Analysis of Polysorbate 80 in IgG Aqueous Solution by Online SPE Using a Shim-pack MAYI Column - Part 2

Introduction

Polysorbates play a role in maintaining the stability of proteins to prevent their denaturation, so they are often added to protein formulations. Therefore, evaluation of the quality and stability of these formulations also requires evaluation of the quality of the polysorbate. A polysorbate is not just a mixture of polyoxyethylene sorbitan fatty acid esters with polyoxyethylene chains of different degrees of polymerization. It has a complex composition which includes a variety of by-products as impurities, and those are said to affect the stability of protein formulations. Thus, it is believed that utilizing the high resolution offered with an HPLC coupled with a mass spectrometer can provide effective monitoring of quality. In Application News No. L486, a method in which automated deproteinization can be conducted followed by quantitative analysis of polysorbate 80 in an antibody model sample was presented. Here, using this system for higher resolution analysis, we conducted detection and mass spectral measurement of possible by-product components of polysorbate 80. After about 3 minutes, switching was made to include the pretreatment column in the analytical flow line, and polysorbate 80 began to elute from that column. In this case, separation with a more gradual gradient elution was achieved using an analytical column longer than that used in quantitative analysis (Application News No. L486). For detection, the LCMS-8050 was used (Fig. 1, Table 2). Fig. 2 shows the TIC chromatogram in which elution is achieved with an especially gradual slope and retention times up to 35 minutes.

April 16, 2015 GMT