1. Proceeding information of the project

 

Based on the important physiological functions of human serum albumin (HSA) in human body, it is mainly used clinically to correct the acute hypovolemia induced by large operation, trauma, organ transplantion, etc; and to treat disequilibrium of water, electrolyte and limb balance induced by large area of burning, respiratory distress to prevent and control shock; it is also used in acute gastrointestinal bleeding, renal dialysis and serious chronic diseases such as malabsorption, cirrhosis, nephrotic syndrome, cerebrovascular accident, cerebral ischemia, etc, especially in the cases combined with edema. Human serum albumin (HSA) is also the necessary protectant and excipient for biological pharmacy.

 

The significance of the project on recombinant human serum albumin (rHSA) is critical. HSA is a kind of blood preparation extracted from human blood plasma, which is a kind of vital drug for the application of modern medicine in prevention and treatment. It is a kind of special drug and its only resource is human blood, firstly, it can not be produced freely due to the restrictions of social, economical and political factors, secondly, it is an important substance to maintain life and the clinical application is limited due to the shortage of raw material so that it belongs to scarce drugs, meanwhile, it is inevitable that the blood may be contaminated by various pathogens, such as human immunoddficiency virus, hepatitis B virus, hepatitis C virus, etc. Thus, the safety of medication of blood products has become an outstanding problem attracting close attention of international society.

 

The “Method for producing recombinant human serum albumin (rHSA)”of our company has acquired national patent of invention. At present, the development of laboratory period has finished and we have obtained highly purified rHSA with clinical significance, the semi-trial amplification period is going to finish. It is anticipated that the necessary works for clinical study and application of new drug certification be finished within two to three years.

 

Since 2004, our country has thought highly of safe production of blood products and performed vigorous adjustment in this trade, and the number of plasma collection centers all around the country has decreased from over 150 to over 110, and the domestic amount of collected plasma has decreased from over 5,000 tons in 2004 to about 3,000 tons in last year, the shortage of plasma resource has become the main bottle neck restricting the development of blood products and directly resulted 50% decrease of HSA in 2007 on year-on-year basis.

HSA lies in the first place on the sales of blood products in international market which is 40% of total sales, our country has over 30 manufactures of blood products now and the actual annual output capacity of HSA is about 30,000,000 bottles. However, the total annual output amount of HSA is about 13,000,000 bottles (calculated based on 10 g albumin/bottle) due to the effect of deficiency of plasma resource so that 3,300,000 bottles are necessary to be imported. The sale of domestic HSA in domestic market in 2006 will surpass RMB 4 billion yuan, and the annual increment of the requirement is over 20%. For the consumption regions of HSA, it is mainly provided to Beijing, Shanghai, Guangzhou, Hangzhou (over 60% of the sale), finally, the contradiction between supply and demand of HSA resulted in gradual increase of its price (from RMB 260 yuan/bottle in early 2007 to RMB 360 yuan/bottle in late 2007).

The “recombinant human serum albumin (rHSA)” project of the company takes the owned patent of invention as the core technique so that it can not only increase the international status of domestic biological pharmacy but also bring great social benefits and economic returns once it is carried out successfully.

 

The techniques of recombinant human serum albumin (rHSA) mainly include: accurate clone of HSA gene, insertion of the gene expressing albumin into the biological producing system for replication, fermentation for secretary expression, collection and separation. The expression systems or producing systems generally include: bacteria, yeasts, cell culture, transgenic animals, etc.

 

In 1981, Lawn et al (Genentch company, America) firstly expressed rHSA gene in E. coli expression system successfully and achieved the patent, however, the expression level is milligram level without the capability of industrialization.

The SIBIA-Pichia yeast expression system constructed by American Philip petroleum group secrets and expresses recombinant human serum albumin to 4-5 g/L which has the prospect of industrialization.

In 1990, the Japanese Green-cross company obtained the technical license of producing recombinant human serum albumin with SIBIA from American Philip petroleum group and the drug went on the market in batches in 1999.

 

At present, only the method of Hygene Biopharma Co., Ltd for producing recombinant human serum albumin obtained national patent of invention in our country. Some other scientific research institutions are also studying rHSA, however, most of them are still in the following and imitating stage.

 

 

The “Method for producing recombinant human serum albumin (rHSA)”of our company has acquired national patent of invention (Patent number: 981025064, primary number of international patent classification: CO7K14/765). It is one of national new drugs type 1.

 

The method for producing rHSA of the company realized the breakthrough of high performance of rHSA through the innovation of technical route.

Japanese Green-cross company obtained whole gene of HSA through mature reverse transcription method HSAmRNA→ cDNA, Philip petroleum group has already applied the patent with this method. However, Zhongshan Hygene Biopharma Co., Ltd fully synthesized HSA gene, which is a new method so that it is not conflicting about the patent, which all forms the company’s creative characteristic.

 

The detailed procedures are as below

 

1. The designation of artificial synthesis of HSA gene replaces condons: Compared with the natural gene, the artificially synthesized HSA gene sequence for HSA amino acid sequence, had 155 bases replaced and 14 condons changed in the synthesized gene. Exclude the sequence that may affect transcription and translation; insert and add some synthesized restriction enzyme cutting sites to easy the clone of the gene and the splicing among various subfragments.

 

2. The applied method and procedure for the artificial synthesis of HSA gene can accurately clone HSA gene. HSA protein is composed of 585 amino acid. Together with the sequences of restriction enzyme cutting sites on two ends, the length of the synthesized HSA gene is 1797 bp, and the full length sequence of HSA gene can be synthesized rapidly with the PCR mediated gene synthesis method. Divide the whole gene into 3 fragments for synthesis, and the three fragments are HSA-I (612 bp), HSA-Ⅱ (562 bp) and HSA-Ⅲ (765 bp), respectively. The two ends of each fragment has BamHI and BtI cutting sites for cloning of each DNA fragment. These three fragments are spliced respectively and the cloning can correct error bases. Joint these three fragments into the full length fragment of the gene through the pre-set HindIII and PstI cutting sites once it is certain that each fragment is correct.

 

We overcame the problem of low expression level of Pichia yeast expression system and constructed a high-performance expression system with expression level of 5-10 g/L, which has reached the international level and makes the industrialization of rHSA feasible.

 

Currently, the HSA expression amount of the strain constructed by the company is comparable to the strains in other international laboratories. The HSA expression level may be 5 g/L through increasing the number of copies of HSA gene in the strain and obtain highly purified rHSA product meeting the requirement for clinical application. The HSA expression level may reach over 10 g/L through the screening and construction of high expression strain.

 

The obtained high-expression strain in the company has the value for industrial production; it is easy to conduct large-scale production. The collected and separated HSA has the features of high purity and safety, and it also has identical biological functions compared with natural HSA.

 

The company owns the domestic and international patent project of production and invention of “recombinant human serum albumin”, which is a critical project that is badly in need of development in our country. This project solved current difficulty of blood collection and the cross infection of acquired immune deficiency syndrome, liver diseases, etc in the courses of blood collection and transfusion resulted from “human serum albumin” produced by blood. “Human serum albumin” is a routine drug for emergency treatment and has billions of dollars of the great market. Now, it is one of the most popular projects in the world. A remarkable medicine and biotechnology city will rise once the industrialization of “recombinant human serum albumin” project become success, which can not only produce great economic benefits and social benefits but also benefit mankind.