Rapid Membrane-Based Digestion and Purification For LC-MS Protein Analysis

Glycosylation plays a critical role in the bioactivity of proteins. For example, glycosylation of the SARS-CoV-2 Spike receptor-binding domain affects viral entry into host cells. Similarly, the glycosylation of erythropoietin and monoclonal antibodies significantly influences their pharmacokinetic and pharmacodynamic properties. The bottom-up approach, which enables the attachment of glycans to peptides, is one of the most straightforward methods for studying protein glycosylation by liquid chromatography-mass spectrometry (LC-MS). In addition to identifying non-glycosylated peptides, it facilitates the analysis of site-specific glycosylation. However, this approach typically requires several hours or even overnight digestion. The digestion process becomes even more prolonged when multiple proteases are needed, which significantly hampers the efficiency of protein glycosylation analysis. The research presented in this dissertation utilized protease-immobilized membranes to enable rapid protein digestion. This method achieves comparable glycan identification and semi-quantitative assessments to conventional overnight in-solution digestion. Further studies explored the use of tandem membranes, consisting of trypsin-immobilized membranes and C18-derivatized membranes, to streamline protein digestion and peptide purification. During centrifugation, proteins are initially digested in the first layer (trypsin-immobilized membranes), and the resulting peptides are subsequently enriched in the second layer (C18-derivatized membranes). Most salts and contaminants pass through the membranes and are effectively removed from the protein digests. This technique has been successfully applied to the sequence and glycosylation analysis of commercial monoclonal antibodies (Kanjinti, Bevacizumab, and Rituximab), as well as to the analysis of host cell proteins and in-house expressed antibodies. This prototype could prove valuable for real-time monitoring of product glycosylation, ensuring the consistency and efficacy of therapeutic agents.