Glutamine Synthetase Promoter Attenuation Modeling Data

Published: 14 March 2022| Version 1 | DOI: 10.17632/jwc5d33wdp.1
Contributor:
Sarah Sacco

Description

The glutamine synthetase (GS) expression 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 on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two “lactate runaway” reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures. This dataset contains all data used as inputs for the 13C MFA modeling as well as the final output fluxes from the 13C MFA modeling.

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Institutions

Vanderbilt University

Categories

Metabolic Flux Analysis

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