Designed Amino Acid Feed in Improvement of Production and Quality Targets of a Therapeutic Monoclonal Antibody

Abstract

Cell culture feeds optimization is a critical step in process development of pharmaceutical recombinant protein production. Amino acids are the basic supplements of mammalian cell culture feeds with known effect on their growth promotion and productivity. In this study, we reported the implementation of the Plackett-Burman (PB) multifactorial design to screen the effects of amino acids on the growth promotion and productivity of a Chinese hamster ovary DG-44 (CHO-DG44) cell line producing bevacizumab. After this screening, the amino acid combinations were optimized by the response surface methodology (RSM) to determine the most effective concentration in feeds. Through this strategy, the final monoclonal antibody (mAb) titre was enhanced by 70%, compared to the control group. For this particular cell line, aspartic acid, glutamic acid, arginine and glycine had the highest positive effects on the final mAb titre. Simultaneously, the impact of the designed amino acid feed on some critical quality attributes of bevacizumab was examined in the group with highest productivity. The product was analysed for N-glycan profiles, charge variant distribution, and low molecular weight forms. The results showed that the target product quality has been improved using this feeding strategy. It was shown how this strategy could significantly diminish the time and number of experiments in identifying the most effective amino acids and related concentrations in target product enhancement. This model could be successfully applied to other components of culture media and feeds.

Fig 1. Predicted values versus actual values plot for the final mAb titre, It helps we detect a value, or group of values, that are not easily predicted by the model.

The near 45 degree line of correlating plot shows the acceptable correlation between predicted and actual response values.

Fig 2. Representative charts of response surface.

Response surface shows the effects and interactions of Glu and Asp, Arg and Asp, Gly and Asp, Arg and Glu, Gly and Glu, Gly and Arg on the final mAb titre response. (For instance in part B, if Asp and Arg concentrations become less or more than the middle point (0), the response will reach the maximum amount. These changes should be the same for both amino acids. It means Asp and Arg should increase or decrease in one direction and if both of them decrease, the response will be better. Also in part C, if Asp and Gly concentrations increase toward +1, the response will reach the maximum amount.)

Fig 3. Comparison of final mAb titre in control and designed amino acid feed groups.

Fig 4. Glycoprofiling of bevacizumab in control and designed amino acid feed groups, glycan species abbreviations are as follow,G0: asialo, agalactose, biantennary complex (common core (Man3GlcNAc2) with terminal two GlcNAc residues), G0F: asialo, agalactose, biantennary complex, core substituted with fucose, G1: asialo, mono-galactosylated, biantennary complex, Man5: terminal two manose residues attached to the common core (Man3GlcNAc2), G1F/G1F’: asialo, mono-galactosylated, biantennary complex, core substituted with fucose, G2F: asialo, galactosylated, biantennary complex, core substituted with fucose.

Fig 5. Charge heterogeneity profiling of bevacizumab in control and designed amino acid feed groups.

Fig 6. Electropherograms of non-reduced bevacizumab in control and designed amino acid feed groups.

Fig 7. Electropherograms of reduced bevacizumab in control and designed amino acid feed groups, (LC: light chain, HC: heavy chain, NGHC: non glycosylated heavy chain).