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Pim Hermans, Frank Detmers, Bruce Dawson and Jessica de Rooij Thermo Fisher Scientific, J.H. Oortweg 21, the Netherlands, 2333 CH Accelerating antibody drug development with subdomain-specific affinity ligands INTRODUCTION With the development of novel bio-therapeutic antibody formats, such as trifunctional and bi-specific monoclonal antibodies or antibody fragments, new purification challenges in the downstream process of these molecules arise. Thermo Scientific™ CaptureSelect™ antibody subdomain-specific affinity products and analytical tools are developed for the discovery and manufacturing of therapeutic antibodies and antibody fragments. The affinity resins provide high target purity in a single step, independent of feedstock. CAPTURESELECT TECHNOLOGY CaptureSelect ligands offer a unique affinity purification solution based on Camelid- derived single domain [V H H] antibody fragments (Fig.1). These small, 14 kD affinity ligands, are the platform solution for many bio-pharmaceutical purification challenges. Fig.1 Regular antibody compared to a Camelid single domain antibody. The V H H antibody fragments offer high specificity, affinity and stability. A unique set of CaptureSelect affinity ligands has been developed (fig 2.), directed against a variety of antibody subdomains, providing tools for researchers and manufacturers to help facilitate purification of a broad range of antibody formats. ANTIBODY SUBDOMAIN TARGETS Product coverage • Isotypes: IgG, IgM, IgA • Subclass specific: IgG1, 3 and 4 • Species human, murine, multi-species • Fragments Fab (C H 1 or LC), Fc-fusions (C H 3) CaptureSelect CH1-XL affinity matrix Binding to the constant domain of the heavy chain, CaptureSelect CH1-XL provides a platform solution for the purification of Fab fragments, irrespective of the type of light chain. CaptureSelect CH1-XL Affinity Matrix is the improved version of the IgG-CH1 Affinity Matrix. Fig.2 Available subdomain-specific affinity ligands CONCLUSIONS CaptureSelect antibody subdomain-specific affinity resins address the purification challenges in therapeutic antibody development by providing unique selectivity, high purity and yields in a one-step purification process. ANALYTICAL TOOLS CaptureSelect ligand conjugates (fig 6) CaptureSelect biotinylated ligands can be used to develop a range of analytical assays, including ELISA, Western Blot and assays for label-free detection platforms such as Surface Plasmon Resonance (SPR). IgG IgG Fab IgG-Fc Free LC kappa Free LC lambda Buffer Fig. 5. Chromatogram showing elution of a protein recovered from CHO-conditioned supernatant spiked to a final concentration of 1 mg/mL. Column: POROS CaptureSelect IgG-Fc. Fig. 6. SPR binding curves showing binding with intact IgG or Fab fragment and no cross binding with IgG-Fc or free light chains. Conjugate: CaptureSelect Biotin anti-IgG-CH1. POROS™ CaptureSelect HPLC columns (fig 5) POROS CaptureSelect affinity columns combine speed, selectivity, method automation and high precision when monitoring antibody titers and yield during manufacturing. A true platform for Fab fragment purification • High selectivity for human IgG-Fc (C H 3 domain) of all subclasses • High dynamic binding capacity • Efficient elution at milder (pH 5 -6) with additives PURIFICATION CaptureSelect FcXL affinity matrix The CaptureSelect FcXL affinity matrix combines high specificity and mild elution for the purification of monoclonal antibodies and Fc-fusion proteins, which makes it ideal for the purification of low pH sensitive Mabs and Fc-fusion constructs. The alternative for more sensitive IgGs and Fc-fusion proteins Fig.3 Purification of human IgG1 monoclonal; efficient clearance of product-related impurities using FcXL Product-related impurities observed in the elution fraction of Prot A purification (due to possible cross- binding to VH and Fab) are mainly present in the flow- through using FcXL IgG1 monoclonal Benefits CaptureSelect technology • The three complementarity determining regions (CDRs) of the V H H ligand provide unique tunable specificity • Unique screening technology for target specificity, mild elution and stability to help ensure process robustness • Animal origin free production process (Saccharomyces cerevisiae) • Technology used in commercial processes • Binds to the constant heavy domain (CH1) of all human IgG subclasses • No co-purification of over-expressed free light chains due to heavy chain binding • Equal specificity as CaptureSelect IgG-CH1 • Wide elution range: pH 3 – 4,5 • High Dynamic Binding Capacity • Improved stability at high pH Fig. 4 Lab chip analysis of Fab fragment purification shows no binding of light chains (LC) or light chain dimers (LC2) in the elution fraction (B) A. Analysis of feed stock. B. Analysis of elution fraction CAPTURESELECT ANTIBODY AFFINITY PRODUCTS HPLC POROS IgG-C H 1 Hu + + - + + CH1-XL (C H 1) Hu + - Contact us + + FcXL (C H 3) Hu + primate + + + IgG-Fc Multi-Species + (human) - + - KappaXL (C L ) Hu + primate - - + + LC-kappa (C L ) Hu + primate + + - - LC-kappa / lambda Murine + - + - LC-lambda Hu + primate + + + - LC-lambda Mouse / Rat + - + - IgM Hu, Mo, Rt + + + - IgA (Fc or C H 1) Hu + + (Fc) + + - IgA Bovine + - + - IgG1 Hu - - + - IgG3 Hu + - + - IgG4 Hu + - + - Fab-kappa kinetics Hu + primate + - - - Fab-lambda kinetics Hu + primate + - - - IgG-Fc PK (C H 2) Hu only + - - - Free LC- kappa (C L ) Hu + - - - CaptureSelect Ligand Species Biotin cGMP resin Research resin Selection of CaptureSelect antibody affinity products. For more information visit: www.thermofisher.com/ Captureselect A B * * * * Intravenous immunoglobulins Caution: For Research Use or further manufacturing, not for diagnostic use or direct administration in humans or animals. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
