Biopharmaceuticals and Protein Analysis

The Column spoke to Richard Shannon from AstraZeneca about his work characterizing monoclonal antibodies (mAbs), why ion-exchange chromatography (IEC) is his technique of choice for analyzing mAbs, and offers his advice for anyone wanting to use the technique.

Glycan isomer expressions have not been well studied, due to inefficient separation and structural identification techniques. Fortunately, with the development of novel separation techniques and liquid chromatography–mass spectrometry (LC–MS) based glycan isomer identification strategies, new efforts have been made to investigate the glycan isomers in various diseases. Here, we review the recent advances of several isomeric separation techniques for both N- and O-linked glycans.

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.

With SEC, special care is required to achieve in practice the chromatographic efficiency that is expected from theory.

Coupling CE with MS presents some challenges. Here, we discuss the advantages of CE–MS over LC–MS, and the parameters that are important to obtain a stable CE–MS profile.

Characterization of mAbs and related products requires the identification of chromatographic peaks with MS. However, the conventional salt- and pH-gradient elution techniques used in IEX are inherently incompatible with MS. Ammonium acetate- and ammonium carbonate-based mobile phase systems have been recently applied in IEX-MS, but the influence of the eluent composition on peak shape and retention has not been discussed nor studied systematically until now. The aim of the present study was to understand the impact of ionic strength, buffer capacity, and pH-response on the retention behaviour and peak shape of mAb species.

This is the first in a series of articles exploring current topics in separation science that will be addressed at the HPLC 2019 conference in Milan, Italy, from 16–20 June. In this preview, Davy Guillarme discusses multidimensional liquid chromatography approaches for the characterization of protein biopharmaceuticals.

This is the first in a series of articles exploring current topics in separation science that will be addressed at the HPLC 2019 conference in Milan, Italy, from 16–20 June.

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.

A discussion of hydrophobic interaction chromatography (HIC) theory and its application to the analysis of proteins and biomolecules is presented.

Determining the higher order structure of a protein pharmaceutical is important. Here, we review the approaches for HOS determination that are currently receiving the most attention in the literature and at scientific meetings.

New strategies for “bottom-up” analysis of therapeutic proteins, using faster enzymes, new buffer systems, and optimal column chemistries, enable analysts to perform these studies much faster and with fewer artifacts.

The Column spoke to Steven Janvier, a PhD student at Sciensano and Ghent University (Belgium), and Celine Vanhee, a scientist at Sciensano, about their work to develop a hydrophilic interaction chromatography (HILIC) methodology capable of detecting counterfeit polar peptide drugs available on the black market.

This article presents two case studies regarding the characterization of protein-DNA complexes using two complementary multi-angle light scattering (MALS) techniques, namely size-exclusion chromatography (SEC–MALS) to determine absolute molar mass of each component, and composition-gradient MALS (CG–MALS) to quantify stoichiometry and affinity at binding sites in solution.

Within the broad scope of analytical techniques required to characterize a protein, chromatographic methods have shifted towards high-flow analyses that can drop development time significantly. However, fast analytical methods for charge heterogeneity have lagged in development because current column technologies are ultrahigh-pressure liquid chromatography (UHPLC)-incompatible. This article will demonstrate the development of a high-flow method for charge variant analysis made possible through a bioinert titanium column flow path.

The analytical techniques used for characterizing biotherapeutics have evolved. We review the utility of the traditional tools and discuss the new, orthogonal techniques that are increasingly being used.

Among all the analytical techniques available for epitope mapping studies, hydrogen–deuterium exchange mass spectrometry (HDX-MS) is usually the fastest and easiest to carry out. We present here the epitope mapping of three distinct monoclonal antibody (mAb) candidates targeting the same antigen, an interleukin receptor. The goal is to establish the binding mode of these mAbs, and explain possible differences observed for in vitro binding and in vivo function.

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.

Much of the conventional wisdom regarding size-phase separations of proteins has been negated thanks to development of superior chemistries and advances in research. In this article, details that the authors have found to be especially beneficial in achieving effective SEC separations are examined.

Why the technique is a handy component of any analyst’s arsenal.

For reversed-phase separations of proteins, you must consider pore size,column temperature, and stationary-phase chemistry. Here are some guidelines.

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).

This instalment of “Perspectives in Modern HPLC” provides an overview of antibody–drug conjugates (ADCs) as a new class of biotherapeutics and describes their analytical characterization for quality assessment with examples from extensive applications libraries.

Comprehensive characterization of ADCs requires increasingly powerful approaches consisting of small- and large-molecule techniques.

To ensure the reliable and accurate characterization of biotherapeutics, an arsenal of orthogonal analytical techniques is needed.

In this study, we compare the performance of plastic and metal materials in UHPLC columns designed for the analysis of biological molecules. We evaluate the performance of these materials in terms of inertness, column chromatographic performance, and reproducibility.

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.

Parameters such as pore size, column dimensions, temperature, flow rate, and mobile phase are important to consider when developing SEC methods.

Characterization of protein modifications is an essential aspect of biopharmaceutical development. Traditionally, the characterization process of chromatographic peaks involves manual, larger-scale fractionation to obtain a sufficient amount of material for further analytical studies. This article presents a fully automated process for online peak fractionation and reduction of therapeutic antibodies with subsequent quadrupole time-of-flight mass spectrometry (QTOF-MS) characterization. This innovative technique significantly accelerates MS peak characterization compared to traditional approaches and avoids the risk of unintended modifications of the variants as a result of the isolation process, for example, deamidation during storage of isoforms. This approach considerably reduces the required sample amount and can be used for the characterization of product-related impurities during early stage development.

A look at techniques for charge-variant analysis of monoclonal antibodies and the question of whether pH gradients are really better than salt gradients