Antibody fragments possess advantageous properties over monoclonal antibodies (mAbs) for certain therapeutic areas due to their smaller size and thus better penetration into tissues. For mAb products, Protein A resins are often used for capture due to their high selectivity for the fragment crystallizable (Fc) region. This selectivity results in a highly purified product with excellent recovery in a single step, significantly minimizing the need for additional steps to achieve the same product quality. Since antibody fragments lack this Fc region, purification for this diverse group of molecules can be more time-consuming and resource intensive.<br/><br/>In an effort to expedite process development and obtain a highly purified product, multiple affinity capture resins for an antigen binding fragment product were evaluated. These resins possessed selectivity towards either the light chain (LC), heavy chain constant region (CH) or variable region (VH) of the Fab molecule. Although each resin demonstrated good binding capacity, there were challenges associated with each. LC binding resin co-purified impurities of free light chain and light chain mispair products, leading to increased burden on the polishing steps. CH-binding resin showed high selectivity towards the intact Fab, but due to its ligand instability under typical alkaline cleaning conditions a multi-faceted approach had to be adopted to improve the resin lifetime. Finally, Protein A resin was evaluated since there are some Protein A resins that possess a VH binding region in addition to Fc binding, thus making them selective for Fabs. The work presented herein provides a detailed comparison of multiple resins screened for their associated challenges and unique benefits to enable efficient process development and ultimately, manufacturing of fab products.

Development of an antigen binding fragment (Fab) affinity capture step: Challenges associated with the various binding domains

ACS Spring 2022

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