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. Unlike previous reports, we found that tyrosine sulfation modestly decreased the mAb cell binding and T cell-mediated killing, primarily by increasing the rate of antigen disassociation as determined from surface plasmon resonance-binding experiments. We also found that, while this acidic modification had no significant impact on the mAb thermal stability, sulfation did modestly increase its rate of aggregation, presumably by lowering the mAb's colloidal stability as indicated by polyethylene glycol induced liquid-liquid phase separation experiments.

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. Despite their prevalence of use in the biopharmaceutical industry, methods for assessing conformational stability such differential scanning calorimetry and isothermal equilibrium unfolding showed little predictive power and we highlight some of the assumptions and technical challenges of their use with mAbs. Conversely, measures of colloidal stability of the native-state such as preferential interaction coefficients measured by vapor pressure osmometry and solubility assessed by polyethylene-glycol induced precipitation correlated reasonably well with the mAb dimerization data and are most consistent with the excipients tested minimizing dimerization by interacting favorably with the residues comprising the protein-protein association interface.

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. METHODS: We utilized small scale incubations to generate adequate amounts of the metabolite of interest. After purification, LC-MS/MS and Proton Nuclear Magnetic Resonance (1H-NMR) were utilized to unequivocally assign the novel site of glutathione conjugation on the purine ring system. RESULTS: A proposed novel site of glutathione conjugation was investigated on a diaminopurine-containing molecule. It was demonstrated that the formation of the glutathione conjugate at the C-6 position of the purine ring system was due to the bioactivation of the compound to a di-imine intermediate by CYP3A4, followed by the nucleophilic addition of glutathione. CONCLUSION: S-glutathionylation at C-6 position of a purine was proven unequivocally. This previously unreported mechanism constitutes a novel biotransformation for purines.