Recombinant protein bioprocess optimization using emerging expression platforms

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Since the first recombinant product in 1982, the biopharma industry has experienced an unprecedented growth in Canada with sales approaching $10billion annually. The conventional platforms expressing many biopharmaceuticals (animal cell lines and Escherichia coli) have some drawbacks. To alleviate these, the engineering assessment and optimization of fed-batch cultures of two alternate, emerging recombinant hosts, namely Pichia pastoris (a eukaryote) and Streptomyces lividans (a prokaryote) expressing human interleukin-3 (huIl-3) are proposed as model systems for study. The proposed research is built upon the applicant's experience with recombinant protein bioprocesses. The objective is to develop a model-based optimization strategy for manipulating the nutrient feed and operating conditions in order to maximize the recombinant protein productivity and the concentration. A structured experimental approach and data analysis will be required to fully optimize the process. As a first step a stoichiometry-based metabolic flux model will be constructed to detail catabolic pathways, biosynthesis, and gene regulation. The full flux model will be simplified and fine-tuned by statistical methods. Potential bottlenecks affecting optimum expression will be identified. The metabolic flux model will be applied, in turn, to establish a balanced medium to reduce metabolic stress that may lead to product degradation.. Control routines will be developed and optimized so as to produce robust process trajectories in batch and fed-batch culture. An important aspect of the experimental work is to identify probable mass transfer limitations in high density culture and apply corrective measures if necessary. The resulting data will then be used as the basis for optimization of the bioprocesses using both in situ and ex-situ instrumentation.