Abstract
Sequence variants (SV) in protein bio therapeutics can be categorized as unwanted impurities and may raise serious concerns in efficacy and safety of the product. Early detection of specific sequence modifications, that can result in altered physicochemical and or biological properties, is therefore desirable in product manufacturing. Because of their low abundance, and finite resolving power of conventional analytical techniques, they are often overlooked in early drug development. Here, we present a case study where trace amount of a sequence variant is identified in a monoclonal antibody (mAb) based therapeutic protein by LC–MS/MS and the structural and functional features of the SV containing mAb is assessed using appropriate analytical techniques. Further, a very sensitive selected reaction monitoring (SRM) technique is developed to quantify the SV which revealed both prominent and inconspicuous nature of the variant in process chromatography. We present the extensive characterization of a sequence variant in protein biopharmaceutical and first report on control of sequence variants to < 0.05% in final drug product by utilizing SRM based mass spectrometry method during the purification steps.
Highlights
Sequence variants (SV) in protein bio therapeutics can be categorized as unwanted impurities and may raise serious concerns in efficacy and safety of the product
The Glutamic acid (E) to Lysine (K) sequence variant described here was first identified during the charge variant characterization of a far basic variant (FBV) in the end-of-fermentation product (EOF) of a monoclonal antibody (Fig. 1a)
While generation sequencing (NGS) and software based SV searches in high resolution liquid chromatography (LC)–mass spectrometry (MS)/MS data generated from the enzymatic peptide map analysis of drug product or upstream products are used widely to identify sequence variants in therapeutic proteins, both of these techniques have challenges, especially when the SV is present in trace amounts
Summary
Sequence variants (SV) in protein bio therapeutics can be categorized as unwanted impurities and may raise serious concerns in efficacy and safety of the product. Identification of each peptide in software assisted MS detection is score based that is affected by the quality of MS/MS data which is again dependent on the abundance of the substitution, instrument sensitivity, degree of chromatographic separation, ionization efficiency of the separated peptides and ionization suppression by more dominant ions co-eluting in the complex matrix of the protein digest[38, 39]. These informatics tools often generate many false positives[3,15,40] primarily due to misinterpretation of chemical modifications, N and C-terminal modifications as sequence variants. In such advanced stages where the product is characterized for its functional advances in efficacy, development of strategies to control sequence variant(s) in the desired product weighs over evaluating new clones
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