Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability

Self Cite

The phenomenon of monoclonal antibody (mAb) interchain disulfide bond reduction during manufacturing processes continues to be a focus of the biotechnology industry due to the potential for loss of product, increased complexity of purification processes, and reduced stability of the drug product. We hypothesized that antibody reduction can be mitigated by controlling the cell culture redox potential and subsequently established a threshold redox potential above which the mAb remained intact and below which there were significant and highly variable amounts of reduced mAb. Using this knowledge, we developed three control schemes to prevent mAb reduction in the bioreactor by controlling the cell culture redox potential via an online redox probe. These control methodologies functioned by increasing the concentration of dissolved oxygen (DO), copper (II) (Cu), or both DO and Cu to maintain the redox potential above the threshold value. Using these methods, we were able to demonstrate successful control of antibody reduction. Importantly, the redox control strategies did not significantly impact the cell growth, viability, mAb production, or product quality attributes including aggregates, C‐terminal lysine, high mannose, deamidation, and glycation. Our results demonstrate that controlling the cell culture redox potential is a simple and effective method to prevent mAb reduction.

Section: Resultsmentioning

confidence: 70%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Online control of cell culture redox potential prevents antibody interchain disulfide bond reduction

Handlogten

Wang

Ahuja

2020

Self Cite

“…Antibody reduction can also impact glycan microheterogeneity (Dionne, Mishra, & Butler, 2017), increase aggregate levels at the low pH viral inactivation step (Chung et al, 2017), and impart color to the product via unwanted reactions with hydroxocobalamin (the active form of vitamin B12) in cell culture media (Derfus et al, 2014;Du, Martin, et al, 2018;Prentice et al, 2013;Vijayasankaran et al, 2013;Vijayasankaran, Varma, Yang, Meier, & Kiss, 2018;Wierzba, Wojciechowska, Trylska, & Gryko, 2016;Xu et al, 2014). Antibody reduction can also impact glycan microheterogeneity (Dionne, Mishra, & Butler, 2017), increase aggregate levels at the low pH viral inactivation step (Chung et al, 2017), and impart color to the product via unwanted reactions with hydroxocobalamin (the active form of vitamin B12) in cell culture media (Derfus et al, 2014;Du, Martin, et al, 2018;Prentice et al, 2013;Vijayasankaran et al, 2013;Vijayasankaran, Varma, Yang, Meier, & Kiss, 2018;Wierzba, Wojciechowska, Trylska, & Gryko, 2016;Xu et al, 2014).…”

mentioning

confidence: 99%

“…Potential strategies to reduce or prevent antibody reduction include lowering the pH of the cell culture (Xie et al, 2016) and clarified harvest , manipulating culture redox potential (Dionne et al, 2017), maintaining a minimum DO in the bioreactor before harvest (Mullan et al, 2011), partially filling the clarified harvest storage bag with air (Mullan et al, 2011), minimizing cell lysis during harvest, sparging of air in the clarified harvest to prevent low DO conditions (Kao, Laird, Schmidt, Wong, & Hewitt, 2009;Mun et al, 2015;Singh, Fishkin, Kitchener, & Meshulam, 2017;Trexler-Schmidt et al, 2010), addition of inhibitors to prevent the Trx system mechanism (Chung et al, 2017;Kao et al, 2009), addition of ethylenediaminetetraacetic acid to chelate Mg 2+ and inhibit hexokinase (HK) (Bruhlmann et al, 2015;Kao et al, 2010;McAtee, Templeton, & Young, 2014;Trexler-Schmidt et al, 2010), addition of copper ions as an oxidizing agent (Chaderjian, Chin, Harris, & Etcheverry, 2005), addition of hydrogen peroxide to maintain oxidative conditions , addition of antireducing agents (Singh et al, 2017), knockdown of Trx-1 expression (Koterba, Borgschulte, & Laird, 2012), and use of low temperature to increase oxygen solubility and lower enzymatic rates (Chung et al, 2017). Potential strategies to reduce or prevent antibody reduction include lowering the pH of the cell culture (Xie et al, 2016) and clarified harvest , manipulating culture redox potential (Dionne et al, 2017), maintaining a minimum DO in the bioreactor before harvest (Mullan et al, 2011), partially filling the clarified harvest storage bag with air (Mullan et al, 2011), minimizing cell lysis during harvest, sparging of air in the clarified harvest to prevent low DO conditions (Kao, Laird, Schmidt, Wong, & Hewitt, 2009;Mun et al, 2015;Singh, Fishkin, Kitchener, & Meshulam, 2017;Trexler-Schmidt et al, 2010), addition of inhibitors to prevent the Trx system mechanism …”

mentioning

confidence: 99%

Impact of depth filtration on disulfide bond reduction during downstream processing of monoclonal antibodies from CHO cell cultures

