Primary Study On Quality Control, Large Scale Production And Purification Of Adenovirus CNHK200-hEndostatin

Gene therapy has been accepted by medical society as one kind of therapy on genetics diseases. However, with the development of gene therapy technology, it has been focused more and more on the different fields, including tumor therapy, cardiovascular disease therapy. The main explaination on the concept of gene therapy is to put human normal genes or genes that have function of therapy into human target cells by a definite way to correct defect of genes or take effect, and finally to treat or cure diseases. At present, the most available way is to import genes through viral vector, but so far, viral vector is still the most effective carrier that to import genes into human cells to treat diseases. Its advantages include, (1) wide host range, less pathogenicity to human; (2) homologous with human genes; (3) the capacity of expressing different genes simultaneously; (4) without the help of helper virus and the capacity of exogenous DNA from several kbs to dozens of kbs; (5) able to infect cells and express genes in both proliferation cells and non- proliferation cells; (6) not integrating into chromosome and without mutagenicity. (7) easy to amplify , purify and gain virus stock with high titer.Endostatin has been recognized as one kind of the most effective suppressive agent to new vessels of tumor. It has characteristics that suppress the formation of new vessels of tumor effectively, and then inhibit the growth and metastasis of tumor.Even if its dose rise up to hundreds of times in human body, it do not display side effects. Moreover, when it has been used repeatedly, it do not produce drug resistance. But when endostatin protein is enrolled to treat diseases, since the amount required is huge and great (50mg/kg/day) and patients need to undergo treatment each day, the cost is very high. To solve this problem, we established a new gene-virus system which can replicate in tumor cells with specificity and express human endostatin effectively. This particular system has been confirmed that it can suppress tumor growth significantly in experimental study, which promotes a great promise of therapy clinically.Nowadays, the research on adenovirus mainly focused on vectors. Other technologies of adenovirus including its production, purification and quality control, have rarely been explored, especially in domestic research references. How to obtain large amounts of adenovirus with GMP quality stably and how to perform quality control, has been an important problem that limits adenovirus to be applied in clinical therapy.To make a good foundation of clinical work in the future, We worked on adenovirus quality control, large scale production and purification using gene-virus vector system CNHK200-hEndostatin which can highly express human Endostatin.Quality control methods of adenovirus were based on'human genes therapy research and preparation quality control technology guideline','Chinese biologic product regulations','Chinese pharmacopoeia'and'good practices in the manufacture and quality control of drugs', and so on. Meanwhile, we consulted guidelines that released from WHO, FDA and ICH. We paid more attention to establishing methods for identification of adenovirus, identification of expression gene, wild type adenovirus detection, quantitation of adenovirus, purity detection and impurity detection.The quality control process of adenovirus contains several stages.Quality control should be performed not only on final products, but also on production procedure and materials used in the production, especially cell bank and virus bank. Quality control of adenovirus is a complicated, important task and is a guarantee for final quality of adenovirus.Sinc quality control methods on work cells of traditional biologic products have been developed completely, this study is focused on the methods establishment of adenovirus control quality. Recombined adenovirus products CNHK200-hEndo, is consisted of vector DNA, namely, adenovirus DNA and therapy gene, namely, human endostatin gene. The basic quality guideline includes identification tests and efficiency tests, which contain identification of adenovirus product, presentation, expression and biologic activity of therapy genes, in detail. Identification tests contain restriction map analysis and PCR identification of therapy gene, efficacy tests contain detection of gene expression and biologic activity detection. Its goal is to validate objective gene in products that mediated by adenovirus vector whether express objective protein in target cells or not and objective protein whether has biological characteristic or not. In this study, adenovirus was identified by SDS-PAGE to analyze its proteins, and by restriction map analysis to analyze its DNA. The results showed that samples were coincidental to the standard of objective adenovirus. Meanwhile, we used PCR to identify endostatin gene whether located correctly in this adenovirus or not, applied ELISA to study its expression and biologic activity. The results demonstrated that this gene was located in DNA of this adenovirus and it could express in the cells successfully with biologic activity. The core index of