Purification of lambda Fab – Characterization of a new affinity chromatography resin Linus Laurin, Maria Ersoy and Pia Thunman GE Healthcare Bio-Sciences AB, Uppsala, Sweden First presented at the Next Generation Protein Therapeutics Summit, in San Francisco, CA June 25-27, 2012
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Purification of lambda Fab Characterization of a new ...€¦ · 2012-07-09 · Characterization of a new affinity chromatography resin Summary •LambdaFabSelect is an affinity
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Purification of lambda Fab – Characterization of a new
affinity chromatography resin
Linus Laurin, Maria Ersoy and Pia Thunman GE Healthcare Bio-Sciences AB, Uppsala, Sweden
First presented at the Next Generation Protein
Therapeutics Summit, in San Francisco, CA June 25-27,
2012
2 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Introduction
• Antibody fragments (Fab, scFv, Dab etc.) are emerging as the next important class of protein-based biotherapeutics after monoclonal antibodies (MAbs).
• Antibody fragments are smaller and more accessible compared to the parent antibody, and they usually retain the target specificity, even though the Fc region is lacking. They also exhibit lower immunogenicity compared to the whole antibody.
3 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Introduction
• The Fc region of the antibody is the main binding site for protein A chromatography media, which has been the platform technology for the capture step in MAb purification for many years. For antibody fragments lacking the Fc region, no such generic solution is available, and since the human immunoglobulin light chain can be of either lambda or kappa type, the situation is more complex in terms of generic purification protocols.
• Here we present the characterization of LambdaFabSelect, a new chromatography medium for affinity purification of antibody fragments containing the lambda light chain, which together with Capto™ L, KappaSelect and MabSelect™ builds up a toolbox for antibody fragment purification.
4 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Different types of fragments and specificity of different ligands
• In humans approximately 60% of antibodies consist of the kappa light chain and approximately 40% of the lambda light chain.
• Approximately 10% of the antibodies have the VH3 heavy chain for which Protein A has an affinity.
• Fab binding ligands developed by BAC BV (recombinant protein, Mr 13000 produced in S. cerevisiae) have affinity for the constant region of the lambda light chain and kappa light chain.
• Protein L has affinity for the variable region of the kappa light chain.
5 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Different types of fragments and specificity of different ligands
6 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Application – capture of lambda Fab from E. Coli lysate
• 99 % recovery in elution fraction determined by off-line spectrophotometric measurement at 280 nm.
• Samples reduced prior to SDS-PAGE, if not stated otherwise.
• Proteins visualized by Deep Purple staining.
7 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Application – capture of lambda Fab from E. Coli lysate
8 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Ligand leakage
• Ligand leakage has been determined using the LambdaFabSelect leakage ELISA (BAC BV).
• 96 well plate with 50 μl resin per well.
• Equilibrated with 3 x 200 μl PBS, pH 7.4.
• Loaded with 10 - 17 mg Fab/ml resin in PBS, pH 7.4, incubation 1.5 hours.
• Elution with 5 x 120 – 200 μl 100 mM acetate buffer, pH 2.8. Samples immediately neutralized in 1 M Tris pH 8 A blank run without Fab was run first. The amount of ligand in elution fractions was found to be below 2.5 ppm.
9 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Compatibility to common cleaning/sanitization solutions
• Compatibility towards various commonly used cleaning and sanitization solutions were determined by 96 well plate studies.
• The capacity of the media was determined after incubation at different times in respective solution and they were compared to reference media stored in 20 % ethanol.
10 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Cleaning in place (CIP) study • Various cleaning protocols were screened in a 96 well plate
study (20 μl resin/well).
• Feed loaded five times according to the protocol below:
• Equilibration: PBS, pH 7.4, 3x 200 μL
• Sample load: Homogenated and clarified E. coli lysate spiked with human Fab lambda, 1x 200 μL (load 15 mg Fab/mL gel media), incubation 30 min
• After fouling, 15 min. incubation with 300 μL CIP solution. PBS and MQ wash performed between steps. The amount of protein contaminants was analyzed with capillary electrophoresis, after boiling the resin at reducing conditions.
11 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Cleaning in place (CIP) study
12 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
LambdaFabSelect characteristics
13 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Suggested approach for capture of antibody fragments
Antibody fragment Chain subtype Recommended product Alternative product
Kappa light
chain
Lambda light
chain
Fab
CaptoTM L
Lambda
FabSelect
KappaSelect
scFv
Dab
Kappa light
chain
Capto L
Kappa light
chain
Capto L
VH3 heavy
chain
MabSelectTM
14 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Summary
• LambdaFabSelect is an affinity BioProcess™ chromatography medium that has high binding capacity and good selectivity for antibody fragments containing the lambda light chain.
• High purity and recovery was obtained using a single chromatography step.
• The ligand allow cleaning with sodium hydroxide.
• The product contains no mammalian derived components.
15 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Summary
• Rigid agarose base matrix allow high flow rates with low back pressures.
• These features suggest LambdaFabSelect for the capture step of a high productivity, industrial scale platform approach to lambda light chain containing antibody fragment purification. Complementing LambdaFabSelect is a capture toolbox including Capto™ L, KappaSelect and MabSelect™, which together covers the majority of antibody fragments.
16 Purification of lambda Fab –
Characterization of a new affinity chromatography resin
Acknowledgments
GE, imagination at work, and GE monogram are trademarks of General Electric Company.
ÄKTAexplorer, BioProcess, Capto, MabSelect, and Tricorn are trademarks of GE Healthcare companies.
All third party trademarks are property of their respective owners.