O’Mara

Gao

Kuruganti

et al. 2019

Monoclonal antibody interchain disulfide bond reduction was observed in a ChineseHamster Ovary manufacturing process that used single-use technologies. A similar reduction has been reported for processes that involved high mechanical shear recovery unit operations, such as continuous flow centrifugation and when the clarified harvest was stored under low dissolved oxygen (DO) conditions (Trexler-Schmidt et al., 2010. Biotechnology and Bioengineering, 106(3), 452-461). The work described here identifies disposable depth filtration used during cell culture harvest operations as a shear-inducing unit operation causing cell lysis. As a result, reduction of antibody interchain disulfide bonds was observed through the same mechanisms described for continuous flow centrifugation. Small-scale depth-filtration models were developed, and the differential pressure (ΔP) of the primary depth filter was identified as the key factor contributing to cell lysis. Strong correlations of ΔP and cell lysis were generated by measuring the levels of lactate dehydrogenase and thiol in the filtered harvest material. A simple risk mitigation strategy was implemented during manufacturing by providing an air overlay to the headspace of a single-use storage bag to maintain sufficient DO in the clarified harvest. In addition, enzymatic characterization studies determined that thioredoxin reductase and glucose-6phosphate dehydrogenase are critical enzymes involved in antibody reduction in a nicotinamide adenine dinucleotide phosphate (NADP + )/NADPH-dependent manner. K E Y W O R D Sair overlay, antibody, cell lysis, depth filtration, differential pressure, dissolved oxygen, disulfide reduction 1 | INTRODUCTION Monoclonal antibodies (mAbs) are commonly manufactured with fed-batch or perfusion mammalian cell culture processes. In such manufacturing processes, the mAb, which is expressed and secreted from the cell, accumulates in the cell culture fluid and is recovered using centrifugation and/or filtration. With the advent of high productivity (>5 g/L) cell culture processes and the availability of improved disposable technologies, the implementation of single-use systems, such as bioreactors, depth filters, columns, membranes, and bioprocess storage bags in the production of biologics has become standard industry practice (Liu, 2005).

Glutathione and thioredoxin systems contribute to recombinant monoclonal antibody interchain disulfide bond reduction during bioprocessing

Handlogten¹,

Zhu

Ahuja³

2017

Self Cite

Antibody interchain disulfide bond reduction during biopharmaceutical manufacturing has received increased attention since it was first reported in 2010. Antibody reduction leads to loss of product and reduced product stability. It is therefore critical to understand the underlying mechanisms of reduction. To date, the thioredoxin system has been reported as the sole contributor to antibody reduction during bioprocessing. In this work, we show that the glutathione system, in addition to the thioredoxin system, is involved in reducing antibody molecules and the contributions of the two systems can vary depending upon the cell culture process. The roles of the glutathione and thioredoxin systems were evaluated for three molecules with different IgG subclass where reduction was observed during manufacturing: mAb A, mAb B, and mAb C representing an IgG , IgG , and IgG respectively. The expression of enzymes for both the thioredoxin and glutathione systems were confirmed in all three cell lines. Inhibitors were evaluated using purified mammalian reductases to evaluate their specificity. The optimized experimental conditions enabled both the determination of reductase activity contributed from as well as the amount of antibody reduced by each enzymatic system. Our results demonstrate that the underlying enzymatic mechanisms are different depending upon the cell culture process; one of the two systems may be the dominant mechanism, or both enzymatic systems may be involved. Specifically, the glutathione system was found to be the major contributor to mAb A reduction while the thioredoxin system was the major contributor to mAb C reduction. Intriguingly, mAb B experienced significant reduction from both enzymatic systems. In summary, we have demonstrated that in addition to the thioredoxin pathway, the glutathione system is a second major pathway contributing to antibody reduction and this knowledge can be leveraged to develop more specific antibody reduction mitigation strategies targeted at the dominant reduction mechanism. Biotechnol. Bioeng. 2017;114: 1469-1477. © 2017 Wiley Periodicals, Inc.