Trehalose is a safe and reliable natural sugar. It is a non-reducing sugar composed of two glucose molecules with α,α,1,1-glycosidic bonds. It is very stable in nature and has many biologically active substances. Magical protection. Trehalose has a magical protective effect on organisms because trehalose forms a unique protective film on the cell surface under harsh environmental conditions such as high temperature, high cold, high osmotic pressure and dry water loss, effectively protecting protein molecules from invariance. Live to maintain the life process and biological characteristics of the living body. This unique functional feature makes trehalose an important ingredient in cell-active, moisturizing cosmetics in addition to its excellent active protective agents for protein drugs, enzymes, vaccines and other biological products.
(1) Trehalose has been successfully used in medicine to replace plasma proteins as stabilizers for biologically active substances such as blood products, vaccines, lymphocytes, and cell tissues. Not only can it be stored dry under normal temperature conditions, but more importantly, it can prevent the spread of deadly diseases such as hepatitis B and AIDS caused by blood pollution. The World Health Organization attaches great importance to this.
(2) The Quadrant Research Foundation in Cambridge, England, after lyophilization of polio vaccine and trehalose, found that its stability at 45 ° C in dry state is comparable to that at 4 ° C in liquid state. The successful research of this project will greatly reduce the vaccine treatment process, reduce the storage and transportation costs of the vaccine, and ensure that the long-distance transport vaccine can still maintain a relatively high activity, which will greatly help the World Health Organization achieve the maximum range. The goal of eradicating polio.
(3) John Crow and his colleagues at the University of California, USA, mixed trehalose with cells that make platelets, dried and dehydrated to dry the cells, and then lyophilized at room temperature for long-term storage. It has been proven that platelets added with trehalose and preserved for a long time are still 85% viable after hydration, and the survival rate is higher than that of platelets stored in most blood banks for a short period of time.
(4) Trehalose can be used for the preservation of biological reagents for research, such as various tool enzymes, cell membranes, organelles, antibodies, antigens and viruses, etc., making life science research more convenient and efficient, and the British University Camilo.C and other details The protective effect of trehalose on DNA restriction endonuclease DNA ligase and DNA polymerase was studied. The results showed that all enzymes added with trehalose were stored at 70 ° C for 35 days or at 37 ° C for 9 months. It has no loss of vitality and can still cut off DNA accurately. The Institute of Microbiology of the Chinese Academy of Sciences applied three kinds of diagnostic tools for the preparation of human serum cholesterol by trehalose drying. After long-term storage at room temperature, the activity retention rate was above 90%, and it has been successfully used in clinical applications. This is an effect that cannot be achieved by other kinds of protective agents at present. The use of trehalose as a stabilizer and a protective agent for biological reagents such as a diagnostic tool enzyme can be dried and stored under normal temperature conditions, which not only simplifies the preparation process of biological reagents. It also brings convenience to the diagnosis and treatment of diseases in the vast rural areas of China.
(5) Bifidobacterium is a bacteria used in the intestinal tract to improve the micro-ecological balance of the human body. The live bacterial preparation of Bifidobacterium is a powerful weapon for disease prevention and treatment, and is popular in developed countries such as Europe, America and Japan. In China, the live bacterial preparation of Bifidobacterium has gradually become a new force in the pharmaceutical industry. Because Bifidobacterium is an anaerobic bacteria that requires extremely harsh living conditions, the environment may change slightly, which may cause the death of the bacteria. Therefore, how to improve the survival rate of Bifidobacterium and ensure the shelf life of the product has been A technical problem that plagues the live bacterial preparation industry. At present, skim milk is generally used as a lyoprotectant, but the effect is not satisfactory. During storage, the survival rate of bacteria decreases rapidly. Recent studies have shown that using trehalose as a protective agent, the survival rate of Bifidobacterium is more than double that of skim milk. It is particularly exciting that trehalose can keep lyophilized Bifidobacterium active at room temperature for a long time. Significantly extend the shelf life of live bacterial preparations. Therefore, the problem of poor storage performance and short shelf life of the products faced by the live bacterial preparation industry can be solved.
