Intact and N-glycan Analysis of Glycoproteins

Glycoprotein Analysis using Immobilized PNGase F and PNGase F

Removal of N-glycans from glycoproteins is widely used for sample preparation for MS analysis – to reduce the protein heterogeneity and enable released glycan analysis – and to study the functional role of the N-glycans. PNGase F (Peptide N-glycosidase F) is a glycoamidase hydrolyzing the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. During the reaction, the asparagine residue from which the glycan is removed is deamidated to aspartic acid, and the released oligosaccharide is left intact and can be used for further analysis.
 


Schematic overview of the PNGase F workflow.

PNGase F Workflow

Immobilized PNGase F and PNGase F

Benefits of using Immobilized PNGase F

  • Increase enzyme to substrate ratio for faster deglycosylation
  • Reduce enzyme peaks in the final sample
  • Easy-to-use microspin columns with capacity of 200 ug glycoprotein
  • Robust and handles both native and denaturing conditions

Benefits of using PNGase F

  • In-solution deglycosylation
  • Lyophilized format
  • Robust and handles both native and denaturing conditions

PNGase F Workflow

Released Glycan Analysis

The N-glycosylation profile of therapeutic proteins is a critical quality attribute. It affects both safety and efficacy of the biopharmaceutical and therefore needs to be characterized and monitored during development and production. A common glycan analysis workflow is to release the N-glycans with PNGase F and then label the generated glycans with a fluorescent label such as 2-AB, 2-AA, procainamide or RapiFluor-MS™ (Waters Corporation). The labelled glycans are then separated using HILIC-HPLC or capillary electrophoresis to characterize and quantify the different glycan structures.

Here, the N-glycans of the therapeutic antibody trastuzumab, the Fc-fusion protein etanercept and the glycoprotein RNase B were analyzed using a released glycan approach. Trastuzumab and etanercept were deglycosylated with PNGase F under native conditions for 1 h at 37°C, while RNase B required denaturation and reduction to be deglycosylated completely. RNase B was therefore treated with RapiGest™ SF surfactant (Waters Corporation) and TCEP before deglycosylation with PNGase F for 10 min at 50°C. The resulting released glycans were labelled with RapiFluor-MS and analyzed by HILIC UPLC-FLD-MS (Fig. 1).
 
 
 
 
 
 
 
 
 
 

Figure 1. RapiFluor-MS™ labelled N-glycans released from trastuzumab (top), etanercept (middle) and RNase B (bottom) were analyzed by HILIC-FLD-MS using a Waters™ BioAccord™ system equipped with a Waters™ ACQUITY Premier Glycan BEH Amide column (130 Å, 2.1 x 150 mm). The fluorescence chromatograms are shown and the glycan structures were annotated using a combination of glucose unit (GU) library search and MS confirmation.


 

PNGase F Workflow

Hydrolysis of N-glycans under Native Conditions

The Fc N-glycan plays a crucial role in the effector function of antibodies but may add complexity to protein characterization assays. Removing the Fc N-glycan with PNGase F under native conditions enables characterization of both the free N-glycan and the function and structure of the deglycosylated antibody.
To demonstrate the efficient removal of Fc N-glycans by Immobilized PNGase F and PNGase F under native reaction conditions, the therapeutic antibody trastuzumab was processed. The mass shift in Fig. 2 demonstrates successful removal of the Fc N-glycans on trastuzumab after incubation with PNGase F or Immobilized PNGase F for 1 h at 37°C, with no enzyme interfering in the analysis of the sample processed with Immobilized PNGase F as compared to the sample processed with PNGase F in solution.

 
 

 

 

 

 
 

 

 
 
 

 

Figure 2. TIC chromatogram (top) and deconvoluted mass spectra (bottom) of the Fc/2 fragment of trastuzumab treated with PNGase F or Immobilized PNGase F under native reaction conditions. Trastuzumab was incubated for 1 h with Immobilized PNGase F using native reaction conditions or PNGase F in-solution for 1 h at 37°. The deglycosylated antibodies were digested with FabRICATOR, reduced and analyzed by reverse-phase LC-MS on a Waters™ BioAccord™ system equipped with a Waters™ BioResolve™ RP mAb column (2.1 x 50 mm). The TIC chromatogram clearly shows the absence of the PNGase F enzyme in the sample treated with Immobilized PNGase F.

Denaturing Conditions Speed up Deglycosylation

Some glycosylation sites are poorly accessible for PNGase F and the deglycosylation reaction is slow or inhibited completely by steric hindrance. Denaturation of the glycoprotein increases the rate of deglycosylation. To demonstrate the increase in reaction speed under denaturing conditions, the fusion protein abatacept was processed with Immobilized PNGase F using either the native or the RapiGest denaturing workflow (Fig. 3). Native conditions allowed for removal of N-glycans without any additives or increased temperature but required overnight (18 h) incubation for complete deglycosylation. Removal of the N-glycans using denaturing conditions was complete within 30 minutes. As can be seen from the MS spectra, all N-glycans are removed while the O-glycans are left intact. The intact protein is too complex to be analyzed by MS directly, but after treatment with Immobilized PNGase F, the complexity of the sample was greatly reduced, resulting in high quality mass spectra.
 
 
Figure 3. Deconvoluted mass spectra of abatacept (top panel) treated with Immobilized PNGase F under denaturing (middle panel) or native conditions (bottom panel). The protein was incubated for 18 h with Immobilized PNGase F using native reaction conditions or 30 min using denaturing conditions. The deglycosylated protein samples were reduced and analyzed by reversed-phase LC-MS on a Waters™ BioAccord™ system equipped with a Waters™ BioResolve™ RP mAb column (2.1 x 50 mm). The mass shifts show the complete removal of N-glycans, leaving only the O-glycoforms.
 
 

PNGase F Activity on an Array of Glycoproteins

To demonstrate the deglycosylation ability of PNGase F and Immobilized PNGase F on various substrates, a selection of glycoproteins was processed with either PNGase F or Immobilized PNGase F (Fig. 4). All N-glycans on trastuzumab and etanercept, and the Fc-glycans on cetuximab were efficiently removed within one hour using native reaction conditions. When adding RapiGest™ and performing the reaction under denaturing conditions, all N-glycans – including the Fab-glycans – of cetuximab were completely removed within 10 min with PNGase F and 15 min with Immobilized PNGase F. The more complex Fc-fusion proteins abatacept and aflibercept were successfully deglycosylated by Immobilized PNGase F in 30 min under denaturing reaction conditions. Using both denaturing and reducing reaction condition, the commonly used glycoprotein standard RNase B was completely deglycosylated in 10 or 15 min, with PNGase F or Immobilized PNGase F, respectively. The deglycosylated samples generated under both native and denatured conditions are compatible with LC-MS analysis.

Figure 4. Key characteristics and SDS-PAGE assay of a selection of glycoproteins deglycosylated using PNGase F and Immobilized PNGase F. The table above specifies important characteristics of the examined glycoproteins, the reaction conditions used for the deglycosylation and the sample preparations performed before SDS-PAGE analysis. The mass shifts on the gels show the successful removal of N-glycans from the various glycoprotein substrates.
 
 


 
* RapiGest™ SF Surfactant from Waters Corporation is included in Immobilized PNGase F Denaturing. RapiGest™ is a trademark of Waters Corporation.

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