Process transfer and optimization of Chinese hamster ovary cell cultivation for monoclonal antibody production

Abstract

Biopharmaceutical market has been progressively expanding, and moving away from small molecular drugs to biotechnologically produced therapeutics such as recombinant proteins. Genetically modified mammalian cells, such as Chinese Hamster Ovary cells, are being used extensively for the production of these proteins. However, a challenge faced by the biopharmaceutical industry is to attain maximum product within a limited culture volume due to volume constraints in the bioreactor. This is overcome by intensifying the process and understanding the fundamentals of cell growth, which varies across different cell strains. The main objective of this study was to successfully transfer and scale the process in different types of bioreactors with a working volume ranging from 15 mL to 50 L. Process attributes, namely, viable cell concentration and final titre quantity were used to evaluate the scalability of the process. It was shown that the process was robust and scalable across different types of bioreactors. The second part of the project was to optimize the cultivation process in terms of testing the process parameters that control cultivation, primarily, the dissolved oxygen (DO) concentration and pH. We identified that reducing the DO to 40% and maintaining the pH at 7.1 not only decreased the requirement of pure oxygen in production scale bioreactors, but also reduced the damage to cultivated cells caused by oxygen driven free radicals. The next part of process optimization was conducted by varying the concentrations of ingredients in production medium and feed media used for the fed batch process. Concentration of carbon (glucose) and nitrogen (glutamine and glutamate) sources in the production medium were altered and the impact on the viable cell concentration and protein production was studied. The results showed that the production medium can be further improved by altering the initial concentration of glutamine and glucose to range between 0.6 to 1.2 g/L and 6 to 12 g/L, respectively. Glutamate was essentially used for protein production and was supplemented to the culture through the feed medium. Therefore, was not required to be added additionally in the production medium. In order to optimize the percentage of feed medium, different concentrations of the two feed media (FMA and FMB) were added to the cell culture. It was shown that increasing the concentration of the FMA beyond 6.52% and FMB beyond 0.62 % of the total working volume had a detrimental effect on the cell growth and protein production. Along with the above mentioned tests, the amino acid consumption across different scales of bioreactor was also studied. The amino acids were divided into two groups: amino acid required for cell growth (glutamine, tyrosine, phenylalanine and isoleucine) and protein production (the remaining essential and non-essential amino acids). This provided an insight into the function of amino acids within the cells.