(6) Application Example 1) Preparation of solid product from liquid product 500 g of anhydrous trehalose, 270 g of egg yolk powder prepared by the above method, 290 g of skim milk powder, 4.4 g of calcium chloride, 1.85 g of potassium chloride, After mixing 0.01 g of thiamine, 0.1 g of sodium ascorbate, 0.6 g of acetic acid and 0.04 g of nicotinic acid, 25 g of each part was placed in a waterproof aluminum foil pouch and heat-sealed to obtain the solid product. Due to the low moisture content in the bag, the product does not need to be refrigerated and can be stored stably for a long time at room temperature. It has good water solubility and dispersibility. Before use, it can be prepared by dissolving 1 sachet of the solid product in about 150-300 ml of water to make a liquid food, inhaling the body or injecting into the nasal cavity, stomach or intestine.
2) Preparation of solid pharmaceuticals In order to perform subcutaneous transplantation of BALL-1 cells, the immune serum prepared by the traditional method is injected into the newly-produced voles to reduce the immune response, and after being fed for 3 weeks according to the general method, it is taken out. The tumor formed under the skin of the voles was cut into small pieces, and then the pieces were dispersed and dissolved in physiological saline. The eluted tumor cells were washed with serum-free RPMI1640 medium (pH 7.2), and then dissolved in the newly prepared same medium, and the concentration of the culture medium was diluted to 2 × 106 cells per ml, and at 35 Store at °C.
200 IU/ml human a-interferon was added to the cell suspension, and after about 2 hours of culture, 300 HA/ml HVJ was added, and culture was further carried out for 20 hours to induce the culture to produce more human a-interferon. The cell culture solution was centrifuged at 1,000 ° C at 4 ° C to remove the precipitate, and the supernatant was filtered through a membrane, and the filtrate was added to a column containing an anti-a-interferon antibody, and then a buffer was added thereto. The unabsorbed component flows out, and then the components absorbed by the column are eluted and concentrated to a human a-interferon solution having a concentration of about 0.01 w/v%, wherein the specific activity of the human a-interferon is about 2 x 108 Iu/mg protein, about 4 ml of a-interferon can be obtained per voles.
6 g of anhydrous trehalose was placed in a 100 ml moisture-proof plastic bottle, and 0.2 ml of a human a-interferon solution containing about 4×10 6 IU was injected into the bottle, and the bottle was aseptically sealed with a rubber stopper. Get solid pharmaceuticals. According to the preparation process, the human a-interferon-containing solution is easily dehydrated and dried by contact with anhydrous trehalose, and the a-interferon of the solid product can be stabilized and efficiently without lyophilization.
The product is easily soluble in water, and the human a-interferon can be used as an anti-allergic agent (such as anti-viral agent, anti-tumor agent and anti-rheumatic agent), and enters the human body by instillation or intramuscular injection. Effectively prevent or treat a variety of diseases. This product is suitable for internal medicine and can also be used as oral reagents and diagnostic agents.
3) Preparation of solid pharmaceuticals BALL-1 cells derived from human lymphocytes were inoculated into Eagle basal medium (pH 7.4) supplemented with 20% fetal calf serum, and cultured in a suspension at 37 ° C according to a conventional method. The cultured cells were washed with serum-free Eagle basal medium (pH 7.4), poured into freshly prepared Eagle's basal medium containing 20% ​​fetal bovine serum, and concentrated to a concentration of 1 × 10 7 cells/ Ml. 10,000 HA/ml HVJ was added to the solution and incubated at 38 ° C for 24 hours at constant temperature to induce HVJ into a-hTNF. The cell suspension containing a-hTNF was centrifuged at 1,000 ° C at 4 ° C, and the supernatant was dialyzed against physiological saline containing 0.01 M phosphate buffer for 15 hours, and then filtered through a membrane. To purify the a-hTNF solution, the filtrate was added to a column containing an anti-interferon antibody, and the fraction not absorbed by the column was poured into a layer containing an anti-tumor necrosis a-gene monoclonal antibody with affinity. In the column, the components absorbed by the column were eluted to obtain a-hTNF solution concentration to 0.01 w/v%, wherein the specific activity of a-hTNF was about 2 x 106 JRU/mg protein. Thus the yield of a-hTNF is approximately 5 x 104 JRU/L of cell culture.
10 g of anhydrous trehalose was placed in a 100 ml bottle, and 0.5 ml of a solution containing 1 x 105 JRU a-hTNF was injected, and the product was obtained by aseptically capping with a rubber stopper. The medicine prepared by the above method, the powdery anhydrous trehalose absorbs water to dehydrate and dry the solution of a-hTNF, and the a-hTNF can be stabilized and efficiently without lyophilization treatment.
