unless otherwise stated.

© 2013 Life Technologies Corporation. All rights reserved.

The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners.

Thank you for your attention!

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