Control strategies for mammalian cell bioprocesses in the production of glycosylated biopharmaceuticals

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Mammalian cell technology is the basis for the production of an increasing number of biopharmaceuticals used as therapeutics for unmet medical needs. This proposal aims to control the production bioprocesses to enable high yield of these glycoproteins with the critical quality attributes that ensure high bioactivity. The research will be conducted in a laboratory which has facilities for mammalian cell culture as well as bioanalytical tools for the analysis of recombinant products and their glycosylation profiles. The proposal will be conducted through a series of projects, each of which will be related to the training of up to 6 graduate students over the course of the program. (a) Chemostat cultures of antibody-secreting Chinese hamster ovary (CHO) cells will be established to assess the effect of culture parameters on the monoclonal antibody (Mab) yield and glycosylation profile. The parameters will include pH, dissolved oxygen and nutrient concentrations, particularly glucose, galactose and glutamine. Steady-state metabolism of the cells will be established following incremental step changes of these parameters. Nutrient uptake rates and steady state intracellular concentrations of critical precursors such as nucleotide sugars will be measured to obtain data that may be used to develop predictive models for product yields and glycosylation profiles. (b) Enzymic re-modelling will be implemented during the purification of Mabs produced in the bioreactor using a solid state technique that we have previously developed. It will be attempted to obtain high yields of single glycoform entities by studying the kinetics of enzymic conversions using selected enzymes that have just been made commercially available. (c) Culture conditions that result in potentially immunogenic glycan forms will be characterized. This will include the glycolyl neuraminic acid and gala1-3gal epitopes. As well as CHO cells, this project will use mouse cells (NS0) because of their propensity to form such epitopes. (d) Chemically-defined, animal-component-free and serum-free medium will be developed for the growth and productivity of difficult cell lines such as the anchorage-dependent cells used in viral vaccine production. These projects will be conducted with the use of industrially-relevant cell lines that are available and have been used in preliminary studies. These include CHO cell lines that secrete a number of recombinant products such as humanized Mabs, chimeric human-llama Mabs and tissue-specific plasminogen activator (t-PA). Each of these products has a distinctive structure that can affect glycosylation profiles that are critical for their function. Where possible the structural profiles obtained in the studies will be related to functional assays. These studies are important to strategic developments in the implementation of platform technologies to be used in industrial bioprocesses.