| Polypeptide drugs have been used in the treatment of many kind diseases,including immune diseases,tumors and diabetes.Lately they have become the hotspot of the drug discovery and development.Nevertheless,peptide drugs have shortcomings too,for instance,they are instable and prone to be affected by external factors such as temperature,pH,and excipient;have short half-life in blood which leads to frequently dosing;can be enzymolysed easily.These characteristics make it a challenge for the personnel engaged in polypeptide drug research.In clinical practice,to improve the stability,most polypeptide drugs are made in lyophilized powder.Recently there are also reports about the development of slow-sustained release preparation microcapsules and new dosage forms of liposomes.Thymosin ?1(T?1)and thymopentin(TP5)are two polypeptides obtained from animal thymus,which have similar immunomodulatory activity.The in vivo half-life time(t1/2)of TP5 is very short(30 s)while the in vivo half-life time of T?1 is long(?100 min).To extend the t1/2 of TP5,T?1 and TP5 with similar biological activities and clinical uses were linked by genetic engineering.In preliminary pharmacological studies,Tal-TP5 fusion peptide showed good immunomodulation and auxiliary antitumor activity,basically possessed the potency to be developed as an auxiliary antitumor drug.Based on extensive previous work,preparation technology of Tal-TP5 fusion peptide was studied on middle-scale,the technical transfer from lab-scale to large-scale production was completed;the preparation technology of T?1-TP5 fusion peptide lyophilized powder for injection was studied and the lyophilization curve was determined;the quality research and methodology validation were completed,and the quality control standard was preliminarily formulated;the long-term stability of the samples of 3 pilot batches for 6 months was inspected.This study provided important theoretical and technical bases for the development of Tal-TP5 fusion peptide as a new auxiliary antitumor drug.1.Optimization of mid-scale expression and purification of T?1-TP5 fusion peptideThis project employed E.coli expression system for the mid-scale production of T?1-TP5 fusion peptide.By optimizing the process,systematically studying of expression condition and purification technology,Tal-TP5 fusion peptide was successfully produced on mid-scale.The technique transfer was completed from lab-scale to large-scale production.Due to the lack of the modification such as glycosylation and phosphorylation,nor disulphide bond in T?1-TP5 fusion peptide,recombinant E.coli expression system is a suitable method for large-scale preparation.Using recombinant E.coli strain ER2566-T?1-TP5 containing pTYB2-T?1-TP5-Sce intein-CBD plasmid and target compounds Tal-TP5 fusion peptide as research objects,the preparation and the intein-mediated purification of T?1-TP5 fusion peptide was optimized for mid-scale production.Our study showed that the optimal conditions for the expression of T?1-TP5 fusion peptide in E.coli strain ER2566-T?1-TP5 were:adding the inducer IPTG when the dry weight of bacteria was 10g/L at a final concentration of 0.1mmol/L and incubating for 5 h at 28?.In order to reduce the cost and improve the efficiency,Tal-TP5 fusion peptide was prepared by intein-mediated purification.The optimal sample size of the recombinant protein T?1-tp5-sce intein-CBD for the chitin affinity column was 15 mg/ml.Elution with two-column volume elution buffer was able to recover more than 90%of the fusion peptide.Based on the optimal conditions for the induced expression and purification of T?1-tp5 fusion peptide,three mid-scale batches were prepared where the sample purity is more than 95%.The yields of these three bathes were 2.16 g,2.10 g and 2.20 g.2.Study on the formulation and preparation process of T?1-TP5 fusion peptide for injectionAccording to the clinical use and prescription of similar commercialized products,the preparation of Tal-TP5 fusion peptide lyophilized powder for injection was studied,where the focus was on the lyophilisation molding process,mainly including the selection of support agent,the determination of pH and lyophilisation process parameters,the amount of activated carbon used,the eutectic point,the temperature of solution preparation.Through screening and optimization of the process parameters,the formulation and preparation parameters of Tal-TP5 fusion peptide for injection were determined as:mannitol as proppant;0.5 mol/L disodium hydrogen phosphate solution as pH regulator;pH value in the range of 6.0?8.0;water temperature controlled at 30?;amount of activated carbon used was 0.1%(g/ml);liquor bottling capacity of 1ml/bottle;prescription:each 1000 bottles,Tal-TP5 fusion peptide 1 g,mannitol 50 g,0.5mol/L disodium hydrogen phosphate solution adjusting pH value to 6.8,the injection water is added to 1000 ml;preparation specifications:1 mg(Tal-TP5 fusion peptide.).Through formulation screening and production process optimization,three batch preparations of T?1-TP5 fusion peptide for injection were successfully prepared on mid-scale(1000 pcs/group),which proved the reasonability of the formulation design.The technology,process and parameters for the lyophilization of Tal-TP5 fusion peptide preparation were determined,and the lyophilization curve of the product was also determined,which provided technical foundation for the industrial production of the product.3.Study on the quality of Tal-TP5 fusion peptideTo establish the quality control method and the limit of Tal-TP5 fusion peptide,to provide guidance and basis for the pharmaceutical drug preparation,the physicochemical properties of three batches of T?1-TP5 fusion peptide APIs(Batch number:TT-20170601,TT-20170602,TT-20170603)were studied,including the clarification of appearance,identification,solubility inspection,pH determination,specific rotation determination,solution clarity and colour,moisture content.A high-performance liquid chromatography(HPLC)method