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.
Size-Exclusion Chromatography for Analyzing Complex and Novel Biotherapeutic Products
November 1st 2019Applications of size-exclusion chromatography (SEC) are presented for characterization and quality control of novel biotherapeutic products, including antibody–drug conjugates, hydrophobic proteins, and coformulations.
Ferritin as a Natural Protein Scaffold: Building a Multivalent Ferritin–Fab Conjugate
November 1st 2019In this study, a conjugation strategy for Fab–ferritin conjugates, used for drug delivery, was successfully optimized using LC–MS. Characterization of the resulting conjugates was performed using SEC-MALS-QELS.
Utilizing Multidimensional LC–MS for Hydroxyl Radical Footprinting Analysis
November 1st 2019The potential of multidimensional online peptide mapping analysis as a strategy for improving a postlabeling workflow for protein–protein interactions is demonstrated using both hydroxy radical footprinting–mass spectrometry (HRF–MS) and LC–MS/MS.
The Benefits of Ion-Exchange Chromatography to Monitor Charge Heterogeneity in Monoclonal Antibodies
August 6th 2019The 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.
A Brief Review of Recent Advances in Isomeric N- and O-Glycomics
July 1st 2019Glycan 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.
Optimization of MS-Compatible Mobile Phases for IEX Separation of Monoclonal Antibodies
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.
Methods and Purposes for Determining Higher Order Structures of Biopharmaceuticals
January 1st 2019Determining 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.
Drawing A Better Map: The Recent Advances in Protein Digestion and Peptide Mapping
December 1st 2018New 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.
Combatting Counterfeit Biopharmaceuticals Using Hydrophilic Interaction Chromatography
November 12th 2018The 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.
Characterizing Protein-Nucleic Acid Conjugates with Light Scattering
November 12th 2018This 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.
High-Flow Weak Cation Exchange for Charge Variant Analysis
November 12th 2018Within 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.
Analytical Characterization of Biotherapeutic Products, Part II: The Analytical Toolbox
November 1st 2018The 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.
Antibody–Drug Conjugates: Perspectives and Characterization
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.
Perspectives and Characterization on Antibody–Drug Conjugates
Comprehensive characterization of ADCs requires increasingly powerful approaches consisting of small- and large-molecule techniques.