The separation of various glycoforms to study the biological relevance of glycosylation is a real challenge for glycoproteins. Although many successful works have been completed to produce therapeutic monoclonal antibodies with specific glycotypes, it is still very difficult to produce optimized IgGs with homogeneous glycotypes. To accomplish this, Creative Biolabs provides a new avenue to remodel Fc N-glycan of IgG antibodies from a heterogeneous N-glycosylation pattern to a homogeneous one based on chemoenzymatic glycosylation methods. This chemoenzymatic glycoengineering is considered to be one of the most promising methods for synthesizing homogeneous glycotypes of a given glycoprotein (including IgG), and holds great potential for generating mAbs with more homogeneous glycans and desired efficacy.
The chemoenzymatic synthesis scheme includes the use of ENG'ase (endo-β-N-acetylglucosaminidase) to deglycosylate the IgG antibody to leave the innermost GlcNAc with or without the N-glycosylation site Core fucose. After preparing the glycan oxazoline as the donor substrate, the transglycosylation step is used together with an ENGase-based sugar synthase, and then an enzymatic reaction is used to prepare homogeneous N-glycans (M3, G0, G2 and A2) glycoengineered monoclonal antibodies.
This technology uses the highly active polyoxazoline (a mimic of the transition state) as the donor substrate, and uses the synthetic polyoxazoline to perform the transglycosylation in a stereo and regiospecific manner. This chemical enzymatic glycoengineering is considered to be one of the most promising methods for synthesizing homogeneous glycotypes, and has been used to synthesize fully sialylated IgG glycotypes, otherwise it will be very difficult.
Fig.1 Chemoenzymatic glycan remodeling of monoclonal antibody rituximab to prepare homogeneous and selectively modified glycoforms.
In order to support our mission to achieve the frontier advancement in life sciences, Creative Biolabs has developed many cutting-edge technologies in the field of glycan engineering based on our GlycoDegree™ platform. Through modern recombinant technology, a group of specific glycosyltransferase and/or glycosidase genes can be specifically knocked out or adjusted to produce glycoengineered antibodies with more humanization and homogenous glycosylation, better pharmacokinetics and enhanced pharmacological efficacy. Our scientists are fully confident to provide effective and economically feasible antibody glycoengineering solutions.
It is worth noting that our chemoenzymatic glycoengineering technology shows many advantages that cannot be ignored.
1. Various ENGase mutants are available (EndoS D233Q, EndoA N171A, EndoA E173Q, EndoMN175A and EndoM N175Q), and all exhibit the function of transglycosylation activity, which have been modified to have different substrate specificities and limitations.
2. Novel developed Endo-S2 glycosynthases (D184M and D184Q) exhibit relaxed substrate specificity and act on transferring three major types (complex, high-mannose, and hybrid type) of N-glycans.
3. LC-ESI-MS analysis can be used to confirm the purity of the homogenous glycoform of the remodeled mAb.
4. Experienced employees and years of project experience provide detailed reports and data interpretation.
Please feel free to send us your project description and requirements, and you will get customized all-in-one services to meet your R&D or cGMP production needs.
Creative Biolabs provides luciferase-based ADCC assay. This Jurkat cell based assay is pioneered by Creative Biolabs, and the methodology is very well accepted by the field. See attached ADCC Reporter Assay Protocol for further details.
All products and services are for Research Use Only. Do Not use in humans.
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