for the determination of Tal-TP5 fusion peptide was established and the methodology was studied from several aspects such as linear range,precision,repeatability and stability of solution.The results showed that this product is freely soluble in water,very slightly soluble in methanol,acetonitrile,insoluble in ethyl acetate;pH value ranged from 3.12 to 3.15;the average value of specific rotation is-96°;the product is a clear and colourless solution;the average moisture content was 1.30%;the Tal-TP5 fusion peptide has a good linear relation(r=0.9998)in the range of 0.479?1.367 mg/ml,the limit of quantification was 19.14 ng/ml,the detection limit was 6.38 ng/ml.The high-performance liquid chromatography(HPLC)parameters:Agilent ZORBAX 80A Extend-C18 column(250 mm×4.6 mm,5?m);using 0.05 mol/L disodium hydrogen phosphate solution(adjusted to pH 7.5 with phosphoric acid)-acetonitrile(9:1,v/v)as the mobile phase;the injection volume was 20 L;the detection wavelength was 208 nm;the flow rate was 0.6 ml/min.The content was calculated by peak area normalization method.The specificity,repeatability and precision ofthe method were all good and the results were accurate.The contents of T?1-TP5 fusion peptide in the three batches were 99.122%,99.125%and 99.190%,respectively.This method can be used to determine the content of Tal-TP5 fusion peptide.The quality standard of Tal-TP5 fusion peptide is preliminarily determined which provides a theoretical basis to control the product quality and to ensure the safety,effectivity and controllable quality of Tal-TP5 fusion peptide.4.Study on the quality of T?1-TP5 fusion peptide preparation for injectionThe quality control method and limit of Tal-TP5 fusion peptide preparation for injection were established,and the quality control of the product was achieved.The physiochemical properties of Tal-TP5 fusion peptide preparation for injection from the three mid-scale production batches(Batch number:20170801S,20170802S,20170803S;Volume:1000 pcs/group)were studied.These properties include appearance,identification,determination of pH,clarity and colour of the solution,the moisture content etc.The method of asepsis examination,determination of bacterial endotoxin and the yield test was verified.The HPLC method for the determination of fusion peptide content was established and the linear range,precision,repeatability,stability and solution durability was also studied.The results showed that the product was white or ivory loose lump;the range of pH value was 6.78?6.80;the solution was clarified and colourless;the average water content was 2.42%,It was determined that the amount of endotoxin should be less than 5.0 EU in every 1 mg fusion peptide injection.Asepsis examination is according to the China Pharmacopoeia 2015 Generak ?1101;the content determination of Tal-TP5 fusion peptide for injection took the HPLC method,which according to methodological research shown to have good specificity,good linear relationship(r=0.9998)in the concentration range of 0.479?1.367 mg/ml,high precision,good reproducibility.5.Study on the stability of Tal-TP5 fusion peptide preparation for injectionThe experimental study on the stability of T?1-TP5 fusion peptide preparation for injection was performed.The preparation changes with time in the effects of temperature and light were studied,which provided the scientific bases for drug manufacturing,packaging,storage,transportation,and at the same time drafted the storage condition and shelf life of products according to the experimental data of the stability study.According to the Principle of Stability Test of API and DrugPreparation to the Guidelines for Stability Test of Bulk Drugs and Pharmaceutical Preparations,influencing factors test,accelerated test and long-term test were done for the samples from the three batches where the examination items were traits,pH value,clarity,related substances and content.The influencing factor study showed that at the temperature of 60?,the contents of single and total impurities were both significantly increased,indicating the instability of the product under high temperature;after stored at the illuminance of 4500±500 Lx for 10 days,all tested characteristics of the product were not changed significantly,indicating the product is relatively stable to light.After six-month acceleration test,related substances increased significantly while no other indexes significantly changed,which met the requirements of the quality standard of this product.The long-term test of six months showed that the indexes did not change significant while the related substances increased slightly and the content decreased slightly.These indicated that the product is stable under long-term test conditions and the proposed package could meet the requirements for launching in market.Subsequent stability tests are still under way to determine the actual duration of the drug.The main achievements and results obtained of this research are as follows:(1)The efficient induced expression of Tal-TP5 fusion peptide in E.coli system was achieved;the intein-mediated Tal-TP5 fusion peptide purification system suitable for mid-scale production was established and optimized;the technique transfer from the lab-scale to the large-scale production was completed.(2)The preparation process of T?1-TP5 fusion peptide preparation for injection was studied.The formulation,processing conditions and lyophilization curve of Tal-TP5 fusion peptide preparation were determined.Three batches of samples with good stability were prepared,providing the technical basis for industrial production.(3)The quality control method and limit of Tal-TP5 fusion peptide material and preparation were established.The quality standard is preliminarily determined.(4)The conditions of production,packaging,storage and transportation,as well as the valid date were determined by studying the stability of T?1-TP5 fusion peptide preparation for injection. |
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