Rapid and Accurate Therapeutic mAb Aggregate Analysis Using TSKgel® UP-SW3000, 2 μm, SEC Column

HPLC analytical size exclusion chromatography (SEC) columns are widely used to determine the ratio of aggregates, dimers, monomers, and fragments in monoclonal antibodies (mAbs). Columns are expected to deliver high resolution, excellent reproducibility in a short analysis time. In order to achieve these parameters, SEC columns must have the appropriate particle size, pore size, good bonding chemistry, and suitable column dimensions. In addition, the columns must be packed well. Traditionally, SEC columns with 30 cm length are used for high resolution analysis because the length allows different molecular sizes to be separated with a longer run time. However, because of the long length, a typical analysis can take up to 30-40 minutes for each analysis. With the demands for high sample throughput, there is a need for shorter analysis time. There are many available SEC columns with 15 cm length currently available for this usage. However, these columns typically suffer from low resolution.

This application describes the use of a 4.6 mm ID × 15 cm TSKgel UP-SW3000 SEC column for fast and accurate mAb aggregate analysis without compromising the quality of the aggregate determination or reproducibility. Unlike many other available 15 cm length SEC columns, these columns are packed such that they can be operated with both HPLC and UHPLC systems. The 4.6 mm ID × 15 cm TSKgel UP-SW3000 SEC column has a particle size of 2 μm and a 25 nm pore size. The particles are coated with a hydrophilic diol-type bonded phase in order to minimize the interaction between the silica surface and proteins. The column is designed to be operated with a simple and well established method (sodium phosphate mobile phase, pH 6.8). A comparison study was done between a TSKgel UP-SW3000, 15 cm column and a 30 cm length column, both 4.6 mm ID. Results show that the run time of the 15 cm column was completed in 4 minutes without compromising the resolution of the chromatogram.

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