Expression And PEGylation Of Anti-CGRP Antibody Fab Fragment

Migraine is a complex,persistent or intermittent headache disease that is triggered by nervous system disorders and it's characterized by periodicity,disability,and recurrent episodes and so on.Moreover,migraine was classified as the fourth disabling disease by the World Health Organization after high paraplegia,schizophrenia,and dementia,which seriously affects the work and life of patients.So,its severity and influence is clear enough.Therefore,the research on the pathogenesis and treatment of migraine is become urgent and cannot be delayed.With decades of research and development,the pathogenesis of migraine has been converted from the original“vascular theory”into a more mature and convincing“neural theory”by now.The calcitonin gene-related peptide?CGRP?was gradually found and confirmed it's important physiological role in triggering migraine in this process.Since then,the monoclonal antibody of CGRP or its receptor as the new alternative drug were expected to prevent and cure migraine,the new type drug also opened a new chapter in the field of migraine's treatment.In this study,we based on the anti-CGRP antibody Fremanezumab to optimize and synthesize the gene sequence of the Fremanezumab's fragment of antigen-binding which is abbreviated as“Fab”according to the codon preferences of E.coli.Then we used our own lab's Fab expression system and based on the specific problems in the process of the experiment to optimize the condition.In the end,we realized efficient and soluble expression of Fab in culture medium,we also ensured the correctness of the disulfide bond between the heavy chain and light chain.According that Protein L purified medium can specificly binding to human antibody's?light chain,we used Protein L to purify the medium after expression and the yield of Fab was about 10 mg/L,the Fab's purity was about 60%.Due to the low purity of Fab,we adopted Nano SP cationic exchange medium for secondary purification,and obtained anti-CGRP antibody's Fab fragment?ACFab?with a purity of 83%and a yield of 62%.The final product anti-CGRP antibody's Fab is abbreviated as ACFab.Based on the Fab's molecular weight is small and it's half-life in vivo is short,combining with the principle and method of PEGylation of protein molecules we adopted the site-specific PEGylation to modificate the Fab fragment.We introduced one cysteine into the end of the heavy chain to provide a free sulfydryl.At first,we used a suitable reducing agent to reduct cysteine.Secondly,the modificate reaction was carried out under the optimal conditions with PEG-maleimide.Finally,the modified reaction product was subjected to cation exchange purification to obtain a Fab-PEG modified product with a purity of 97%.Finally,the antigen-antibody binding curve and antibody's affinity constant of ACFab and ACFab-PEG modified product were detected by non-competitive ELISA.Wherein the ACFab's affinity constant was?7.52±1.57?*10⁷M^-1,ACFab-PEG modified product's affinity constant was?2.90±0.25?*10⁷M^-1.Thereby the binding ability of the ACFab and its PEG modified product to the antigen CGRP were confirmed and demonatrated the feasibility of ACFab's prokaryotic expression and purification and PEG modification in this study,which laid a foundation for subsequent research and large-scale production.