Model‐based investigation of intracellular processes determining antibody Fc‐glycosylation under mild hypothermia

Abstract

Despite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (q mAb) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc‐glycosylation and the mechanism behind changes in the glycan composition are not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimization. To address this issue, a mathematical model that quantitatively describes Chinese hamster ovary (CHO) cell behavior and metabolism, mAb synthesis and mAb N‐linked glycosylation profile before and after the induction of mild hypothermia is constructed. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N‐linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggests the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc‐glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimization, for example, by tailoring feeding strategies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering. Biotechnol. Bioeng. 2017;114: 1570–1582. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.

Metabolomic profiling of CHO fed‐batch growth phases at 10, 100, and 1,000 L

“…Most commonly performed by liquid or gas chromatography coupled to mass spectrometry, metabolomic analysis is quickly finding its way into the biopharmaceuticals industry for in‐depth understanding of physiological processes underlying the recombinant drug production in CHO. Metabolomics was already applied to study differences between recombinant and parental CHO cell line (Dietmair et al, ), differences between high and low producing CHO clones (Chong et al, ), CHO cell metabolism during fed‐batch (Ahn & Antoniewicz, , Dean & Reddy, ; Sengupta, Rose, & Morgan, ; Templeton, Dean, Reddy, & Young, ), metabolic background of two distinct lactate phenotypes observed in the cultivation of the recombinant CHO cells (Luo et al, ), effect of temperature shift on antibody glycosylation (Sou et al, ; Sou et al, ), and characterization of the intracellular nucleotide sugar pools and their connection to product glycosylation profiles (Fan et al, ; Sou et al, ).…”

Constrained global sensitivity analysis for bioprocess design space identification

“…Briefly, the cell growth model uses Monod kinetics to describe growth dependence on key metabolites. Intracellular NSD metabolism is described based on a reduced set of reactions and as a function of the extracellular environment as in [23]. The Golgi glycosylation model employed herein was developed specifically to describe the reactions relevant to N-linked glycosylation of the Fc region of monoclonal antibodies [24].…”

Kinetic Modeling of Mammalian Cell Culture Bioprocessing: The Quest to Advance Biomanufacturing

“…In a related work by the same group, an unstructured cell culture model with a similar antibody assembly equations was expanded to include N‐glycosylation of the secreted mAb . In a more recent work, a nucleotide sugar synthesis network was further incorporated into the model structure to investigate the impact of mild hypothermia on cell growth, mAb productivity, and product quality in terms of N‐glycans . Without considering the antibody assembly mechanisms, Jedrzejewski et al coupled the biosynthetic pathways of nucleotide and nucleotide sugars with an unstructured Monod ‐type model and a pre‐existing Golgi model to simulate the glycan structures of a monoclonal antibody produced in a fed‐batch culture of hybridoma cells .…”