Isolation and quantification of N-glycans from immunoglobulin G antibodies for quantitative glycosylation analysis
- TRIS (Fisher Scientific, Cat. # BP152)
- Sodium phosphate monobasic (NaH2PO4) (Fisher Scientific, Cat. # BP329)
- Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, Cat. # BP332)
- Glycine (Fisher Scientific, Cat. # BP381)
- Acetonitrile (Fisher Scientific, Cat. # A998)
- HCl (Fisher Scientific, Cat. # AC124210010)
- Ammonium hydroxide (NH4OH) (Fisher Scientific, Cat. # AC255210010)
- Formic acid (Fisher Scientific, Cat. # AC270480010)
- Acetic acid (Fisher Scientific, Cat. # AC222140010)
- Dimethyl sulfoxide (DMSO) (Fisher Scientific, Cat. # AC397600010)
- 2-Aminobenzamide (2-AB) (Sigma-Aldrich, Cat. # A89804)
- Sodium cyanoborohydride (NaBH3CN) (Sigma-Aldrich, Cat. # 156159)
- Maltotetraose (DP4) (Sigma-Aldrich, Cat. # 47877)
- PNGase F (Promega Corporation, Cat. # V4831)
- 2-AB-Dextran ladder (Waters Corporation, Cat. # 186006841)
- Note: Unlabelled dextran standard can be purchased from other sources and 2-AB labeled stock can be prepared using the procedure presented in section 4 of this protocol
2-AB labeling solution
Elution buffer (100 mM glycine-HCl)
Neutralizing buffer (1 M TRIS-HCl)
Buffer A (50 mM ammonium formate solution)
- Protein-A HP SpinTrap columns (GE Healthcare Life Sciences, Cat. # 28-9031-32)
- HyperSep Diol SPE cartridges (Thermo Fisher Scientific, Cat. # 60108-571)
- Nanosep 10K Omega filters (Pall Corporation, Cat. # OD010C33)
- 2 ml sample tubes (Corning Life Sciences, Cat. # MCT-200-C)
- 1.5 ml sample tubes (Corning Life Sciences, Cat. # MCT-150-C)
- 0.5 ml tube rack (Eppendorf, Cat. # 022364243)
- 1.5 ml tube rack (Eppendorf, Cat. # 022364227)
1.Antibody capture and glycan cleavage
1.1.Remove the plug at the bottom of new Protein A column, place it in 2 ml sample tube and centrifuge at lowest possible speed on micro-centrifuge to remove storage solution.
1.2.Wash the column by adding 600 µl of phosphate buffer and centrifuge to remove the buffer.
1.3.Plug the bottom of the column and add antibody solution.
1.4.Incubate at room temperature for 15 min on a rocker or orbital shaker.
1.5.Remove the plug and centrifuge to remove the solution.
1.6.Repeat the steps 1.3–1.5, if necessary, to load desired amount of antibody (50–100 µg).
1.7.Wash the columns 3 times with 600 µl of phosphate buffer.
1.8.Plug the bottom of column and add 150 µl of phosphate buffer and 10–20 units of PNGase F (1–2 µl).
1.9.Incubate the columns at 37°C on a rocker or rotary shaker for 24 h.
2.1.Add 1 or 2 nmol of DP4 standard to the protein-A column containing released glycans and mix gently (optional).
2.2.Remove the plug and place the column immediately in fresh 2 ml tube.
2.3.Centrifuge to collect the filtrate containing glycans.
2.4.Plug the bottom of the column and add 250 µl of ultrapure water, place the cap and rinse the column by gently inverting the column with hand.
2.5.Remove the plug and collect the filtrate into the same tube as in step 2.3.
2.6.Plug the bottom of the column and add 600 µl of phosphate buffer and keep it aside for eluting antibody as described in section 3.
2.7.Filter the glycans through 10K filter and collect the filtrate into 1.5 ml micro-centrifuge tube.
2.8.Dry the glycans in a vacuum concentrator.
3.Elution of antibody (optional)
3.1.Take the column from step 2.6 and wash it three times with phosphate buffer.
3.2.Plug the bottom of column and add 300 µl of elution buffer.
3.3.Gently mix by shaking with hand.
3.4.Collect the filtrate into a 2 ml tube.
3.5.Repeat steps 19 and 20 with 100 µl of elution buffer and collect the filtrate in the same tube as in step 3.4.
3.6.Neutralize the eluted antibody with neutralizing buffer to pH ~7.2.
3.7.Store it to quantify later by absorbance at 280 nm using UV-Vis spectrophotometer.
4.2-AB labeling and clean-up of glycans
4.1.Add 5 µl of 2-AB solution to the dried glycans tube from step 2.8 and mix.
4.2.Incubate at 65°C for 2 h.
4.3.Wash the HyperSepTM Diol cartridge with 1 ml of ultrapure water.
4.4.Condition the cartridge with 4 ml of acetonitrile (1 ml each time).
4.5.Add 40 µl of acetonitrile to the labeled glycans, mix the solution and transfer the glycans on to the top of the cartridge.
4.6.Let it sit for 15 min to allow the glycans to bind.
4.7.Wash the cartridge with 6 ml of acetonitrile (1 ml at each time).
4.8.Elute the glycans with 3 × 400 µl of ultrapure water and collect them into 1.5 ml micro-centrifuge tube.
4.9.Dry the labeled glycans in a vacuum concentrator.
5.Analysis of glycans by HILIC-HPLC
5.1.Add 20 µl of ultrapure water to each glycan sample from step 4.9 and mix well.
5.2.Prepare samples with HPLC vials by mixing 6 µl of sample with 24 µl of acetonitrile in HPLC auto-sampler tubes.
5.3.Prepare 30 µl of dextran ladder standard in HPLC vial with 80% of acetonitrile.
5.4.Place the vials and run the HPLC with the above mentioned settings.
5.5.Integrate the dextran ladder standard chromatogram with empower software and obtain retention times for at least 12 glucose unit (GU) polymers.
5.6.Fit the GU value as a function of retention time (t) of dextran standard data with a polynomial equation: GU = a × t + b × t2 + c × t3.
5.7.Integrate the chromatograms of samples and record the retention times and peak areas for each sample as outlined in Table 3.
5.8.Calculate the GU values for each peak and assign structure based on reference GU values from Glycobase database (https://glycobase.nibrt.ie)
5.9. If needed, perform exo-glycosidase digestion of the labeled glycan sample and rerun on HPLC to confirm the structures assigned.
5.10.If internal standard is used, record the area of internal standard and sum of the areas of all N-glycans of a sample as outlined in Table 4
5.11.Perform the calculations for the samples as outlined in Table 4 to quantify the molar yield of glycan units per molar quantity of antibody sample.
Kinetics of glycan release
Glycan recovery efficiency and reproducibility
1.Samples contain only internal standard peak with no trace of glycans.
1.1No antibody captured: Check pH of mAb solution.
1.2Antibody captured: Check PNGase F activity or verify the reaction buffer pH.
1.3Antibody with uncommon Fc sequence or N-glycan structures: Verify with a glycan analysis method involving reduction of antibody (in-solution or gel-based method)
2.Neither internal standard nor glycans peaks were detected.
2.1Glycans were not labeled: Check the labeling solution.
2.2Glycans were lost during processing: Check the glycan clean-up cartridges.
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