Integrated cell and process engineering for improved transient production of a "difficult-to-express" fusion protein by CHO cells.

Based on an optimized electroporation protocol, we designed a rapid, milliliter-scale diagnostic transient production assay to identify limitations in the ability of Chinese hamster ovary (CHO) cells to produce a model "difficult-to-express" homodimeric Fc-fusion protein, Sp35Fc, that exhibited very low volumetric titer and intracellular formation of disulfide-bonded oligomeric aggregates post-transfection. As expression of Sp35Fc induced an unfolded protein response in transfected host cells, we utilized the transient assay to compare, in parallel, multiple functionally diverse strategies to engineer intracellular processing of Sp35Fc in order to increase production and reduce aggregation as two discrete design objectives. Specifically, we compared the effect of (i) co-expression of ER-resident molecular chaperones (BiP, PDI, CypB) or active forms of UPR transactivators (ATF6c, XBP1s) at varying recombinant gene load, (ii) addition of small molecules known to act as chemical chaperones (PBA, DMSO, glycerol, betaine, TMAO) or modulate UPR signaling (PERK inhibitor GSK2606414) at varying concentration, (iii) a reduction in culture temperature to 32°C. Using this information, we designed a biphasic, Sp35Fc-specific transient manufacturing process mediated by lipofection that utilized CypB co-expression at an optimal Sp35Fc:CypB gene ratio of 5:1 to initially maximize transfected cell proliferation, followed by addition of a combination of PBA (0.5 mM) and glycerol (1% v/v) at the onset of stationary phase to maximize cell specific production and eliminate Sp35Fc aggregation. Using this optimal, engineered process transient Sp35Fc production was significantly increased sixfold over a 12 day production process with no evidence of disulfide-bonded aggregates. Finally, transient production in clonally derived sub-populations (derived from parental CHO host) screened for a heritably improved capability to produce Sp35Fc was also significantly improved by the optimized process, showing that protein-specific cell/process engineering can provide a solution that exceeds the limits of genetic/functional diversity within heterogeneous host cell populations. .

Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture.

Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post-translational insertion of a sulfur atom into disulfide bonds at the heavy-heavy and heavy-light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run-to-run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench-scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody.

Novel cholesterol feeding strategy enables a high-density cultivation of cholesterol-dependent NS0 cells in linear low-density polyethylene-based disposable bioreactors.

We have developed a perfusion-based high cell density (HD) cell banking and inoculum expansion procedure for a cholesterol-dependent NS0 myeloma cell line using linear low-density polyethylene-based disposable bioreactors. Challenges associated with cholesterol-polymer interactions, which suppress cholesterol-dependent NS0 myeloma cell growth, were overcome using a novel cholesterol feeding protocol that included a combination of two cholesterol formulations: an ethanol-based formulation and an aqueous formulation. Using a cholesterol feed optimized for HD cell culture in a disposable bioreactor perfusion system, cell densities of >25 × 10(6) viable cells/ml at ≥ 90 % cell viability were achieved. Vials of high density cell banks were created by filling 90-100 × 10(6) viable cells/ml in 5 ml cryotube vials. Implementation of the HD cell banks enabled a significant reduction in the number of step operations in the inoculum expansion phase in a large-scale manufacturing setting.

Development and implementation of a perfusion-based high cell density cell banking process.

A perfusion-based high cell density (HD) cell banking process has been developed that offers substantial advantages in time savings and simplification of upstream unit operations. HD cell banking provides the means to reduce the time required for culture inoculum expansion and scale-up by eliminating the need for multiple small to intermediate scale shake flask-based operations saving up to 9 days of operation during large-scale inoculum expansion. HD perfusion cultures were developed and optimized in a disposable Wave bioreactor system. Through optimization of perfusion rate, rocking speed and aeration rate, the perfusion system supported peak cell densities of >20 × 10(6) cells/mL while maintaining high cell viability (≥ 90%). The cells were frozen at HD (90-100 × 10(6) viable cells/mL) in 5-mL CryoTube vials. HD cell banks were demonstrated to enable direct inoculation of culture into a Wave bioreactor in the inoculum expansion train thus eliminating the need for intermediate shake flask expansion unit operations. The simplicity of the disposable perfusion system and high quality of the cell banks resulted in the successful implementation in a 2000 L scale manufacturing facility.