effectivity and coincidence of adenovirus products are adenovirus particle number, active infectious unit, activity ratio and purity. At present, both FDA and SFDA recommend that quantitation of adenovirus is tested by virus particle number, not virus titer, and particle number as marker of the amount of virus in clinicle. Meanwhile, activity ratio is ristricted to guarantee activity quality of adenovirus products. Traditional detection method of virus particle number still shows some defects in technic. Traditional method is depended on measurement of optical absorption value of DNA viral genome. This required high concentration and high purity virus. If not, its results are not accurate. The methods can not differentiate infectious virus particles from defective ones. We established an anion-exchange chromatography method to detect virus particle number, which does not require large amount samples and high purity samples. The result is more stable and accurate. Although this method needs further evaluation, it has a great feasibility to replace with traditional method. The detection of viral infection activity is taken by biological methods and its results are affected by some variable factors.So it always shows that less stability and repetitiveness. These methods include, report gene assay, virus plaque assay and TCID50 assay. At present, TCID50 assay is applied mostly. This method detects faster, results show more predictable and stable in different individuals. We modified TCID50 assay to make it more stable. Each sample was detected twice by two technicists and average value was caculated as the final value. At present, the commonly used detection methods of adenovirus purity are OD260/OD280 ratio analysis, HPLC analysis and SDS—PAGE electrophoresis. OD260/OD280 ratio analysis is more simply. As a semi-quantitation method, the result is affected by host cell residue protein and DNA, and it is also easily affected by manipulative factors. SDS—PAGE electrophoresis is one kind of detection that measure the purity of vrial protein, and easily affected by operative influence factor, it is suitable for laboratory research. When the sample contains low amount of protein or foreign protein similar to viral proteins, the result will deviate easily. High performance liquid chromatographic analysis performs purity analysis by ion exchange chromatography. The process is controlled by automative program, which could basically remove the interference of human factor, and spend less time. It also can detect virus yield during the whole process quickly and accurately, and lead to quality control of large scale production. We applied established HPLC method to detect the purity of purified adenovirus CNHK200-hEndo and the purity was up to 99%, which achieved the standard of clinical application completely. We detected adenovirus wide type by PCR through amplifing adenovirus E1B area preserved in adenovirus wild type while defected in adenovirus CNHK200-hEndo. The results implied that this method is adapted to detect existence of wide type adenovirus completely and wide type adenovirus was not detected in adenovirus product CNHK200-hEndo. Both host cell DNA residue and BSA residue are two important items of adenovirus impurity, it is significant to be safely applied in clinical use in the coming days. In this study, probe of digoxin was applied to hybride with adenovirus sample. This method showed high sensitivity, which could detect 1pg/ml contents of DNA. But it easily appeared false negative and false positive, and further optimization was needed. To detect the content of BSA residue, we took the routine detection method supplied by China drug and biologicals agency. This method was simple and could be easily performed. The results were stable and could be applied to quality control of adenovirus. Other quality control items, including PH, content of endotoxin, sterility test, abnormal toxicity test, are presented routine quality control methods of common biologicals, well-rounded agents, equipments. So we did not discuss these points. In this study, according to international and country laws and regulations, standards and reference, we established the whole quality control method of adenovirus CNHK200-hEndo.At present, packaging cells of adenovirus mainly contain 293, 911 and PER.C6. The most commonly used cell is 293. Although this cell line has the danger of producing adenovirus wild type, it can produce higher virus titer. We adopted DMEM medium plus 10% FCS as growth medium of 293 cells. Owing to FCS containing inactivative ingredient of adenovirus, in general, during the process of viral amplification, medium with fewer serum was applied. Meanwhile, cell growth would be affected without supporting ingredient from serum, certain amount of serum must be preserved. We consulted the documents from home and abroad, and according to our own practical experience, the medium of adenovirus production was determinated as DMEM medium plus 2% FCS. During the process of adenovirus amplification in bioreactor, since cell growth situation decided the production of virus, making a right choice to cell carrier is critical. The growth characteristic of 293 cell is low adherence ability, which causes cell easy to desquamate and