The product is easily soluble in water, and a-hTNF can be used as an anti-allergic agent (such as antiviral agents, anti-abdominal agents and anti-immunological agents), and can be effectively prevented by instillation or intramuscular injection into the human body. Treat a variety of diseases. This product is suitable for internal medicine, but also for oral reagents and diagnostics.
(2) The Quadrant Research Foundation in Cambridge, England, after lyophilization of polio vaccine and trehalose, found that its stability at 45 ° C in dry state is comparable to that at 4 ° C in liquid state. The successful research of this project will greatly reduce the vaccine treatment process, reduce the storage and transportation costs of the vaccine, and ensure that the long-distance transport vaccine can still maintain a relatively high activity, which will greatly help the World Health Organization achieve the maximum range. The goal of eradicating polio.
(3) John Crow and his colleagues at the University of California, USA, mixed trehalose with cells that make platelets, dried and dehydrated to dry the cells, and then lyophilized at room temperature for long-term storage. It has been proven that platelets added with trehalose and preserved for a long time are still 85% viable after hydration, and the survival rate is higher than that of platelets stored in most blood banks for a short period of time.
(4) Trehalose can be used for the preservation of biological reagents for research, such as various tool enzymes, cell membranes, organelles, antibodies, antigens and viruses, etc., making life science research more convenient and efficient, and the British University Camilo.C and other details The protective effect of trehalose on DNA restriction endonuclease DNA ligase and DNA polymerase was studied. The results showed that all enzymes added with trehalose were stored at 70 ° C for 35 days or at 37 ° C for 9 months. It has no loss of vitality and can still cut off DNA accurately. The Institute of Microbiology of the Chinese Academy of Sciences applied three kinds of diagnostic tools for the preparation of human serum cholesterol by trehalose drying. After long-term storage at room temperature, the activity retention rate was above 90%, and it has been successfully used in clinical applications. This is an effect that cannot be achieved by other kinds of protective agents at present. The use of trehalose as a stabilizer and a protective agent for biological reagents such as a diagnostic tool enzyme can be dried and stored under normal temperature conditions, which not only simplifies the preparation process of biological reagents. It also brings convenience to the diagnosis and treatment of diseases in the vast rural areas of China.
(5) Bifidobacterium is a bacteria used in the intestinal tract to improve the micro-ecological balance of the human body. The live bacterial preparation of Bifidobacterium is a powerful weapon for disease prevention and treatment, and is popular in developed countries such as Europe, America and Japan. In China, the live bacterial preparation of Bifidobacterium has gradually become a new force in the pharmaceutical industry. Because Bifidobacterium is an anaerobic bacteria that requires extremely harsh living conditions, the environment may change slightly, which may cause the death of the bacteria. Therefore, how to improve the survival rate of Bifidobacterium and ensure the shelf life of the product has been A technical problem that plagues the live bacterial preparation industry. At present, skim milk is generally used as a lyoprotectant, but the effect is not satisfactory. During storage, the survival rate of bacteria decreases rapidly. Recent studies have shown that using trehalose as a protective agent, the survival rate of Bifidobacterium is more than double that of skim milk. It is particularly exciting that trehalose can keep lyophilized Bifidobacterium active at room temperature for a long time. Significantly extend the shelf life of live bacterial preparations. Therefore, the problem of poor storage performance and short shelf life of the products faced by the live bacterial preparation industry can be solved.
(6) Application Example 1) Preparation of solid product from liquid product 500 g of anhydrous trehalose, 270 g of egg yolk powder prepared by the above method, 290 g of skim milk powder, 4.4 g of calcium chloride, 1.85 g of potassium chloride, After mixing 0.01 g of thiamine, 0.1 g of sodium ascorbate, 0.6 g of acetic acid and 0.04 g of nicotinic acid, 25 g of each part was placed in a waterproof aluminum foil pouch and heat-sealed to obtain the solid product. Due to the low moisture content in the bag, the product does not need to be refrigerated and can be stored stably for a long time at room temperature. It has good water solubility and dispersibility. Before use, it can be prepared by dissolving 1 sachet of the solid product in about 150-300 ml of water to make a liquid food, inhaling the body or injecting into the nasal cavity, stomach or intestine.
