Washing with alkaline solutions in protein A purification improves physicochemical properties of monoclonal antibodies.

Protein A affinity chromatography has been widely used for both laboratory scale purification and commercial manufacturing of monoclonal antibodies and Fc-fusion proteins. Protein A purification is specific and efficient. However, there still remain several issues to be addressed, such as incomplete clearance of impurities including host cell proteins, DNA, aggregates, etc. In addition, the effects of wash buffers in protein A purification on the physicochemical characteristics of antibodies have yet to be fully understood. Here we found a new purification protocol for monoclonal antibodies that can improve physicochemical properties of monoclonal antibodies simply by inserting an additional wash step with a basic buffer after the capture step to the conventional protein A purification. The effects of the alkaline wash on monoclonal antibodies were investigated in terms of physicochemical characteristics, yields, and impurity clearance. The simple insertion of an alkaline wash step resulted in protection of antibodies from irreversible aggregation, reduction in free thiols and impurities, an improvement in colloidal and storage stability, and enhanced yields. This new procedure is widely applicable to protein A affinity chromatography of monoclonal antibodies.

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

Improved production of recombinant human antithrombin III in Chinese hamster ovary cells by ATF4 overexpression.

To improve the production of recombinant human antithrombin III (AT-III) in Chinese hamster ovary (CHO) cells, the genes encoding transcription factors, ATF4 (activating transcription factor 4) and XBP-1s (the spliced form of X-box binding protein 1), which were involved in the mammalian unfolded protein response (UPR), were cloned from CHO-K1 cells. Overexpression of ATF4 significantly enhanced the production of recombinant AT-III in CHO 13D-35D cells. The specific rate of AT-III production in the ATF4-overexpressed CHO 13D-35D cells reached approximately 23 pg/cell/day. After 144 h of incubation, the AT-III concentration in the culture supernatant was twofold greater compared to that observed with parental CHO 13D-35D cells. In contrast, ectopic expression of XBP-1s failed to enhance the production of recombinant AT-III in CHO 13D-35D cells. RT-PCR analysis revealed that high levels of XBP-1s mRNA were present in the CHO cells, regardless of ectopic expression of XBP-1s. Our results indicate that overexpression of the UPR transcription factor ATF4 is a promising means for improving the production of secreted protein pharmaceuticals in CHO cells.

Overexpression of GADD34 enhances production of recombinant human antithrombin III in Chinese hamster ovary cells.

To improve the production of recombinant human antithrombin III (AT-III) in Chinese hamster ovary (CHO) cells, the gene encoding growth arrest and DNA damage inducible protein 34 (GADD34), which is a transcription factor involved in the unfolded protein response (UPR), was cloned from CHO-K1 cells. Overexpression of GADD34 significantly enhanced the production of recombinant AT-III in CHO 13D-35D cells. The specific rate of AT-III production in the GADD34-overexpressing CHO 13D-35D cells reached approximately 28 pg/cell/d. After 144 h of incubation, the AT-III concentration in the culture supernatant was approximately 40% higher than that observed in the case of the parental CHO 13D-35D cells. The mRNA expression, specific activity, and fucosylation of AT-III were not affected by GADD34 overexpression. Overexpression of GADD34 is a promising method of improving the production of secreted protein pharmaceuticals in CHO cells.

Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells.

Antithrombin is a serine protease inhibitor that inactivates several coagulation proteases, primarily thrombin and factor Xa. The Chinese hamster ovary (CHO) cell line transfected with a vector expressing recombinant human antithrombin (rAT) and a selectable marker, glutamine synthetase (GS), was cultivated in a 2-l fed-batch culture process using serum-free, glutamine-free medium. To maximize the rAT yield, effects of culture pH, balanced amino acid feeding, and an increased glutamate concentration on cell metabolism and rAT production were investigated. When cells were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the maximum cell density and maximum lactate concentration decreased with decreasing pH. The highest production level of rAT was obtained at culture pH 6.8 due to the extended culture lifetime. Compared to the imbalanced amino acid feeding at culture pH 6.8, the balanced amino acid feeding increased the amount of rAT activity by 30% as a result of an increased viable cell number. A decrease in the specific glucose consumption rate (q(Glc)) with increasing culture time was observed in all the above-mentioned experiments, while the glucose concentration was maintained above 0.7 g l(-1). In addition, a decrease in the specific rAT production rate (q(rAT)) was observed after the depletion of lactate in the late cultivation stage. Taken together, these results suggest that the reduced availability of cellular energy caused by the decrease in q(Glc) and depletion of lactate led to the decrease in q(rAT). This decrease in q(rAT) was partially prevented by increasing the residual glutamate concentration from 1 mM to 7 mM, thus resulting in an additional 30% increase in the amount of rAT activity. The optimized fed-batch culture process yielded 1.0 g l(-1) rAT at 287 h of cultivation.