Impact of Cysteine and Tyrosine Dipeptides on CHO Cell Performance in a Perfusion Mimic

Author

Corrin L. Pruitt, Clemson UniversityFollow

Date of Award

8-2025

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Bioengineering

Committee Chair/Advisor

Sarah Harcum

Committee Member

Agneta Simionescu

Committee Member

Dan Simionescu

Abstract

Perfusion cell cultures typically achieve higher cell densities and generate higher volumetric productivities than fed-batch cultures due to continuous nutrient supply and spent media removal. Despite these advantages, perfusion cell cultures remain underutilized for licensed product manufacturing, primarily due to the lack of small-scale perfusion models for high-throughput process development and the complexities involved in media formulation capable of supporting high cell densities during long-term continuous operation. To avoid nutrient limitation at high cell densities, current perfusion processes rely on using higher perfusion rates, which results in significant media wastage and downstream product dilution. Therefore, there has been an effort to formulate perfusion media by enriching amino acids that can meet the nutrient demands at lower perfusion rates to lower overall media consumption while generating higher volumetric productivity. However, critical amino acids, such as cysteine and tyrosine, that are important for cellular redox homeostasis, metabolism, and biomass synthesis in CHO cultures, cannot be concentrated in liquid media at neutral pH due to poor solubility. To address these challenges, this project focused on developing a small-scale perfusion mimic for high-throughput process development and enriching essential amino acids in perfusion media, with particular emphasis on evaluating cysteine- and tyrosine-containing dipeptides to overcome solubility limitations. This enrichment strategy aimed to enhance cellular glucose uptake and increase productivity by alleviating amino acid v bottlenecks in CHO cell cultures. A 50 mL spin tube perfusion mimic was developed by optimizing working volume and bleed strategy. The system maintained a steady-state culture at ~19 million cells/mL for 13 days, with an average bleed rate of ~0.33 d⁻¹ with a working volume of 7.5 mL. Amino acid analysis during steady-state operation revealed ten amino acids (Asn, Cys, Val, Ser, Leu, His, Tyr, Phe, Met, and Trp) depleted to limiting levels. An enriched perfusion medium was formulated by increasing the concentrations of these amino acids. Cysteine and tyrosine, which could not be supplemented directly due to poor solubility, were instead delivered using dipeptide variants. Alanyl-cystine (AC) and lysyl-cystine (KC) were used as cysteine sources, while alanyl-tyrosine (AY) and glycyl-tyrosine (GY) were evaluated for tyrosine. Dipeptides were supplemented in combinations (AC–AY, AC–GY, KC–AY, and KC– GY) to identify optimal pairing. The effects of limiting amino acid enrichment and dipeptide supplementation were assessed using the spin tube model. Overall, amino acid enrichment significantly improved glucose uptake, titer, and cell productivity; however, no major differences were observed between the dipeptide combinations and the control without dipeptides. In conclusion, this study advances the understanding of nutrient limitations in CHO perfusion cultures and presents a systematic approach for media optimization through targeted amino acid enrichment and dipeptide supplementation. Additionally, the establishment of a small-scale perfusion model offers a cost-effective platform for accelerating media development through high-throughput screening studies.

Recommended Citation

Pruitt, Corrin L., "Impact of Cysteine and Tyrosine Dipeptides on CHO Cell Performance in a Perfusion Mimic" (2025). All Theses. 4597.
https://open.clemson.edu/all_theses/4597