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Polysorbates (PS) are added to biopharmaceutical formulations to stabilize proteins and monoclonal antibodies (mAbs) and to prevent aggregation, denaturation, and surface adsorption. Polysorbate analysis can be challenging because of their inherent complexity, polydispersity, and their presence in matrices containing high concentrations of proteins and other excipients such as sugars, amino acids, salts, and buffers.This instalment of 'Biopharmaceutical Perspectives' provides an overview of different approaches for the qualitative and quantitative analysis of polysorbates, its impurities and degradants in raw material, intermediate dilutions, drug substance, and drug products. Various gas (GC) and liquid chromatography (LC) methods and accompanying detection techniques are briefly discussed and illustrated with results obtained at the authors' laboratory., Polysorbates (PS) are a class of nonionic surfactants mainly used in the food and pharmaceutical industry. In the biopharmaceutical field, polysorbate 20 (PS20) and 80 (PS80) are widely accepted and [...]

Analytical requirements in the biopharmaceutical, pharmaceutical, and food industries, among several others, are more demanding than ever. Chromatographic techniques are excellent tools to acquire detailed information on a vast number of molecules and sample types.The present challenge in research and development (R&D) as well as in quality control (QC) laboratories is to collect as much sample information as possible. However, even with the current one-dimensional (1D) analytical portfolio, it is not possible to fully ensure that all the relevant information from a sample is captured.This article illustrates the power of an online two-dimensional liquid chromatographic (2D-LC) setup to unravel the complexity of biopharmaceutical and pharmaceutical samples. This technology tremendously increases the resolving power in all areas where LC is applied and drastically reduces the risk of missing an unknown in the samples., Chromatographic instrumentation and column technology are continuously evolving and the state-of-the-art equipment used regularly today is quite impressive. Ultrahigh-pressure liquid chromatography (UHPLC) has enabled laboratories to boost the chromatographic resolution, [...]

The production process of in vitro transcribed messenger RNA (IVT-mRNA)-based vaccines has matured in recent years, partly due to the fight against infectious diseases such as COVID-19. One key to success has been the use of modified, next to canonical, nucleotides and the efficient addition of a Cap-structure and poly A tail to the 5' and 3' end, respectively, of this massive biomolecule. These important features affect mRNA stability and impact translation efficiency, consequently boosting the optimization and implementation of liquid chromatography-mass spectrometry (LC-MS)-based oligonucleotide profiling methods for their characterization. This article will provide an overview of these LC-MS methods at a fundamental and application level. It will be shown how LC-MS is implemented in mRNA-based vaccine analysis to determine the capping efficiency and the poly A tail length, and how it allows, via RNA mapping, (i) to determine the mRNA sequence, (ii) to screen the fidelity of the manufactured modifications, and (iii) to identify and quantify unwanted modifications resulting from manufacturing or storage, and sequence variants resulting from mutation or transcription errors., When dealing with the coronavirus pandemic, academia and pharmaceutical companies have been investing greatly in vaccine development and production during the last two years (1,2). Noticeably for the first time, [...]

The publication of the first LCGC Europe issue back in 1987 followed a major milestone in biology, the FDA approval of the first therapeutic monoclonal antibody (mAb). This event, together [...]

Hydrophobic interaction chromatography (HIC) is one of the historical analytical methods applied for the separation and purification of proteins. In the first part of this series, the elution and separation mechanisms were discussed. In this second part, some practical considerations such as method development, selection of the phase system, combined salt systems, and possibilities to combine HIC with other chromatographic modes are explained. Applications are critically discussed and the hyphenation to mass spectrometry (MS) briefly tackled., Historically, a kosmotropic salt--typically 1.5-2 M ammonium-sulfate--has been used as a mobile phase additive in hydrophobic interaction chromatography (HIC) (as well as 50-100 mM phosphate buffer). However, various salts can [...]

Hydrophobic interaction chromatography (HIC) is one of the historical analytical methods applied for the separation and purification of proteins. The main advantage of HIC is that separations are performed under mild--in most cases non-denaturing--conditions (ambient mobile phase temperature, aqueous mobile phase, and physiological pH). Thanks to the intense developments in the field of protein-based therapeutics, HIC has recently gained a huge interest and is currently considered as a reference method for the characterization of cysteine-linked antibody drug conjugates (ADCs) and widely used for the analysis of therapeutic monoclonal antibodies (mAbs) and bispecific antibodies (bsAbs), at both intact and sub-unit levels. In the first part of this two-part series, the current state of HIC is reviewed with a focus on retention and separation mechanism., Hydrophobic interaction chromatography (HIC) is considered to be the reference technique to separate hydrophobic variants of mAbs and their subunits, and also to analyze the different populations of cysteine-linked ADC [...]

Protein biopharmaceuticals have substantially reshaped the pharmaceutical market and today over 350 products have been approved for human use in the United States and the European Union. Protein biopharmaceuticals are [...]

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 [...]

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. [...]

Comprehensive two-dimensional liquid chromatography (2D-LC) was used for detailed profiling of various nonionic ethoxylated surfactants applied in pharmaceutical formulations. Hydrophilic-interaction chromatography (HILIC) and reversed-phase liquid chromatography (LC) were used as [...]

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 antibody--drug 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., The discovery of monoclonal antibodies (mAbs) in the 1970s and the realization of their therapeutic potential in the following decades has been a key milestone in medicine (1-3). Today, over [...]

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 [...]

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 [...]

Protein biopharmaceuticals are commonly produced recombinantly in mammalian, yeast, or bacterial expression systems. In addition to the therapeutic protein, these cells also produce endogenous host cell proteins (HCPs) that can [...]

Monoclonal antibodies (mAbs) have emerged as important therapeutics for the treatment of life-threatening diseases including cancer and autoimmune diseases. With the top-selling mAbs evolving out of patent there has been [...]