Ala-Cys-Cys-Ala dipeptide dimer alleviates problematic cysteine and cystine levels in media formulations and enhances CHO cell growth and metabolism.

Cysteine and cystine are essential amino acids present in mammalian cell cultures. While contributing to biomass synthesis, recombinant protein production, and antioxidant defense mechanisms, cysteine poses a major challenge in media formulations owing to its poor stability and oxidation to cystine, a cysteine dimer. Due to its poor solubility, cystine can cause precipitation of feed media, formation of undesired products, and consequently, reduce cysteine bioavailability. In this study, a highly soluble cysteine containing dipeptide dimer, Ala-Cys-Cys-Ala (ACCA), was evaluated as a suitable alternative to cysteine and cystine in CHO cell cultures. Replacing cysteine and cystine in basal medium with ACCA did not sustain cell growth. However, addition of ACCA at 4 mM and 8 mM to basal medium containing cysteine and cystine boosted cell growth up to 15% and 27% in CHO-GS and CHO-K1 batch cell cultures respectively and led to a proportionate increase in IgG titer. 13C-Metabolic flux analysis revealed that supplementation of ACCA reduced glycolytic fluxes by 20% leading to more efficient glucose metabolism in CHO-K1 cells. In fed-batch cultures, ACCA was able to replace cysteine and cystine in feed medium. Furthermore, supplementation of ACCA at high concentrations in basal medium eliminated the need for any cysteine equivalents in feed medium and increased cell densities and viabilities in fed-batch cultures without any significant impact on IgG charge variants. Taken together, this study demonstrates the potential of ACCA to improve CHO cell growth, productivity, and metabolism while also facilitating the formulation of cysteine- and cystine-free feed media. Such alternatives to cysteine and cystine will pave the way for enhanced biomanufacturing by increasing cell densities in culture and extending the storage of highly concentrated feed media as part of achieving intensified bioproduction processes.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling.

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Self-assembly of a 5-fluorouracil and camptothecin dual drug dipeptide conjugate.

Nano-formulated, combinatory therapeutics that control the spatiotemporal aspects of drug release have potential to overcome many of the challenges faced in cancer therapy. Herein, we describe a peptide nanotube functionalized with two anticancer drugs, 5-fluoruracil (5-FU) and camptothecin (CPT). The nanotube was formed via peptide self-assembly, which positioned 5-FU on the surface at the aqueous interface; whereas, CPT was sequestered within the hydrophobic walls. Thus, two different release profiles were observed: rapid release of 5-FU, followed by slower, sustained production of CPT. This profile emerged from the rapid hydrolytic cleavage of 5-FU at the aqueous/nanotube interface, which produced a smaller nanotube comprised of the peptide fragment.