Edouard S. P. Bouvier

Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates.

In recent years, the use and number of biotherapeutics has increased significantly. For these largely protein-based therapies, the quantitation of aggregates is of particular concern given their potential effect on efficacy and immunogenicity. This need has renewed interest in size-exclusion chromatography (SEC). In the following review we will outline the history and background of SEC for the analysis of proteins. We will also discuss the instrumentation for these analyses, including the use of different types of detectors. Method development for protein analysis by SEC will also be outlined, including the effect of mobile phase and column parameters (column length, pore size). We will also review some of the applications of this mode of separation that are of particular importance to protein biopharmaceutical development and highlight some considerations in their implementation.

Advances in sample preparation in electromigration, chromatographic and mass spectrometric separation methods.

The quality of sample preparation is a key factor in determining the success of analysis. While analysis of pharmaceutically important compounds in biological matrixes has driven forward the development of sample clean-up procedures in last 20 years, todays chemists face an additional challenge: sample preparation and analysis of complex biochemical samples for characterization of genotypic or phenotypic information contained in DNA and proteins. This review focuses on various sample pretreatment methods designed to meet the requirements for the analysis of biopolymers and small drugs in complex matrices. We discuss the advances in development of solid-phase extraction (SPE) sorbents, on-line SPE, membrane-based sample preparation, and sample clean-up of biopolymers prior to their analysis by mass spectrometry.

Ultra-high performance size-exclusion chromatography of synthetic polymers: demonstration of capability.

Ultra-high performance size-exclusion chromatography (UHP SEC) is a newly developed disruptive technology that allows the high-resolution separation of synthetic polymers in as little as 2 min. The capability of UHP SEC for the characterization of synthetic polymers in organic solvents has been demonstrated. Using the Waters ACQUITY UPLC® H-Class system and ethylene-bridged hybrid size-exclusion chromatography (SEC) columns packed with 1.7 to 2.5-μm particles with pore sizes ranging from 45 to 900 Å, size-based separations of polystyrene and poly(methyl methacrylate) standards in tetrahydrofuran and poly(ethylene oxide) standards in 20 mM ammonium acetate in methanol are achieved within 2-4 min. The speed of analysis is about ten times faster than conventional SEC separations, and greater resolution is achieved. Average molecular weights of selected commercial polymers have been determined using ultra-high performance and conventional SEC. Average M data of analyzed samples are in good agreement using the two approaches. An inherent limitation of SEC in UHP mode is the characterization of very high M polymers (above ca. 2 million Da) due to the deformation and/or mechanical shearing of large molecules at high flow rates.