background:

Currently, more and more antibodies and antibody fusion proteins are used as biotherapeutics, and the properties of these biotherapeutics are significantly affected by their glycosylation status. For example, the pharmacokinetics and efficacy of recombinant erythropoietin are severely affected by its glycosylation status, and the conserved N-linked glycans at Asn297 in the Fc region of IgG are essential for its functional activity. In addition, some antibodies have additional N-linked glycans, as shown in Figure A, which together with the conserved N-glycans at Asn297 in the Fc region affect the recognition, half-life and immune response of the antibody. However, various variables in the cell culture process may lead to changes in the degree of antibody glycosylation and increase its glycan diversity, so monitoring the glycosylation state of the antibody during the production process is crucial to obtaining the correct glycoprotein form.

^™ PNGase F-Plus from Rhinogen® is an improved recombinant reagent that is fast, efficient, and retains intact deglycosylated proteins (under non-reducing conditions). It is suitable for N- linked glycan analysis and proteomics analysis during the development and quality control of therapeutic monoclonal antibodies . The fast processing time reduces the risk of impurities and unwanted protein modifications (oxidation, deamidation) that may be introduced by the standard
PNGase F long enzymatic deglycosylation reaction protocol.

Product Packaging:

Rhinogen ^® Quick™ PNGase F-Plus packaging specifications are as follows:

Product Source:

Rhinogen ^® Quick™ PNGase F-Plus is a recombinant glycosidase expressed by Escherichia coli with a molecular weight of 36kDa.

Product quality:

The purity was ≥95% by SDS-PAGE analysis; no contaminating exoglycosidase, endoglycosidase, or protease activity was detected.

Product Features:

Heat inactivation conditions: 75°C for 10 min.

Features:

High efficiency: rapid and non-preferential release of N-linked glycans;
High purity: no contaminating proteases/glycosidases, purity ≥95%;
High applicability: effective for a variety of structural antibodies or glycoproteins;
High stability: each batch of Quick™ PNGase F-Plus products undergoes strict quality control to achieve high stability;
Retaining intact disulfide bonds: Retaining intact deglycosylated proteins makes protein characterization more convenient and reduces deviations (under non-reducing conditions).

Storage conditions:

is transported in ice packs. Please store the enzyme at 2-8℃ immediately after receiving it. Freezing is strictly prohibited. The supporting reagents are stored at -20℃. After the Quick™ reaction enhancer lyophilized powder is dissolved, it can be stored at -70℃ for one month and at 2~8℃ for one week. The enzyme solution does not contain preservatives. Be sure to ensure sterile operation to avoid contamination.

application:

rapid deglycosylation of antibodies or antibody fusion proteins within minutes ;

2. Monitoring of glycosylation status during the production of antibodies and antibody-fusion proteins;

3. Product quality control of antibodies and antibody-fusion protein biotherapeutics;

4. Application and research of proteomics.

Instructions:

This product is for research use only and is not intended for diagnostic or therapeutic use in humans or animals.

Frequently asked questions

^Q1 : Rhinogen® QuickTM What product is obtained after PNGase F-Plus deglycosylation ?

A1: After deglycosylation of antibodies and antibody fusion proteins using Rhinogen® QuickTM PNGase F-Plus , all N-linked oligosaccharide chains will be released quickly and completely, and at the same time, asparagine residues on the amino acid characteristic sequence will be converted into aspartic acid residues.

Q2: What is the difference between the operating methods under reducing and non-reducing conditions?

and completely deglycosylated within minutes , making them ideal for downstream N-glycan analysis and applications in the development and quality control of therapeutic monoclonal antibodies. Under non-reducing conditions, the integrity of antibody disulfide bonds can be maintained, and fully deglycosylated multimeric proteins (i.e., antibodies) can be obtained, making them ideal for mass spectrometry studies of deglycosylated proteins.

Q3 ^: Rhinogen® QuickTM What is the ideal control substrate for PNGase F-Plus?

A3: Anti-MBP monoclonal antibody is a good control substrate for QuickTM PNGaseF -Plus.

Q4: How to detect Rhinogen ^® QuickTM Does PNGase F-Plus completely deglycosylate within 10 minutes ?

A4: For monoclonal antibodies, the change in the position of glycosylated and deglycosylated antibodies on the gel can be observed on SDS-PAGE after the removal of N-glycans. If a more precise detection of whether a small amount of glycosylated antibody remains is required, a more sensitive assay (e.g., LS-ESI-TOF) can be used.

Q5: Is downstream HPLC and MS analysis compatible with Rhinogen ^® QuickTM PNGase F-Plus compatible?

A5: Yes, for samples prepared for LC and/or MS, N-glycans can be separated by conventional solid phase extraction methods such as C18 and graphitized carbon; deglycosylated proteins can be used for mass spectrometry analysis after buffer exchange by microdialysis or microfiltration. Of course, you can also decompose the QuickTM reaction enhancer into non-interfering byproducts under acidic conditions without inhibiting peptide ionization.

Q6: Can protease inhibitor cocktails be used in Rhinogen ^® QuickTM Is PNGase F-Plus reactive?

A6: Yes, the following protease inhibitor cocktail is compatible with the QuickTM PNGase F-Plus reaction: Aprotinin (10 μg/ml final concentration); Benzamidine (1 mM final concentration); Pepstatin (10 μg/ml final concentration); Leupeptin (1 μM final concentration); EGTA (1 mM final concentration); EDTA (1 mM final concentration).

Q7 ^: Rhinogen® QuickTM Is the PNGase F-Plus reaction compatible with trypsin?

A7: Yes, trypsin can be added directly to samples digested with QuickTM PNGaseF -Plus.