Advanced Affinity Purification Solutions for Next-Generation Antibody Therapeutics

Learn more at thermofisher.com/antibodypurification Pim Hermans, Frank Detmers, Kevin Sleijpen, Simon Adema, Hendrik Adams, Anja Overweel, Paul Janszen & Laurens Sierkstra Thermo Fisher Scientific, Leiden, the Netherlands Innovative capture purification solutions for therapeutic antibody manufacturing Introduction For decades, affinity purification platforms such as Protein A and Protein L have been crucial in the manufacturing processes of therapeutic monoclonal antibodies. However, as engineered modalities such as bispecific antibodies, fragments, and Fc-fusion proteins emerge, new challenges arise in the downstream processing of these complex molecules. To address these challenges, affinity chromatography resins designed to target specific antibody subdomains offer a promising alternative for purifying these novel formats. This advancement is crucial in enhancing the commercial manufacturing of next-generation antibody therapeutics. In summary The Thermo Scientific antibody purification toolbox enables process developers to address the challenges that arise during the production of new therapeutic antibody modalities. This portfolio of resins helps to: + Obtain high purity and yields in a single capture step + Reduce process steps + Easily upscale for larger manufacturing batches Trademarks/licensing © 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. CDR1 Camelid Ig VHH CDR2 CDR3 CaptureSelect Technology  Technology based on single-domain antibody fragments [VHH]  High target purity in a single step, independent of feedstock  Unique screening technology to determine final resin properties:  target specificity  mild pH elution  ligand stability  Scalable & animal origin free technology  Suitable for cGMP manufacturing processes A unique antibody affinity purification portfolio Supporting manufacturers in the purification of novel antibody formats MabCaptureC Affinity Matrix and CaptureSelect FcXP Affinity Matrix. The complete platform for IgG-based molecules and Fc-fusion proteins The MabCaptureC affinity resin is a high-performance Protein A resin specifically designed to improve the purification efficiency of mAbs with intact Protein A binding sites. For antibody molecules that lack or have an altered Protein A binding site, or for Fc-fusion proteins, the CaptureSelect FcXP CH3 subdomain-specific resin is an ideal solution. 41.2 47.1 54.4 70.6 72.1 38.3 44.1 55.9 63.3 67.7 30.0 40.0 50.0 60.0 70.0 80.0 2.5 3.3 4.8 7.5 10.0 Dynamic Binding Capacity (mg/mL) Residence time (min) DBC 10% BT comparison using monoclonal IgG (Herceptin, IgG1) MabCapture C Resin B 35 36 37 38 39 40 41 42 43 40 50 60 70 80 90 100 110 0 20 40 60 80 100 Relative recovery (%) Cycle # 19.0 29.3 44.5 59.4 11.9 23.2 36.9 50.8 0 10 20 30 40 50 60 70 Dynamic Binding Capacity (mg/m) 0 1 2 3 4 5 6 7 8 9 Residence time (minutes) DBC 10% BT using Monoclonal and polyclonal IgG Monoclonal IgG Polyclonal IgG Start FT EL ST 150 kDa 100 kDa 50 kDa 25 kDa Thermo Scientific CaptureSelect CH1-XL Affinity Matrix A scalable platform solution designed to purify Fab-fragments Fig. 4. Ranibizumab feed from HEK293 cells (Fab fragment). Purification shows high yield and purity in a single step • Binds to the CH1 domain • No co-purification of free light chains (only correctly assembled Fabs) • Efficient elution at milder pH (4 – 4,5) Left: SDS-PAGE silver staining of the load (L), flow-through (FT) and elution (E) fractions, showing no presence of light chains in the elution pool. Right: Gel filtration analysis showing 98% purity of the Fab fragment in the elution fraction with a yield of 86% CaptureSelect KappaXP Affinity Matrix & CaptureSelect LambdaXP Affinity Matrix Solving purification challenges for bi-specific formats • Binds to constant domain of Kappa or Lambda light chain • High dynamic binding capacity (tested with Bi-specific mAbs) • Efficient elution at milder pH Fig.1. Affinity capture resin selectivity. Infographic showing the binding regions of the Thermo Scientific MabCaptureC affinity matrix (Protein A) and all Thermo Scientific CaptureSelect antibody affinity resins. Fig.2A MabCaptureC Resin Dynamic Binding Capacity comparison at increasing residence times. The resin was compared to commercially available, alternative Protein A resin (Resin B). Fig.2B MabCaptureC Resin Reusability study. The resin shows excellent alkaline stability. No decline is observed after cleaning with 0.2M NaOH over 100 cycles. CHO expressed Rituximab. Fig.3A CaptureSelect FcXP Resin Dynamic Binding Capacity. The resin shows a high DBC; > 40 g/L (10% BT/ 5 min residence time) - Rituximab. Fig 3B. One-step purification from crude material with high purity. Overexpressed light chain dimers are present in the flow through (FT) but not in the elution fraction (E). ST = strip pH 2 Thermo Scientific resin Dynamic Binding Capacity (human IgG) Elution properties CaptureSelect KappaXP resin 40 g/L at 2 min residence time Efficient elution at milder conditions (pH 5-6) with additives CaptureSelect LambdaXP resin > 35 g/L at 4 min residence time Efficient elution at pH 3.5-4 – small elution pool volume Fig.5. Elution performance of CaptureSelect LambdaXP resin. Efficient elution (3 CV) of a bi-specific antibody at pH 3.6 using a load concentration of 32 mg/mL  Start - Rituximab, monoclonal human/mouse chimeric IgG1 (1.7 g/l)  FT – flowthrough fraction  EL – 10 x diluted pH 4.0 elution fraction,  ST – pH 2.0 strip For Research Use or Further Manufacturing. Not for diagnostic use or direct administration into humans or animals.