Glycoform analysis of alpha1-acid glycoprotein by capillary electrophoresis

Abstract

A relatively fast and reproducible CE separation was developed for the glycoform analysis of α₁-acid glycoprotein (AGP). Factors that were considered included the pH for this separation and various techniques for coating the capillary and/or to minimize electroosmotic flow and protein adsorption. Optimum resolution of the AGP glycoforms was obtained at pH 4.2 with a running buffer containing 0.1% Brij 35 and by using static and dynamic coatings of PEO on the capillary. These conditions made it possible to separate nine AGP glycoform bands in about 20min. The limit of detection (based on absorbance measurements) ranged from 0.09 to 0.38μM for these AGP glycoform bands, and the linear range extended up to a total AGP concentration of at least 240μM. The migration times for the glycoform bands had typical within-day and day-to-day precisions of ±0.16-0.23% or less, respectively, on a single treated capillary and the variation between capillaries was ±0.56% or less. A charge ladder approach was employed to examine the mass or charge differences in the glycoforms that made up these bands, giving a good fit to a model in which the neighboring bands differed by one charge (e.g., from a sialic acid residue) and had an average mass difference of approximately 0.7-0.9kDa. The approaches used to develop this separation method are not limited to AGP but could be extended to the analysis of other glycoproteins by CE.

Keywords: Alpha(1)-acid glycoprotein; Capillary coating; Capillary electrophoresis; Charge ladder; Glycoform analysis; Glycoproteins.

Figure 1

Effect of pH on the resolution of AGP glycoforms. Conditions: sample, 4.0 g/L AGP in water; capillary conditions, 5 min rinse with 1.0 M sodium hydroxide, 3 min rinse with water, 5 min rinse with 1.0 M hydrochloric acid, 5 min rinse with 0.2% (w/v) PEO containing 0.1 M hydrochloric acid, 5 min rinse with buffer (pH 5.0, 4.6, 4.4, 4.2, 4.0, or 3.8, 20 mM sodium acetate); injection, 0.5 psi, 3 s; separation voltage, −30 kV.

Figure 2

Separation of AGP glycoforms on an untreated silica capillary. Conditions: sample, 4.0 g/L AGP in water; capillary conditions, 5 min rinse with 1.0 M sodium hydroxide, 3 min rinse with water, 5 min rinse with pH 4.2, 20 mM sodium acetate buffer; injection, 0.5 psi, 3 s; separation voltage, +30 kV. A rinse with 1.0 M sodium hydroxide for 20 min was performed between the third and fourth injections.

Figure 3

Structures of poly(vinyl alcohol), or PVA; poly (ethylene oxide), or PVA; and dextran. The structure shown for dextran is based on an α(1,6)-type linkage; less than 5% of the dextran had an α(1,3)-type linkage.

Figure 4

Separation of AGP glycoforms by capillaries that had been modified with various types of coatings: (a) static coating of PVA (range of peak resolution values, R_s, 0.53–0.74), (b) static and dynamic coatings of PVA (R_s, 0.41–0.84); (c) permanent coating of PVA (R_s, 0.48–0.85); (d) permanent and dynamic coatings of PVA (R_s, 0.68–0.76); (e) static coating of dextran (R_s, 0.43–0.98); (f) static and dynamic coatings of dextran (R_s, 0.42–0.87); (g) static coating of PEO (R_s, 0.71–0.95); and (h) static and dynamic coatings of PEO (R_s, 0.44–1.08). Conditions: sample, 4.0 g/L AGP in water; injection, 0.4 psi for 3 s; applied voltage, −30 kV.

Figure 5

(a) Electropherograms obtained for AGP glycoforms at various concentrations of Brij 35 in the running buffer, expressed as %(w/v), and (b) the average resolution of the AGP glycoform bands as a function of the concentration of Brij 35 that was placed into the running buffer. Conditions: running buffer, pH 4.2 acetate buffer containing 0.05% (w/v) PEO and various concentrations of Brij 35, sample, 4.0 g/L AGP in water; injection, 0.5 psi, 5 s; separation voltage, −30 kV.

Figure 6

Validation of CE method for the separation of AGP standards: Sample, 4 g/L AGP dissolved with water; Background electrolyte, pH 4.2 acetate buffer with 0.05% (w/v) PEO and 0.1% (w/v) Brij 35; Separation voltage, −30 kV.

Figure 7

Observed relationship for a plot of $\frac{1}{{\mathrm{\mu }}_{\mathrm{n}}-{\mathrm{\mu }}_0}$ versus $\frac{1}{\mathrm{n}}$ for AGP glycoforms, where μ₀ is the electrophoretic mobility of AGP glycoform with the least negative charge; μ_n is the electrophoretic mobility of an AGP glycoform that has an additional number (n) of charges (e.g., due to sialic acids) versus the glycoform with the least negative charge. The best-fit line obtained for this fit was y = −1810 (± 20) x − 29 (± 9), with a correlation coefficient of 0.9997 (n = 90).