(393d) A Case Study of In-Process Modifications to the Unpaired Sulfhydryl of An N-Terminal Cysteine to Achieve Homogeneous Vaccine Antigen

A protein antigen for a vaccine candidate produced in an insect cell system was found to contain one unpaired sulfuhydryl on the N-terminal cysteine residue. The sulfuhydryl was reactive with components in the cell culture media, such as glutathione and free cysteine, as well as to sulfuhydryls present on other antigens resulting in dimerization. Such propensity to react led to an undesirable heterogenous antigen population. In an effort to eliminate molecular heterogeneity without genetic mutation to eliminate the cysteine residue altogether, in-process modifications to the unpaired residue were incorporated during downstream processing.  These modifications included removal of the cysteine residue by proteolytic digestion or capping with N-ethylmalemide (NEM) or free cysteine. The proteolytic digestion was performed by way of natural proteases present in the cell culture and was shown to yield consistent removal of the sulfuhydryl along with eight subsequent residues thereafter. Capping conditions were optimized using multi variable experimentation. Capping with cysteine was shown to be challenging due to slow reaction rate and the reversibility of the attachment. Conversely, capping with NEM was permanent but could lead to potential multiple NEM attachments if the reaction occurs outside of the optimal ranges. The use of TCEP as reducing agent for capping with NEM was found to be most suitable. The molecular properties of the resulting products were monitored using various analytical methods such as LC/MS, charge-based and size-based separations, and biophysical techniques to show the modification extent and stability. In addition, the biological activities of the modified products were also investigated in an animal model.