Importance of Developing a Robust UPLC-Based BEH SEC Method
a. Choosing the Appropriate ACQUITY UPLC Protein BEH SEC Column
A number of factors need to be evaluated in the development of a UPLC Protein BEH SEC separation method that can reliably separate and accurately quantitate the various constituents contained in a protein sample. SEC separates compounds primarily based on their relative size in solution. Figure 3 shows calibration curves generated on ACQUITY UPLC Protein BEH SEC Columns of different pore sizes. This data is based on the use of proteins and peptides of known molecular weights. Chromatographers can use this data to select the most appropriate Protein BEH SEC column for a specific application performed on an appropriately configured Waters ACQUITY UPLC System.
Figure 3. Calibration curves for ACQUITY UPLC Protein BEH SEC, 125Å, 200Å, and 450Å Columns.
b. Method Development
A number of factors need to be evaluated in SEC method development. Ideally, SEC separations are based on differences in the size of native proteins in solution. For this reason, size-exclusion chromatography of biomolecules is usually performed under aqueous, "physiological" conditions. However, the presence of secondary interactions can obscure the desired size-based separation and accurate quantitation of the resolved species. To minimize secondary interactions, the mobile phase and separation conditions need to be evaluated. It should be noted that the conditions used for the SEC separation can alter the protein structure and state. The concentration and type of salt used, as well as, the mobile-phase pH, can affect the three-dimensional structure and the protein-protein interactions. For these reasons, evaluation of a SEC method should be performed with the actual sample to be analyzed. (For more information, reference "Method Development for Size-Exclusion Chromatography of Monoclonal Antibodies and Higher Order Aggregates.")
Figure 4. The effect of pH and ionic strength on the separation of a mAb using an ACQUITY UPLC Protein BEH SEC, 200Å, 1.7 µm, 4.6 x 300 mm Column.
