Advances in Biopharmaceutical Analysis

An introduction from the guest editors, Pat Sandra and Koen Sandra of the Research Institute for Chromatography, focusing on recent developments in biopharmaceutical analysis.

When we were asked to edit a follow-up to the LCGC Europe May 2013 supplement “Advances in Pharmaceutical Analysis,” we immediately wanted to highlight the challenges in biopharmaceutical analysis. Indeed, within the pharmaceutical industry and also within our own research activities related to pharmaceutical analysis, there has been a remarkable shift from small to large molecules. On the market since the early 1980s, protein biopharmaceuticals have seen an enormous growth in the last decade. It is even expected that within the current decade, more than 50% of new drug approvals will be biological in nature. A dominant role is thereby played by monoclonal antibodies (mAbs), of which a substantial number have reached blockbuster status. The top 10 bestselling pharmaceuticals are currently heavily populated by mAbs.

Protein biopharmaceuticals are large and heterogeneous and their in-depth analysis during development and also during their lifetime requires the best of both chromatography and mass spectrometry (MS). In editing this special issue, we have therefore selected authorities in the field to illustrate the state of the art in biopharmaceutical analysis.

The first contribution, authored by Szabolcs Fekete, Jean-Luc Veuthey, and Davy Guillarme, provides an overview of the different liquid chromatography (LC) column formats recently introduced in the market for reversed-phase, size-exclusion (SEC), ion-exchange, and hydrophobic interaction (HIC) chromatographic analyses of therapeutic proteins, mAbs, and antibody–drug conjugates (ADCs).

In the May 2013 supplement we described the features of liquid chromatography coupled to mass spectrometry (LC–MS) in the characterization of protein biopharmaceuticals. With the patents of the first generation protein biopharmaceuticals expired and blockbuster mAbs appearing on the market, activities in biosimilars have exploded in recent years. More than 15 biosimilars have already been approved in Europe, and a version of filgrastim was launched in the United States as the first biosimilar toward the end of 2015. Analytical methods to compare originators with biosimilars are highlighted in the second contribution from our team at the Research Institute for Chromatography.

The antibody market has been reshaped by various next-generation formats (biospecific mAbs, antibody mixtures, nanobodies, brain penetrant mAbs, glyco-engineered formats), and in recent years the ADCs brentuximab vedotin and trastuzumab emtansine have been approved by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA). In ADCs, a cytotoxin is coupled to an antibody that specifically targets a certain tumor marker. As such, highly toxic drugs can be delivered in a targeted fashion to tumor cells without affecting healthy cells. Compared to naked mAbs, the conjugation of cytotoxic drugs further adds to the complexity. The power of MS to unravel this complexity is illustrated in the paper authored by Alain Beck and by Sarah Cianferani (available online at: www.chromatographyonline.com/harnessing-benefits-mass-spectrometry-depth-antibody-drug-conjugates-analytical-characterization-0).

The previous two contributions clearly illustrate the importance of MS in the elucidation of the primary structure of therapeutic proteins. Higher order elements, on the other hand, can be derived from special MS technologies such as native MS, ion mobility MS, hydrogen–deuterium exchange MS, and chemical cross-linking MS. In the third contribution, Christian Huber describes the basic principles of these techniques and illustrates their features for the characterization of higher order structures of some protein biopharmaceuticals.

Traditionally, ligand-binding assays (LBAs) are applied to study the pharmacokinetic behavior of protein biopharmaceuticals in biological fluids. LBAs are characterized by a high throughput and sensitivity, but may suffer from long development times and potential interferences from other proteins present in the matrix. In addition, the generation of drug-specific antibody tools is a time-consuming process. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) methods are used more and more as alternatives to LBAs, often offering improved figures-of-merit while at the same time being generically applicable. Some of the technicalities and advantages and disadvantages of LC–MS/MS compared to LBAs for monitoring biopharmaceuticals in biological fluids are addressed in the fourth contribution by Nico C. van de Merbel.

The presence of residual host cell proteins (HCPs) is a potential safety risk in any biopharmaceutical product. Despite enormous purification efforts, these HCPs may be left behind from the expression hosts. HCPs are normally dosed during downstream processing and in the final biopharmaceutical product by enzyme-linked immunosorbent assays (ELISA). As mentioned in the previous paper, LBAs are more and more complemented or even replaced by LC–MS/MS and this is illustrated in the last contribution by our group. The use of off-line two-dimensional LC–MS/MS in the characterization of HCPs is described and the added value of using multidimensional chromatography is clearly demonstrated.

We hope that the contributions in this supplement are of interest and even a source of inspiration to the numerous analysts in the biopharmaceutical industry. It was a pleasure for us to edit and review the contributions of outstanding (preselected) colleagues. We would like to thank all of them for their excellent work.