Introduction
Biopharmaceuticals are drugs made from biological molecules such as proteins. Protein activity is often defined by its molecular weight and aggregation state. Light scattering is more and more commonly being used to measure the molecular weight of proteins, their oligomers and their aggregates. SEC-MALS is one of the more common implementations of light scattering for protein measurements. Malvern’s Viscotek SEC-MALS 20 is a multi-angle light scattering instrument with 20 detection angles for the measurement of protein absolute molecular weight and oligomeric state. It can also be used to study the molecular weight and size of protein aggregates, as well as for the analysis of synthetic and natural polymers.The Waters Alliance® system is frequently used in protein separations and is popular throughout the biopharmaceutical environment, particularly due to its powerful software, Waters Empower®. Adding advanced detectors to most SEC systems is usually possible thanks to the generic analog and digital connections that can be used to transfer signals between devices but software packages are often incompatible. In this application note, we show that Malvern’s OmniSEC software can now communicate with Waters Empower® for the seamless exchange of sample sequences. This allows OmniSEC to follow the injections performed by a Waters SEC system and to synchronize data collection. A selection of proteins was used to demonstrate the measurement of protein molecular weight, oligomeric and aggregation state.
Materials and methods
The Viscotek SEC-MALS 20 was connected to the Waters Alliance® system comprising an e2695 pump, degasser and autosampler module and an e2414 RI detector (figure 1). The autosampler trigger signal was connected to synchronize injections and data collection. The RI analog signal out from the RI detector was connected to one of the analog inputs on the SEC-MALS 20. The fluidic connections went from the column, to the SEC-MALS 20 and then to the RI detector. The RI detector was plumbed last in line to protect the flow cell from any increase in back-pressure. Waters Empower® and Malvern’s OmniSEC software were installed on the same PC. The ‘sample set method’ was created in Empower® and set to run. OmniSEC then loaded the sequence information from Empower® and data collection was initiated. OmniSEC then waits for injections in the Waters Alliance® system and collects data as those injections are made. The separations were performed on a Malvern PLS5030 SEC column running PBS at 1 mL/min. These columns are silica-based columns for the separation of proteins. Their high resolution and low particle shedding makes them ideal for protein light scattering measurements. Bovine serum albumin (BSA) was run as a molecular weight standard and used for detector response calibration and normalization. Two test proteins, pepsin and trypsin inhibitor, were dissolved in the mobile phase. The trypsin inhibitor was heated in a microwave for 10 seconds to stimulate severe aggregation. From experience, pepsin samples already contain significant levels of aggregates. The two samples were then injected (100 µL) at concentrations of approximately 2 mg/mL. The results were then analyzed in OmniSEC. To read 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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