A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production

Abstract

We present an integrated microfluidic bioreactor for fully continuous perfusion cultivation of suspended microbial cell cultures. This system allowed continuous and stable heterologous protein expression by sustaining the cultivation of Pichia pastoris over 11 days. This technical capability also allowed testing the impact of perfusion conditions on protein expression. This advance should enable small-scale models for process optimization in continuous biomanufacturing.

Microbioreactors and Perfusion Bioreactors for Microbial and Mammalian Cell Culture

“…Expression of recombinant human growth hormone (rhGH) or recombinant interferon alfa-2b (rIFNα-2b) by Pichia pastoris was found to be maintained after 11 days of growth which is close to the duration of many industrial processes with duration lasting from 7 to 30 days. Success of this study establishes the feasibility of use of perfusion-capable bioreactors to study impact of perfusion culture on expression and process optimization of many other microbial systems [38] at microfluidic levels, thus saving the initial process development costs [39].…”

Pharmacy on demand: New technologies to enable miniaturized and mobile drug manufacturing

“…Microfluidics-based bioreactors have the demonstrated capability to perform stable two-week fermentations (in P. pastoris) of interferon alfa-2b and human growth hormone, with crude protein being produced within a few hours. 41 These microfluidic bioreactors have unique channels and chambers enabling the user to collect cells from the growth chambers, perfused material, and waste independently and allowing for diffusion of molecules between gas and liquid phases. It is conceivable, given the genomic stability of P. pastoris, that genetic circuitry can be developed to enable tunable production of two or more biologicals from the same P. pastoris organism (as opposed to using separate strains for different biologicals) in a sequential manner.…”

“…There are many types of PTMs that can introduce functional groups to the polypeptide chain, including but not limited to phosphates, amide groups, and carbohydrates (through glycosylation). 41 These PTMs can affect a protein's folding and stability and, thus, its tertiary and quaternary structures, thereby affecting the protein's fundamental ability to exert its biological activity. Specifically, achieving humanlike Nglycosylation so that structural and functional activity is preserved will be a unique challenge for both mammalian in vitro translation-produced and yeast-secreted proteins.…”

A microfluidic platform for physical entrapment of yeast cells with continuous production of invertase

“…There have been recent studies developing microreactors for purposes of screening of cell culture conditions. These platforms could also potentially be used for production …”

“…These platforms could also potentially be used for production. 12,14,15,27 In this work, we propose a system that can be used for the controlled cell-growth and continuous culture of a hydrodynamically-trapped non-adherent microbial cell type in order to produce an extracellular enzyme. The system should allow both screening of conditions and the production of the extracellular enzyme under optimized conditions.…”