Improved purification of protein-based

biotherapeutics using CaptureSelect

affinity ligands

Hendrik Adams, Ph.D.

BAC BV Leiden, the Netherlands
 Outline presentation

Company overview
Business areas
CaptureSelect technology – molecular basis
Ligand Discovery program
Applications in Life sciences & Bioprocess
– CaptureSelect products
– New plasma protein ligands

Confidential © BAC BV 2009

 BAC BV – Overview

BAC’s Core Business

Affinity products for purification of biopharmaceuticals: CaptureSelect®

1995 – BAC BV started as subsidiary of Unilever

1999 – PoP for use of single domain antibody technology in separation
2002 – BAC’s spin-off facilitated by Unilever Ventures
2005 – Private financing round completed
2008 – Secured additional investment for R&D financing

Based in Naarden, NL
– 37 FTE
– Dedicated R&D (Leiden, NL)
– Microbial Biotech manufacturing site (Naarden, NL)
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Confidential © BAC BV 2009

 Locations

Leiden Naarden

Confidential © BAC BV 2009

 Outline presentation

Company overview
Business areas
CaptureSelect technology – molecular basis
Ligand Discovery program
Applications in Life sciences & Bioprocess
– CaptureSelect products
– New plasma protein ligands

Confidential © BAC BV 2009

 BAC’s Business Areas

Custom ligand design

– A unique platform for the development of ligands for biotherapeutic purification

CaptureSelect for life science research

– Providing new specificities and formats for bench-scale purification, proteomics
and analysis

CaptureSelect for Bioprocess

– Addressing the emerging purification needs of biopharmaceuticals, such as
viruses, antibodies and (blood) proteins, with off-the-shelf affinity products

Confidential © BAC BV 2009

 Affinity chromatography is an excellent
protein purification technology
Selectivity
- high purity in single step / feed stock independent

Mild elution conditions

+
- retaining biological activity of target Immobilized Ligand Impurities Purification
target

Reduction of process steps

- higher yields, reduced costs
Complex Impurities

Efficient clearance of HCP, DNA, virus

- high selectivity in capture step
Purified sample

Confidential © BAC BV 2009

 Outline presentation

Company overview
Business areas
CaptureSelect technology – molecular basis
Ligand Discovery program
Applications in Life sciences & Bioprocess
– CaptureSelect products
– New plasma protein ligands

Confidential © BAC BV 2009

 Discovery of Camelid antibodies

2 spin-off companies
– BAC BV CaptureSelect® Affinity Ligands use
– Ablynx the Uniqueness of VHH antibody
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fragments

Confidential © BAC BV 2009

 Analysis of Camelid IgG

150 kDa

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© BAC BV 2009
 Features of VHHs

Advantages:
‰ Specificity - Broad / Narrow
‰ Recognition of enzyme active sites
‰ Stability - Cleaning agents (NaOH)
‰ Screening - Operating conditions (e.g.
elution)
‰ High Affinity - nmol Kd
‰ High level production in yeast

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Confidential © BAC BV 2009

 Outline presentation

Company overview
Business areas
CaptureSelect technology – molecular basis
Ligand Discovery program
Applications in Life sciences & Bioprocess
– CaptureSelect products
– New plasma protein ligands

12

Confidential © BAC BV 2009

 Affinity Ligand Discovery

From Target to Lead Ligand in less than 8 months (phase 1)

¾ Immunization with target

~ 3 months
Milestone 1
¾ Construction ligand expression
libraries

¾ Library screening
Milestone 2
9 Binding, Specificity, Elution, Stability

~ 5 months
¾ Small scale Affinity Chromatography
Milestone 3

Non-Bound Elution
mAU UV1 214nm

UV2 280nm

¾ Lead ligand
800
pH

600

Optical Density (214 nm)

400

200

13
0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 ml

(ml)

Confidential © BAC BV 2009

 Affinity Ligand Discovery

From Target to Lead Ligand (phase 1)

I TARGET LIBRARY Immunization with target ~ 3 months

Construction ligand expression libraries

● Lama Immune Response against Target ● Target reactive VHH Expression Libraries

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BAC BV © 2009
 Affinity Ligand Discovery

From Target to Lead Ligand (phase 1)

II LIBRARY SCREENING Library screening at monoclonal level

on application conditions

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

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

- Binding & Elution Conditions (mild)

- Ligand Stability (acidic / caustic cleaning)

15 Capture-ELISA Biacore / Octet (binding kinetics)

BAC BV © 2009
 Affinity Ligand Discovery

From Target to Lead Ligand (phase 1)

III LEAD IDENTIFICATION Cloning into yeast expression system

Small scale affinity chromatography under
process conditions

Non-Bound Elution
mAU UV1 214nm 1 start
UV2 280nm 2 Marker
800
pH 3 FT
4 Wash
600
5 Eluate
Optical Density (214 nm)

400

200

0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 ml
Column: Tricorn 5/100, 2 cm bed height
Ligand density: 2.5 mg/ml (ml)

