Malvern Instruments Ltd.
A purified polyclonal antibody (IgG) is separated and fully characterized using the Viscotek SEC-MALS 20, allowing calculation of molecular weight and radius of gyration (Rg).
Therapeutic recombinant antibodies represent a growing proportion of biopharmaceuticals and are primarily classed as Immunoglobulin G (IgG). However, proteins have a tendency to aggregate over time and one challenge for biologic drugs is that the presence of aggregates will stimulate an immune response. Size-exclusion chromatography (SEC) is a powerful tool that is commonly used to look at the aggregation of proteins. While most SEC systems use a single concentration detector such as ultraviolet (UV), the addition of light scattering allows the molecular weight of the protein to be measured independent of its retention volume. The new SEC-MALS 20 detector, which uses multi-angle light scattering (MALS), is ideal for this application. In addition, the MALS detector makes it possible to measure the radius of gyration (Rg) of molecules that scatter light anisotropically.
In this application note, a purified polyclonal antibody (IgG) is separated using SEC and characterized using the Viscotek SECMALS 20.
Figure 1: Overlay of MALS detector responses for IgG.
Samples were analysed using a Viscotek TDAmax system connected to Viscotek SEC-MALS 20. The mobile phase was phosphate buffered saline, which was also used to prepare the IgG for analysis.
Table 1: Measured molecular weights of the different peaks of the IgG sample.
The SEC-MALS results are presented in Table 1. The monomer (15.80 mL) and dimer (14.00 mL) peaks are clearly identified by the measured molecular weights and low polydispersity (Mw/Mn). No size (Rg) can be measured for these peaks as they are below the isotropic scattering threshold of 10–15 nm. Studying Figure 1, it is just possible to see that the SEC-MALS 20 show the same response for the monomer peaks at all angles. The aggregate peak (13.23 mL) is clearly different. The molecular weight is higher and more polydisperse, which shows that there is a variable composition of molecules within the aggregate peak. Because it is large, the light scattering response varies with angle and can be used to measure the Rg.
Malvern Instruments Ltd.
Enigma Business Park, Grovewood Road, Malvern, UK
Tel: +44 (0) 1684 892456
E-mail: salesinfo@malvern.com
Website: www.malvern.com
Inside the Laboratory: The Gionfriddo Group at the University at Buffalo
March 28th 2024In this edition of “Inside the Laboratory,” Emanuela Gionfriddo, PhD, an associate professor of chemistry at the University at Buffalo, discusses her group’s current research endeavors, including using solid-phase microextraction (SPME) coupled to liquid chromatography (LC) and gas chromatography (GC) to further understand the chemical relationship between environmental exposure and disease and elucidate micropollutants fate in the environment and biological systems.
Transferring Methods to Compact and Portable HPLC
February 14th 2024The current trend in laboratory equipment design is the miniaturization of laboratory instruments. Smaller-scale HPLC instruments offer benefits that cannot be matched by analytical-scale equipment, especially in the areas of portability, reduced fluid volumes, and reduced operating costs. Yet, the miniaturization of laboratory equipment has brought with it a unique set of challenges, including transferring methods to compact LC. Capillary LC expands the use of LC to applications not currently done using conventional LC in a wide array of application areas, including pharmaceutical, food and beverage, petrochemical, environmental, and oil and gas. Greg Ward, Axcend’s CEO wrote, “Customers want an HPLC system with a small footprint, low flow rates and green chemistry.” Join his podcast where he shares method transfer in these application areas.