|Yao Chen, Ph.D., received his Ph.D. in the Department of Pharmaceutical Chemistry, University of Kansas, where he worked on LC-MS based quantifications of membrane-bound drug-metabolizing enzymes or drug-transporters. Upon graduation, he joined Catalent Biologics in Bloomington, IN and started a career as an analytical scientist, with the routine work of identifications and quantification of pharmaceutical impurities, host-cell proteins, post-translational modifications, sequence variance / mis-substitutions, etc. with LC-MS. Global characterization of chemical modification is the main topic of his recent first author manuscript submitted to MCP.
Global Characterization of Human IgG1 under Oxidative Stresses—Independent Digestion with Two Protease Enzymes Combining LC-HRMS Data Independent Acquisition (DIA)Peroxide and leachable metals induced chemical modifications are among the most import quality attributes in process development. However, overall characterization covering all common modifications in a therapeutic protein has not been previously reported. We describe a global characterization of a leachable metal- and/or peroxide- stressed human IgG1 mAb using bottom-up, liquid chromatography-high-resolution mass spectrometry to monitor 59 chemical modifications with maximum coverage. Read More
Skyline was used to generate all the combinations of modified peptides and to filter false positive identifications. Each identification, which allowed up to 3 modifications per peptide and maximum 4 miscleavages, went through 5 and 10 ppm of precursor or product ion mass filters, obtained by as much as 4 rounds of exhaustive searching, survived 3 barriers of false positive eliminations, and cross-validated by trypsin and chymotrypsin digests. Dual digestions enhanced the sequence coverage and modification visibility. Up to 54 modifications sites were found and the rank of amino acids’ propensity to react in trastuzumab were: Met> Asn> Leu/Ile> Glu> Arg> Cys(C-C)> His > Trp> Tyr> Phe> Thr> Val. Two potential undiscovered metal binding sites and one previously hypothesized binding site were found differentially modified in H2O2 and/or leachable metals stressed conditions. Modifications on trastuzumab binding interphases (Leu H238, His H272, His H437 and AsnH438) to FcɣRIII, complement protein C1q, and FcRn were observed, which could affect their binding activities or even their corresponding physiological functions. Global characterization assay not only offers a broader and deeper views into IgG1 PTMs, but also shows a more complete picture on how oxidative stresses might potentially impact mAb’s functionalities.