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Background: Untargeted proteomics enables quantitative host cell protein (HCP) determination in biotherapeutics, yet no workflow has been validated under ICH Q2(R2) for regulated quality control. Methods: A prospective total-error (TE) validation of label-free ddaPASEF proteomics was performed. A stable isotope-labeled whole-proteome standard was spiked into NISTmAb at seven levels (20-80 ng) and analyzed in four independent assays (198 injections), supporting one-way random-effects ANOVA with...

The selection of host cell lines and culture media plays a central role in influencing the quality attributes of monoclonal antibodies, particularly in biosimilar development and process comparability. This study used standardized upstream conditions to evaluate the impact of host cell line selection on product quality attributes of a non-originator version of the NISTmAb by comparing results from antibodies made in murine myeloma NS0 or Chinese Hamster Ovary (CHO) cells. NISTCHO and NS0 clones...

Labeling of proteins with deuterium is an essential tool in overcoming size limitations in the application of nuclear magnetic resonance (NMR) spectroscopy to proteins larger than 30 kilodaltons (kDa). A non-originator antigen-binding fragment (Fab) of NIST RM 8671 NISTmAb, so called yNIST-Fab, is a ~ 50 kDa protein, with 5 native disulfide linkages, that can be expressed in properly folded form in methylotrophic Komagataella phaffii (formerly Pichia pastoris). Further, the K. phaffii host can...

Monoclonal antibody N-glycosylation is a critical quality attribute influencing therapeutic safety and efficacy, and is strongly influenced by bioprocess design. NISTCHO, a publicly available Chinese hamster ovary producer cell line, is increasingly encouraged for use as a reference system. However, the impact of feeding strategies on cellular performance and N-glycosylation has not been assessed. Here, we applied multivariate analysis of compositional N-glycan data to assess how feeding...

During early-stage biotherapeutic development, analytical methods play an important role in candidate screening, process development, formulation screening and stability determination. However, developing sensitive and robust analytical methods is challenging in the early stages as there is often insufficient product knowledge and limited sample amount. Here we present a high-sensitivity multi-attribute method (MAM) centred on nano-flow LC-MS/MS for the characterisation of mAb-based molecules....