Native peptide mapping – A simple method to routinely monitor higher order structure changes and relation to functional activity

Abstract

In the biopharmaceutical environment, controlling the Critical Quality Attributes (CQA) of a product is essential to prevent changes that affect its safety or efficacy. Physico-chemical techniques and bioassays are used to screen and monitor these CQAs. The higher order structure (HOS) is a CQA that is typically studied using techniques that are not commonly considered amenable to quality control laboratories. Here, we propose a peptide mapping-based method, named native peptide mapping, which could be considered as straightforward for HOS analysis and applicable for IgG4 and IgG1 antibodies. The method was demonstrated to be fit-for-purpose as a stability-indicating assay by showing differences at the peptide level between stressed and unstressed material. The unfolding pathway induced by a heat stress was also studied via native peptide mapping assay. Furthermore, we demonstrated the structure–activity relationship between HOS and biological activity by analyzing different types of stressed samples with a cell-based assay and the native peptide mapping. The correlation between both sets of results was highlighted by monitoring peptides located in the complementary-determining regions and the relative potency of the biotherapeutic product. This relationship represents a useful approach to interrogate the criticality of HOS as a CQA of a drug.

Limited Proteolysis Coupled with Mass Spectrometry for Simultaneous Evaluation of a Large Number of Protein Variants for Their Impact on Conformational Stability

“…This report describes a workflow, based on limited proteolysis followed by mass spectrometry (MS), to assess the conformational stability of different variants of a therapeutic protein. Limited proteolysis has been demonstrated to be useful in conformation analysis of recombinant proteins in design, optimization, and development of biopharmaceuticals. − The method is based on the fact that a less stable domain is more susceptible to proteolytic degradation, and therefore the conformational stability of a domain can be evaluated from the release rate of the peptide variant in the domain. A kinetic model is established to quantitatively determine the degree of domain stabilization/destabilization.…”