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Analysis of human erythropoietin and erythropoietin N-linked glycans using capillary electrophoresis and mass spectrometry

University of British Columbia

Glycoproteins are of interest as therapeutic agents. Characterization of the glycan component of glycoproteins is challenging due microheterogeneity, but necessary as glycans can cause negative immune system reactions. Erythropoietin is a glycoprotein hormone that stimulates the production of red blood cells and is used as a therapy for anemic patients. However, erythropoietin is also used as a doping agent in sports to boost performance by increasing the oxygen-carrying capacity of blood. Techniques for studying the glycans of erythropoietin and for differentiating between endogenous and exogenous erythropoietin are of interest in the scientific community. This thesis presents work on the development of techniques for the study of erythropoietin and its glycans for differentiation between endogenous and exogenous erythropoietin. Chapter 2 presents a capillary electrophoresis-mass spectrometry method that separates the terminal residue found on glycans, sialic acid. The presence of one type of sialic acid indicates an exogenous origin since humans cannot produce that sialic acid. By manipulating the post-column chemical conditions of the separation, the sensitivity of the method was increased. The most common sialic acid and the sialic acid that cannot be produced by humans were separated, and the feasibility of the method to detect low abundance levels of sialic acids from erythropoietin was shown. A key factor in lowering the LOD was the ability to inject reproducibly from a 1 µL sample volume. In chapter 3, different sialic acids from the producing conditions of erythropoietin (e.g. in the body or in cell culture) change the isoelectric point. A capillary isoelectric focusing method was developed with ultraviolet and laser induced fluorescence detection to analyze erythropoietin produced both endogenously and exogenously. The method was able to separate erythropoietins produced from different sources, and the isoelectric points of the various glycoforms of recombinant erythropoietin were determined. Chapter 4 focuses on simulations of an electro-fluid-dynamic microfluidic device for the feasibility of desalting glycoproteins with the device. Simulations concerning the ability of the device to selectively separate and purify analytes using a single power supply by manipulating the dimensions of the lengths and widths of the collection channels of the device.

Text

2020-06-16

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/74706

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/