Elucidating the effects of pH shift on IgG1 monoclonal antibody acidic charge variant levels in Chinese hamster ovary cell cultures

Abstract

Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies' properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography. It is found that acidic charge variant contents were significantly decreased by pH downshift. Then, to reveal the mechanism by which the content of acidic charge variants is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift.

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

“…Operational changes and processing conditions are usually the first modifications (Table ) employed to reduce charge heterogeneity as these are easily implemented in manufacturing. Temperature control, dissolved oxygen (DO), pH control, media concentration, and glucose control all impact acidic and basic charge species and are already optimized by PD teams (Abu‐Absi et al, ; Horvath et al, ; Kishishita et al, ; Mallaney, Wang, & Sreedhara, ; Xie et al, ; Yang et al, ; Yoon et al, ; Zhang et al, ). DOE studies are often employed to assess the combination of parameter interactions and the impact of these interactions on cell performance and product quality.…”

“…Operational changes and processing conditions are usually the first modifications (Table 4) Kishishita et al, 2015;Mallaney, Wang, & Sreedhara, 2014;Xie et al, 2016;Yang et al, 2016;Yoon et al, 2003;Zhang et al, 2015). DOE studies are often employed to assess the combination of parameter interactions and the impact of these interactions on cell performance and product quality.…”

“…Although pH is an effective factor to control charge heterogeneity, tight pH control may lead to excessive amount of CO 2 sparging and may negatively impact other process performance such as titer. Alternatively, Xie et al (2016) employed a pH downshift on day 6 and observed reduced acidic charge variants as a result of decreased disulfide bond reduction, glycosylation, and Asn deamination. Glucose and galactose control is important for controlling glycation and its subsequent contribution to the acidic peak species (Gramer, 2013).…”

Main Quality Attributes of Monoclonal Antibodies and Effect of Cell Culture Components

“…It has been revealed that they can change the binding to proteins or cell membrane targets, thereby affecting the tissue penetration, tissue distribution, and pharmacokinetics of the antibodies[20,55-58]. There is enough evidence in the literature to recommend that the existence of acidic species variants on mAbs can at least have an effect on the resulting protein’s efficacy and function[59-61]. The impacts of the charge variants depend highly on the nature, site, and the amount of post-translational modifications that cause the acidic and basic variants’ formation[62].…”

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

“…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 …”