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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....

Chinese hamster ovary (CHO) cells are widely utilised in the biopharmaceutical industry to produce therapeutic proteins. Understanding the mechanisms of endoplasmic reticulum (ER) stress and its interplay with protein degradation pathways remains pivotal for improving production efficiency and product quality. In this study, we investigated the proteomic responses of CHO-K1 (non-producer), CHO DP-12 (IgG-producer), and NISTCHO (IgG-producer) cell lines under ER stress induced by a combination of...

Accurate characterization of the amino acid sequence and post translational modifications (PTMs) of monoclonal antibodies (mAbs) is essential for evaluating product quality. Peptide mapping through bottom-up LC/MS analysis is a key methodology for this purpose. While trypsin is commonly the first choice for mAb digestion, it typically yields high but incomplete sequence coverage. As a result, supplementary endoproteases such as Asp-N, chymotrypsin, Glu-C or Lys-C are often employed to enhance...