(W1030-09-56) Peptide-Based-Solutions to Reduce Undesired Cell Culture Media Chemistry – New Options for Stabilized Media Formulations

Purpose: Cell culture media (CCM) optimization has been a key element in realizing high yields and productivity while ensuring product quality in biopharmaceutical production throughout the last decade. However, further bioprocess intensification with chemically defined media is limited by undesired CCM chemistry such as formation of reactive oxygen species (ROS) that negatively impacts cellular metabolism and final protein quality. ROS are formed during aerobic metabolism but also by chemical reactions of various media components such as transition metal ions (e.g., Fenton-based reactions) and photosensitive vitamins. Intra- and extracellular, ROS react with and damage biomolecules, including DNA, lipids, and proteins as well as single amino acids. Therefore, the oxidation and degradation of media components and cellular biomolecules can strongly impact the productivity and product quality of bioprocesses [1]. Studies have shown that the essential amino acid l-tryptophane (Trp), as a single media component or as an individual residue in the final product, can be rapidly oxidized and degraded with some end products showing toxicity as well as contributing to undesirable CCM and product colorization, and product micro heterogeneity [2]. Similarly, l-cysteine is highly reactive and can generate hydroxyl free radicals and sulfide free radicals that promote oxidative stress leading to an insufficient process performance. Moreover, it can oxidize to l-cystine, which can precipitate in CCM due to its low solubility. In this regard, various antioxidants as well as cysteine/cystine derivatives such as s-sulfocysteine or N-acetyl-cysteine have already been tested to secure CCM stability through ROS scavenging and/or oxidative stress prevention [3]. Chemically defined (di-)peptides such as l-alanyl-l-tyrosine (Ala-Tyr) and glycyl-l-tyrosine (Gly-Tyr) as well as N,N'-di-l-alanyl-l-cystine [(Ala-Cys)₂] and N,N'-di-l-lysyl-l-cystine [(Lys-Cys)₂] are now commonly used to formulate more concentrated media due to their superior solubility but their involvement in media chemistry has not been investigated in detail.

Methods: In this study, stability and reactivity studies were carried out in model systems to better understand the roles l-tyrosine and l-cystine peptides play in CCM chemistry. Their effects on CCM stabilization and their impact on the performance of an industrial cell line producing a recombinant protein were assessed.

Results: Light induced colorization of Trp-containing model solutions and CCM could be strongly reduced by addition of Tyr-dipeptides. This could be explained by photoprotective effects and the natural ROS scavenger ability of the Tyr residue in combination with a much higher solubility of the Tyr-dipeptides compared to the free amino acid Tyr. ROS formation and cell viability was also studied as a function of Cu(II) ion concentration and increasing concentrations of l-cysteine or highly soluble l-cystine-peptides. Viability strongly decreased with increasing l-cysteine concentration in the presence of Cu(II) ions whereas no such effect was observed in the presence of peptides.

Conclusion: Tyr-dipeptides can be used as light stabilizers while Cys-peptides can be applied in CCM to reduce / prevent ROS formation catalyzed by free Cys. We demonstrated that their application in bioprocesses resulted in improved cell culture performance. Therefore, in addition to their nutritional function, these peptides can be used to control media chemistry and stabilize CCM to further improve media formulations and enable more efficient bioprocessing.

References: [1] Chevallier et. al., (2019). Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells. Biotechnol. Bioeng. 117(4):1172-1186
[2] Bellmaine S., et. al., (2020). Reactivity and degradation products of tryptophan in solution and proteins. Free Radic. Biol. Med. 160:696-718
[3] Chevallier et. al., (2021). Use of novel cystine analogs to decrease oxidative stress and control product quality. J. Biotech. 327:1-8