Introduction
Multi-detector size-exclusion chromatography (SEC) is a development beyond single detector or ‘conventional’ systems. Single-detector systems might typically include an ultraviolet (UV) detector, and would typically be used to study proteins and to identify the presence of any aggregates, either oligomers or larger molecules, and then quantify them. They are also used to make measurements of molecular weight by comparing a protein’s elution time against that of some standards. Single-detector conventional systems have been available since the development of liquid chromatography and are used throughout industry and academia in diverse applications including protein purification, research, biopharmaceutical manufacture and QC. There are a number of reasons why they are so popular, including, most importantly, their repeatability and reproducibility, their ease-of-use and robustness, and their simplicity and accessibility.In recent years, however, technology has advanced and with additional detectors comes a wealth of additional information. Light scattering detectors, whether SEC-MALS (Multi-angle light scattering), RALS (right-angle light scattering), or LALS (low-angle light scattering), measure absolute molecular weight with excellent accuracy, which allows oligomers and larger aggregates to be easily differentiated. Conjugate analyses allow the characterization of PEGylated proteins, antibody-drug-conjugates (ADCs) and membrane proteins, and a viscometer is occasionally used to study large changes in protein conformation. With these capabilities in mind, this white paper lists the top ten reasons your laboratory should consider an advanced multi-detector SEC system.
1. Absolute molecular weight
The greatest limitation of conventional SEC measurements is the way in which they calculate molecular weight. Since molecular weight is calculated from retention volume (which is directly related to the sample’s size) and a comparison with molecular weight standards (such as a series of globular proteins), the calculated results are only accurate if the protein’s structure is globular and happens to fit on the calibration curve. Conventional calibration is blind to countless possibilities of a protein’s structure and the effects this have on its elution time. With so many proteins under study, the molecular weight of only very few can be accurately measured with such a technique. It is far more common for researchers to simply assume that the primary peak in a separation is the monomer and that anything else present is some form of aggregate. While this may be sufficient for a few applications, it is an extremely limited amount of information. The addition of a light scattering detector means that from now on, all of your molecular weight measurements will be accurate, irrespective of the protein being measured. Absolute molecular weight means a better understanding of your protein and the types of aggregates formed, providing better control over its behavior and stability. Ultimately, this ensures a better quality product. In research, absolute molecular weight means better knowledge about a newly purified or recombinant protein, and greater accuracy in data used for publication purposes. One of the key reasons for measuring the absolute molecular weight of a protein is to reliably assess its oligomeric state. Many proteins come together in multimers of two or more sub-units to control activity and/or stability. For instance, insulin, while active as a monomer, forms an inactive hexamer for stability during production and storage. In a biopharmaceutical, the difference between oligomeric states can mean the difference between stability and rapid aggregation, or between activity and inactivity. In research, it can mean the difference between a functioning experiment and useful data, and a significant amount of wasted time and effort. Multi-detector SEC and light scattering, in particular, allow accurate measurement of protein oligomeric state, which allows you to better understand the differences between stability and aggregate formation, or activity and inactivity. To access the full article, click hereMalvern provides the materials and biophysical characterization technology and expertise that enables scientists and engineers to investigate, understand and control the properties of dispersed systems. These systems range from proteins and polymers in solution, particle and nanoparticle suspensions and emulsions, through to sprays and aerosols, industrial bulk powders and high concentration slurries. Used at all stages of research, development and manufacturing, Malvern’s instruments provide critical information that helps accelerate research and product development, enhance and maintain product quality and optimize process efficiency. Our products reflect Malvern’s drive to exploit the latest technological innovations. They are used by both industry and academia, in sectors ranging from pharmaceuticals and biopharmaceuticals to bulk chemicals, cement, plastics and polymers, energy and the environment. Malvern systems are used to measure particle size, particle shape, zeta potential, protein charge, molecular weight, mass, size and conformation, rheological properties and for chemical identification, advancing the understanding of dispersed systems across many different industries and applications. www.malvern.com Material relationships http://www.malvern.com/en/ [email protected]
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