Non-enzymatic chemical modifications such as deamidation, disulfide bond scrambling and oxidation have negatively affect efficacy and stability of biotherapeutic proteins. Peptide mapping is the primary analytical tool used to monitor these modifications, and the process involves proteolysis of the biotherapeutic protein into peptides followed by downstream reverse phase HPLC and mass spectrometry analyses. Unfortunately, steps involved in peptide mapping sample preparation are also a source of PTMs. In fact, deamidation and disulfide bond scrambling are induced at alkaline pH, which is favored by trypsin and other proteases used in peptide mapping. Excipients and impurities possessing protein oxidation activity cause the third major non-enzymatic PTM, oxidation. These issues complicate analysis of the PTMs.
To address these problems, we developed a sample preparation procedure according to which all sample preparation steps are performed at acidic conditions. To achieve efficient reduction and alkylation at these conditions, we selected suitable modifying chemicals and introduced special procedural modifications. The proteolytic step has represented the major bottleneck since trypsin, the most commonly used protease in peptide mapping, is inhibited at acidic pH. We solved this problem by supplementing trypsin with a specialized, low pH resistant Lys-C protease. Using this approach we achieved robust digestion at acidic conditions while suppressing deamidation and disulfide bond scrambling. We were able to further optimize digestion by introducing a pre-digestion step under strong denaturing conditions. The pre-digestion step assured digestion of tightly folded proteins and protein domains. To suppress artificial protein oxidation during sample preparation, we selected a compound with high oxygen scavenging activity. In a model study, we successfully applied the compound to suppress protein oxidation caused by hydrogen peroxide.
Sergei Saveliev, PhD
Senior Research Scientist
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