A target affinity enrichment workflow to characterize critical post-translational modifications within therapeutic antibodies

The therapeutic efficacy of a monoclonal antibody (mAb) relies on tight and specific binding to its intended target. This interaction may be abrogated or influenced by antibody fragmentation and/or post-translational modifications (PTMs) on or near the paratope. PTMs with reduced target affinity are considered impactful to drug quality and should be well-characterized during pharmaceutical development. The task of identifying and characterizing these PTMs can be facilitated by employing a strategy which utilizes semi-preparative affinity chromatography using an immobilized ligand target. Here, we present a proof-of-concept application of this strategy for a therapeutic antibody targeting a Type I cytokine receptor. Briefly, a sub-molar equivalent of the therapeutic antibody was applied to a column containing the immobilized receptor target. Fractions containing antibody variants with differential affinity to target were collected and evaluated by a panel of extended characterization assays, including size, charge, target-binding affinity, and cell-based potency. This approach specifically targets variants based on ligand affinity and enabled the identification of novel and specific PTMs, including Fab glycosylation, which were shown to be impactful to drug quality and could be considered critical quality attributes (CQAs). Furthermore, characterization of affinity-enriched fractions using assays that are orthogonal and complimentary to those used for release could guide or support the development of such assays which are sufficiently sensitive to detect these PTMs during product release.