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The authors describe a new microfluidic-based workflow that integrates and automates glycan cleaveage, purification, and chromatographic separation onto a microfluidic liquid chromatography–mass spectrometry chip for MS detection.
Figure 5: Extracted compound chromatogram of the glycans released from antibody sample Ab1. The β -glycosylamine intermediates
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 three predominant
glycan (G) peaks are annotated with the glycan cartoon structures and color coded: red = G0, green = G1, and blue = G2. The
inset shows the mass spectra for the doubly charged -glycosylamine form (top) and the free-reducing-end glycan form (bottom).
The mass difference is 0.984 atomic mass units (amu), which is the mass difference between –OH and –NH2.
CHO cell-derived monoclonal antibody A1 and Ab2 and mouse NS0 cell-derived Ab3 were analyzed to evaluate the integrated microfluidic
LC–MS chip workflow. Figure 5 shows the extracted compound chromatogram (ECC) with separation of the dominant glycan peaks.
The β-glycosylamines eluted between 2.2 and 2.4 min, and the free-reducing-end glycans eluted later, between 2.5 and 2.7 min.
In the -glycosylamine form, each glycan is represented by a single peak in the 1.5-min. separation. In contrast, the free-reducing-end
form is represented by pairs of peaks. This pair is due to the anomeric carbon at the reducing end and is characteristic of
PGC chromatography. In a conventional deglycosylation experiment, a reduction step would have been used to simplify the chromatogram.
The inset in Figure 5 shows the mass spectra of the doubly charged -glycosylamine form and the free-reducing end for the
glycan G0. The difference in mass–to–charge ratio (m/z) of the doubly charged monoisotopic ions is shown to be 0.492, which
corresponds to the 0.984 atomic mass units (amu) mass difference between the –OH and –NH2 forms. Because The β-glycosylamines exist as single peaks, a reduction step is not needed. An important advantage of coupling
TOF-MS to the microfluidic chip is that it enables detection of -glycosylamine intermediates prior to conversion to free-reducing-end
glycans. The acidification step used with traditional HPLC approaches involving labeling or derivatizing the free glycans
by reductive animation is not needed.
Figure 6: Extracted compound chromatogram for the glycan profile of Ab2. In addition to the fucosylated glycans seen in Ab1
(see Figure 5), Ab2 contains lower abundance nonfucosylated glycans that were separated from the other glycans (eluting earlier,
between 2.1 and 2.2 min).
Figure 6 shows the glycan profile of Ab2. In addition to the fucosylated glycans found in A1 (see Figure 5), Ab2 contains
lower abundance nonfucosylated glycans that were separated from the other glycans, eluting earlier between 2.1 and 2.2 min.
Figure 7: Extracted compound chromatogram of Ab3 made in a mouse NSO cell line. The -glycosylamine G1 (green trace) and G2
isomers (blue trace) were separated using a longer liquid chromatographic gradient. In addition to the more commonly observed
G0, G1, and G2 glycans, many others were found and identified.
As shown for Ab3 in Figure 7, by using a longer 10-min, LC gradient, it is possible to separate isomeric glycan forms. The
data show three peaks with masses equal to the G2 glycan, which are likely to be G2 isomers with the structures labeled G2
and G2 isomer shown in Table I. Antibody Ab3 was expressed in a NS0 mouse cell line that is known to produce G2 isomers.
Mass spectral data were generated by an Agilent 6224 Accurate-Mass TOF LC–MS. Data analysis was performed using Agilent MassHunter
Workstation software. The molecular feature extraction (MFE) algorithm in MassHunter identifies all charge states and adducts
for each compound and combines these into one compound or molecular feature. Following extraction, the mass spectral results
are plotted as an ECC and exported into Microsoft Excel. An Excel macro was created to automatically compute the ratios for
each glycan as a percentage of the total.
Maggie Bynum*is a scientist at Agilent Technologies' Agilent Laboratories, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, tel. 408.553.3152, fax 408.553.2161
Articles by Maggie Bynum
