L-Cysteine and its oxidized form L-Cystine are essential amino acids used in cell culture and are present in all chemically defined media formulations. Both forms can be taken up by most cell lines, including those used in monoclonal antibody production. L-Cysteine is not only an important building block of biomass and recombinant proteins but has a range of additional functions. For example, it is the rate limiting substrate in the biosynthesis of glutathione, which is the main mediator of intracellular redox homeostasis and helps to reduce oxidative stress. Thus, providing sufficient Cys-equivalents is critical to achieve the best bioprocess performance during the cultivation of cells in serum-free, chemically defined conditions.
Understanding solubility challenges and performance of L-Cystine
While the solubility of free L-Cysteine is high at a neutral pH, the applicable concentration in basal and feed media can be limited by several factors. The main challenge relates to its rapid oxidization to L-Cystine. This reaction is catalyzed by the presence of trace elements such as copper and iron which are typically present in cell culture media. L-Cystine has a low solubility and can precipitate at concentrations of greater than 1 mM. The high reactivity of the free thiol group of L-Cysteine can also promote a number of radical reactions that result in toxic and growth inhibitory chemical species.
How to overcome the solubility and performance challenges of L-Cystine
To overcome the solubility limitations, separate alkaline feeds have traditionally been used. Often, these feeds are used together with L-Tyrosine, which also has a low solubility at neutral pH. However, this approach can have several disadvantages including increased process complexity, the risk of pH-spikes, the introduction of higher salt concentrations, and the precipitation of L-Cystine and L-Tyrosine in the bioreactor. Most importantly, it cannot be used to increase the concentration in the bioreactor to overcome nutrient limitations. It is also not easily applicable when developing perfusion media concentrates.
cQrex® AC significantly enhances the solubility and performance of L-Cystine
Evonik has developed a reliable solution to address these solubility and performance challenges of L-Cysteine based on its dipeptide platform. By coupling two molecules of L-Alanine to a molecule of L-Cystine, cQrex® AC (N,N'-di-L-alanyl-L-cystine, (Ala-Cys)2) was created. This oxidized Cys-dipeptide is more than 20 times more soluble than L-Cystine, and efficiently metabolized. It can be used as part of a neutral feed, or to increase the concentration of Cys-equivalents in the bioreactor to provide optimum nutrition to further improve process performance. Based on the success of cQrex® AC and our understanding of Cysteine chemistry in cell culture media, Evonik has also developed a second generation Cys-Peptide - cQrex® KC (N,N'-di-L-lysyl-L-cystine, (Lys-Cys)2), which has outstanding properties. The solubility is more than 1000x higher than L-Cystine and it has been shown to extend growth, maintain high viability and increase antibody titers.