May 21, 2026
Enzymatic Release of N-Glycans from Monoclonal Antibodies Using PNGase F
- Kimmy Park1,2,
- Ron Orlando1,3
- 1Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602;
- 2Current address: Incyte, Wilmington, DE, 19803;
- 3GlycoScientific, Athens, GA, 30602
- Orlando Lab Protocols
Protocol Citation: Kimmy Park, Ron Orlando 2026. Enzymatic Release of N-Glycans from Monoclonal Antibodies Using PNGase F. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjk5d5gk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 20, 2026
Last Modified: May 21, 2026
Protocol Integer ID: 317615
Keywords: glycans from monoclonal antibody, release of glycan, glycan analysis, enzymatic method, released glycan, glycan, enzymatic release, safety of monoclonal antibody, antibody, using peptide, monoclonal antibody, mg of antibody, main enzyme, peptide backbone
Funders Acknowledgements:
Ron Orlando
Grant ID: R42GM113666
Abstract
N-glycan influences the efficacy and safety of monoclonal antibodies. (1-3) N-glycan analysis is a critical quality attribute during drug development. This process involves the release of glycans from the peptide backbone. Two popular methods used in glycan analysis are chemical and enzymatic release. The chemical approach is less popular due to the use of highly toxic chemicals and the potential production of unpredictable modifications to the released glycans. (4-6) Most current studies employ enzymatic methods, using Peptide: N-Glycosidase F (PNGase F) as the main enzyme to release N-glycans.(7-9) PNGase F can cleave most N-glycans, including large and complex species, with the only exceptions for non-mammalian glycans containing a 1,3-core fucose. (10) This protocol provides a detailed, step-by-step method for releasing N-glycans from monoclonal antibodies using PNGase F. This workflow is optimized for 1 mg of antibody but can be scaled proportionally.
Materials
**Reagents**
- Monoclonal antibody (1 mg)
- PNGase F. This protocol has been evaluated with PNGase F purchased from New England Biolabs (catalog # P0704S/ P0704L, Ipswich, MA) and Lectenz Bio (SKU # GE0101, Athens, GA).
- Dithiothreitol (DTT), 40 mM or 40 mM DTT in 0.5% SDS (if using denaturing buffer kit purchased from New England Biolabs)
- NP-40, 1% (required only when SDS is present) (provided in the denaturing buffer kit purchased from New England Biolabs)
- Ultrapure water
- 50 mM ammonium bicarbonate
**Equipment**
- Heat block capable of 100°C
- Pipettes and low-retention tips
- Vortex mixer
- Incubator at 37°C
- Lyophilizer or SpeedVac (optional)
Before start
**Reagent Preparation**
**50 mM ammonium bicarbonate**
1. Weigh 40 mg of ammonium bicarbonate
2. Add 10 mL water.
3. Mix until fully dissolved
**40 mM DTT**
1. Weigh 6.2 mg of DTT
2. Add 1 mL of water.
3. Mix until fully dissolved
Optional: 40 mM DTT 0.5% SDS is provided when purchasing PNGase F from New England Biolabs or can be purchased separately from available denaturing kit.
Step-by-Step Procedure
Denaturation of the Antibody: Mix 1 mg monoclonal antibody with 50 mM Ammonium bicarbonate to 1 mg/mL concentration.
Add 4 µL of 40 mM DTT or 4 µL of denaturing buffer (40 mM DTT, 0.5% SDS).
Heat at 100°C for 12 minutes to fully denature the protein.
Cool the Sample: Immediately place the tube on ice or in a freezer for 5 minutes to dissipate residual heat.
Neutralize SDS (Only if SDS Was Used in the denaturation step): Add 4 µL of 1% NP-40 to neutralize SDS and prevent PNGase F inhibition.
Add PNGase F: Add 2–4 µL of PNGase F (or the equivalent recommended enzyme units).
Incubation: Incubate at 37°C for 16–20 hours.
Collection of Released Glycans: After incubation, collect the reaction mixture containing released N-glycans and residual protein.
