Non-enzymatic chemical modification such as deamidation and oxidation is a major source of recombinant monoclonal antibody instability and microheterogeneity. They can also affect the stability and efficacy of biotherapeutic proteins. In order to obtain an accurate assessment of site-specific Asparagine (Asn) deamidation and Methionine (Met) oxidation, it is critical to reduce artifactual modifications that occur during sample preparation steps. Various analytical methods have been used to monitor these modifications and peptide mapping is one of them. However, significant amount of deamidation and oxidation will be induced during traditional protein digestion. Here, we have developed an optimized protocol utilizing an improved digestion buffer which allows efficient protein digestion in less than 6 hours with minimized artificial modifications such as deamidation and oxidation. NIST mAb was used as a model antibody and digested with our optimized protocol and with a stressed condition (e.g., in-solution with over 24hour digestion time, basic pH, and high temperature). Dual Peptide C-18 columns in series with shallow gradient were used in order to obtain better separation and resolution for deamidated species. Also, the sequence coverage was improved because small hydrophilic peptides, which in most cases eluted in void volume, were retained on the column and analyzed by LC-MS/MS. The experiments were performed in two systems Thermo QE Plus and Waters XEVO. Data processing was handled with two platforms in parallel, BioPharma Finder 3.0 and UNIFI software.

Development of a peptide mapping protocol to minimize oxidation and deamidation for biotherapeutic characterization

2019 Fall National Meeting

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