Utilization of tyrosine‐ and histidine‐containing dipeptides to enhance productivity and culture viability

Abstract

Adequate supply of nutrients, especially providing a sufficient level of specific amino acids, is essential for cell survival and production. Complex raw materials such as soy hydrolysates or yeast extracts are the source for both free amino acids and peptides. However, typical chemically defined (CD) media provide amino acids only in free form. While most amino acids are highly soluble in media and can be provided at fairly high concentrations, certain amino acids such as tyrosine have poor solubility and thus, only a limited amount can be added as a media component. The limited solubility of amino acids in media can raise the risk of media precipitation and instability, and could contribute to suboptimal culture performance due to insufficient nutrient levels to meet cellular demands. In this study, we examine the use of chemically synthesized dipeptides as an alternative method for delivering amino acids to various monoclonal antibody producing cell lines. In particular, we focus on tyrosine-containing dipeptides. Due to their substantially higher solubility (up to 250-fold as compared with free tyrosine), tyrosine-containing dipeptides can efficiently provide large amounts of tyrosine to cultured cells. When tested in fed-batch processes, these supplemental dipeptides exerted positive effects, including enhanced culture viability and titer. Moreover, dipeptide-supplemented cultures displayed improved metabolic profiles including lower lactate and NH 4(+) production, and better pH maintenance. In bioreactor studies using two-sided pH control, a lactate spike occurring on Day 10 and the concomitant high levels of base addition could be prevented with dipeptide supplementation. These beneficial effects could be obtained by one-time addition of dipeptides during inoculation, and did not require further feeds during the entire 11-15-day process. Non-tyrosine-containing dipeptides, such as His-Gly, also showed improved productivity and viability over control cultures.

Control of amino acid transport into Chinese hamster ovary cells

“…SLC7A11, responsible for Cys import, is of specific interest as only inhibition of SLC7A11 by SAS induced a rapid loss of cell viability in all cell populations, consistent with findings that SLC7A11 activity is strongly associated with GSH synthesis and SAS‐mediated inhibition of cystine uptake via SLC7A11 induces apoptotic cell death (Chung et al, ; Gout et al, ; Shih et al, ; Timmerman et al, ). Cystine transport might therefore be a competitive bottleneck to production of cell and MAb product biomass that may itself induce stress responses (Lee et al, ) which may be amenable to either genetic cell engineering or improved media design such as supplementing cultures with cysteine containing dipeptides or tripeptides that have a higher solubility as reported by others (Kang, Mullen, Miranda, & Deshpande, ; Kishishita et al, ). However, evidence would suggest that Cys levels in mammalian cells are tightly regulated as excess Cys is cytotoxic (Stipanuk, Dominy, Lee, & Coloso, ).…”

Metabolic Alterations in Lung Cancer–Associated Fibroblasts Correlated with Increased Glycolytic Metabolism of the Tumor

“…Some possibilities include increased CAF autophagy as a means of sparing nutrients in the tumor microenvironment for use by the cancer cells, or the secretion of nutrients from CAFs generated by autophagy to enrich the nutrient milieu of the tumor microenvironment. The latter is of particular interest with regards to dipeptides since supplementation of media with certain dipeptides enhances in vitro cell viability (33). …”

Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: History, key components, and optimization strategies

“…Maintaining tyrosine at a concentration above 1 mM in the culture medium can effectively eliminate misincorporation at tyrosine sites . However, tyrosine is a poorly soluble amino acid, which complicates the use of high tyrosine concentrations in cell culture media …”