Glenn P. Godwin

Recombinant Protein Production by CHO Cells Cultured in a Chemically Defined Medium

Serum-free culture of Chinese hamster ovary (CHO) cells has become increasingly common as a way of obtaining high levels of expression of recombinant proteins while simplifying recovery and downstream processing of the product. However, serum-free media may still contain one or more of a variety of animal-derived components including albumin, fetuin, various hormones and other proteins. We have demonstrated that it is possible to eliminate animal-derived proteins from a CHO medium formulation. Plasma protein fractions like albumin and fetuin may be replaced by plant-derived hydrolysates, resulting in medium that is protein-free but still undefined (CHO III PFM). CD CHO Medium is a chemically defined formulation which contains no protein or hydrolysates of either plant or animal origin. Peak cell densities and recombinant protein expression in CD CHO cultures compared favorably to expression in other media, although the maximal cell density and the highest levels of expression were observed at later time points. We were able to successfully supplement the culture with sodium butyrate to increase expression levels at the expense of peak cell density, so for recombinant cell lines showing an inverse relationship between growth and expression of recombinant product, strategies which limit the peak cell density may be useful for increasing expression.

Technical Approaches to Minimize Regulatory Hurdles to Fed-Batch and Continuous Medium Supplementation

Mammalian cell culture biological production applications have traditionally implemented batch production techniques to minimize real or perceived obstacles to product approval by regulatory agencies. Analysis of the bioreactor environment during the course of a batch fermentation run demonstrates significant fluctuations in levels of critically-limiting nutrients, accumulation of catabolic products and increased medium osmolality. These factors can be demonstrated to impact specific productivity and to result in product microheterogeneity. Extended bioreactor campaigns may exhibit superior productivity and longevity by controlled nutrient delivery via fed batch or continuous perfusion of concentrated supplements. Regulations defining criteria for “well-characterized products” and international efforts to harmonize standards may minimize barriers to extended production campaigns and process modifications. This paper examines the evolution of these regulatory guidelines and their application to process changes which permit productivity enhancement through supplementation with salt-free nutrient concentrates.