Infoscience

Thesis

Development of high-titer transient gene expression processes for manufacturing recombinant proteins

The market for recombinant therapeutic proteins (including antibodies) is estimated to be greater than $50-billion and has a compounded annual growth rate of around 20%. More than 50% of all approved processes for manufacturing recombinant proteins use mammalian cells as expression hosts due to their ability to carry out complex assembly and processing, human-like glycosylation and secretion of proteins. This percentage is not expected to change and should even increase with time. Classically, these manufacturing processes use stable cell lines for protein expression, wherein the plasmid coding for the protein of interest is stably integrated into the host cell chromosomes (Stable Gene Expression, SGE). An alternative method for expressing recombinant proteins is Transient Gene Expression (TGE). Herein, the expression of the protein of interest takes place from plasmids which are transfected into the cells and are maintained extra-chromosomally. TGE has become a rapid and easy method for obtaining proteins for structural, biochemical or pre-clinical studies, where only small amounts of one or more proteins might be required. As yet, TGE has not been used for manufacturing recombinant proteins for clinical testing or approved clinical use due to the requirement of large amounts of protein of consistent quality. Even though TGE has been scaled-up to the 100-liter scale and is being attempted at the 1000-liter scale, due to low specific productivity, low volumetric yields and the high cost of DNA, TGE processes are not able to economically produce sufficient amounts of proteins for such purposes. Therefore, the goal of this thesis was to improve TGE in order to create high-titer processes, which would be economically more feasible for manufacturing large-amounts of recombinant proteins. This was achieved by: Improving and simplifying gene delivery – by the combination of high cell density at time of transfection and in-situ complex formation (as apposed to a-priori complex formation), we could enhance protein expression levels and gene delivery. This made implementation more robust and easy, with greater choice of media which could be used and cell density which could be maintained. Improving gene expression – by using a combination of inhibitors of histone deacetylases like valproic acid, and growth factors like acidic fibroblast growth factor, we could enhance specific productivity by ∼20-fold. Enhancing the cell densities at which cells could be maintained after transfection – by the combination of cell cycle regulators like human p18 and p21 and treatment with inhibitors of histone deacetylases, we blocked cell growth which allowed cells to be maintained at significantly higher cell densities, allowing us to enhance volumetric productivity by ∼100-fold. Overall these improvements allowed antibody titers in excess of 1 g/l. The gram per liter range of volumetric productivity has previously only been reported for optimized SGE based manufacturing processes. We therefore improved the economic feasibility of TGE for manufacturing recombinant proteins and reduced the difference in manufacturing costs between TGE and SGE, for a given amount of protein, by an estimated 50-fold. With some further development work and study of related issues like protein quality, TGE based processes can be expected to become part of mainstream recombinant protein manufacturing in the future.

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