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

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.

Bax and Bak knockout apoptosis‐resistant Chinese hamster ovary cell lines significantly improve culture viability and titer in intensified fed‐batch culture process

“…Compared to standard mAb molecules, the production of bispecific or complex antibodies is more challenging due to their non‐standard format, which can pose additional manufacturing problems such as product instability, undesired byproduct species, higher product fragment or aggregation levels, and low expression levels. The non‐natural format of bispecific antibodies or complex molecules increases the chance of molecule misfolding and disulfide bond mispairing, causing higher levels of intracellular reactive oxygen species (ROS) accumulation and oxidative stress inside the cell, which eventually leads to low VCD, viability and productivity 15 . We hence tested whether Bax/Bak DKO cell lines can help to mitigate these issues.…”

“…Apoptosis happens more frequently during the intensified production processes when the cell density is high, likely due to elevated risk of hypoxia, shear stress, nutrient deprivation, and faster accumulation of toxic metabolic by-products produced by the cells 20 including inhibitory metabolites (e.g., isovalerate and formic acid) 30 and ROS. 31,32 Perfusion techniques can be used to reduce these cellular stresses by reducing intracellular ROS accumulation, 15 and removing inhibitory metabolites from the cell culture while continuously providing the culture with oxygen and nutrients. 7 However, because of the complex process control and large volumes of media required, perfusion cell culture is generally less ideal.…”

“…The non-natural format of bispecific antibodies or complex molecules increases the chance of molecule misfolding and disulfide bond mispairing, causing higher levels of intracellular reactive oxygen species (ROS) accumulation and oxidative stress inside the cell, which eventually leads to low VCD, viability and productivity. 15 We hence tested whether Bax/Bak DKO cell lines can help to mitigate these issues.…”

“…5 In recent studies, process intensification was demonstrated to improve titer in both continuous 6,7 and fed-batch processes. 4,[7][8][9][10][11][12] Continuous cell culture with perfusion (i.e., perfusion production) [13][14][15] and semi-continuous cell culture or hybrid fed-batch with perfusion culture 16 have all been successfully validated for process intensification purposes. However, perfusion cell culture in production bioreactors has not been widely applied because of the complex process control, large volumes of perfusion media, and challenges for process development and process characterization.…”

CDMOs Play a Critical Role in the Biopharmaceutical Ecosystem

“…Continuous culture may improve product quality. The perfusion process produces significantly lower bispecific antibody aggregates compared to the fed-batch process ( Sinharoy et al, 2020 ).…”