Multivariate data analysis of process parameters affecting the growth and productivity of stable Chinese hamster ovary cell pools expressing SARS-CoV-2 spike protein as vaccine antigen in early process development.

The recent COVID-19 pandemic revealed an urgent need to develop robust cell culture platforms which can react rapidly to respond to this kind of global health issue. Chinese hamster ovary (CHO) stable pools can be a vital alternative to quickly provide gram amounts of recombinant proteins required for early-phase clinical assays. In this study, we analyze early process development data of recombinant trimeric spike protein Cumate-inducible manufacturing platform utilizing CHO stable pool as a preferred production host across three different stirred-tank bioreactor scales (0.75, 1, and 10 L). The impact of cell passage number as an indicator of cell age, methionine sulfoximine (MSX) concentration as a selection pressure, and cell seeding density was investigated using stable pools expressing three variants of concern. Multivariate data analysis with principal component analysis and batch-wise unfolding technique was applied to evaluate the effect of critical process parameters on production variability and a random forest (RF) model was developed to forecast protein production. In order to further improve process understanding, the RF model was analyzed with Shapley value dependency plots so as to determine what ranges of variables were most associated with increased protein production. Increasing longevity, controlling lactate build-up, and altering pH deadband are considered promising approaches to improve overall culture outcomes. The results also demonstrated that these pools are in general stable expressing similar level of spike proteins up to cell passage 11 (~31 cell generations). This enables to expand enough cells required to seed large volume of 200-2000 L bioreactor.

A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens.

Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.

Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells.

Recombinant adeno-associated virus (rAAV) has emerged in recent years as a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broadening the usage of rAAV vectors remains the limited capacity of available production systems to provide sufficient rAAV quantities for preclinical and clinical trials. The impracticality of expanding commonly used adherent cell lines represents a limitation to large-scale production. This paper describes successful productions of rAAV type 2 using suspension-growing human embryonic kidney (HEK293) cells in serum-free medium. The developed process, based on triple transfection employing polyethylenimine (PEI) as DNA transporter, allowed for a serum-free production of AAV, yielding viral vector titer up to 4.5x10(11) infectious viral particles (IVP) in a 3.5-L bioreactor. A maximum ratio of VG:IVP in the order of 200:1 was obtained, indicating the efficient encapsidation of viral vectors in HEK293 cells. The effect of varying the ratio of three plasmids and the influence of cell density at transfection were studied. The conditioned medium did not limit or inhibit the rAAV production; therefore, the elimination of the medium exchange step before or after transfection greatly simplified the scale-up of rAAV production. The cell-specific viral titers obtained in bioreactor suspension cultures were similar or higher than those obtained with control adherent cell cultures which further supported the scalability of the process. From multiple aspects including process simplicity, scalability, and low operating costs, this transfection method appears to be the most promising technology for large-scale production of rAAV.

Large-scale transfection of mammalian cells for the fast production of recombinant protein.

Recombinant proteins (r-proteins) are increasingly important in fundamental research and for clinical applications. As many of these r-proteins are of human or animal origin, cultivated mammalian cells are the host of choice to ensure their functional folding and proper posttranslational modifications. Large-scale transfection of human embryonic kidney 293 or Chinese hamster ovary cells is now an established technology that can be used in the production of hundreds of milligram to gram quantities of a r-protein in less than 1 mo from cloning of its cDNA. This chapter aims to provide an overview of large-scale transfection technology with a particular emphasis on calcium phosphate and polyethylenimine-mediated gene transfer.

Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme.

Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%.

Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency.

Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.