Chinese hamster ovary (CHO) cells are the industry standard cell line for high yield expression of biopharmaceuticals. However, leveraging the influencers of specific productivity to expedite generation of high productivity clones remains a challenge. One factor impacting specific productivity is the strong, constitutive activity of the CMV promoter used in most vectors for transgene applications. In this study, we sought to understand the influence of DNA methylation along the CMV promoter in driving transcription in higher vs lower productivity cell lines. Bisulfite pyrosequencing was used to characterize the CMV methylation pattern in parental (lower productivity) and DHFR-amplified (higher productivity) monoclonal antibody-expressing cell lines. In-silico analysis of promoter sequence revealed a large CpG island covering the transcription factor binding sites for CREB1 and NFκB. Variable methylation of specific CpG sites appears correlated with transcription factor binding. Treatment of the cells with 5’-azacytidine, a known DNA hypomethylation agent, led to methylation reduction in the CMV promoter, particularly upstream of the NFκB binding consensus region. This induced DNA hypomethylation correlated with cell line-specific increases in RNA expression and specific productivity. These encouraging findings suggest that site-specific methylation along the CMV promoter plays an underutilized, and important role in improving transgene transcription using the CMV promoter and de-bottlenecking the production capacity of CHO cell lines.

Currently, stable Chinese hamster ovary cell lines producing therapeutic, recombinant proteins are established either by antibiotic and/or metabolic selection. Here we report a novel technology, PTSelect™ that utilizes an siRNA cloned upstream of the gene of interest (GOI) that is processed to produce functional PTSelect™-siRNAs, which enable cell selection. Cells with stably integrated GOI are selected and separated from cells without GOI by transfecting CD4/siRNA mRNA regulated by PTSelect™-siRNAs and exploiting the variable expression of CD4 on the cell surface. This study describes the PTSelect™ principle and compares the productivity, doubling time and stability of clones developed by PTSelect™ with conventionally developed clones. PTSelect™ rapidly established a pool population with comparable stability and productivity to pools generated by traditional methods and can further be used to easily monitor productivity changes due to clonal drift, identifying individual cells with reduced productivity.

Clinical use of pancreatic beta islets for regenerative medicine applications requires mass production of functional cells. Current technologies are insufficient for large-scale production in a cost-efficient manner. Here, we evaluate advantages of a porous cellulose scaffold and demonstrate scale-up to a wicking-matrix bioreactor as a platform for culture of human endocrine cells. Scaffold modifications were evaluated in a multi-well platform to find the optimum surface condition for pancreatic cell expansion followed by bioreactor culture to confirm suitability. Preceding scale-up, cell morphology, viability and proliferation of primary pancreatic cells were evaluated. Two optimal surface modifications were chosen and evaluated further for insulin secretion, cell morphology and viable cell density for human induced pluripotent stem cell-derived pancreatic cells at different stages of differentiation. Scale-up was accomplished with uncoated, amine-modified cellulose in a miniature bioreactor, and insulin secretion and cell metabolic profiles were determined for 13 days. We achieved 10-fold cell expansion in the bioreactor along with a significant increase in insulin secretion compared with cultures on tissue-culture plastic. Our findings define a new method for expansion of pancreatic cells on wicking-matrix cellulose platform to advance cell therapy biomanufacturing for diabetes.