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

Abstract

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.

Glycosylation of biosimilars: Recent advances in analytical characterization and clinical implications

“…While current one-dimensional (1D) separation techniques are reaching their optimal performance, novel two-dimensional (2D) separation techniques provide an interesting alternative approach for the challenging mAb materials [114,115]. 2D approaches allow the combination of two chromatographic techniques, such as IEX, HILIC, RPLC, hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) in a single analytical platform.…”

Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

“…There are several ways to implement 2D-LC, which can be ranked in the order of increasing complexity: off-line 2D-LC involving time-consuming fraction collection (out of the scope of the present review article); on-line heart cutting or multiple heart cutting 2D-LC (LC-LC), when only one or few parts of the 1D chromatogram are sent to the second dimension; and on-line comprehensive 2D-LC (LCxLC), which is considered today as the most powerful and informative 2D-LC mode [173].…”

Recent Advances in Chromatography for Pharmaceutical Analysis

“…278 For this type of demanding application, 2D-LC in combination with MS and even IM-MS 279 will certainly continue its expansion. 24,136,280…”