Introduction
All biopharmaceuticals are subject to extremely high regulatory burdens which must be met to show their efficacy and safety. One particular area of regulatory scrutiny for biologics is their stability and the level of aggregate material that they contain. Tools that can be used to study protein stability and aggregation provide valuable information for regulators and assist developers to produce safe, reliable, high quality products. Using orthogonal technologies for complementary analyses increases confidence in the resulting data.Size exclusion chromatography (SEC) and light scattering are two workhorse techniques for biopharmaceutical stability and aggregation analysis. SEC is regularly used to assess the aggregation state of protein samples throughout the drug development pipeline. Static light scattering (often referred to as multi-angle light scattering (MALS) is used to measure protein molecular weight (MW). Dynamic light scattering (DLS) is used to detect early aggregates and also to monitor aggregate formation in response to stimuli such as time or raised incubation temperatures. These two techniques used together provide a wealth of important information about the stability and aggregation profile of a sample, whilst conserving sample volume and operator time and effort.
SEC and Light Scattering: Individually powerful; together formidable
The Viscotek SEC-MALS 20 is a modular multi-angle light scattering detector that is easily integrated into any SEC system, allowing measurement of the absolute MW of a protein as well as the radius of gyration (Rg) of any aggregates more than 15 nm in radius. This provides vital information on the stability of the protein. The 20 measurement angles, particularly the additional low angles, generate the most accurate MALS data.
But sometimes measuring MW is not enough, especially when conformational changes that do not have an effect on the protein’s MW may be occurring. The addition of an online DLS detector to an SEC system provides the hydrodynamic radius (Rh) across the entire size range of the sample. In combination with the absolute MW supplied by the SEC-MALS 20, Rh data can give invaluable insight into protein conformation.
The Malvern Zetasizer µV (ZMV) is a highly sensitive dual capability light scattering detector which can be integrated with the SEC-MALS 20 detector to provide Rh data in flow mode during SEC experiments. Alternatively, it can be used independently in cuvette mode for detecting and monitoring aggregate formation. The system allows investigation of the effect of temperature on the stability of a sample, as well as measurements of the second virial coefficient (B22 or A2) and DLS interaction parameter (KD), indicators of the protein’s stability and propensity to aggregate.
When the SEC-MALS 20 and ZMV are used in flow mode, a single powerful software package - OmniSEC - is used to control the system and analyze data. When the ZMV is used in cuvette mode, the Zetasizer software is used for data acquisition and analysis.
In this application note the use of combined inline SEC-MALS and DLS to assess a protein sample’s stability and tendency to aggregate is explored.
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Malvern Instruments provides the materials and biophysical characterization technology and expertise that enable scientists and engineers to 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 materials characterization 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 and our commitment to maximizing the potential of established techniques. 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. Headquartered in Malvern, UK, Malvern Instruments has subsidiary organizations in all major European markets, North America, Mexico, China, Japan and Korea, a joint venture in India, a global distributor network and applications laboratories around the world.
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