2) Preparation of solid pharmaceuticals In order to perform subcutaneous transplantation of BALL-1 cells, the immune serum prepared by the traditional method is injected into the newly-produced voles to reduce the immune response, and after being fed for 3 weeks according to the general method, it is taken out. The tumor formed under the skin of the voles was cut into small pieces, and then the pieces were dispersed and dissolved in physiological saline. The eluted tumor cells were washed with serum-free RPMI1640 medium (pH 7.2), and then dissolved in the newly prepared same medium, and the concentration of the culture medium was diluted to 2 × 106 cells per ml, and at 35 Store at °C.
200 IU/ml human a-interferon was added to the cell suspension, and after about 2 hours of culture, 300 HA/ml HVJ was added, and culture was further carried out for 20 hours to induce the culture to produce more human a-interferon. The cell culture solution was centrifuged at 1,000 ° C at 4 ° C to remove the precipitate, and the supernatant was filtered through a membrane, and the filtrate was added to a column containing an anti-a-interferon antibody, and then a buffer was added thereto. The unabsorbed component flows out, and then the components absorbed by the column are eluted and concentrated to a human a-interferon solution having a concentration of about 0.01 w/v%, wherein the specific activity of the human a-interferon is about 2 x 108 Iu/mg protein, about 4 ml of a-interferon can be obtained per voles.
6 g of anhydrous trehalose was placed in a 100 ml moisture-proof plastic bottle, and 0.2 ml of a human a-interferon solution containing about 4×10 6 IU was injected into the bottle, and the bottle was aseptically sealed with a rubber stopper. Get solid pharmaceuticals. According to the preparation process, the human a-interferon-containing solution is easily dehydrated and dried by contact with anhydrous trehalose, and the a-interferon of the solid product can be stabilized and efficiently without lyophilization.
The product is easily soluble in water, and the human a-interferon can be used as an anti-allergic agent (such as anti-viral agent, anti-tumor agent and anti-rheumatic agent), and enters the human body by instillation or intramuscular injection. Effectively prevent or treat a variety of diseases. This product is suitable for internal medicine and can also be used as oral reagents and diagnostic agents.
3) Preparation of solid pharmaceuticals BALL-1 cells derived from human lymphocytes were inoculated into Eagle basal medium (pH 7.4) supplemented with 20% fetal calf serum, and cultured in a suspension at 37 ° C according to a conventional method. The cultured cells were washed with serum-free Eagle basal medium (pH 7.4), poured into freshly prepared Eagle's basal medium containing 20% ​​fetal bovine serum, and concentrated to a concentration of 1 × 10 7 cells/ Ml. 10,000 HA/ml HVJ was added to the solution and incubated at 38 ° C for 24 hours at constant temperature to induce HVJ into a-hTNF. The cell suspension containing a-hTNF was centrifuged at 1,000 ° C at 4 ° C, and the supernatant was dialyzed against physiological saline containing 0.01 M phosphate buffer for 15 hours, and then filtered through a membrane. To purify the a-hTNF solution, the filtrate was added to a column containing an anti-interferon antibody, and the fraction not absorbed by the column was poured into a layer containing an anti-tumor necrosis a-gene monoclonal antibody with affinity. In the column, the components absorbed by the column were eluted to obtain a-hTNF solution concentration to 0.01 w/v%, wherein the specific activity of a-hTNF was about 2 x 106 JRU/mg protein. Thus the yield of a-hTNF is approximately 5 x 104 JRU/L of cell culture.
10 g of anhydrous trehalose was placed in a 100 ml bottle, and 0.5 ml of a solution containing 1 x 105 JRU a-hTNF was injected, and the product was obtained by aseptically capping with a rubber stopper. The medicine prepared by the above method, the powdery anhydrous trehalose absorbs water to dehydrate and dry the solution of a-hTNF, and the a-hTNF can be stabilized and efficiently without lyophilization treatment.
The product is easily soluble in water, and a-hTNF can be used as an anti-allergic agent (such as antiviral agents, anti-abdominal agents and anti-immunological agents), and can be effectively prevented by instillation or intramuscular injection into the human body. Treat a variety of diseases. This product is suitable for internal medicine, but also for oral reagents and diagnostics.
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