Advantages of MabSelect VH3, a novel VH3-specific Protein A resin, over its two conventional counterparts in purifying a VHH-based trispecific antibody

Background: Protein A affinity chromatography represents the most extensively used technique for initial product capture in antibody purification. The ligands of commercial Protein A resins from different, or even the same, vendors may exhibit distinct binding specificity. For example, MabSelect SuRe LX and MabSelect PrismA bind the antibody's fragment crystallizable region and both the fragment crystallizable and variable heavy chain 3 (VH3) regions, respectively, while MabSelect VH3, a newly launched Protein A resin, binds the VH3 region exclusively. Objective: This study aimed to compare the capabilities of three Protein A resins-namely, MabSelect SuRe LX, MabSelect PrismA, and MabSelect VH3-in removing byproducts associated with a variable domain of heavy chain-only antibody-based trispecific antibody (TsAb). Methods: Clarified culture harvest containing the TsAb was processed separately using MabSelect SuRe LX, MabSelect PrismA, and MabSelect VH3 columns. For each column, byproduct separation was monitored by analyzing relevant elution fractions with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography-high-performance liquid chromatography. Results: MabSelect VH3 demonstrated superior byproduct removal performance compared with the other two Protein A resins. Conclusion: MabSelect VH3 is particularly suitable for the purification of bispecific antibodies and TsAbs, where the product and byproducts contain different numbers of VH3 domains. For multispecific antibody purification, screening different affinity resins with distinct binding specificity is recommended, as this can help identify the most effective option for separation.