Flow rate: 150 cm/h

16 Lead ligand
BAC BV © 2009
 Product Development and Supply

Phase-1 Phase-2 Solid supports Strategic

Library construction Product Partnerships
Library Screening Development
Lead Identification

Ligand Strain &

Selection & Ligand Affinity
Process Production
Screening Performance Products
Development

> 20 Patents and licenses

Non-Bound Elution
mAU UV1 214nm

UV2 280nm

800
pH

600
Optical Density (214 nm)

400

200

0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 ml

17 (ml)

Confidential © BAC BV 2009

 Ligand Manufacturing

Production Biomass Removal & Concentration Purification

Fermentation Microfiltration & Ultrafiltration Chromatography

Microbial production of the ligands using Baker’s yeast

ISO9001 but with increased quality level
Audited by GE and end-customers
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Confidential © BAC BV 2009

 Outline presentation

Company overview
Business areas
CaptureSelect technology – molecular basis
Ligand Discovery program
Applications in Life sciences & Bioprocess
– CaptureSelect products
– New plasma protein ligands

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Confidential © BAC BV 2009

 Products: Life Science Research

Life Sciences
Research

BAC products incorporated in all major supplier

Proteomics kits for proteomics for sample treatment of
Pre-purification
plasma in drug discovery

Webshop for immobilized finished products

Separation
• Antibody ToolboxTM media
Antibody Toolbox
• Leakage ELISA

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Confidential © BAC BV 2009

 Antibody ToolboxTM Media

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BAC BV © 2009
 Antibody Toolbox: IgM and IgA Affinity Ligands

CaptureSelect IgM affinity matrix CaptureSelect IgA affinity matrix

1 Human serum 2 Flow-through 1 Human serum 2 Flow-through

3 Elution M Marker 3 Elution M Marker

1 2 3 M M 1 2 3

IgM
IgA

Binds human IgM, μ chain Binds human IgA, α chain

Binds rat and mouse IgM No binding to:
No binding to: - human IgG, IgM
- human IgG, IgA - IgA from other species
- bovine IgM

Ligands and resin characteristics:

• High IgM / IgA purity in a one-step process
22 • Mild elution conditions between pH 3 and 4

BAC BV © 2009
 Products: Bioprocess

Biopharma
Manufacturing
• IgSelect
Multi-Customer • AVB Sepharose HP
Ligands End-users
• IgG • VIIISelect
• Fab’s • Fab kappa
• AAV
• recFactor VIII

Custom ligands
• Recombinant proteins
• Vaccines
• Plasma proteins

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Confidential © BAC BV 2009

 Purification of human IgG1-4

Purification of human IgG from different feed streams

● Cell culture (rec Mabs)
● Transgenic

IgSelect Properties 1 2 3 4 5 6
1 Cow milk
- Human IgG-1 to 4 2 Cow milk spiked with Human IgG

- Human specific 3 Tissue culture medium

- Mild elution 4 Tissue culture medium spiked with

Human IgG
- Acid / Caustic stable
5 Elution fraction from Cow milk

- 20 – 25 mg/ml DBC 6 Elution fraction from Tissue culture

medium
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Confidential © BAC BV 2009

 Viruses: Purification of AAV

l
ke tro

h
ug
n
M :co

Custom ligand project

ro

n
r

n
io
1

io
th
AV

ad

ut

ut
ar

el

el
lo

flo
rA

BAC successfully identified ligands

for purification of AAV

BAC was able to change a

VP1 complex multi-step procedure into a
VP2
VP3
high yield, high purity, 2-step
purification process

Now widely distributed by GE

under the name “AVB Sepharose High
Performance”

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Confidential © BAC BV 2009

 Proteins: Purification of recFVIII

Molecular Wt
Custom ligand project with
Bayer Healthcare
188 kDa

BAC successfully identified

98 kDa
recFVIII ligands
62 kDa

49 kDa
FVIII
REF STD
Now widely distributed via
38 kDa
LIGAND GEHC under the name
ELUATE
28 kDa “VIIISelect”
MAb
17 kDa ELUATE
Currently in use for
6 kDa production of CTM
3 kDa

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Confidential © BAC BV 2009

 One-step purification of human Fab fragments

M1 2 3 4 5
M Marker
1 Tissue culture medium
2 (1) + Fab-κ
3 Flow-through
4 Elution
5 Fab control

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Confidential © BAC BV 2009

 Next Generation Antibody Purification
NEXT GENERATION ANTIBODY PURIFICATION
A diverse set of libraries available against different antibody domains

Broad Ig
VL Fab
scFv
Broad Ig (kappa / lambda specific)
Fab
scFv VH
CL
Broad Ig
Fab
CH-1 (kappa / lambda
specific )
Broad IgG
Subclass specific (e.g. IgG1)
Isotype specific(e.g. IgM, IgA)
Fab Broad IgG
Subclass specific (e.g. IgG1)
Isotype specific(e.g. IgM, IgA)
Fc-fusions
Currently in development
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Confidential © BAC BV 2009

