(489c) Engineering Yeast for the Manufacturing of Human Proteins (Faculty Candidate) | AIChE

(489c) Engineering Yeast for the Manufacturing of Human Proteins (Faculty Candidate)


Dalvie, N. C. - Presenter, Northwestern University
Yang, Y., MIT
Brady, J. R., Massachusetts Institute of Technology
Love, K. R., Massachusetts Institute of Technology
Love, J., Massachusetts Institute of Technology
Microorganisms have tremendous potential for the manufacturing of proteins, boasting fast growth to high cell densities from cheap feedstocks. While microbes are routinely used for the production of industrial enzymes and proteins at lab scale, they are seldom used for the production of therapeutic proteins. Products destined for the clinic have stringent quality requirements and often specific post-translational modifications. Eukaryotic microorganisms like yeast and filamentous fungi are strong candidates as hosts because they can secrete the protein of interest into the culture medium, and are capable of post-translational modifications like glycosylation that are common in higher eukaryotes. Despite lower specific (per cell) productivities, these hosts yield volumetric productivities similar to traditional manufacturing hosts like CHO cells (~10 mg/L/day). Engineering is required, however, to produce high quality therapeutics suitable for the clinic, like the engineering of Komagataella phaffii (Pichia pastoris) for humanized glycosylation. These engineering efforts may consist of many genetic changes to host cells, with severe impact on growth and cell viability, subsequently reducing volumetric productivity. What is needed are methods to improve the titer and quality of complex proteins without sacrificing cell viability and volumetric productivity. The cost of both manufacturing and development of protein therapeutics could be reduced with microbial hosts by accelerating development timelines and reducing the footprint and length of manufacturing campaigns.

We demonstrate three methods to improve the utility of genetic engineering in manufacturing strains of K. phaffii. First, we developed a markerless genome editing tool, eliminating energetically expensive resistance or auxotrophic markers. Second, we applied our engineering tool to humanize the glycosylation in K. phaffii without sacrificing cell viability. We utilize native promoters to precisely control the expression of endogenous and exogenous glycosyltransferases and glycosidases, balancing glycan homogeneity with cell viability. Third, we improve the quality and titer of monoclonal antibody production by introducing human chaperones to assist with protein folding and secretion. Chaperones are essential for antibody production in human cells, but themselves are large, complex molecules that require machinery for efficient folding. We demonstrate that despite adding to the recombinant protein load, chaperones work together with glyco-engineering to enable the production of high quality monoclonal antibodies at industrially relevant titers.