Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests

Vanhoenacker, Gerd; Vandenheede, Isabel; David, Frank; Sandra, Pat; Sandra, Koen

doi:10.1007/s00216-014-8299-1

Cited by 76 publications

(61 citation statements)

References 21 publications

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“…Now, this prior knowledge in the literature is being applied and extended as researchers use 2D-LC to detect and understand small differences between therapeutic proteins. For example, Vanhoenacker, Sandra, et al have used 2D-LC to rapidly determine which amino acid residues of trastuzumab were deamidated as a result of pH stress of the protein molecule [16]. As part of this work, 2D-LC separations involving RPLC separations in both dimensions were shown to be superior to those involving 1D IEX separations.…”

Section: Peptide Level Analysismentioning

confidence: 88%

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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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Section: Peptide Level Analysismentioning

confidence: 88%

“…This is very attractive for biopharmaceutical analysis. 2D-LC-MS has already been evaluated and implemented in many R&D groups of biopharmaceutical companies [4,[12][13][14]16,22]. Future development of 2D-LC-MS will further simplify the instrumentation, method development and data analysis.…”

Section: Discussionmentioning

confidence: 99%

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

Wang¹,

Buckenmaier²,

Stoll³

2017

J Appl Bioanal

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show abstract

“…These fractions are then either infused directly into the mass spectrometer, or further separated using a different mode of LC, resulting in a two-dimensional separation [46]. An example of such an experiment involves analysis of a reduced, alkylated, and digested isolate of a mixture of proteins first by IEX LC, followed by online RPLC-ESI-MS/MS analysis [75]. Identification of individual protein groups is performed by comparing the identified peptides to sequences of the theoretical proteins contained within a genome-specific database.…”

Section: Bottom-up Proteomicsmentioning

confidence: 99%

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

Belov¹

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“…This combination requires fast second dimension separations (typically <1 min). The possibilities of using LC × LC for peptide mapping of therapeutic mAbs has recently been described [32]. As exemplified using Herceptin and a biosimilar in development, LC × LC was shown to be an attractive technique for identity, purity and comparability assessment.…”

Section: Introductionmentioning

confidence: 99%