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August/September 2011
relative glycan ratios in the range 1.6 to 3.9 % show robustness and the stability of the on- chip deglycosylation workflow.
Comparison: On-chip versus standard in-solution deglycosylation experiment Figure 8 shows a comparison of mAb-Glyco Chip based N-glycan analysis to results obtained from an in-solution PNGase F deglycosylation workflow*. Table 5 summarises experimental details.
Figure 7: Long-term stability and robustness of the mAb-Glyco Chip: (A) Extracted glycan pattern of the analysed antibody at injection number 1 and 200. (B) Relative glycan ratio as function of the number of injections (4 most intense N-glycans considered). Sample: IgG from bovine serum (Sigma), 75ng on-column.
In this current case 5 to 6 hours, as required with standard analyses, were reduced to 12 min. The most substantial time saving was due to the reduction of the deglycosylation time from 180 in standard analyses to 4 min with the mAb-Glyco Chip. In standard analyses long exposure times of the mAb to the PNGase F enzyme are needed in order to cleave all the glycans quantitatively. This is crucial since quantification occurs relative to the total amount of glycans. The strong reduction in deglycosylation time with the mAb-Glyco Chip is due to the large amount of PNGase F enzyme that is immobilised on a highly macroporous, large surface support material, leading to a high enzyme to substrate ratio and thus to a fast deglycosylation rate.
Figure 8: Chromatograms obtained (A) from analysis with mAb-Glyco Chip and (B) in-solution workflow. (C) Identified N-glycans together with calculated ratios, relative to glycan-total. Sample: IgG1 Kappa from human myeloma plasma (Sigma), 75ng and 100ng on-column for mAb-Glyco Chip and standard workflow, respectively.
Amount of antibody per sample Deglycosylation time
Protein (deglycosylated mAb and PNGase F) removal
Glycosylamine hydrolysis
Glycan seperation and detection Total workflow time
mAb-Glyco Chip 1µg
4 min not required (immobilized PNGase F)
not required (analysis on glycosylamine level)
6 min 12 min 2 hour 6 min 5-6 hours Table 5: Summary of experimental details: mAb-Glyco Chip and standard in-solution deglycosylation workflow.
5% RSD for glycans > 1% relative ratio. Spray stability was maintained for more than 200 hours. Typical lifetime was in the range of 200-300 injections (10 chips evaluated). Under appropriate storing conditions (-20 °C, wet ER), the immobilised PNGase F conserved 90% of its original activity after a period of three months. Multiple freeze/thaw cycles do not severely affect PNGase F
activity after immobilisation. Figure 7 (A): Glycan chromatograms obtained from injection 1 and 200. Retention time, relative glycan distribution and absolute signal intensities of the identified glycan pattern remain well comparable demonstrating full catalytic activity of the PNGase F reactor over the course of 200 injections. Figure 7 (B): %RSD values for
In-Solution Deglycosylation 20µg
3 hours 10 min
Additional time is saved because the chip based approach does not require conversion of glycosylamines to free glycans. The chromatogram in Figure 8 (A) shows that the mAb-Glyco Chip predominately detects glycosylamines, whereas the standard workflow, the later eluting free reducing end glycans (Figure 8 (B)). The graphic in Figure 8 (C) demonstrates that relative ratios of all glycans are well comparable between the two analyses. Moreover, the mAb-Glyco Chip analysis typically results in a higher number of identified low abundant glycans, which could be attributed to: 1.) Higher ionisation efficiency of glycosylamines, 2.) Conversion of one glycosylamine into two, free reducing end glycans, which could lower the limit of detection in the in-solution workflow, and 3.) potential losses of low abundant glycans in the multi-step in-solution procedure in contrast to the integrated on-chip workflow.
*In-solution deglycosylation experiments were done using a PNGase F kit from New England Biolabs. 20µg of the protein was combined with 4µl of G7 Reaction Buffer, 10µl of PNGase F and water to make a total reaction volume of 40µL. This mixture was incubated for 3h at 37ºC. Deglycoslyated antibody was removed by centrifugation using Vivaspin 4 vials with a molecular cut-off
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