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 it also has additional functions. 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, when cultivating cells in serum-free, chemically defined conditions.

However, while the solubility of free L-cysteine is high at neutral pH, the applicable concentration in basal and feed media is limited by several factors. The main challenge is its rapid oxidization to L-cystine, a reaction that is catalyzed by the presence of trace elements such as copper and iron that 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.

To overcome the solubility limitations of L-cystine, separate alkaline feeds have traditionally been used, often together with L-tyrosine, which also has a low solubility at neutral pH. However, this approach has 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. Most importantly, it cannot be used to increase the concentration in the bioreactor to overcome substrate limitations. It is also not easily applicable when developing perfusion media concentrates.

Evonik has developed a solution to address these solubility and performance challenges based on its peptide platform. "By coupling two molecules of L-Alanine to a molecule of L-Cystine, cQrex® AC (N,N'-di-L-alanyl-L-cystine) was created. This oxidized Cys-peptide is around 30 times more soluble than L-Cystine, and efficiently metabolized. The next-generation cystine peptide cQrex® KC (N,N'-di-L-lysyl-L-cystine dihydrochloride) was designed by coupling L-lysine and L-cystine. cQrex® KC is around 1000 times more soluble than free L-cystine. The cystine peptides 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.

Like all cQrex® peptides and keto acids that can be used as supplements for the cultivation of animal and human cells, cQrex® AC and cQrex® KC are chemically defined and animal-origin free. Samples are available for free from Evonik upon request. All cQrex^® products are supplied as pure substances in crystalline form. Depending on the intended use, they can either be dissolved in water, a buffer of choice, or directly in the medium / feed. Recommendation on handling and formulation can also be provided on demand. To order a sample, please contact us at healthcare@evonik.com.