Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range: Validation of the method parameters

Lingg, Nico; Berndtsson, Martina; Hintersteiner, Beate; Schuster, Manfred; Bardor, Muriel; Jungbauer, Alois

doi:10.1016/j.chroma.2014.11.021

Cited by 30 publications

(27 citation statements)

References 29 publications

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“…The flow rate was 10 ml/min (158 cm/h). The mobile phase used was a pH gradient buffer system as described in Lingg et al 16 . and shown in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors

Hintersteiner

Lingg

Zhang

et al. 2016

mAbs

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We identified active isoforms of the chimeric anti-GD2 antibody, ch14.18, a recombinant antibody produced in Chinese hamster ovary cells, which is already used in clinical trials.1,2,3 We separated the antibody by high resolution ion-exchange chromatography with linear pH gradient elution into acidic, main and basic charge variants on a preparative scale yielding enough material for an in-depth study of the sources and the effects of microheterogeneity. The binding affinity of the charge variants toward the antigen and various cell surface receptors was studied by Biacore. Effector functions were evaluated using cellular assays for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Basic charge variants showed increased binding to cell surface receptor FcγRIIIa, which plays a major role in regulating effector functions. Furthermore, increased binding of the basic fractions to the neonatal receptor was observed. As this receptor mediates the prolonged half-life of IgG in human serum, this data may well hint at an increased serum half-life of these basic variants compared to their more acidic counterparts. Different glycoform patterns, C-terminal lysine clipping and N-terminal pyroglutamate formation were identified as the main structural sources for the observed isoform pattern. Potential differences in structural stability between individual charge variant fractions by nano differential scanning calorimetry could not been detected. Our in-vitro data suggests that the connection between microheterogeneity and the biological activity of recombinant antibody therapeutics deserves more attention than commonly accepted.

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“…The flow rate was 10 ml/min (158 cm/h). The mobile phase used was a pH gradient buffer system as described in Lingg et al 16 . and shown in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors

Hintersteiner

Lingg

Zhang

et al. 2016

mAbs

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“…The flow rate was 10 mL/min (158 cm/h). The mobile phase used was a pH gradient buffer system as described in Lingg et al [24] (listed in Supporting information). For Herceptin buffer system 1 was used, for Arzerra, Avastin, Erbitux and Xolair buffer system 2 was used.…”

Section: Preparative and Analytical Scale Cationexchange Chromatograpmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

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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“…Operating parameters which were examined in this study include salt concentration (50 mM–150 mM NaCl), linear pH gradient (0.1, 0.15, and 0.2 pH units/CV), equilibration salt (NaCl, Na 2 SO 4 , and (NH 4 ) 2 SO 4 ), and resin chemistry (Poros HS and Eshmuno CPX). Elution in the CEX column was achieved using a linear pH gradient as it has been shown to deliver a superior separation of charge variants . To compare the selectivity toward separation of charge variants and HMWI across different resins and operating conditions, cumulative plots for acidic species, basic species, and HMWI were constructed.…”

Section: Resultsmentioning

confidence: 99%

Integrated Chromatographic Platform for Simultaneous Separation of Charge Variants and Aggregates from Monoclonal Antibody Therapeutic Products

Kateja

Kumar

Godara

et al. 2017

Biotechnology Journal

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Achieving consistent product quality of a biotherapeutic is a major target for any biopharmaceutical manufacturer, even more for a biosimilar producer as comparability with the innovator product is a regulatory expectation. The complexity of biotherapeutic products and their tedious manufacturing processes, however, make this a non-trivial exercise. The primary motivation of this work is to develop an integrated chromatographic platform for purification of monoclonal antibody (mAb) therapeutics that can deliver the desired separation of both charge variants and aggregates, in addition to the process related impurities like host cell proteins (HCP) and host cell DNA. To achieve the same, an integrated two-stage chromatographic process platform consisting of cation exchange chromatography and multimodal chromatography is being proposed. The versatility of the proposed platform has been successfully demonstrated for three different mAbs. It have been shown that in each case charge variant separation is achieved with the required clearance of aggregates (<1%), HCP (<10 ppm), and DNA (<5 ppb). Moreover, the proposed platform is conducive to use for development of a continuous process and offers smaller process time, lower buffer utilization, and decreased operational costs when compared to the conventional purification platforms.

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Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range: Validation of the method parameters

Cited by 30 publications

References 29 publications

Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors

Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors

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

Integrated Chromatographic Platform for Simultaneous Separation of Charge Variants and Aggregates from Monoclonal Antibody Therapeutic Products

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