Comparison of Different Brands of Carrier Ampholytes for Monoclonal Antibody Charge Heterogeneity Analysis by Capillary Isoelectric Focusing

This Application Note compares the performance of four commercially available brands of wide-range pH 3 to 10 carrier ampholytes, Pharmalyte, Servalyt, HR, and SH AESlyte, for high-resolution capillary isoelectric focusing on fluorocarbon-coated capillaries. The carrier ampholytes-specific background was increased for Servalyt, and slightly increased for SH AESlyte in comparison to Pharmalyte and HR AESlyte. With all tested brands of carrier ampholytes, it was possible to analyze monoclonal antibody charge isoforms with high resolution and precision. The peak pattern of the test sample showing multiple isoform peaks in the pH range 6 to 7 was similar with Pharmalyte and HR AESlyte. A slightly increased resolution was observed with SH AESlyte, and the best resolution with Servalyt. The intermediate precision of experimentally determined isoelectric points obtained with three different capillary batches on three different days was better than 0.1 %RSD for all brands of carrier ampholytes and all isoforms. In terms of percent area, the observed intermediate precision was better than 3 %RSD for Pharmalyte and HR AESlyte, and better than 9 %RSD for SH AESlyte and Servalyt, with one exception.