Chinese Hamster Ovary (CHO) cells are used extensively throughout the biopharmaceutical industry, producing nearly 70% of all protein therapeutics. The glutamine synthetase (GS) selection system is commonly used to ensure stable transgene integration and amplification in CHO host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter would allow for a more efficient metabolic phenotype. Herein, we characterized CHO cell lines featuring either an attenuated promoter or an SV40 promoter, selected with or without MSX. CHO cells that featured an attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to an SV40-driven GS promoter. Selection with MSX increased both specific productivity and glutamine production, regardless of promoter type. <sup>13</sup>C metabolic flux analysis (MFA) was performed to assess metabolic differences between cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate or glutamine varied depending on promoter and MSX selection. This study highlights the ability to optimize the GS selection system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central carbon metabolism.

Attenuation of the glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells

ACS Spring 2022

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