β-glycosylamines versus free-reducing-end glycans
Because direct coupling of the microfluidic LC–MS chip to the TOF-MS enables detection of -glycosylamine intermediates, it
is important to understand the kinetics of hydrolysis from the -glycosylamine to free-reducing end forms. By varying the
glycan residence time on the PGC trapping column, it is possible to monitor the consistency of the distribution of G0, G1,
and G2 during hydrolysis.
In the time-course experiment summarized in Figure 8, the glycans remained on the trapping column for 0, 30, 60, and 120 min.
The ECC's depict the decrease in abundance of the -glycosylamine forms and the corresponding conversion (increase) to the
free-reducing-end forms over time. With no wait time on the trapping column, as the chip would be used in a real-time process-monitoring
application, The β-glycosylamines were eluted between 2.2 and 2.4 min, and the free-reducing-end glycans were eluted between
2.5 and 2.7 min. The ECC showed that, with no wait time, the abundance counts of The β-glycosylamines were more than double
those of the free-reducing-end glycans. With the microfluidic LC–MS chip approach, measuring β-glycosylamines, rather than
the free-reducing-end forms, doubles the sensitivity of analysis. After 120 min, The β-glycosylamines were completely hydrolyzed
to free-reducing-end forms. The relative percent of the three most abundant glycans was maintained throughout the experiment.
Figure 8: Kinetics of hydrolysis of β-glycosylamines to free-reducing-end forms. By varying the gradient delay time, the time
the glycans were trapped on the porous graphitized chromatographic column, the glycans were allowed to hydrolyze for 0, 30,
60, and 120 min. The β-glycosylamines eluted between 2.2 and 2.4 min, and their peak intensity decreased over time. The free-reducing-end
glycans eluted between 2.5 and 2.7 min, and their peak intensity increased over time. By 120 min, The β-glycosylamines were
completely hydrolyzed to the free-reducing-end forms. The calculated distribution of the glycans G0, G1, and G2 was maintained.
Analysis of intact and deglycosylated antibodies
Using a different chip configuration, it is possible to analyze deglycosylated antibody. For this analysis, the microfluidic
chip was constructed of two layers rather than three: the PNGase enzyme reactor chamber and the LC–MS C8 packed chip layer.
Because laser ablation of polyimide film is used, it is possible to create chips with a wide variety of features and functionality.
By varying the residence time in the reactor chamber, it is possible to determine the time required to completely deglycosylate
the antibody. Figure 9 shows the results from the analysis of intact and degylcosylated antibody A1. Figure 9 (a) shows deconvoluted
mass spectrum of the intact antibody without deglycosylation (no time in the reactor chamber). Three main peaks were found.
The mass difference between consecutive peaks is 163.9 and 161.8 amu, which corresponds to the characteristic mass difference,
162.05, of the terminal galactose on the G0, G1, and G2 glycans. Because A1 has two glycosylation sites, the peaks suggest
a combination of G0, G1, and G2 glycans attached. Specifically, the masses indicate that A1 has the following glycan attachment
configurations: G0 and G0, G0 and G1, G1 and G1, or G0 and G2.
Figure 9: Characterization of intact and deglycosylated antibody Ab1. Figure 9 (a) shows the deconvoluted mass spectrum of
the intact antibody Ab1 prior to deglycosylation. The three peaks suggest a combination of G0, G1, and G2 glycan modifications.
The deconvoluted mass spectrum in Figure 9 (b) shows the partially deglycosylated antibody following a 3-s deglycosylation
in the reactor chamber. Figure 9 (c) shows the single-peak mass spectrum of the completely deglycosylated antibody following
a 6-s residence time in the reactor chamber. mAb is monoclonal antibody.
Figure 9 (b) shows the partially deglycosylated antibody following a 3-s deglycosylation in the reactor chamber. Partially
and completely deglycosylated A1 was measured. The peaks represent the partially deglycosylated forms as follows: 147,645.93
amu is from the A1 G0 form; 147,806.94 amu is the A1 G1 form; 146,201.21 amu is the deglycosylated form. The peak measuring
149,089.41 amu is intact A1 with two G0 glycans. The high mass accuracy of the TOF-MS enables detection of glycans differing
by only one sugar monomer on very high molecular weight antibodies. Figure 9 (c) shows the deglycosylated antibody peak after
6-s residence time in the reactor chamber. Nearly all of the antibody was deglycosylated. The data indicated that this second
chip configuration can be used to rapidly analyze deglycosylated antibody without the need for time-consuming sample preparation,
deglycosylation, and separation steps.