Abstract
Current biopharmaceutical manufacturing systems are not compatible with portable or distributed production of biologics, as they typically require the development of single biologic-producing cell lines followed by their cultivation at very large scales. Therefore, it remains challenging to treat patients in short time frames, especially in remote locations with limited infrastructure. To overcome these barriers, we developed a platform using genetically engineered Pichia pastoris strains designed to secrete multiple proteins on programmable cues in an integrated, benchtop, millilitre-scale microfluidic device. We use this platform for rapid and switchable production of two biologics from a single yeast strain as specified by the operator. Our results demonstrate selectable and near-single-dose production of these biologics in <24 h with limited infrastructure requirements. We envision that combining this system with analytical, purification and polishing technologies could lead to a small-scale, portable and fully integrated personal biomanufacturing platform that could advance disease treatment at point-of-care.
Highlights
Current biopharmaceutical manufacturing systems are not compatible with portable or distributed production of biologics, as they typically require the development of single biologic-producing cell lines followed by their cultivation at very large scales
The basic components of this platform are as follows: (1) a biologics expression system engineered to secrete multiple therapeutic proteins in response to programmable cues and (2) an integrated, small overall footprint, millilitre-scale perfusion microfluidic platform capable of supporting rapid and flexible biomanufacturing process control that enables portable operations in mobile units, such as vehicles (Fig. 1 and Supplementary Movie 1). We demonstrate that this system produces near-single-dose levels of recombinant human growth hormone and interferon-a2b (IFNa2b) in o24 h
The long time needed to go from inoculation of biologic-producing Chinese Hamster Ovary (CHO) cells to release of a drug product, which meets established quality standards and an Food and Drug Administration (FDA)-approved safety profile, is incompatible with a rapid production system
Summary
Addition of b-estradiol displaces HSP90 and permits translocation of the ZF-TF into the nucleus, where it activates expression of genes regulated by a minimal promoter placed downstream of multiple ZF-binding sites (Fig. 2b) This system offers a highly flexible architecture that can be tuned by modifying different parameters, including the following: (1) affinity of the DNA-binding domain to DNA; (2) strength of the transcriptional activation domain; (3) number of binding sites for the ZF; (4) promoter driving expression of the ZF; (5) minimal promoter driving expression of the output; (6) dose of inducer; and (7) integration site. Using the synthetic expression cassettes that were optimized with intracellular fluorescent reporters 245R, 246R and 255R (Fig. 5b), we built a proof-of-concept system to controllably produce two biologic drugs, rHGH and IFNa2b This resulted in three strains capable of selectable expression of two different biologics: strain 245B
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