The Controllability Of Vinyl Sulfone-based Bioconjugate Reactions And Its Applications In Protein Purification And Immobilization

Bioconjugate reactions are the key to functionalization of pharmaceutical materials.The precise control of surface molecular density and orientation,widely applied in protein chromatography,biosensor,enzyme immobilization,drug delivery and medical imaging,etc.,can significantly improve the biological performance of the materials.However,the controllable bioconjugation chemistry have several disadvantages such as low reactivity,poor controllability,poor selectivity,low universality and difficulty in simultaneously controlling density and orientation.To address these challenges,in this dissertation,we used vinyl sulfone(VS)-based bioconjugate reaction as an example to realize the controllability of molecular conjugation.Compared with that of traditional methods and the resulting commercial products,the performance of chromatographic resins,biosensor chips,and active targeting nanoparticle has been significantly improved.In this dissertation,first,a new activation strategy for hydroxyl of resins was developed using catalyzed Michael addition in a controllable manner.Among a series of organic bases,PPh3 was found to have the highest catalytic activity.The resulting maximum activation density is 157.5 ± 1.2 μmol/g resin,which is 2.5 times that of reactions carried out in traditional aqueous conditions.This method also effectively avoided the addition reaction of VS groups with water in alkaline solutions.Using IgG and BSA as model proteins,the studies on MEP density indicated that,the affinity of hIgG increased gradually with the increasing of MEP density,while the specificity first increased and then decreased.Then,the adsorption isotherms and dynamic binding behavior of the resin samples were examined.Higher affinity,specificity and dynamic binding capacity of IgG was observed on our resin samples with high MEP density.In the purification experiments of omalizumab,our resin sample demonstrated a 66%higher purification yield than that of the commercial resin(MEP HyperCel?).On this basis,we developed a density control method for amino-containing ligands on VS surface.Using N,N-bis(carboxymethyl)-L-lysine(ab-NTA)as a model ligand,pyridine dicarboxylic acid(DPA)was screened to be the one with the highest catalytic activity.The abNTA density can be controlled in the range of 0.33-1.88 molecules/nm2.Using His-tagged protein and BSA as model proteins,the amount of immobilized protein through our method is 1.5 times higher than that of NHS ester method,while the high specificity remained.Then,taking advantage of the nucleophilicity difference of amino acid residues,a new site-specific immobilization method for His-tagged proteins was developed.Reactions carried out either in solution or on surface showed that the VS group could specifically react with the imidazole groups in histidine at pH 7.0.Two His-tagged proteins(enzyme and antibody fragment)and BSA were selected as model proteins.Compared to the existing methods using covalent(NHS/EDC(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide)activated carboxyl)and coordinate(Ni2+-NTA)linkages,our method offers the highest density,specificity and stability.The ordered orientation of the enzyme and the antibody fragment on the surface was confirmed by enzyme activity and antigen binding experiments,respectively.Finally,an active targeting nanoparticle toward HER2-positive breast cancer cells was successfully constructed.Through the site-specific immobilization method of His-tagged proteins,the antigen-binding activity of antibody Fab fragment was mostly preserved.By regulating the DVS activation conditions,Fab density was controlled in the range of 50-180 molecules per nanoparticle.Integration of polyethylene glycol(PEG)chains and high Fab density was shown to suppress protein corona formation and macrophage uptake.Three breast cancer cell lines with different HER2 expression levels were selected as models for in vitro experiments.Both high Fab density and high HER2 expression level could accelerate the response of cancer cells as well as enhance the targeting efficacy of nanoparticles,ultimately leading to high cytotoxicity.To sum up,the controllability of vinyl sulfone-based bioconjugate reactions was realized by a series of reactions occurring on surfaces,resulting in improved performance in protein purification and immobilization.The controllable density and even molecular orientation were achieved.This study meets the requirements of bioconjugate chemistry in preparing pharmaceutical materials,and makes it more applicable in biosensing,drug delivery,protein purification,enzyme immobilization and medical imaging.