Design Of Alkali-Resistant Bionic Affinity Ligands

Immunoglobulin G(Ig G)is the main antibody type in blood and extracellular fluid,which has important clinical applications.Protein A is a protein first found in the cell wall of Staphylococcus aureus.It can specifically bind to Ig G and is often used as a ligand for affinity chromatography of Ig G.However,it is expensive and difficult to regenerate,resulting in high purification cost.Therefore,looking for substitute which have similar activity to biological affinity ligands and are replaced by synthetic small molecular compounds has become an important way to overcome the shortcomings of biological affinity ligands.In this paper,starting from the fragment library of drug synthesis intermediates and molecular building blocks,various drug design methods were used to screen small molecule biomimetic affinity ligands.A variety of potential Ig G affinity ligands were screened by drug design software with mature theoretical basis and verified experimentally.This paper mainly includes four aspects:Preparation of drug synthesis intermediate fragment library,Screening based on physicochemical and biological properties,Evaluation of affinity between molecular fragment analysis and Ig G Fc,Development of Ig G purification process and evaluation of chromatography efficiency.Firstly,based on the economic feasibility,this subject integrates the Building Block database of several small molecule suppliers,and establishes the molecular fragment library for biomimetic ligand screening through de duplication and standardization,including 1045326 molecules;Then,according to the properties and affinity principle of biomimetic ligands,a series of physical and chemical properties and biological characteristics parameters for screening were designed,including molecular weight,log P,p K_a,coupling group,toxicity,albumin binding rate,etc.a biomimetic ligand screening process based on molecular properties was constructed,and 23764molecules meeting the requirements were screened from the above molecular library;Then,using the molecular docking method,based on empirical screening and rational screening,17 molecules with biomimetic ligand potential were finally determined for experimental verification.A total of 42 coupling experiments were designed for the different coupling groups contained in 17 different molecules,and the loading of Ig G by different molecules was detected by gravity column detection method,Finally,four molecules with affinity for Ig G were obtained,one of which can reach 90%of protein a load;Finally,the chromatographic process is developed and optimized for the small molecule with the highest loading.The gradient elution method is used to find the appropriate elution p H of the molecule,and the binding rate between the molecule and the filler is greatly improved by changing the reaction time and temperature.The molecule is expected to be a biomimetic alternative to protein A.In conclusion,this paper designs a virtual screening process for affinity biomimetic ligands based on the Building Block database,and obtains a promising small molecule biomimetic ligand.This study provides a research idea for the development of small molecular bionic ligands,which contributes to the discovery of new bionic ligands,and also has important guiding significance for the construction of antibody purification process.