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In Vitro Protein Glycosylation 

Protein stabilization continues to be an important problem and is an accepted method for enhancing the performance of a target protein, enzyme, or antibody.  Glycosylated proteins are typically more stable than their unglycosylated counterparts.[1-5]


Our patented in vitro glycosylation platform is general and gives rise to emergent properties for proteins, enzymes, and immunogens modified using this chemistry.  This technology provides for a universal glycosylation method that does not compromise the activity of an enzyme.  Our technology can further glycosylate proteins that are naturally N- or O-glycosylated.  In vitro glycosylation can enhance an enzyme’s initial activity and lifetime. 

The process uses an N-acyl moiety on the reducing end of a carbohydrate (linear or branched) and no special protecting group chemistry is required.  The unprotected carbohydrate is activated in aqueous media into a carbohydrate oxazoline, which is then reacted with the target protein under mild, protein/enzyme friendly conditions, to form an amidine linkage between the carbohydrate and the protein.  This linkage is very stable at physiological pH.  The choice of carbohydrate can be tailored to produce a desired outcome. 

There are a number of emergent properties that arise when a protein is glycosylated in vitro.  These include, but are not limited to:

  • Enhanced stability

  • Improved degradation resistance

  • Improved shelf-life

  • Cryo-protection and lyo-protection

  • Better cofactor residency for enzymes with prosthetic groups


Effect of in vitro protein hyper-glycosylation on the functional lifetime of commercially available glycosylated glucose oxidase. 


1.         Kajiwara, S., et al. (2020) 10.1016/j.enzmictec.2019.109416

2.         Yang, X., et al. (2019) 10.1016/j.jchromb.2019.04.031

3.         Higel, F., et al. (2016) 10.1016/j.ejpb.2016.01.005

4.         Zhang, Q., et al. (2019) 10.1080/10408398.2018.1507995

5.         Zhou, Q., et al. (2019) 10.1016/j.xphs.2018.11.029

6.         Petillo, P.A., et al. (2019) US Patent Application Pub. No. 2019/0185900

7.         Petillo, P.A., et al. (2021) US Patent #11,021,730 B2

8.         Petillo, P.A., et al. (2021) US Patent 17/301,590 allowed 12-19-2022

9.         Petillo, P.A., et al. (2022) US Patent Application 63/365,432

10.       Petillo, P.A. (2022) US Patent Application 63/382,020

11.       Petillo, P.A., et al. (2022) US Patent Application 63/373,155

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