Description:

A growing number of antibodies andantibody fusions are currently used as therapeuticagents. A conserved N-glycan at Asn297 of the Fcregion of IgG is critical for functional activity. Moreover,some antibodies have additional N-glycansthat, together with the conserved site, affectrecognition, half-life, and immune reactions.Antibody glycosylation is heterogeneous, and variablesin cell culture can increase glycan diversity.Monitoring glycosylation during production isessential to obtain the correct glycoprotein forms.PNGase F is the most effective enzymatic methodfor removing almost all N-linked oligosaccharidesfrom glycoproteins. PNGase F digestion deaminatesthe aspargine residue to aspartic acid, and leavesthe oligosaccharide intact, keeping it suitable forfurther analysis.Obtaining an accurate N-glycan profile in the shortesttime possible is essential for effective processcontrol. Typically, enzymatic release of antibodyN-glycans using PNGase F requires an incubationtime of several hours, followed by glycan derivatizationand analysis by liquid chromatography and/or mass spectrometry. In addition, incompletedeglycosylation can lead to biased results. Someglycans are easier to remove than others and unlessdeglycosylation is extensive, the profile obtainedwill not represent the correct composition of thetherapeutic antibody.Rapid PNGase F is an improved recombinant reagent that allowsthe complete and rapid deglycosylation of antibodiesand fusion proteins in minutes. All N-glycansare released rapidly and without bias, and are readyto be prepared for downstream chromatographyor mass spectrometry analysis. Rapid PNGase Fcreates an optimized workflow which reducesprocessing time without compromising sensitivityor reproducibility.Figure 1: Possible structures of lgG and lgG-fusion proteinsESI-TOF analysis of an antibody before and after treatment with Rapid PNGase F

Specificity

Rapid PNGase F cleaves all complex, hybrid andhigh-mannose type glycans from antibodies andrelated proteins (1). Core α1-3 fucosylation (foundin immunoglobulins expressed in plant or insectcells) is resistant to both PNGase F and RapidPNGase F.

Reagents Supplied

The following reagents are supplied with this product:

	Store at (°C)	Concentration
Rapid PNGase F Buffer	4	5X

Notes:

Storage: It is recommended to store this kit at 4°C.All components will be stable for at least one year if stored correctly. Do not freeze.