Rapid PNGase F enables complete and rapid deglycosylation of antibodies and immunoglobulin fusion proteins, as well as other glycoproteins, in minutes. All N-glycans are released rapidly and without bias, and are ready to be prepared for downstream chromatography or mass spectrometry analysis. Rapid PNGase F creates an optimized workflow, reducing processing time without compromising sensitivity or reproducibility.
Complete deglycosylation of antibodies and immunoglobulin fusion proteins in 10 minutes
Recombinant enzyme; guaranteed endoglycosidase F1, F2 or F3 free
All reaction components are compatible with HPLC and mass spectrometry analysis
≥ 95% purity, as determined by SDS-PAGE and intact ESI-MS
Optimal activity and stability ensured for up to 12 months
A growing number of antibodies and
antibody fusions are currently used as therapeutic
agents. A conserved N-glycan at Asn297 of the Fc
region of IgG is critical for functional activity. Moreover,
some antibodies have additional N-glycans
that, together with the conserved site, affect
recognition, half-life, and immune reactions.
Antibody glycosylation is heterogeneous, and variables
in cell culture can increase glycan diversity.
Monitoring glycosylation during production is
essential to obtain the correct glycoprotein forms.
PNGase F is the most effective enzymatic method
for removing almost all N-linked oligosaccharides
from glycoproteins. PNGase F digestion deaminates
the aspargine residue to aspartic acid, and leaves
the oligosaccharide intact, keeping it suitable for
further analysis.
Obtaining an accurate N-glycan profile in the shortest
time possible is essential for effective process
control. Typically, enzymatic release of antibody
N-glycans using PNGase F requires an incubation
time of several hours, followed by glycan derivatization
and analysis by liquid chromatography and/
or mass spectrometry. In addition, incomplete
deglycosylation can lead to biased results. Some
glycans are easier to remove than others and unless
deglycosylation is extensive, the profile obtained
will not represent the correct composition of the
therapeutic antibody.
Rapid PNGase F is an improved recombinant reagent that allows
the complete and rapid deglycosylation of antibodies
and fusion proteins in minutes. All N-glycans
are released rapidly and without bias, and are ready
to be prepared for downstream chromatography
or mass spectrometry analysis. Rapid PNGase F
creates an optimized workflow which reduces
processing time without compromising sensitivity
or reproducibility. Figure 1: Possible structures of lgG and lgG-fusion proteins ESI-TOF analysis of an antibody before and after treatment with Rapid PNGase F
Specificity
Rapid PNGase F cleaves all complex, hybrid and high-mannose type glycans from antibodies and related proteins (1). Core α1-3 fucosylation (found in immunoglobulins expressed in plant or insect cells) is resistant to both PNGase F and Rapid PNGase F.
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
P0710S
4
Rapid™ PNGase F
P0710SVIAL
4
1 x 0.05 ml
50 reactions
Rapid PNGase F Buffer
B0718SVIAL
4
1 x 0.2 ml
5 X
Properties & Usage
Reaction Conditions
1X Rapid PNGase F Buffer
Incubate at 50°C
Heat Inactivation
75°C for 10 minutes
Advantages and Features
Application Features
Rapid PNGase F is an improved recombinant reagent that
allows the complete and rapid deglycosylation
of antibodies and fusion proteins in only minutes.
All N-glycans are released rapidly and without
bias, ready to be prepared for downstream
chromatography or mass spectrometry analysis.
A variety of therapeutic monoclonal antibodies
were used to validate Rapid PNGase F: different
subclasses (IgG 1 to 4), isotypes (IgA, IgM, IgE),
organisms (mouse, human, and humanized),
sources (CHO, murine myeloma), and structures
(IgG, IgG-fusions).
Rapid PNGase F can effectively remove all
N-glycans from both conserved (i.e. Fc Asn297)
and non-conserved (i.e. Fab N-glycans) glycosylation
sites. Validation was in accordance with
published data. Sensitivity and specificity are not
compromised by a faster and more convenient
glycoprotein characterization workflow using
Rapid PNGase F.
Before using the Rapid PNGase F, make sure all components are in solution.
Before incubation, make sure the Rapid Buffer, IgG, and rapid PNGase F are properly mixed.
If needed, reaction volumes can be scaled up or down, adjusting the amount of Rapid PNGase F accordingly. Altering the components ratio will lead to suboptimal results (for instance, using a larger reaction volume with only 1ul of enzyme).
For high throughput applications reactions can be prepared at room temperature. The deglycosylation reaction will begin once the temperature is raised to 50ºC.
Typically, the reaction is completed after 3-5 minutes at 50ºC. Incubation longer than 15 minutes will not result in further deglycosylation. If the reaction is still incomplete, try a 2 step protocol (pre-denaturation at 80C).
If inactivation of Rapid PGNase F is required after deglycosylation, heat for 20 minutes at 70C. Higher temperatures might result in coagulation of target antibody.
Avoid buffers containing SDS, as it inhibits PNGase F. Common stabilizing reagents such as Tween, Triton X-100, NP-40, octyl glucoside and non-detergent sulfobetaine, as well as traces of organic solvents, can prevent optimal rapid deglycosyl¬ation.
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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