Lyophilize or speedvac to dryness if preparing for downstream cleanup/labeling or store at -80°C.
Troubleshooting
Low glycan yield: Possible causes include incomplete denaturation, SDS inhibition, or insufficient PNGase F. Solutions: Increase incubation time at 100°C to achieve effective denaturation, ensure NP-40 was added when SDS is present, or increase enzyme units or extend incubation.
Protein precipitation: Possible causes include overheating or high SDS. Solutions: Reduce SDS or ensure proper cooling.
Protocol references
(1) Iida, S.; Kuni-Kamochi, R.; Mori, K.; Misaka, H.; Inoue, M.; Okazaki, A.; Shitara, K.; Satoh, M. Two mechanisms of the enhanced antibody-dependent cellular cytotoxicity (ADCC) efficacy of non-fucosylated therapeutic antibodies in human blood. BMC Cancer 2009**, 9_, 58. DOI: 10.1186/1471-2407-9-58.
(2) Planinc, A.; Bones, J.; Delaeghe, B.; Van Antwerpen, P.; Delporte, C. Glycan characterization of biopharmaceuticals: Updates and perspectives. Anal Chim Acta 2016**, 921_, 13-27. DOI: 10.1016/j.aca.2016.03.049.
(3) Castaneda-Casimiro, J.; Vallejo-Castillo, L.; Peregrino, E. S.; Hernandez-Solis, A.; Vazquez-Flores, L.; Chacon-Salinas, R.; Wong-Baeza, I.; Serafin-Lopez, J. N-Glycosylation of Antibodies: Biological Effects During Infections and Therapeutic Applications. Antibodies (Basel) 2025**, 14 (4). DOI: 10.3390/antib14040093.
(4) Patel, T.; Bruce, J.; Merry, A.; Bigge, C.; Wormald, M.; Parekh, R.; Jaques, A. Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins. Biochemistry 1993**, 32 (2), 679-693. DOI: 10.1021/bi00053a037.
(5) Fischler, D. A.; Orlando, R. N-linked Glycan Release Efficiency: A Quantitative Comparison between NaOCl and PNGase F Release Protocols. J Biomol Tech 2019**, 30 (4), 58-63. DOI: 10.7171/jbt.19-3004-001.
(6) Figl, R.; Altmann, F. Reductive Alkaline Release of N-Glycans Generates a Variety of Unexpected, Useful Products. Proteomics 2018**, 18 (3-4). DOI: 10.1002/pmic.201700330.
(7) Onigbinde, S.; Solomon, J.; Gutierrez-Reyes, C. D.; Daramola, O.; Fowowe, M.; Adeniyi, M.; DuBois, K. N.; Bakulski, K. M.; Kanaan, N. M.; Lubman, D. M.; et al. Serum N-Glycan Profiling Identifies Candidate Glycan Biomarkers for Early Detection and Prediction of Alzheimer's Disease. J Proteome Res 2025**, 24 (9), 4417-4436. DOI: 10.1021/acs.jproteome.5c00018.
(8) Lou, D.; Fan, J.; Fan, L.; Song, Y.; Zhang, J. Glycan profiling of therapeutic monoclonal antibodies: Rapid, non-denaturing single-step digestion via pH-tuned enzymatic cleavage. J Chromatogr B Analyt Technol Biomed Life Sci 2026**, 1270_, 124892. DOI: 10.1016/j.jchromb.2025.124892.
(9) Liu, X.; Chen, J.; Fu, B.; Han, S.; Zheng, D.; Zhang, Y.; Lu, H. A high-throughput N-glycan analysis strategy with targeted mass spectrometry (HTnGQs-target) for liver disease diagnosis. Chem Sci 2026**, 17 (1), 364-372. DOI: 10.1039/d5sc06867c.
(10) Tarentino, A. L.; Gomez, C. M.; Plummer, T. H., Jr. Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry 1985**, 24 (17), 4665-4671. DOI: 10.1021/bi00338a028.