April 26th 2024
Scientists from the University of Córdoba in Córdoba, Spain recently used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to comprehensively profile human ceramides to determine their reactions to diseases.
Online Hydrophobic Interaction Chromatography–Mass Spectrometry (HIC–MS) Analysis of Proteins
February 1st 2021Recent advances in stationary phases for hydrophobic interaction chromatography (HIC) permit HIC–MS analysis of intact antibodies and other proteins using direct flow to the mass spectrometer.
The Multi-Attribute Method (MAM) for the Characterization of Biopharmaceuticals
January 1st 2021New analytical workflows are needed to address the advances in biopharmaceutical product composition. A description of the multi-attribute method (MAM) is given, which has been developed to monitor critical quality attributes (CQAs) simultaneously and directly.
In Search of The Needle in The Mariana Trench: Host Cell Proteins and the Problem of Dynamic Range
October 9th 2020This article explores the analytical challenges associated with HCP monitoring and reviews recent advances in HCP characterization, with special emphasis on high performance liquid chromatography mass spectrometry (HPLC–MS)-based methods and HCP enrichment techniques.
Analytical Separation Methods for Therapeutic Oligonucleotides
October 9th 2020The diverse nature of oligonucleotide therapeutics leads to the requirement for multiple separation methods to characterize quality attributes. Highly chemically-modified to improve efficacy and resilience to nucleases, they are challenging to analyse using a single method. This article discusses multiple chromatographic separation methods, and the benefits of mass spectrometry (MS).
Physicochemical Methods for Vectors and Ancillary Materials in Cellular and Gene Therapies
October 9th 2020Cellular and gene therapies (CGTs) have contributed significantly to the improvement of clinical outcomes for patients in the recent years. This paper discusses a range of physicochemical methods that play an important role in the difficult characterization of viral vectors, to meet the unique needs of CGT manufacturing process development, process and product characterization, and the quality control testing of these materials.
Rapid Adeno-Associated Virus Genome Integrity Analysis by Capillary Gel Electrophoresis
October 9th 2020In this paper, two adeno-associated virus (AAV) genome integrity analysis workflows are introduced; a standard sample preparation protocol and an accelerated procedure, both utilizing capillary gel electrophoresis with laser induced fluorescent detection (CGE–LIF) to analyze the released nucleic acids.
Troubleshooting LC Separations of Biomolecules, Part II: Passivation and Mobile-Phase Additives
August 1st 2020If you are analyzing metal-sensitive biomolecules, and a bioinert instrument is unavailable, or insufficient, passivation or mobile-phase additives may help. Here’s how to use those solutions, with tips for avoiding potential pitfalls.
Practical Two-Dimensional Liquid Chromatography in Drug Metabolism Studies and Bioanalysis
June 30th 2020Filip Cuyckens from Janssen R&D in Belgium spoke to LCGC Europe about recent innovative approaches he and his team developed to support drug metabolism and pharmacokinetic studies, and the inventive role that two-dimensional liquid chromatography (2D-LC) plays in his laboratory to boost sensitivity, solve recovery issues, and increase overall efficiency.
Boosting the Purification Process of Biopharmaceuticals by Means of Continuous Chromatography
Single-column (batch) chromatography, involving two or more successive single-column (batch) chromatographic steps, is a standard approach for purifying biopharmaceuticals. Step one, known as the capture step, is used to remove product-related impurities, and step two, the polishing step, is used to remove product-related impurities. Here we present and illustrate the advantages of continuous chromatography for these separations: capture simulated moving bed (captureSMB) for the capture step and multicolumn countercurrent solvent gradient purification (MCSGP) for polishing.
Multidimensional Separation Techniques for Characterization of Biotherapeutics
June 1st 2020Multidimensional separations, in which two or more separation methods are coupled, are a valuable analytical tool for higher peak capacity and improved selectivity for the analysis of complex samples like biotherapeutics.
Glycosylation Analysis Through Released N-Glycan Workflows
May 6th 2020Post-translational modifications are potential critical quality attributes (pCQAs) routinely assessed in biotherapeutic development. Glycosylation is one of the most important attributes to assess because it affects protein function as well as antigen receptor binding. N-glycosylation of asparagine residues is the most common pCQA assessed during monoclonal antibody (mAb) therapeutic development. There are a few protocols to assess and quantitate N-glycans, but the most common approach is through an enzymatic release and labelling procedure, followed by separation and detection. This article demonstrates the method development considerations for sample preparation and chromatographic analysis of N-glycans of therapeutic mAbs.
A method was developed for the molecular weight characterization of heterogeneous polymer mixtures, such as heparins and glatiramer acetate, noting that single molecular structures are not adequate for creating a molecular weight calibration curve. That limitation is overcome in this work, which demonstrates method validation and application to process samples.
The Basics of HPLC Peptide Analysis
December 10th 2019To fully characterize a protein biopharmaceutical, it must be broken down into smaller segments (peptides). Several high performance liquid chromatography (HPLC) techniques can be used to provide a wealth of information on everything from post-translational modifications (PTMs) to the glycoprofile to information on similarity when characterizing biosimilars.
High Sensitivity Native Mass Spectrometry with Cation Exchange Chromatography
November 6th 2019The utility of native high-resolution mass spectrometry (HRMS) in intact protein characterization is rapidly growing because of advances in both ion-exchange chromatography (IEC) as well as MS-compatible buffer systems. MS is a critical component of biotherapeutic characterization, but its combination with traditional chromatographic separations, such as size-exclusion chromatography (SEC) and IEC, has been slow because of the predominant use of high salt mobile phases, which are incompatible with MS. Recently reported methods using cation-exchange chromatography (CEX) with volatile buffer systems for pH gradient elution has given researchers the ability to use these chromatographic techniques with MS detection. In this article a robust, MS-compatible buffer system for high sensitivity IEC with pH gradient elution for charge variant analysis of intact monoclonal antibodies (mAbs) is described.
The versatile size-exclusion ultrahigh‑performance liquid chromatography (SE-UHPLC) platform method described here provides superior separation for bispecific monoclonal antibody formats compared to a previous method.