Monoclonal antibodies (mAbs) consist of four polypeptide chains that are covalently linked by disulfide bonds formed between cysteine residues. However, low levels of free thiols derived from under-processed/reduced cysteines are commonly present. Because free thiols may increase the risk of undesired immunogenicity in mAb therapeutics, monitoring free thiol occupancies during drug development is crucial to ensure drug quality and safety. Multiple assays have been established to identify and...

Host cell proteins (HCPs) in biotherapeutics can present potential safety risks or compromise product stability at trace levels. Therefore, removal, testing, and characterization of HCPs are critical throughout biotherapeutic process development. While enzyme-linked immunosorbent assay (ELISA) is the gold standard for quantifying HCPs, it does not provide information on HCP identity. As a result, HCP analysis by liquid chromatography-mass spectrometry (LC-MS) has gained prominence for its...

Integration of Asymmetrical Flow Field-Flow Fractionation (AF4) with online native mass spectrometry (MS) is an attractive concept that offers significant value for the analytical characterization of protein therapeutics. Its execution, however, is challenged by the incompatibility of mobile phase components and the difficulties of achieving sensitive MS detection without compromising the separation efficiency. Herein, we describe in detail the successful coupling of a commercial AF4 instrument...

In the biopharmaceutical industry, the sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) assay is often used to assess therapeutic critical quality attributes (CQAs). Traditional detection methods for SDS-CGE methods, such as ultraviolet (UV) absorbance and laser-induced fluorescence (LIF), are widely used but come with limitations. A native fluorescence detection (NFD) scheme was previously developed to enhance sensitivity and reduce gel matrix interference without requiring sample...

Cell lines that produce non-originator versions of the National Institute of Standards and Technology (NIST) monoclonal antibody reference material 8671 (NISTmAb) are invaluable to the biopharmaceutical industry because, unlike typical commercial cell lines, they can be used on a collaborative and noncompetitive basis for bioprocess development. NIST has generated NS0 clones, NISTCHO research-grade test material 10197 and reference material 8675 NISTCHO to fill this need. We set out to optimize...