Columns | Column: Biopharmaceutical Perspectives

In this article, the use of comprehensive two-dimensional liquid chromatography (LC×LC) coupled to mass spectrometry (MS) for characterizing glycosylation of therapeutic enzymes is presented.

The purpose of this short tutorial article is to review the terms and official nomenclatures for size-exclusion separations and to provide some guidance and recommendations for practicing chromatographers. The interconversion between the different metrics is explained and some examples are presented.

An overview of different approaches for the qualitative and quantitative analysis of polysorbates.

An overview of mRNA-based vaccine production and a discussion on the methods that are currently in use to check the quality of these vaccines.

There is a need to use more descriptive and precise terms to describe some of the properties of the products used for biopharmaceutical analysis. It is probably more correct to say “low adsorption” and “corrosion-resistant” systems.

In the second part of this review of the current state of HIC, some practical considerations are explained, including method development, selection of the phase system, combined salt systems, and possibilities to combine HIC with other chromatographic modes.

A review of the current state of HIC, focusing on retention and separation mechanisms with the aim of developing more robust methods. Can HIC be considered as a non-denaturing and non-destructive technique with advantages for protein analysis?

This article investigates host cell protein analysis using micro-pillar array columns combined with mass spectrometry.

Monoclonal antibodies (mAbs) are being developed at an explosive rate and have attracted great interest from both smaller biotech firms and big pharmaceutical companies. Developing mAbs and next-generation antibody–drug conjugates (ADCs) is highly demanding in many ways. From an analytical perspective, handling mAbs and ADCs presents many new challenges. This article describes how size-exclusion chromatography (SEC) combined with high-resolution mass spectrometry (HRMS) can be applied to the detailed characterization of mAbs and ADCs.

CZE–ESI‑TOF‑MS for the characterization of the mAb infliximab and its variants is presented. Infliximab was analyzed using a middle-up approach involving either reduction or digestion with the enzyme IdeS. A multilayer capillary coating of PB-DS‑PB in combination with a background electrolyte of 40% acetic acid provided efficient separation of the obtained antibody fragments, that is, LC and HC, as well as F(ab’)2 and Fc/2 parts. C-terminal lysine variants were also resolved. Recorded mass spectra of HC and Fc/2 fragments permitted assignment of 13 glycoforms and provided a quantitative profile, with G0F the most abundant glycoform (~50%). CZE–ESI-TOF-MS represents an efficient means for the straightforward analysis of a monoclonal antibody and its proteoforms.

Glycosylation is a critical quality attribute (CQA) that can impact on product safety and efficacy of protein biopharmaceuticals. Characterization of N-glycans is therefore of paramount importance for the pharmaceutical industry. Hydrophilic interaction liquid chromatography (HILIC) combined with fluorescence detection (FLD) and 2-aminobenzamide (2-AB) labelling is the golden standard for the analysis of N-glycans enzymatically liberated from biopharmaceuticals. However, for phosphorylated N-glycans, that is, those attached on lysosomal enzymes, irreproducible data and recovery issues are observed on conventional liquid chromatography (LC) instrumentation and columns, which can be attributed to the interaction of the phosphate moieties with stainless steel components in the flow path. This article demonstrates the analysis of phosphorylated glycans with full recovery on a bio-inert LC system and PEEK-lined HILIC column.

Federal regulations concerning the safety and efficacy of biopharmaceuticals require the implementation of a comprehensive toolbox of physicochemical and biological characterization methods. In order to demonstrate consistent overall structure, even minute differences in primary structure and post‑translational modifications (PTMs) have to be detectable in therapeutic proteins. Because of their remarkable capability of revealing small changes in molecular structure, high performance liquid chromatography (HPLC) and mass spectrometry (MS) rate among the most powerful technologies for comprehensive protein analysis. This article details the potential of both methods with regard to revealing methionine oxidation, a chemical modification that may be induced during downstream processing and storage of biopharmaceuticals. The benefits and limitations of bottom-up, middle-down, and top‑down HPLC–MS analysis will be demonstrated for the detection of oxidation variants in a therapeutic monoclonal antibody (mAb).

Monoclonal antibodies are becoming a core aspect of the pharmaceutical industry. Together with a huge therapeutic potential, these molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry (MS) to its limits. This article discusses the use of micro-pillar array columns in combination with mass spectrometry for peptide mapping of monoclonal antibodies (mAbs) and antibodyÐdrug conjugates (ADCs). Micro-pillar array columns are produced by a lithographic etching process creating a perfectly ordered separation bed on a silicon chip. As a result of the order existing in these columns, peak dispersion is minimized and highly efficient peptide maps are generated, providing enormous structural detail. Using examples from the author’s laboratory, the performance of these columns is illustrated.

These are exciting times to be involved in monoclonal antibody (mAb) and biopharmaceutical analysis. Advances in instrumentation, column technology, and reagents are providing analysts with a new set of tools to broaden their understanding of the highly complex products they are studying. A good example is hydrophilic interaction chromatography (HILIC). While the technique has been used for more than 20 years to profile enzymatically released and fluorescently labelled N-glycans, the introduction of new columns (sub-2-µm and widepore) has paved the way to explore the technique further. Remarkable separations at all levels of analysis, including protein, peptide, and glycan levels, have been demonstrated. With data from the authors’ laboratories, the versatility of HILIC in mAb analysis will be demonstrated in this month’s “Biopharmaceutical Perspectives”.

Two-dimensional liquid chromatography (2D-LC) has in recent years seen an enormous evolution, and with the introduction of commercial instrumentation, the technique is no longer considered a specialist tool. One of the fields where 2D-LC is being widely adopted is in the analysis of biopharmaceuticals, including monoclonal antibodies (mAbs) and antibody–drug conjugates (ADCs). These molecules come with a structural complexity that drives state-of-the-art chromatography and mass spectrometry (MS) to its limits. Using practical examples from the authors’ laboratory complemented with background literature, the possibilities of on-line 2D-LC for the characterization of mAbs and ADCs are presented and discussed.