Perfusion reduces bispecific antibody aggregation via mitigating mitochondrial dysfunction-induced glutathione oxidation and ER stress in CHO cells

Pritam Sinharoy,Sanjeev Ahuja,Aaron H. Aziz,Natalia I. Majewska,Michael W. Handlogten

doi:10.1038/s41598-020-73573-4

Pritam Sinharoy, Sanjeev Ahuja + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-73573-4

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Abstract

One major challenge observed for the expression of therapeutic bispecific antibodies (BisAbs) is high product aggregates. Aggregates increase the risk of immune responses in patients and therefore must be removed at the expense of purification yields. BisAbs contain engineered disulfide bonds, which have been demonstrated to form product aggregates, if mispaired. However, the underlying intracellular mechanisms leading to product aggregate formation remain unknown. We demonstrate that impaired glutathione regulation underlies BisAb aggregation formation in a CHO cell process. Aggregate formation was evaluated for the same clonal CHO cell line producing a BisAb using fed-batch and perfusion processes. The perfusion process produced significantly lower BisAb aggregates compared to the fed-batch process. Perfusion bioreactors attenuated mitochondrial dysfunction and ER stress resulting in a favorable intracellular redox environment as indicated by improved reduced to oxidized glutathione ratio. Conversely, mitochondrial dysfunction-induced glutathione oxidation and ER stress disrupted the intracellular redox homeostasis, leading to product aggregation in the fed-batch process. Combined, our results demonstrate that mitochondrial dysfunction and ER stress impaired glutathione regulation leading to higher product aggregates in the fed-batch process. This is the first study to utilize perfusion bioreactors as a tool to demonstrate the intracellular mechanisms underlying product aggregation formation.

Highlights

Parameters Temperature (°C) pH ± dead band % Dissolved oxygen Agitation (RPM) Glucose levels Feed timing Perfusion rate
Our data demonstrates that the perfusion process increased peak viable cell density (VCD) by ~ threefold when compared to the fed-batch process (Fig. 1A)
The average cell volume in fed-batch bioreactors were comparatively higher than perfusion bioreactors during the stationary phase, we did not observe any significant difference in cell size between the processes overall (Fig. 1F)

Summary

Introduction

Parameters Temperature (°C) pH ± dead band % Dissolved oxygen Agitation (RPM) Glucose levels Feed timing Perfusion rate. Our hypothesis was that oxidative stressinduced aberrant redox homeostasis contributes to higher aggregate formation in a fed-batch process than a perfusion process This is the first study to investigate and compare fed-batch and perfusion bioreactors at the intracellular levels to gain a perspective on the key regulators contributing to intracellular aggregate formation. We compared the intracellular redox environment of both processes by investigating mitochondrial contribution to ROS, intracellular glutathione levels and protein folding in the ER. Imbalanced intracellular glutathione regulation coupled with ER stress led to mis-paired disulfide bond formation, leading to increased intracellular BisAb aggregate formation in fed-batch bioreactors. Mitigation of mitochondrial dysfunction improved intracellular glutathione homeostasis and attenuated mis-paired disulfide bond formation, causing decreased intracellular BisAb aggregate formation in perfusion bioreactors

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