Characterization of therapeutic antibodies and related products by two-dimensional liquid chromatography coupled with UV absorbance and mass spectrometric detection

Stoll, Dwight R.; Danforth, John; Zhang, Kelly; Beck, Alain

doi:10.1016/j.jchromb.2016.05.029

Cited by 75 publications

(46 citation statements)

References 73 publications

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“…[17][18][19][20][21] For analytical purposes, powerful two-dimensional liquid chromatography mass spectrometry (2D-LC-MS) methods are emerging combining various LC modes, such as CEX, size exclusion chromatography (SEC), hydrophilic and hydrophobic interaction chromatography (HILIC and HIC), or reversed phase (RP), and RP-LC as second dimension, which can be coupled to MS for detection. [22] The antibody to be analyzed can either be the intact antibody (top down), or be enzymatically cleaved into larger parts (middle down), that is, Fc and Fab' fragments with specific proteases such as IdeS, or be cleaved into peptides (bottom up). The most powerful multi-dimensional method for antibody microheterogeneity combines CEX as the first dimension and RP as the second chromatographic dimension.…”

Section: Methods For Microheterogeneity Analysismentioning

confidence: 99%

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%

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Beyer

Schuster

Jungbauer

et al. 2017

Biotechnology Journal

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“…Similar 2D methods can also probably be applied (or at least can be a good starting point) for ADCs as for their parent mAbs [75]. However, up to now, only a limited number of applications have been published.…”

Section: Multidimensional Separationmentioning

confidence: 99%

Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates

Bobály

Fleury-Souverain²,

Beck

et al. 2018

Journal of Pharmaceutical and Biomedical Analysis

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Antibody Drug Conjugates (ADCs) are innovative biopharmaceuticals gaining increasing attention over the last two decades. The concept of ADCs lead to new therapy approaches in numerous oncological indications as well in infectious diseases. Currently, around 60 CECs are in clinical trials indicating the expanding importance of this class of protein therapeutics. ADCs show unprecedented intrinsic heterogeneity and address new quality attributes which have to be assessed. Liquid chromatography is one of the most frequently used analytical method for the characterization of ADCs. This review summarizes recent results in the chromatographic characterization of ADCs and supposed to provide a general overview on the possibilities and limitations of current approaches for the evaluation of drug load distribution, determination of average drug to antibody ratio (DAR), and for the analysis of process/storage related impurities. Hydrophobic interaction chromatography (HIC), reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC) and multidimensional separations are discussed focusing on the analysis of marketed ADCs. Fundamentals and aspects of method development are illustrated with applications for each technique. Future perspectives in hydrophilic interaction chromatography (HILIC), HIC, SEC and ion exchange chromatography (IEX) are also discussed.

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“…Third, a well-developed 2D-LC separation may yield more analytically useful information than two different separation modes operated independently. Several different combinations of separation modes have been applied to separations of therapeutic proteins and related materials in creative ways; these have been discussed in detail elsewhere [3]. Here, we briefly discuss a few examples that are representative of recent work in this area.…”

Section: Protein Level Analysismentioning

confidence: 99%

“…• First, in liquid chromatography (LC) two-dimensional liquid chromatography (2D-LC) has emerged as one of the most active areas of technology advancement [1][2][3]. In 2D-LC, a conventional separation is carried out in the first dimension and aliquots of the effluent are collected and injected to a second-dimension column that has very different separation selectivity compared to the first-dimension column.…”

Section: Introductionmentioning

confidence: 99%

The growing role of two-dimensional LC in the biopharmaceutical industry

Wang¹,

Buckenmaier²,

Stoll³

2017

J Appl Bioanal

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IntroductionOver the past two decades, three major trends have been observed in the bioanalysis world.• First, in liquid chromatography (LC) two-dimensional liquid chromatography (2D-LC) has emerged as one of the most active areas of technology advancement [1][2][3]. In 2D-LC, a conventional separation is carried out in the first dimension and aliquots of the effluent are collected and injected to a second-dimension column that has very different separation selectivity compared to the first-dimension column. Therefore, much higher peak capacity, and thus resolving power, can be achieved in 2D-LC compared to 1D-LC. This increased resolving power can be used to increase the likelihood of separating a complex mixture, or decrease the time required to fully separate simpler mixtures. In addition, 2D-LC allows the coupling of two completely different separation modes (e.g. reversed-phase and ionic exchange) in one method. This makes it possible to measure multiple attributes of a target analyte in one method instead of two separate methods. Although 2D-LC research has been going on for more than three decades, the speed of innovation and commercialization has accelerated in the last ten years due to efforts at both universities and instrument companies.• Second, Mass Spectrometry (MS) has become an indispensable tool in bioanalysis [4]. The ability of new MS instruments to more accurately characterize large molecules keeps improving. However, several challenges still remain. MS works best with volatile buffers of limited concentration. The ionization suppression effect still occurs when compounds with very different concentrations (e.g. several orders of magnitude) co-elute in chromatographic separations. These challenges to MS make LC separation a critical part of any bioanalysis workflow.• Finally, in the application area, biopharmaceutical research has become the fastest growing area in the pharmaceutical industry. In particular, monoclonal antibodies (mAbs) are currently the most important class of biotherapeutic molecules. As of 2016, seven of the top-ten-selling drugs were biologics, and six of these were mAb related [5]. In particular, the number one drug Humira (adalimumab) had an annual sales of $15.7 Billion dollars in 2016. Due to the large size of these antibodies at about 150 kDa molecular weight, analyzing them is very challenging. It often takes a suite of analytical tools to fully characterize the molecule and ensure good quality control of drug products involving these molecules. These three trends are developing at the same time and at high speed. It is our opinion that the combination of 2D-LC with MS is emerging as an exciting and essential tool for efficient, high quality biopharmaceutical analysis. In this article, we will discuss examples that demonstrate the power of 2D-LC-MS in this application area.

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Characterization of therapeutic antibodies and related products by two-dimensional liquid chromatography coupled with UV absorbance and mass spectrometric detection

Cited by 75 publications

References 73 publications

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates

The growing role of two-dimensional LC in the biopharmaceutical industry

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