Identification of tyrosine sulfation in the variable region of a bispecific antibody and its effect on stability and biological activity.

Despite tyrosine sulfation being a relatively common post-translational modification (PTM) on the secreted proteins of higher eukaryotic organisms, there have been surprisingly few reports of this modification occurring in recombinant monoclonal antibodies (mAbs) expressed by mammalian cell lines and even less information regarding its potential impact on mAb efficacy and stability. This discrepancy is likely due to the extreme lability of this modification using many of the mass spectrometry methods typically used within the biopharmaceutical industry for PTM identification, as well as the possible misidentification as phosphorylation. Here, we identified sulfation on a single tyrosine residue located within the identical variable region sequence of a 2 + 1 bispecific mAbs heavy and heavy-heavy chains using a multi-enzymatic approach in combination with mass spectrometry analysis and examined its impact on binding, efficacy, and physical stability.

Isotonic concentrations of excipients control the dimerization rate of a therapeutic immunoglobulin G1 antibody during refrigerated storage based on their rank order of native-state interaction.

Inert co-solutes, or excipients, are often included in protein biologic formulations to adjust the tonicity of liquid dosage forms intended for subcutaneous delivery. Despite the low concentration of their use, many of these excipients alter protein-protein interactions such as dimerization and aggregation rates of high concentration monoclonal antibody (mAb) therapeutics to varying extents during long-term refrigerated clinical storage, challenging the formulation scientist to make informed excipient selections at the earliest stages of development when protein supply and time are often limited. The objectives of this study were to better understand how isotonic concentrations of excipients influence the dimerization rates of a model mAb stored at refrigerated and room temperatures and explore protein sparing biophysical methods capable of predicting this dependence.

Formation of A Novel Purine Metabolite through CYP3A4 Bioactivation and Glutathione Conjugation.

Background: The study of novel sites of metabolism is important in understanding new mechanisms of biotransformation of a particular moiety by metabolic enzymes. This information is valuable in designing metabolically-stable compounds with drug-like properties. It may also provide insights into the existence of active and reactive metabolites.