ThioMabs are recombinant monoclonal antibodies that have been engineered to have a point mutation by replacing one amino acid residue with cysteine. Antibody-drug conjugates (ADC) for targeted chemotherapeutics can be formed by attaching cytotoxic drugs of interest via a maleimide functional group to these residues. Chinese Hamster Ovary (CHO) stable cell lines have been developed for the production of ThioMabs in suspension cell culture.

During cell culture, the additional ThioMab cysteine residues are usually capped with cysteine or glutathione from the media. However, in some cases ThioMabs composed of two heavy chains (HC) and three light chains (LC) have also been found. These antibodies, known as Triple Light Chain Antibodies (3LC), are formed due to an improperly capped engineered cysteine that disulfide bonds to an extra LC. In this work, the underlying causes for the formation of these 3LC antibodies in CHO cell lines were studied.

Stable cell lines expressing several types of ThioMabs were cultured in suspension in shake flasks with a temperature shift after 24-72 hours, and harvested after 14 days. The Harvest Cell Culture Fluid (HCCF) was analyzed for titer and percentage of 3LC antibodies. Additionally, cell lysates were analyzed for mRNA levels and protein expression for light and heavy chains. Correlations between LC/HC mRNA ratio, free LC expression, concentrations of capping agents and 3LC levels were determined.

Methods for decreasing 3LC formation were also investigated. Analyses of the impact of cell culture conditions such as temperature and pH on decreasing amount of 3LC yielded cell-line specific results. Addition of extra cysteine to the cell culture fluid for facilitating the capping process of ThioMabs was evaluated and it was found to decrease 3LC levels in a time-dependent manner.