Research And Application Of The Key Quality Indexes Of Extracted Biochemical Drugs

Extracted biochemical drugs(EBD) are produced from organism through separation,extraction,and purification.Extracted biochemical drugs have been applied widely in clinic and are playing an active role in the fighting against coronavirus-19 pneumonia.However,safety and effectiveness of some long been marketed EBD products remains uncertain because of the biodiversity and uncertainty in composition.A scientifically reasonable quality evaluation system becomes an imperative requirement for both manufacturing enterprise to improve the quality of the products and for drug regulators in our country to supervise scientifically the post-marketing drugs.Affiliated to Chinese Pharmacopoeia Committee(CPC),the current study is part of the 13 th Five-Year Plan of CPC.Four typical EBDs,urokinase,pancreatin,phospholipid,and succinylated gelatin,which cover the three main types and two main preparations of extracted biochemical drugs,were chosen as entry point to do further research in difficulties and problems existing in both domestic and overseas official standards of EBD products.Modern analytical technologies were utilized,including liquid chromatography,enzyme kinetics,molecular biology,and cytobiology.Biochemically characterized key quality control items were included,such as purity,dissolution,molecular weight and molecular weight distribution,pyrogen,species identification,virus detection,enzyme activity,and composition analysis.Relative methods were developed and validated to improve the current standard after systemic research.Theories and methods developed in this study have been applied in the quality evaluation and analysis of the above mentioned products and for the first time,potential risks in quality were detected.Four new methods developed in the current study,fatty acid composition,sterol composition,polymerase chain reaction,and monocyte activation test,have been confirmed by Chinese Pharmacopeia Committee and are to be recorded for the first time in the General Chapter of Chinese Pharmacopoeia(2020 edition).The national quality standards made in this study for the relative products are in process of reporting to Chinese Pharmacopeia Committee and publicity.For the first time,this study innovatively established basic quality evaluation system and general eligibility criteria of technology for the EBD on the basis of experimental research.Having summarized the key points of quality control for the evaluation of the EBD products from a strategic perspective,this study suggests a comprehensive analysis with a combination of technical measures according to the each variety and preparation,and application of new technologies and methods.This provides a solution and guidance for this kind of products domestically and internationally.Innovative reach of each variety are as follows:(1)Urokinase Two methods,size exclusion chromatography(SEC)and capillary electrophoresis(CE),were separately developed to determine molecular fractions of urokinase raw material for the first time.Results indicated a small amount of high molecular weight impurity existed in the samples,which should be controlled.To remove the impact of human albumin excipient in EBD analysis,ion exchange chromatography(IEC)was developed as sample pretreatment method in urokinase for injection.This helps to solve the problem for the first time internationally that molecular fractions in urokinase preparation is always beyond control.Chromogenic substrate method(CSM)was developed to determine the activity of raw material and preparation of urokinase.Using the developed methods,molecular fractions of in urokinase,both some of the raw material and preparation,were for the first time found not confirming to the standard.Difference was found of products between batches in some enterprises,which indicated the possibility of feeding with disqualified raw materials.(2)Pancreatin Based on the fact that activities of pancreatin varied,this study for the first time internationally applied self-contrasted method to the analysis of EBD and developed a dissolution test innovatively using activity of trypsin as index.The absolute activity was replaced by the relative activity.Species identification and nucleic acid of porcine parvovirus were carried out using polymerase chain reaction(PCR)technology.This helps to control the quality of this product from the source.Consistency evaluation between domestic preparation and imported reference preparation of pancreatin was carried out using the established method.Between the products of each domestic enterprises and the imported reference standard,large gap was found in dissolution rate and stability of different batches.Disqualification rate of domestic products was 57%,indicating question in clinical effectiveness of these products.Porcine parvovirus was detected in raw material of each enterprise,which indicated the insufficiency of the implication of GMP for biomedical drugs(new edition).(3)Phospholipid The four most extensively used phospholipids,egg lecithin,soya lecithin,egg phosphatidylcholine and soya phosphatidylcholine,were investigated.Content of eight kinds of phospholipid component were determined by supercritical fluid chromatography(SFC).Twenty kinds of fatty acids were determined by gas chromatography(GC)after boron trifluoride-methanol derivatization.For the first time internationally,nine kinds of sterols were determined by GC after separation with HPLC and derivatization of the unsaponifiable matter.The results showed that phospholipids of different kind or different source had their distinctive characteristic content and composition of phospholipids,fatty acids and sterols.Using the mentioned three components as indicators,significant difference of type and source between the phospholipids was found by hierarchical cluster analysis and principal component analysis.The three indicators can therefore be used to distinguish different properties and sources of phospholipids.For the first time,this paper proposes that egg phospholipid and soybean phospholipid to be regulated by classification.(4)Succinylated Gelatin Size exclusion chromatography and multiangle laser light scattering(SEC-MALS)was developed to detect directly the absolute molecular weight and molecular weight distribution of succinylated gelatin injection.Monocyte activation test(MAT)on the basis of HL-60/IL-6 assay was employed to assess pyrogen in succinylated gelatin injection.For the first time internationally,the MAT method is proved to be suitable for pyrogen detection of extracted biochemical drugs.