conglobate. We applied fixed bed reactor as the 293 cell culture system because cells with low adherence ability could also grow well in this system. We applied this method by infusion mode and cells inoculated could adhere fast. The adherence rate was up to 98% or even more after 1h of inoculation. The culture cycle was about 7-10 days and the amount of cell proliferation was 25-50 times more. The production of adenovirus not only depends on the amount and growth conditions of host cells, but also on infectious conditions. Among these infectious conditions, virus MOI(Multiplity Of Infection)is the most important critical factor. In this study, when MOI more than 50, virus production would be affected significantly, but if MOI less than 5, the time of harvest would be affected significantly. On the basis of determined range of MOI, we applied 5,10,15,20 of MOI to infect 293 cells during the process of amplification, results showed that production of virus was highest under the condition of MOI=10 and the harvest time of virus was suitable. During the infusion, one of the most important parameters was infusion volume. Because the amplification is a dynamic process, after cells were infected by virus, its metabolism would change, and the demand of nutrition would change also. We detected the content of glucose to trail the metabolism of cells, and maintain different level of glucoses to study infusion influence on virus amplification. The results showed that when infusion volume was lower, keeping low concentration of glucose, cell growth would be slower and the ability of viral amplification would be lower; but, if infusion volume was too high, keeping high level of glucose concentration, speed of cell metabolism would be faster and the condition would not be beneficial to viral amplification and growth, the whole viral production would not be improved. Only by suitable infusion volume and keeping appropriate glucose concentration could obtain higher production of adenovirus. In addition, we determined other parameters that involved with adenovirus amplification in the bioreactor, including carrier amount, agitation rotation speed, infection time, diameter of filtration and established the bioreactor method of adenovirus production. According to this method, we amplificated three batches of adenovirus CNHK200-hEndo, the results showed the total production was up to 1015vp or more, activity ratio was also met with demand, which can satisfied the demand of clinical treatment.CsCl gradient ultracentrifugation has always been employed as the method of adenovirus purification. Traditional CsCl gradient ultracentrifugation to purify virus has been developed completely and widely used in laboratories. But this method is limited, firstly it can only deal with small samples and its recovery rate is low, just about 10-30%; secondly, it needs several times of ultracentrifugation to obtain pure product and the process is complicated; thirdly, viral activity is not stable and the result can not easily repeated. Along with the advance of clinical experiment, demand of clinical grade adenovirus increases. To meet these demands of gene therapy, it is essential to setup the purification technology suitable for large production. Ion-exchange chromatography is a branch of chromatography technology, by differrent affinity of exchangeable ion and detached ion to change balance to separate components. This method can analyze many kinds of ionic compounds simultaneously, which shows high sensitivity, good repetitiveness and selectivity and fast detachment. It is one of the most commonly used methods. It is applied in detachment of many kinds of ionic bio-molecular, including proteins, amino acids, multiple peptides and nucleic acids. We choosed three different material for ion exchange chromatography in the test, which are DEAE Sepharose F.F., SOURCE 30 Q and Q Sepharose XL. Adenovirus was purified at the same condition with these three material and the final purity of adenovirus was examined. The results showed that the purity of adenovirus was up to 85% with SOURCE 30Q, which was the best purity in those three tests, and the recovery ratio of adenovirus was 80%. So we choosed SOURCE 30Q in the further experiments. We used SOURCE 30Q to purify adenovirus crude, and set up the parameters: (1) the volume was 300ml; (2) balance buffer was 20mM Tris, 150mM NaCl, 2mM MgCl2, PH7.5 and detaching buffer was 20mM Tris, 1M NaCl, 2mM MgCl2, PH7.5; (3) flow rate was 10ml/min; (4) flow program was ten times volume of column by gradual flow program. According to such program, we got purified adenovirus whose purity was 99.7% and the recovery ratio of adenovirus was beyond 80%. We found that it met the quality and quantity of big bulk adenovirus production. Moreover, how to preserve adenovirus is an important problem in its application. We made three different preservative buffer to determin which is the best one for adenovirus preservation. We found in recipe A and C, adenovirus could be stable for up to six months. All in all, we built up a new method, which was called anion exchange chromatography, for adenovirus purifying and it could be used in large scale production of adenovirus. Evenmore, it met the requirements of clinical adenovirus product.