 Multi target Immune Libraries: human plasma

Providing VHH expression libraries covering a broad range of

plasma proteins

Protein distribution
in human plasma 1.0% 0.6% 0.4%
1.1%
0.3%
1.2%
2.0%
1.0% Other
2.0%
2.9%
ALB
Removal of most abundant
IgM

3.3% IgG
AAT plasma proteins by
3.3% A2M
IgA CaptureSelect affinity ligands:
3.5% TF
IgA Albumin FIB

3.6%
HPT ● anti human albumin
54.0%
IgM
ApoAI/II
● anti human kappa LC
3.8%
A1AG ● anti human lambda LC
IgG C3
HPX
16.0% AACT
C4
TTR
Other

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Confidential © BAC BV 2009

 Human plasma depleted from Immunoglobulins
and Albumin

Human Plasma (1)

1.0% 0.6% 0.4%

Capture-Select
- kappa 1.1%
- lambda Ig HSA 1.2%
0.3%

2.0%
1.0% Other
FT (2) 2.0%
FT (5) 2.9%

3.3%

Elution (3) Elution (6) 3.3%

3.5%
Albumin
1 2 3 5 6 3.6%

54.0%
3.8%

16.0%

Plasma depleted from albumin, IgG, IgM, IgA

(±75% of total protein content in plasma)

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Confidential © BAC BV 2009

 Functional Ligands identified against diverse set
of plasma proteins

Plasma Protein level in

plasma
(%)

a1-Antitrypsin (AAT) (3.8)

a2-Macroglobulin (3.6)

Transferrin (TF) (3.3)

Fibrinogen (FIB) (3.3)

Haptoglobulin (HPT) (2.9)

Complement C3 (2)
ApoA1 (1)
a1-Acid glycoprotein (1)
Factor H (0.5)
Complement C4 (0.5)
C1 Inhibitor (0.3)
Complement C1q (0.25)

..

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Confidential © BAC BV 2009

 Screening overview

Stage 1 ELISA
Stage 3 Small scale
Affinity Chromatography

Stage 2 Immunoprecipitation

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 Alpha 1 acid glyco protein

Stage 2 Immunoprecipitation

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 Transferrin

Stage 3 Small scale

Affinity Chromatography

Figure 1. Purification of Transferrin from serum

Column: Tricorn 5/100, 2 cm bed height

Ligand density: 2.5 mg/ml
Flow rate: 150 cm/h
Loading material: human serum 20x diluted with 10 ml equilibration buffer Figure 2. Analysis of fractions. (A) Coomassie stained SDS-PAGE gel
Equilibration: 20 mM Tris-HCl, 150 mM NaCl, pH 7, 10 column volumes
and (B) Western blotting/immunodetection with anti-TF antibodies of the
Wash: Same as equilibration buffer
Elution: phosphate-buffered saline, pH 2 fractions collected after purification. Lanes: 1, starting material; 2,
molecular weight marker (kDa); 3, flowthrough; 4, peak 1

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Confidential © BAC BV 2009

 Apo lipoprotein A1 purification from Cohn IV

Stage 3 Small scale

Affinity Chromatography
ApoA1 purification

1 2 3 4 5
MW (kD)

180
115
82
64
49

37

26

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Confidential © BAC BV 2009

 Human serum albumin

New population of anti-HSA sequences

36 Stage 2 Immunoprecipitation
Confidential © BAC BV 2009
 A platform for downstream processing of
biopharmaceuticals

CaptureSelect: Protein A equivalent for non antibody targets

Feed
Stock

CaptureSelect
chromatography

Platform downstream process for Mabs Platform downstream process for

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biopharmaceutical
Confidential © BAC BV 2009
 BAC’s Pipeline

Lead Lead Customer Clinical Commercial

Target Library
Identification Optimization Evaluation Design-in Manufacturing

IgG (Fc), AAV, FVIII

AAT, IgA, IgM, IgG4, Fab kappa,

Fab lambda, ApoA1, Fibrinogen,
Transferrin, HSA, Multi-IgG,
Multi-Albumin

Toxins/LPS, Haptoglobin, α1-acid GP,

α-2 Macroglobulin, IgG1

Blood factors, Prothrombin

anti-Thrombine III
Complement C3/C4/C1q, C1
inhibitor, HSA-fusion, EPO

ApoA2, hVWF, IFNa-2b, IgG2/3, IgG

(CH1), scFv (VH/VL), AACT,
Ceruloplasmin

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Confidential © BAC BV 2009

 Conclusion

Use of Multi target libraries speed up the timings of the screening

phase strongly
Internal pipeline is filled with new (potential) lead ligands
High specificity / selectivity / stability
High affinity (low nM range)
High sequence diversity within pool of binders

Next steps: preparation of multi target libraries for the generation of

ligands against the minor plasma proteins (e.g. cytokines and
chemokines)

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Confidential © BAC BV 2009

 Visit us at:

http://www.bac.nl
http://www.captureselect.com