Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function

B, Antes; Amon, Sabine; Rizzi, Andreas; Wiederkum, Susi; Kainer, Manuela; Szolar, Oliver H. J.; Fido, Markus; Kircheis, Ralf; Nechansky, Andreas

doi:10.1016/j.jchromb.2007.01.024

Cited by 78 publications

(57 citation statements)

References 12 publications

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“…[23] Electrophoretic techniques, including flat gel IEF or CE, again combined with mass spectrometry for identification, can be useful in some cases as an alternative to chromatographic separation. [24,25] A comprehensive list of methods for heterogeneity analysis is given in Table 1.…”

Section: Methods For Microheterogeneity Analysismentioning

confidence: 99%

“…[27] Cell based effector function assays are often used to assess the significance that any previously identified differences in antigen or receptor binding may have for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). [24,28] 3. Sources and Effects of Microheterogeneity 3.1.…”

Section: Methods For Microheterogeneity Analysismentioning

confidence: 99%

“…High resolution separation of charge variants, IEX chromatography as orthogonal method to electrophoretic techniques [24,25] Bottom-up LC-MS [24,25] Incomplete signal peptide cleavage…”

Section: N-glycosylationmentioning

confidence: 99%

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Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Beyer

Schuster

Jungbauer

et al. 2017

Biotechnology Journal

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Antibodies are typical examples of biopharmaceuticals which are composed of numerous, almost infinite numbers of potential molecular entities called variants or isoforms, which constitute the microheterogeneity of these molecules. These variants are generated during biosynthesis by so‐called posttranslational modification, during purification or upon storage. The variants differ in biological properties such as pharmacodynamic properties, for example, Antibody Dependent Cellular Cytotoxicity, complement activation, and pharmacokinetic properties, for example, serum half‐life and safety. Recent progress in analytical technologies such as various modes of liquid chromatography and mass spectrometry has helped to elucidate the structure of a lot of these variants and their biological properties. In this review the most important modifications (glycosylation, terminal modifications, amino acid side chain modifications, glycation, disulfide bond variants and aggregation) are reviewed and an attempt is made to give an overview on the biological properties, for which the reports are often contradictory. Even though there is a deep understanding of cellular and molecular mechanism of antibody modification and their consequences, the clinical proof of the effects observed in vitro and in vivo is still not fully rendered. For some modifications such as core‐fucosylation of the N‐glycan and aggregation the effects are clear and should be monitored, but with others such as C‐terminal lysine clipping the reports are contradictory. As a consequence it seems too early to tell if any modification can be safely ignored.

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Section: Methods For Microheterogeneity Analysismentioning

confidence: 99%

Section: Methods For Microheterogeneity Analysismentioning

confidence: 99%

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Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Beyer

Schuster

Jungbauer

et al. 2017

Biotechnology Journal

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“…These variants can either be the result of post-translational modifications, happening during antibody production in cell culture or they can originate from chemical and physical modifications during product purification, formulation or storage [1]. In fact, such modifications have been shown to affect all sorts of quality characteristics of monoclonal antibodies including structural stability, biophysical properties, serum halflife, and biological activity [2,[4][5][6][7][8][9][10][11][12]. Therefore microheterogeneity pattern associated with a given antibody product i.e.…”

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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“…Various modifications are determined by analyzing recombinant monoclonal antibodies at different levels, depending on the molecular weight differences of the modifications. Modifications, such as N-terminal glutamine and glutamate cyclization [3][4][5][6][7][8][9], different types of the conserved N-linked oligosaccharides [5,[7][8][9][10][11][12][13], amino acid truncation and insertion [8,11], cysteinylation [14], C-terminal lysine processing [5,7,11,15,16], fragmentation [12,15,17], glycation [18], oxidation [19,20], and nitration [21] can be directly determined by measurements of the molecular weights of intact antibodies, antibody light chain and heavy chain, and Fab and Fc fragments after papain or lys-C digestion [14]. On the other hand, analysis at the peptide level is normally required to determine the sites of modifications and modifications with small molecular weight differences, such as deamidation [22,23] and amidation [11], which results in a molecular weight difference of only 1 Da.…”

mentioning

confidence: 99%

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

Liu

Gaza-Bulseco

Chumsae

2009

J. Am. Soc. Mass Spectrom.

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Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly. (J Am Soc Mass Spectrom 2009, 20, 2258 -2264) © 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry M ass spectrometry is one of the most indispensable techniques for the characterization of recombinant monoclonal antibodies because most of the modifications that result in heterogeneity and degradation are involved in molecular weight differences [1,2]. Various modifications are determined by analyzing recombinant monoclonal antibodies at different levels, depending on the molecular weight differences of the modifications. Modifications, such as N-terminal glutamine and glutamate cyclization [3][4][5][6][7][8][9], different types of the conserved N-linked oligosaccharides [5,[7][8][9][10][11][12][13], amino acid truncation and insertion [8,11], cysteinylation [14], C-terminal lysine processing [5,7,11,15,16], fragmentation [12,15,17], glycation [18], oxidation [19,20], and nitration [21] can be directly determined by measurements of the molecular weights of intact antibodies, antibody light chain and heavy chain, and Fab and Fc fragments after papain or lys-C digestion [14]. On the other hand, analysis at the peptide level is normally required to determine the sites of modifications and modifications with small molecular weight differences, such as deamidation [22,23] and amidation [11], which results in a molecular weight difference of only 1 Da.Mass spectrometry is commonly coupled with reversedphase high-performance liquid chromatography (RP-HPLC), which desalts the samples and separates different components based on hydrophobicity. The molecular weights of intact antibodies [12,15] and their light chains and heavy chains can be readily measured by on...

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Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function

Cited by 78 publications

References 12 publications

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

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

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

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