HILIC-MS Analysis of Protein Glycosylation Using Nonporous Silica

Rachel Elizabeth Jacobson, Purdue University

Abstract

1.1 Protein chromatography1.1.1 Importance of protein chromatographyBiologics are the fastest growing segment among novel pharmaceutical actives and will only increase with a greater number of biosimilars coming onto the market.1,2 Figure 1.1 shows the increase in FDA approvals of biologic new molecular entities (NMEs) from 2011-2017. Since proteins and other biological molecules are produced by cellular machinery, an inherently imprecise process, these products will be heterogeneous. Furthermore, heterogeneity can be introduced during the purification process, storage, or stress conditions (temperature, light, chemical modifications).3 Protein heterogeneity has a great effect on function, stability, immunogenicity, and clearance; therefore it is necessary to control these processes.4,5 The control process must first begin, however, with characterization.6 Chromatography can aid in this analysis, including providing orthogonal information to mass spectrometry (MS). As biologics continue to increase in number of regulatory approvals, chromatography is expected to be used in their characterization.1.1.2 Basics of chromatographyChromatography is a broad range of techniques used to fractionate molecules in a mixture by their differing properties. The molecules separated are analytes, and the mixture is a sample. The sample is dissolved in the mobile phase, which carries the analytes through the packed bed. In liquid chromatography, which is the focus of this dissertation, the mobile phase is a liquid and the stationary phase is a solid. The constituents of the sample travel through the mobile phase at different rates, depending on their attraction to the stationary phase. The method used in this dissertation is column chromatography, where the mobile phase moves through the stationary phase packed in a cylindrical column. Silica particles are modified to produce a stationary phase and packed inside the column, producing a packed bed. To observe the physical separation of the analytes, a detector is placed at the end of the column. Detection techniques include mass spectrometry, UV absorbance, and fluorescence.Figures of merit in chromatographyA chromatographic separation can be judged by a variety of metrics including resolution, column efficiency, selectivity, retention time, and peak broadening. These figures of merit are not independent of one another but are useful descriptions of specific separation mechanisms. A wide range of conditions can affect these metrics and can be changed to improve the separation.1.1.2.1.1 ResolutionResolution (Rs) is a description of how far apart two peaks are in a separation, calculated in Equation 1, where tRB is the retention time of analyte B, tRA is the retention time of analyte A, WA is the peak width of analyte A, and WB is the peak width of analyte B. The equality on the right can be used to calculate Rs from a chromatogram. To show a mathematical relationship between resolution, the analyte retention factors, efficiency, and selectivity, use the center equality as follows where N is the number of theoretical plates, α is a selectivity term equaling k2'⁄k1', k1 is the retention factor for analyte 1, and k2 is the retention factor for analyte 2.

Degree

Ph.D.

Advisors

Wirth, Purdue University.

Subject Area

Analytical chemistry|Chemistry|Pharmaceutical sciences|Polymer chemistry

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