Study On The Sameness Between Generic And Branded Enoxaparin Sodium Using Top-down And Bottom-up Analysis Strategies

[Background and purpose]Enoxaparin sodium is a new generation of heparins anticoagulant drugs prepared by alkaline alkaline ? elimination with porcine intestinal mucosa as the starting material,is the blockbuster drug in anticoagulants.Its patent has expired and can be legally copied.Based on the complexity of biogenic drugs,FDA had proposed that generic enoxaparin sodium should be studied in consistency with the original drug,including physicochemical properties;heparin source material and mode of depolymerization;disaccharide building blocks,fragment mapping and sequence of oligosaccharide species;biological and biochemical assays;and vivo pharmacodynamic profile.China's generic of enoxaparin sodium consistency research has also been launched.According to the research requirements of FDA,using the top-down and bottom-up strategies to study the consistency between the generic enoxaparin sodium and the original drug in terms of physicochemical properties;disaccharide building blocks and fragment mapping.The consistency of the generic enoxaparin sodium produced by different processes conditions with the original drug is investigated,at the same time,the differences of the original drugs in different listing are investigated.Therefore,the consistency research method and criterion are established to analyze the consistency difference between generic drugs and original drugs,and to provide reference for the process optimization and quality improvement of generic drugs,it also laid the foundation for meeting the new registration requirements of enoxaparin sodium in China and the international registration.[Methods]This study adopts two strategies:top-down and bottom-up,the consistency of the structure and composition of 9 batches of the domestic generic enoxaparin sodium and 3 batches of original enoxaparin sodium from different origins are studied.All the analytical methods adopted have been verified.(1)The top-down strategy is a method to directly comparative analyze the structure of the generic and original enoxaparin sodium.?Dynamic coating strong anion exchange chromatography(cetyltrimethylammonium strong anion exchange high performance liquid chromatography,CTA-SAX-HPLC)is used to separate and compare the enoxaparin sodium based on charge density and polarity difference;? The composition of enoxaparin oligosaccharides is analyzed by gel exclusion chromatography(size exclusion chromatography,SEC)based on different molecular weights.(2)The bottom-up strategy is to degrade the enoxaparin sodium to oligosaccharide fragments by chemical or enzymatic methods,and then comparing with the structure of the oligosaccharide fragments between the original and generic enoxaparin sodium.This topic adopts respectively?heparinase ?,? and ? in combination with;?heparinase I partially enzymatic;?heparinase ? partially enzymatic;? heparinase III partially enzymatic,the four methods are used to degrade enoxaparin sodium from two sources.And then SAX-HPLC method is used to separate and compare the oligosaccharide fragments obtained from enoxaparin sodium degradation based on the difference in charge density.[Results]Research on top-down strategy.Enoxaparin sodium is separated into 25 oligosaccharide fragments by CTA-SAX-HPLC.The differences between the domestic generic and the original enoxaparin sodium are mainly concentrated in the top 10 oligosaccharide peaks;there are only slight differences in the oligosaccharide content of the original drugs in different markets;enoxaparin sodium is separated to 11 oligosaccharide peaks with different polymerization degrees by SEC-HPLC,the differences between the generic and original enoxaparin sodium are mainly concentrated in the small fragments,such as trisaccharides and tetrasaccharides units,the content of oligosaccharides is the same between the original drugs in different markets.Research on bottom-up strategy.Enoxaparin sodium after heparin ?,? and ?combined with enzymolysis is separated by SAX-HPLC to obtain a total of 19 components,the ??A,?Glyserox1,??S gal of the generic drugs are significantly different from those of the original,there are four differential peaks of ??A,?Glyseroxl,??S Gal and 1,6-Anhydro ??S in different marketsAt least 12 oligosaccharide fragments are achieved by heparinase I partially enzymatic of enoxaparin sodium,1 to 2 oligosaccharide fragments between the generic and original enoxaparin sodium,there are 2 differences pesks between the original drugs in different markets.At least 13 oligosaccharide fragments are achieved by heparinase ? partially enzymatic of enoxaparin sodium,1 to 2 oligosaccharide fragments between the generic and original enoxaparin sodium,there are 3 differences pesks between the original drugs in different markets.At least 25 oligosaccharide fragments are achieved by heparinase III partially enzymatic of enoxaparin sodium,1 to 6 oligosaccharide fragments between the generic and original enoxaparin sodium,there are 6 differences pesks between the original drugs in different markets.[Conclusion]By means of top-down and bottom-up analysis strategies,the consistency relationship of physicochemical properties;disaccharide building blocks and fragment mapping between the generic and original enoxaparin sodium is comprehensively analyzed,the oligosaccharide fragments of enoxaparin sodium are significantly different from that of the original drug,but there are only slight differences between the original drug in different markets.This study suggests that to further improve the quality of domestic generic enoxaparin sodium,research should be carried out from three aspects,such as controlling the quality of the starting raw material heparin sodium,optimizing the production process of enoxaparin sodium and expanding the consistency standard of the original drug from multiple batches.