Affinity Membranes Enable Rapid Quantitation of Therapeutic Antibodies and Analysis of Glycosylation

This research develops peptide-functionalized porous membranes to capture therapeutic monoclonal antibodies (mAbs) for rapid analysis of their concentrations and N-glycosylation patterns. Established approaches for determining these mAb critical quality attributes are typically complex and time consuming. Techniques that rapidly analyze mAb concentrations and glycan profiles will enable monitoring of these attributes during manufacturing and development. To quantify mAb concentrations in minutes, this work employs membrane-containing 96-well plates that capture most classes of mAbs from fermentation broths. Flow-induced mass transport in membrane pores enables immobilized peptide ligands to rapidly bind to the fragment crystallizable (Fc) region of mAbs. Subsequent passage of a fluorophore-labelled secondary antibody generates a fluorescence signal, which is proportional to the target mAb concentration. Because the Fc regions of immunoglobulin G (IgG) antibodies are very similar, this method is effective for various IgG subclasses. The assay gives the accuracy, precision, and limit of detection (LOD) needed for analysis of mAbs in expression systems and requires < 5 min. Thus, membrane-based analyses could aid in quality control and early intervention during mAb production. Analysis of mAb glycans relies on a workflow that includes enriching mAbs from cell culture supernatant, eluting and digesting the captured mAbs, and analyzing their glycan patterns using liquid chromatography – tandem mass spectrometry (LC-MS/MS). The process uses in-membrane pepsin digestion that is fast (< 5 min) and does not require alkylation and desalting, greatly simplifying the workflow. This strategy requires < 2 h and could help to monitor the continuity of mAb glycosylation during fermentation. Demonstrations of the technique include examination of glycosylation in different batches and subclasses of mAbs, in biosimilars, and in mAbs from different expression systems. Overall, membrane-based devices show great promise for rapid analyses of therapeutic mAbs.