The culture of Chinese Hamster Ovary (CHO) cells for modern industrial applications, such as expression of recombinant proteins, requires media that support growth and production. Such media must support high viable cell densities while also stimulating the synthesis and extracellular transport of biologic products. Early media development efforts in this area yielded basic formulations to sustain growth, viability, and cellular function, albeit comprising animal sourced components, and complex constituents used in batch culture mode. Subsequent improvements included the development of serum‐free and chemically defined (CD) media, the identification of critical nutrients, growth factors, and potentially inhibitory or toxic cellular metabolites, and the use of fed‐batch and perfusion culture techniques to optimize nutrient delivery while minimizing accumulation of unwanted waste products. This review is comprised of sections covering milestones in the evolution of mammalian cell culture media, nutrient composition and formulation requirements, optimization strategies, consistency and scalability of powder and liquid media preparation for industrial applications, and key recent advances driving progress in CHO cell culture medium design and development. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1407–1426, 2018
A recombinant monoclonal antibody produced by Chinese hamster ovary (CHO) cell fed-batch culture was found to have amino acid sequence misincorporation upon analysis by intact mass and peptide mapping mass spectrometry. A detailed analysis revealed multiple sites for asparagine were being randomly substituted by serine, pointing to mistranslation as the likely source. Results from time-course analysis of cell culture suggest that misincorporation was occurring midway through the fed-batch process and was correlated to asparagine reduction to below detectable levels in the culture. Separate shake flask experiments were carried out that confirmed starvation of asparagine and not excess of serine in the medium as the root cause of the phenomenon. Reduction in serine concentration under asparagine starvation conditions helped reduce extent of misincorporation. Supplementation with glutamine also helped reduce extent of misincorporation. Maintenance of asparagine at low levels in 2 L bench-scale culture via controlled supplementation of asparagine-containing feed eliminated the occurrence of misincorporation. This strategy was implemented in a clinical manufacturing process and scaled up successfully to the 200 and 2,000 L bioreactor scales.
The importance of speed to clinic for medicines that may address unmet medical needs puts pressure on product development timelines. Historically, both toxicology and first-in-human clinical materials are generated using the same clonal-derived cells to ensure safety and minimize any development risks. However, cell line development with single cell cloning is time consuming, and aggravated by the time needed to screen for a lead clone based on cell line stability and manufacturability. In order to achieve faster timelines, we have used pools of up to six clones for earlier production of drug substance for regulatory filing-enabling toxicology studies, and then the final single clone was selected for production of clinical materials. This approach was enabled by using platform processes across all stages of early development, including expression vectors, host cell lines, media, and production processes. Through comprehensive cell culture and product quality analysis, we demonstrated that the toxicology material was representative of the clinical material for all six monoclonal antibody programs evaluated. Our extensive development experience further confirmed that using a pool of clones for toxicology material generation is a reliable approach to shorten the early development timeline.
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