(69d) Addition of Butyrate in CHO Fed?Batch Culture of Branched-Chain Amino Acid Transaminase 1 Knock out Cell Lines Enhances Their Specific Productivity

Chinese hamster ovary (CHO) cells in fed‐batch cultures produce several metabolic byproducts derived from amino acid catabolism, some of which, including butyrate has proven to enhance specific productivity¹. Limiting glucose supply to suppress glucose uptake and lactate production, as in the case of fed‐batch cultures employing high‐end pH‐controlled delivery of glucose (HiPDOG) technology, has been demonstrated to effectively enhance branched chain amino acid (BCAA) catabolic byproduct accumulation, resulting in higher specific productivities¹.

Engineering attempts to knock out (KO) the gene encoding the first enzyme in the BCAA catabolic pathway, namely branched-chain amino acid transaminase 1 (BCAT1) from the CHO cells, eliminated the production of the downstream inhibitory byproducts short chain fatty acid (SCFA)². As a result, HIPDOG strategy does not increase the specific productivities of BCAT1 KO cell lines but leads to higher lactate production post HiPDOG stage, potentially due to lack of ability to make SCFAs as q_P enhancers post HiPDOG.

In this study, the addition of butyrate through feed in HiPDOG culture of BCAT1 KO cell lines results in higher specific productivities at both bench scale (2L) and large scale (500L), likely due to epigenetic changes induced by butyrate³. This method has proven applicable to five BCAT1 KO cell lines producing monoclonal antibodies and other therapeutic proteins, resulting in 50 to 150% increase in titers as compared to a conventional fed-batch process. To date there have been no significant differences in levels of high or low molecular weight species, acidic species, or N-linked glycosylation patterns for those proteins that contain glycosylation sites, when compared with a conventional fed-batch process.

1. Harrington, C., Jacobs, M., Bethune, Q., Kalomeris, T., Hiller, G. W., & Mulukutla, B. C. (2021). Production of butyrate and branched-chain amino acid catabolic byproducts by CHO cells in fed-batch culture enhances their specific productivity. Biotechnology and Bioengineering, 118, 4786–4799. https://doi.org/10.1002/bit.27942
2. Mulukutla, B. C., Mitchell, J., Geoffroy, P., Harrington, C., Krishnan, M., Kalomeris, T., Morris, C., Zhang, L., Pegman, P., Hiller, G. W. (2019), Metabolic engineering of Chinese hamster ovary cells towards reduced biosynthesis and accumulation of novel growth inhibitors in fed-batch cultures. Metab Eng. 54: 54-68. Doi:10.1016/j.ymben.2019.03.001.
3. McBain, J. A., Eastman, A., Nobel, C. S., & Mueller, G. C. (1997). Apoptotic death in adenocarcinoma cell lines induced by butyrate and other histone deacetylase inhibitors. Biochemical Pharmacology, 53(9), 1357–1368. https://doi.org/10.1016/s0006-2952(96)00904-5