Use of S-sulfocysteine (SSC) as a L-cysteine source in Chinese hamster ovary (CHO) suspension batch and fed-batch cultures

Numerous proteins being used as therapeutic agents such as therapeutic antibodies are produced within cell cultures. This work sought to develop a single nutrient solution (single feed) for fed-batch processes comprising all nutrients in one complex feed at neutral pH without impacting or improving cell culture performance, titer and monoclonal antibody (mAb) characteristics. The amino acid L-cysteine was shown to rapidly dimerise into poorly soluble L-cystine via oxidation mechanisms. Addition of L-cysteine to a complex, chemically defined, highly concentrated neutral pH nutrient solution (feed) resulted in rapid production of L-cystine from L-cysteine indicating poor stability of L-cysteine in the feed. To keep L-cysteine stable in the standard fed-batch process at Merck, the amino acid was maintained in an alkaline pH environment separated from the other feed nutrients (two feed strategy). The addition of the alkaline L-cysteine nutrient solution to fed-batch cultures resulted in pH peaks while feedings. To achieve the aim of establishing the single feed strategy, screening of several L-cysteine derivatives was carried out in the part 5.1 of this work. Out of several L-cysteine derivatives, the derivative S-sulfocysteine (SSC) was chosen since it showed the desired physico-chemical properties. In part 5.3 of this work, SSC was shown to be stable over three months at 4°C in chemically defined, complex, highly concentrated neutral pH feed. In part 5.5 of this work, L-cysteine was replaced by SSC in batch cultures with Chinese hamster ovary (CHO) suspension clone 1. It was shown that SSC was able to replace L-cysteine in batch mode indicating that SSC was used as a L-cysteine source. The use of SSC in fed-batch experiments (spin tubes, bioreactors) with CHO suspension clone 2 was examined in parts 5.6 and 5.7. Application of 15 mM SSC (single feed strategy) revealed prolonged growth and increased titer compared with the standard process (two feed strategy). The antibody produced with 15 mM SSC showed no differences in N-glycosylation, charge variant and mAb sequence compared with antibody produced from the standard process as shown in part 5.8. The mechanism of SSC use as a L-cysteine source was described in part 5.9. Due to its interaction with glutathione (GSH), mixed disulfides were generated. After their enzymatic reduction, L-cysteine was released and ready for metabolization. Further, increased intracellular total GSH pools, superoxide dismutase 1 and 2 (SOD-1, SOD-2) levels and decreased intracellular reactive oxygen species (ROS) concentrations were observed in the single-feed containing 15 mM SSC compared with the standard process indicating anti-oxidant related mechanisms of SSC. Toxic effects of 20 mM SSC in fed-batch and 1.5 mM SSC in batch mode on cell growth were correlated to the external pH in part 5.10. The observed drops in pH with toxic SSC concentrations were assumed to be responsible for the monitored reproducible, rapid cell death in batch and fed-batch cultures compared with the standard processes. Further, it was shown that different CHO suspension clones reacted differently to the derivative indicating clone dependent effects of SSC. Overall, the results of this work confirmed the concept of the single-feed strategy in CHO suspension fed-batch culture. For the first time, SSC was successfully used as a L-cysteine source in cell culture. The successful application of SSC in cell culture was shown to be dependent on its applied concentration, the used cultivation mode and clone