Biopharmaceuticals and Protein Analysis

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

The discovery and development of biopharmaceuticals that target specific diseases can be transformative for people living with illness. However, bringing a new therapy to market is a prolonged and costly process mired in uncertainty. Ensuring safety, efficacy, and product quality is paramount. Biopharmaceuticals, by their nature, are highly complex. A myriad of heterogeneity can be intentionally functional, an unwanted consequence of manufacturing and storage, or generated by biological modification in vivo. Not all, but some post-translational modifications or biotransformations can impact development, manufacturing, safety, efficacy, and overall product quality. These critical quality attributes (CQAs) need to be identified, characterized, controlled, and monitored throughout the drug discovery and development cycle. Specialty measurement using mass spectrometry (MS) continues to play an ever‑increasing role across the continuum.

Colloidal interactions arising from surface-exposed moieties on therapeutic proteins, monoclonal antibodies, antibody–drug conjugates, and other biopharmaceuticals lie at the heart of drug product stability. Therefore, it is not surprising that much effort has been devoted to finding effective means to characterize these interactions and to rapidly screen drug candidates and formulations for optimal colloidal properties. The most common techniques for performing these analyses are based on analytical light scattering, in its two primary flavours: static light scattering (SLS) and dynamic light scattering (DLS). Recent advances in light scattering instrumentation, analytical methods, and algorithms provide developers of biologics with powerful tools to perform these studies.

Tips for effective use of chromatography and mass spectrometry (MS) for the analysis of antibody–drug conjugates, glycoengineered proteins, and biosimilars.

Researchers from University College London have developed a novel method of characterizing the mechanical strength of agarose-based chromatography resins used in the manufacturing of biopharmaceuticals.

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”.

An increasing number of drugs coming onto the market are proteins rather than small molecules. A major portion of these are produced using a host cell system. Host cells express many of their own proteins that can easily contaminate the recombinant protein drug. Traditionally, these host cell proteins (HCPs) have been measured using immunoassays, but recently, orthogonal analytical methods, particularly mass spectrometry (MS), have started to be used. This article considers some of the current methods for HCP detection, with a focus on MS.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are heterogeneous and exist as variant mixtures of structurally similar molecules. The heterogeneity of monoclonal antibodies is revealed by charge-sensitive methods, such as ion exchange chromatography (IEX). Changes in charge profile can significantly impact the structure, stability, binding affinity, and efficacy of the biotherapeutic drug. It is therefore necessary to understand the profile of the drug so that variants are identified and controlled. This article describes advances in ion exchange column chemistries, elution buffers, and ultrahigh-pressure liquid chromatography (UHPLC) instruments to meet the needs for modern, robust analysis of charge variants in monoclonal antibodies and therapeutic proteins.

The HPLC symposium series is recognized as “the forum” where new developments in liquid phase separations and their hyphenation to mass spectrometry (MS) for the analysis of (bio)pharmaceutical compounds and their metabolites are presented.

Gel permeation chromatography/size-exclusion chromatography (GPC/SEC) is the standard method to separate samples by molecular size. In protein analysis, size-exclusion chromatography is either applied to detect and quantify aggregation, or to measure the complete molar mass distribution. However, method development is not trivial and the choice of suitable detection options is crucial.

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.

All agencies have issued varying guidances for the approval of recombinant biosimilars of biopharmaceuticals, and all submittals are considered on a case-by-case basis. This instalment of “Focus on Biopharmaceutical Analysis” looks at the best methodologies for demonstrating their analytical comparability.

All agencies have issued varying guidances for the approval of recombinant, biosimilars of biopharmaceuticals. However, their impact or meaning is in our understanding and that all submittals are considered on a case-by-case basis.

The recent trends in column technology for reversed-phase LC, SEC, ion-exchange chromatography, and HIC for analysis of biopharmaceuticals are critically discussed.

An outline of the basic principles of MS techniques used to investigate higher order structural features of biopharmaceuticals, as well as some insights into applications relevant to the pharmaceutical industry.

The technical requirements for a successful LC–MS/MS method for the quantitation of biopharmaceuticals are presented and the advantages and disadvantages compared to ligand-binding assays are evaluated.

Mass spectrometry (MS) is emerging as a critical tool in biopharmaceutical late stage development, manufacturing, and quality control (QC) environments. The rapid growth of biologics in development, the increasing demand for more robust analytical technologies to directly monitor the critical quality attributes (CQAs) of these new drugs, and longer term industry initiatives aimed at improving quality and productivity, such as quality by design (QbD) regulatory submissions and continuous manufacturing, are all fueling a greater need for mass monitoring with MS.

Leading separation scientists discuss their approaches to improving the analysis of large molecules.