High Intracellular Seed Train BiP Levels Correlate With Poor Production Culture Performance in CHO Cells.

Consistent cell culture performance is a prerequisite to ensure product quality consistency and achieve productivity goals for the manufacture of recombinant protein therapeutics, including monoclonal antibodies. Here a peculiar observation is reported where high levels of intracellular BiP in seed train cultures are consistently predictive of poor cell culture performance in the subsequent inoculum and production cultures for a monoclonal antibody produced in CHO cells. This investigation suggests that in this cell line the high intracellular BiP levels in the seed train are triggered by a slightly lower culture pH, which interferes with proper antibody folding and secretion. While the seed train culture does not display any obvious signs of the problem at slightly lower culture pH, inoculum trains, and production cultures sourced from these low pH seed trains display significantly lower cell growth and cell size. High intracellular BiP levels may interfere with UPR signaling, thereby hampering a proper and timely UPR response in the production media. Studies of other problematic cell lines have shown a similar correlation between intracellular BiP accumulation and poor production performance. The authors believe intracellular BiP levels in seed train should hence be low in order to increase the success rate in production.

Effects of copper on CHO cells: cellular requirements and product quality considerations.

Recent reports highlight the impact of copper on lactate metabolism: CHO cell cultures with higher initial copper levels shift to net lactate consumption and yield lower final lactate and higher titers. These studies investigated the effects of copper on metabolite and transcript profiles, but did not measure in detail the dependences of cell culture performance and product quality on copper concentrations. To more thoroughly map these dependences, we explored the effects of various copper treatments on four recombinant CHO cell lines. In the first cell line, when extracellular copper remained above the limit of detection (LOD), cultures shifted to net lactate consumption and yielded comparable performances irrespective of the differences in copper levels; when extracellular copper dropped below LOD (∼13 nM), cultures failed to shift to net lactate consumption, and yielded significantly lower product titers. Across the four cell lines, the ability to grow and consume lactate seemed to depend on the presence of a minimum level of copper, beyond which there were no further gains in culture performance. Although this minimum cellular copper requirement could not be directly quantified, we estimated its probable range for the first cell line by applying several assumptions. Even when different copper concentrations did not affect cell culture performance, they affected product quality profiles: higher initial copper concentrations increased the basic variants in the recombinant IgG1 products. Therefore, in optimizing chemically defined media, it is important to select a copper concentration that is adequate and achieves desired product quality attributes.

Effect of cell culture medium components on color of formulated monoclonal antibody drug substance.

As the industry moves toward subcutaneous delivery as a preferred route of drug administration, high drug substance concentrations are becoming the norm for monoclonal antibodies. At such high concentrations, the drug substance may display a more intense color than at the historically lower concentrations. The effect of process conditions and/or changes on color is more readily observed in the higher color, high concentration formulations. Since color is a product quality attribute that needs to be controlled, it is useful to study the impact of process conditions and/or modifications on color. This manuscript summarizes cell culture experiments and reports on findings regarding the effect of various media components that contribute to drug substance color for a specific monoclonal antibody. In this work, lower drug substance color was achieved via optimization of the cell culture medium. Specifically, lowering the concentrations of B-vitamins in the cell culture medium has the effect of reducing color intensity by as much as 25%. In addition, decreasing concentration of iron was also directly correlated color intensity decrease of as much as 37%. It was also shown that the color of the drug substance directly correlates with increased acidic variants, especially when increased iron levels cause increased color. Potential mechanisms that could lead to antibody coloration are briefly discussed.

Identification of cell culture conditions to control N-glycosylation site-occupancy of recombinant glycoproteins expressed in CHO cells.

The effect of different cell culture conditions on N-glycosylation site-occupancy has been elucidated for two different recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells, recombinant human tissue plasminogen activator (t-PA) and a recombinant enzyme (glycoprotein 2-GP2). Both molecules contain a N-glycosylation site that is variably occupied. Different environmental factors that affect the site-occupancy (the degree of occupied sites) of these molecules were identified. Supplementing the culture medium with additional manganese or iron increased the fraction of fully occupied t-PA (type I t-PA) by approximately 2.5-4%. Decreasing the cultivation temperature from 37 to 33 degrees C or 31 degrees C gradually increased site-occupancy of t-PA up to 4%. The addition of a specific productivity enhancer, butyrate, further increased site-occupancy by an additional 1% under each cultivation temperature tested. In addition, the thyroid hormones triiodothyronine and thyroxine increased site-occupancy of t-PA compared to control conditions by about 2%. In contrast, the addition of relevant nucleoside precursor molecules involved in N-glycan biosynthesis (e.g., uridine, guanosine, mannose) either had no effect or slightly reduced site-occupancy. For the recombinant enzyme (GP2), it was discovered that culture pH and the timing of butyrate addition can be used to control N-glycan site-occupancy within a specific range. An increase in culture pH correlated with a decrease in site-occupancy. Similarly, delaying the timing for butyrate addition also decreased site-occupancy of this molecule. These results highlight the importance of understanding how cell culture conditions and media components can affect the product quality of recombinant glycoproteins expressed in mammalian cell cultures. Furthermore, the identification of relevant factors will enable one to control product quality attributes, specifically N-glycan site-occupancy, within a specific range when applied appropriately.

Chemometric evaluation of on-line high-pressure liquid chromatography in mammalian cell cultures: analysis of amino acids and glucose.

An on-line high-pressure liquid chromatography (HPLC) system capable of measuring amino acids and carbohydrates was used to study metabolism in mammalian cell culture systems. The HPLC method utilized anion-exchange chromatography followed by integrated pulsed amperometric detection. The method is capable of measuring 19 amino acids plus glucose with a complete method time of 65 min. In actual cell cultures, the method was shown to be useful for monitoring 17 amino acids plus glucose. The two amino acids that were not accurately monitored were arginine and lysine, possibly due to their elution near the void volume of the column. The HPLC system was used to study variability in metabolism across different cell culture processes, as well as the effect of glucose and glutamine limitation on a single cell culture process. Chemometric analysis was used to draw statistically meaningful conclusions from the highly correlated, multivariate data set that resulted from these experiments. Using chemometrics, variation between processes was linked to differences in uptake rates of seven amino acids. Similarly, lactate concentration, cell density, and aspartate uptake rate were linked to glucose and glutamine limitation. The effect of nutrient limitation on glutamate, alanine, and ammonium was also considered.