captureselect™ and poros captureselect™ affinity resins for … · 2017-07-21 · life...
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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
+1 (910) 256-5756
3 Life Technologies™ |
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
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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
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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
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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
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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
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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
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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
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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)
11 Life Technologies™ |
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
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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
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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
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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
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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
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Case Study: Recombinant Factor VIII
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
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Case Study: Recombinant Factor VIII
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
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Case Study: Recombinant Factor VIII
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
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Case Study: Recombinant Factor VIII
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
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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
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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)
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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
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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
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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
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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
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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)
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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
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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)
30 Life Technologies™ |
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
31 Life Technologies™ |
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
32 Life Technologies™ |
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.
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