A recent technique in antibody drug conjugate development is the use of engineered cysteine residues in antibodies for drug addition. While engineered cysteines make for an attractive platform for drug addition, they present new challenges in process development, as the exposed cysteine groups will readily bind between molecules in solution, causing aggregation. To combat this, a cap can be added to the engineered cysteines in the cell culture process. Many naturally occurring thiols will cap these residues in culture; however, this requires that the caps be removed to add the linking agent and cytotoxic drug later in the process. The heterogeneous natural capping that occurs in cell culture means that a harsh reduction step must be used to fully remove the caps, which results in reduction of the antibody, requiring subsequent reoxidation and product losses. In this study we develop a cell culture process to make antibodies with nearly 100% homogeneously capped engineered cysteines, which can later be removed with a more delicate reduction step for improved performance. We first explored different capping agents to add in excess in the culture and their performance in outcompeting the preexisting caps present in the cell culture process. We found cysteine to be the best candidate for capping due to its high binding, preexistence in the culture media, and limited impact on the culture performance. We explored the timing of the cap addition and found that adding excess capping agent post clarification performed the best. We then studied the parameters around the capping reaction including, time, temperature, pH, cap concentration and cysteine to cystine ratio. Increasing pH for short duration capping reactions led to the highest homogeneity. Interestingly, increasing cysteine concentration improved capping performance; however, increasing cystine concentration beyond a small background addition did not show additional improvements, even with changing cysteine concentrations. These parameters effects on capping efficiency were mirrored in a decrease in oxidation reduction potential (ORP). Findings from these studies were applied to a pilot batch, demonstrating the scalability of this capping process to produce a scale batch of 97% cysteine capped engineered cysteines with minimal losses or negative product quality impacts.
