Cysteine-condensed thiazolidine as an effective cysteine source for CHO cell culture.

Cysteine is an essential amino acid for Chinese hamster ovary (CHO) cell culture, yet its poor stability and solubility at neutral pH complicate media and feed formulation, particularly in fed-batch and intensified processes. Thiazolidines, formed by condensation of cysteine with α-ketoacids, offer enhanced chemical stability; however, there are no published applications of their use as cysteine sources. This may be due to limitations by their slow reaction kinetics and poor compatibility with industrial feed preparation. Here, we reported a rapid, high-yield, and manufacturing-compatible cysteine-to-thiazolidine condensation method using α-ketoacids naturally present in mammalian metabolism. By optimizing reaction pH, substrate concentration, and molar ratios, complete conversion was achieved within 1-2 h under near-neutral conditions, as indicated by depletion of free thiols to negative control levels. This enabled the preparation of highly concentrated and storage-stable thiazolidine solutions suitable for direct incorporation into single-feed formulations. Using this optimized approach, thiazolidines were deployed as the sole cysteine source in CHO cell culture feeds and supported cell growth, productivity and product quality comparable to established cysteine delivery approaches across multiple CHO cell lines in both standard and intensified fed-batch processes. Mechanistically, cysteine-condensed thiazolidines function as reversible reservoirs that release cysteine through non-enzymatic equilibrium, maintain low free thiol levels while ensuring sufficient intracellular cysteine availability. Together, these results demonstrate that optimized cysteine-condensed thiazolidines provide a manufacturing-compatible alternative for single-feed cysteine delivery in CHO cell culture processes.