Biopharmaceuticals and Protein Analysis

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.

The recent trends in column technology for reversed-phase LC, SEC, IEX, and HIC for analysis of biopharmaceuticals at the protein level is critically discussed.

Modern Column Technologies for the Analytical Characterization of Biopharmaceuticals in Various Liquid Chromatographic Modes

Higher Order Mass Spectrometry Techniques Applied to Biopharmaceuticals

An analytical methodology for the characterization of the primary structure of biotherapeutic proteins using sheathless CE–ESI-MS-MS instrumentation is presented. For the first time, complete sequence coverage can be achieved using a bottom-up proteomic approach from a single analysis of a tryptic digest. In a biosimilarity assessment, a single amino acid substitution was detected.

The biopharmaceutical industry continues to expand in response to a demand for novel biopharmaceuticals. LCGC spoke to José Paulo Mota from the Faculty of Science and Engineering (FCT-UNL) of Universidade Nova de Lisboa, Portugal, and Institute of Experimental and Technological Biology (IBET), Portugal, about the development of novel downstream purification strategies.

This article explores how multiple detectors, particularly light scattering detectors, may be used for size-exclusion chromatography (SEC) in protein analysis.

This article describes the development of a new data-independent acquisition (DIA) workflow for protein quantification that uses a mass spectrometer that combines three types of mass analyzers to achieve lower limits of detection (LOD), higher sensitivity, more accurate quantitative results, wider dynamic range, and better reproducibility than existing high-resolution accurate-mass (HRAM) tandem mass spectrometry (MS-MS) DIA workflows.

An overview of the analytical approaches used for the analysis of biomolecules

Translational proteomics has not been as successful as originally anticipated. Because mass spectrometry (MS) can separate proteins at the sequence level, it provides the selectivity needed for this application; however, traditional challenges still exist, including time-to-result, throughput, and sample-size requirements.

The modern usage and specific applications of HILIC for two other major classes of analytes - glycopeptides and glycoproteins - are discussed.

Answers to common questions about HILIC, and an overview of its application to biopharmaceutical analysis.

This article highlights some selected examples of the power of liquid chromatography combined with mass spectrometry (LC–MS) in the development of protein biopharmaceuticals.

In biopharmaceutical development the stability of biological molecules in drug formulations is important. This article describes three different approaches to the measurement and quantification of aggregates in protein solutions, and highlights the range of information that each technique can provide about the sample.