Rapid analysis of charge variants of monoclonal antibodies using non-linear salt gradient in cation-exchange high performance liquid chromatography

Joshi, Varsha; Kumar, Vijesh; Rathore, Anurag S.

doi:10.1016/j.chroma.2015.06.015

Cited by 43 publications

(28 citation statements)

References 37 publications

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“…IEX is the preferred technique for preparative and process scale separation for charge variants. While bench‐top analytical scale resolution has improved tremendously (Amand et al, ; Farnan & Moreno, ; Fekete et al, , ; Follman & Fahrner, ; Joshi, Kumar, & Rathore, ; Kang, Kutzko, Hayes, & Frey, ; Lingg et al, ; Ng & Snyder, ; Nordborg et al, ; Rea, Moreno, Lou, & Farnan, ; Santora, Kaymakcalan, Sakorafas, Krull, & Grant, ; Talebi et al, ; Talebi, Shellie, Hilder, Lacher, & Haddad, ; Tao, Carta, Ferreira, & Robbins, , ; Tao, Perez, Carta, Ferreira, & Robbins, ; Weitzhandler et al, ; Zhang, Patapoff, Farnan, & Zhang, ), industrial separation of charge variants remains challenging due to the scale and capacity required. For most industrial processes, the stationary phase, physical parameters of the columns, are already fixed.…”

Section: Separation and Purification Of Charge Variants In Downstreammentioning

confidence: 99%

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: Separation and Purification Of Charge Variants In Downstreammentioning

confidence: 99%

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Chung

Tian

Tan

et al. 2018

Biotech & Bioengineering

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“…(Fig. 1 clearly recognizable more acidic and more basic peaks [27][28][29]. In the Prolia sample only very limited amounts of acidic and basic variants were visible.…”

Section: Initial Screening Of Charge Variants Profile Of Therapeutic mentioning

confidence: 99%

“…(Fig. 1) Arzerra, Avastin, Erbitux, Herceptin and Xolair showed the typical profile, expected for IgG molecules, consisting of one main peak, flanked by some clearly recognizable more acidic and more basic peaks [27][28][29]. In the Prolia sample only very limited amounts of acidic and basic variants were visible.…”

Section: Initial Screening Of Charge Variants Profile Of Therapeutic mentioning

confidence: 99%

Microheterogeneity of therapeutic monoclonal antibodies is governed by changes in the surface charge of the protein

Hintersteiner

Lingg

Janzek

et al. 2016

Biotechnology Journal

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It has previously been shown for individual antibodies, that the microheterogenity pattern can have a significant impact on various key characteristics of the product. The aim of this study was to get a more generalized understanding of the importance of microheterogeneity. For that purpose, the charge variant pattern of various different commercially available therapeutic mAb products was compared using Cation-Exchange Chromatography with linear pH gradient, antigen affinity, Fcreceptor affinity, antibody dependent cellular cytotoxicity (ADCC) and conformational stability. For three of the investigated antibodies, the basic charge variants showed a stronger binding affinity towards FcγRIIIa as well as an increased ADCC response. Differences in the conformational stability of antibody charge variants and the corresponding reference samples could not be detected by differential scanning calorimetry. The different biological properties of the mAb variants are therefore governed by changes in the surface charge of the protein and not by an altered structure. This can help to identify aspects of microheterogeneity that are critical for product quality and can lead to further improvements in the development and production of therapeutic antibody products. Keywords: ADCC · Bio-better · Cation exchange chromatography · Effector functions · FcγRIIIa · Therapeutic mAbsCorrespondence: Prof. Alois Jungbauer, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria E-mail: alois.jungbauer@boku.ac.at Abbreviations: ADCC, antibody dependent cellular cytotoxicity; ApHGE, analytical ionexchange chromatography with pH gradient elution; CEX, Cation exchange chromatography; EGFR, epidermal growth factor receptor; mAb, monoclonal antibody; DSC, differential scanning calorimetry; SPR, surface plasmon resonance ants it is still difficult to decide, how precisely a product produced by different methods must match [14][15][16][17][18]. Cation exchange chromatography (CEX) with pH gradient elution has repeatedly been reported as a suitable method for high-resolution separation of antibody variants exhibiting different surface charge characteristics [19][20][21][22]. In this study we used this technique for the analysis and the large-scale separation of several highselling therapeutic antibodies available on the market today, to obtain one acidic (A), one main (M) and one basic (B) charge variant fraction, a methodology previously developed in our group [23,24]. This did not only provide new information on the charge variant pattern of individual monoclonal antibody products, it also gave us the chance to characterize the charge variants found in these antibody products with a wide variety of additional analytical methods and thereby study the overall effect of the presence of charge variants on the stability and biological activity of therapeutic mAbs in general.As starting material, we obtained clinical doses of the seven monoclonal antibodies listed in Table 1, all of w...

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“…The flow rate used was of 0.2 mL/min with total analysis time of 30 min. For mAb charge variant analysis, CEX eluted fractions were analyzed using Cation Exchange Ultra Performance Liquid Chromatography (CE‐UPLC) using MAbPac SCX‐10 RS, BioLC™ (5 μm, 4.6 × 250 mm) procured from Thermo Fisher Scientific with a sigmoidal gradient method that has been published elsewhere …”

Section: Methodsmentioning

confidence: 99%

Design of experiments applications in bioprocessing: Chromatography process development using split design of experiments

Shekhawat

Godara

Kumar

et al. 2018

Biotechnology Progress

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Development of a chromatographic step in a time and resource efficient manner remains a serious bottleneck in protein purification. Chromatographic performance typically depends on raw material attributes, feed material attributes, process factors, and their interactions. Design of experiments (DOE) based process development is often chosen for this purpose. A challenge is, however, in performing a DOE with such a large number of process factors. A split DOE approach based on process knowledge in order to reduce the number of experiments is proposed. The first DOE targets optimizing factors that are likely to significantly impact the process and their effect on process performance is unknown. The second DOE aims to fine‐tune another set of interacting process factors, impact of whom on process performance is known from process understanding. Furthermore, modeling of a large set of output response variables has been achieved by fitting the output responses to an empirical equation and then using the parametric constants of the equation as output response variables for regression modeling. Two case studies involving hydrophobic interaction chromatography for removal of aggregates and cation exchange chromatography for separation of charge variants and aggregates have been utilized to illustrate the proposed approach. Proposed methodology reduced total number of experiments by 25% and 72% compared to a single DOE based on central composite design and full factorial design, respectively. The proposed approach is likely to result in a significant reduction in resources required as well as time taken during process development. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2730, 2019

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Rapid analysis of charge variants of monoclonal antibodies using non-linear salt gradient in cation-exchange high performance liquid chromatography

Cited by 43 publications

References 37 publications

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Microheterogeneity of therapeutic monoclonal antibodies is governed by changes in the surface charge of the protein

Design of experiments applications in bioprocessing: Chromatography process development using split design of experiments

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