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The Power of Multi-Detector SEC in the Analysis of Antibodies

Image Credit: Malvern Instruments Limited

Antibodies have generated keen interest from the pharmaceutical / biopharma / biotherapeutic industries due to their ability to direct their neutralization attack toward specific structures in harmful agents. As a naturally occurring class of molecules, developments have been made that now allow scientists to produce antibodies in laboratory settings. Since much of an antibody’s functionality depends on its molecular structure and integrity, full characterization, including molecular weight, size, and purity of these materials is critical to ensure efficacy.

Size-exclusion chromatography (SEC), also referred to as gel permeation chromatography (GPC), is a widely used technique to characterize a range of samples, from bulk manufactured materials to proteins and other biological macromolecules, such as antibodies. This technique can be used to measure the molecular weight moments, molecular weight distribution, intrinsic viscosity and hydrodynamic size of these materials.

A brief overview of how SEC works: a solvated sample is carried by a liquid mobile phase through an analytical column full of porous gel particles, where diffusion controlled separation of the macromolecular components occurs and is ultimately observed by different detectors as each slice of sample elutes. The samples elute based on molecular size, with the larger molecules eluting first. It is important to remember that the elution order is based on molecular size and not molecular weight.

The potency of the SEC analysis depends on which detectors are coupled to the chromatography. Two of the common types of SEC configurations are a column calibration SEC setup, and an advanced detection SEC setup. A column calibration SEC system employs a single concentration detector, usually a refractive index (RI) or UV detector, to compare the retention volume of analyte molecules through a SEC column against that of a series of standards with known molecular weights.

A common advanced detection SEC configuration utilizes a combination of RI, UV/Vis photodiode array (PDA), viscometer, and light scattering detectors to make direct measurements of samples’ molecular weight, intrinsic viscosity and molecular size, independent of retention volume or elution order.

This application note will describe the analysis of three antibody samples using column calibration and advanced detection SEC. Comparisons will be made between the data derived from both methods highlighting the advantages of advanced detection.