Preparation Of Glycan Substrates And Antibody Glycoengineering

Glycosylation is an important post-translational modification of an IgG.The heterogeneous glycan on Fc conserved glycosylation site influences antibodies' bio-activity greatly,the affinity of Fc and Fc receptors specially.Consequently,it is critical to prepare homogeneous glycoform antibody via antibody glycoengineering technologies,for instance in vitro chemoenzymatic glycoengineering technology.We studied at first,in this paper,the industrial technology of sialylglycopeptide extraction and purification from egg yolk.A lot of researches have been conducted,and finally a simplified procedure which mainly uses ethanol and water has been established,giving an economical and scale-uped extraction protocol.We also synthesized glycan oxazoline substrate for transglycosylation via simple one-pot strategy from SGP as starting material.In addition,we studied the transglycosylation of various species antibodies taking advantage of Endo-S,Endo-S D233 Q and,oxazoline substrate.Our results showed broad scope of Endo-S recognition substrates and in another hand,we firstly introduced azido as a good biorthogonal group which can be furtherly reacted with alkynyl containing fluorescence dyes onto Fc glycan of an antibody.In the paper,chapter 1 discussed the research background in detail,including antibody structure,glycoengineered antibody and its preparation,sialylglycopeptide and application,etc.Extraction and purification of SGP via ethanol and water was detailed discussed in chapter 2,and the one-pot synthesis of oxazoline substrate was included.We introduced,in chapter 3,chemoenzymatic glycan re-modulation and purification of different antibodies from various species,which utilized the hydrolysis activity of Endo-S and transglycosylation activity of Endo-S D233 Q in the presence glycan oxazoline substrate,achieving series of glycoengineered antibodies.