Characterization of charge variants of a monoclonal antibody using weak anion exchange chromatography at subunit levels

Ponniah, Gomathinayagam; Nowak, Christine; Neill, Alyssa; Liu, Hongcheng

doi:10.1016/j.ab.2016.12.017

Cited by 36 publications

(37 citation statements)

References 45 publications

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“…Interestingly, even IEX-based techniques may produce conflicting results. Ponniah, Nowak, Neill, and Liu (2017) saw the succinimide species eluted as acidic species in AEX column, which is contrary to previous reports Yan et al, 2009;Yu et al, 2011). The authors suggest this may be due to succinimide formation, which causes conformational changes that affect surface charge distributions, although there are no follow-up reports to this study.…”

Section: Isomerization Aspcontrasting

confidence: 94%

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Chung

Tian

Tan

et al. 2018

Biotech & Bioengineering

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Controlling the charge profile of therapeutic protein is a critical challenge in the current quality-by-design (QbD) paradigm, throughout all phases of biologics process development (PD): cell line development, upstream cell culture, recovery process, downstream purification, and analytical characterization. Charge variant profiles may influence efficacy and/or lead to unintended side-effects. Thus, maintaining a consistent charge profile is of tremendous importance, and increasingly, researchers have focused efforts toward developing strategies to mitigate variability during cell culture and to improve separation and detection of charge variants. Current understanding of factors affecting charge variant formation during manufacturing remains inadequate, and sometimes, even substantial commitment of resources may still not fully achieve the desired or consistent profiles. As such, this review attempts to provide a comprehensive resource for the biologics community by summarizing the impact of charge variants and CQA management, analytical methods for charge variant detection, as well as strategies in downstream and upstream PD for controlling charge variant profiles.

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Section: Isomerization Aspcontrasting

confidence: 94%

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Chung

Tian

Tan

et al. 2018

Biotech & Bioengineering

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“…The presence of two peaks indicating single deamidation could be explained by the formation of both, aspartic acid and isoaspartic acid, which have previously been shown to be distinguishable via charge sensitive separation methods. 26,27,31 The presence of deamidated forms is not only suggested on the intact level, but also confirmed via peptide mapping of the two most abundant acidic variants after preparative peak collection. Compared to the main peak these charge variants showed a total incident of asparagine (Asn)329 deamidation of~31% and~25%, respectively (Figure 7d).…”

Section: Deamidation and Isomerisationmentioning

confidence: 75%

“…Succinimide formation in mAbs has previously been described to yield in an increase in protein pI and was also positively correlated to exposure of the molecule to certain environmental conditions, such as acidic pH and elevated temperature. [25][26][27] Succinimide formation of Asp would result in the loss of one acidic residue and would, in consequence, explain an increase in retention on the SCX stationary phase. To investigate our hypothesis further, LC-MS based peptide mapping of the drug substance was performed and revealed the occurrence of succinimide Asp on several residues of up to 1.4% (data not shown).…”

Section: Lysine Truncationmentioning

confidence: 99%

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Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry

Füssl

Trappe

Cook

et al. 2018

mAbs

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Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. We further explored and adapted the chromatographic buffer system to expand the application range. Moreover, we observed no salt adducts on the native protein, also supported by the optimal choice of MS parameters, resulting in increased data quality and mass accuracy. Here, we demonstrate the utility of this improved method by performing an in-depth analysis of adalimumab before and after forced degradation. By combining molecular mass and retention time information, we were able to identify multiple modifications on adalimumab, including lysine truncation, glycation, deamidation, succinimide formation, isomerisation, N-terminal aspartic acid loss or C-terminal proline amidation and fragmentation along with the N-glycan distribution of each of these identified proteoforms. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation.

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“…29 These control strategies modulated the charge profile typically by reducing the deamidation, glycation, or oxidation levels, based on information from in-house peptide mapping and literature reports. 7,[30][31][32] However, a clear and direct link is still missing on how exactly these factors influence these modifications. These three reactions are all chemically oxidative reactions that result in the loss of one electron and formation of ammonia (deamidation) or water (oxidation and glycation).…”

Section: Introductionmentioning

confidence: 99%

Modulating cell culture oxidative stress reduces protein glycation and acidic charge variant formation

Chung

Tian

Tan

et al. 2019

mAbs

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Controlling acidic charge variants is critical for an industrial bioprocess due to the potential impact on therapeutic efficacy and safety. Achieving a consistent charge variant profile at manufacturing scale remains challenging and may require substantial resources to investigate effective control strategies. This is partially due to incomplete understanding of the underlying causes for charge variant formation during the cell culture process. To address this gap, we examined the effects of four process input factors (temperature, iron concentration, feed media age, and antioxidant (rosmarinic acid) concentration) on charge variant profile. These factors were found to affect the charge profile by modulating the cell culture oxidative state. Process conditions with higher acidic peaks corresponded to elevated supernatant peroxide concentration, intracellular reactive oxygen species (ROS) levels, or both. Changes in glycation level were the primary cause of the charge heterogeneity, and for the first time, supernatant peroxide was found to positively correlate with glycation levels. Based on these findings, a novel mathematical model was developed to demonstrate that the rate of acidic species formation was exponentially proportional to the concentrations of supernatant peroxide and protein product. This work provides critical insights into charge variant formation during the cell culture process and highlights the importance of modulating of cell culture oxidative stress for charge variant control.

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Characterization of charge variants of a monoclonal antibody using weak anion exchange chromatography at subunit levels

Cited by 36 publications

References 45 publications

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry

Modulating cell culture oxidative stress reduces protein glycation and acidic charge variant formation

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