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Humulin and it’s production.pdf

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Kristu Jayanti College
Kristu Jayanti College

Humulin and it’s production by genetic engineering

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HUMULIN AND IT’S PRODUCTION
HISTORY Insulin from cattle and pigs was used for many years to treat diabetes and saved millions of lives, but it wasn’t perfect, as it caused allergic reactions in many patients. The first genetically engineered, synthetic “human” insulin was produced in 1978 using E. coli bacteria to produce the insulin. Eli Lilly went on in 1982 to sell the first commercially available biosynthetic human insulin under the brand name Humulin.
WHAT IS INSULIN ? Insulin is a peptide hormone made and secreted by the pancreas to cause excess glucose to be taken up from the blood, thereby lowering blood-glucose levels that could otherwise be toxic . Insulin is made in beta cells, which cluster into islets. The insulin gene encodes 51 amino acids, a very small protein, composed of two polypeptides linked by two disulfide bonds . The A chain consists of 21 amino acids and the B chain consists of 30 amino acids
EXPRESSION SYSTEMS USED IN HUMULIN PRODUCTION Recombinant human insulin is mainly produced either in E. coli or Saccharomyces cerevisiae. 1. Escherichia coli has always been preferred for production of recombinant proteins as it offered several advantages including high growth rate, simple media requirement, easy to handle, high yield and very cost effective. 2. Saccharomyces cerevisiae is the most preferred and predominant yeast for large scale commercial production of insulin, however several other alternate yeast strains have been explored for insulin production . Among yeast strains, Saccharomyces cerevisiae, Hansenulla polymorpha and Pichia pastoris are very commonly used for production of recombinant proteins . Like E. coli, they grow rapidly and are very easy to handle and amenable to various genetic manipulations. Besides, E.coli and yeast, mammalian cells, transgenic animals and plant expression systems are also employed as a host for large-scale production of recombinant insulin
s Source E. coli E. coli S. cerevisiae P. pastoris Destination of product Cytoplasm Secreted Secreted Secreted Biomass cell dry weight (g/l) 80, in bioreactor with fed-batch culture 1.2, in shake flask with batch culture 5, in shake flask with batch culture 59, in bioreactor with fed-batch culture Typical spec. growth rate (1/h) 0.08– 0.12 not specified < 0.33 <0.03 Typical spec. production rate (mg/gh) 14.2 3.4 0.21 0.375 Product concentratio n (g/L) 4.34 0.009 0.075 3.075 Productivity (mg/l h) 1,085 4.01 1.04 17
STEPS INVOLVED IN THE PRODUCTION OF INSULIN
STEPS INVOLVED IN THE PRODUCTION OF INSULIN 1. One method of manufacturing insulin is to grow the two insulin chains separately. This will avoid manufacturing each of the specific enzymes needed. Manufacturers need the two mini-genes: one that produces the A chain and one for the B chain. 2. These two DNA molecules are then inserted into plasmids, small circular pieces of DNA that are more readily taken up by the host's DNA. 3. Manufacturers first insert the plasmids into a non-harmful type of the bacterium E. coli. They insert it next to the lacZ gene. LacZ encodes for 8-galactosidase. Next to this gene is the amino acid methionine, which starts the protein formation. 4. The recombinant, newly formed, plasmids are mixed up with the bacterial cells. Plasmids enter the bacteria in a process called transfection. Manufacturers can add to the cells DNA ligase, an enzyme that acts like glue to help the plasmid stick to the bacterium's DNA.
1. The bacteria synthesizing the insulin then undergo a fermentation process. They are grown at optimal temperatures in large tanks in manufacturing plants. The millions of bacteria replicate roughly every 20 minutes through cell mitosis, and each expresses the insulin gene. 2. After multiplying, the cells are taken out of the tanks and broken open to extract the DNA. The bacterium's DNA is then treated with cyanogen bromide, a reagent that splits protein chains at the methionine residues. This separates the insulin chains from the rest of the DNA. 3. The two chains are then mixed together and joined by disulfide bonds through the reduction-reoxidation reaction. An oxidizing agent is added. The batch is then placed in a centrifuge, a mechanical device that spins quickly to separate cell components by size and density. 4. The DNA mixture is then purified so that only the insulin chains remain. Procedures used include an ion-exchange column, reverse-phase high performance liquid chromatography, and a gel filtration chromatography column. Manufacturers can test insulin batches to ensure none of the bacteria's E. coli proteins are mixed in with the insulin.
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Humulin and it’s production.pdf

  • 2. HISTORY Insulin from cattle and pigs was used for many years to treat diabetes and saved millions of lives, but it wasn’t perfect, as it caused allergic reactions in many patients. The first genetically engineered, synthetic “human” insulin was produced in 1978 using E. coli bacteria to produce the insulin. Eli Lilly went on in 1982 to sell the first commercially available biosynthetic human insulin under the brand name Humulin.
  • 3. WHAT IS INSULIN ? Insulin is a peptide hormone made and secreted by the pancreas to cause excess glucose to be taken up from the blood, thereby lowering blood-glucose levels that could otherwise be toxic . Insulin is made in beta cells, which cluster into islets. The insulin gene encodes 51 amino acids, a very small protein, composed of two polypeptides linked by two disulfide bonds . The A chain consists of 21 amino acids and the B chain consists of 30 amino acids
  • 4. EXPRESSION SYSTEMS USED IN HUMULIN PRODUCTION Recombinant human insulin is mainly produced either in E. coli or Saccharomyces cerevisiae. 1. Escherichia coli has always been preferred for production of recombinant proteins as it offered several advantages including high growth rate, simple media requirement, easy to handle, high yield and very cost effective. 2. Saccharomyces cerevisiae is the most preferred and predominant yeast for large scale commercial production of insulin, however several other alternate yeast strains have been explored for insulin production . Among yeast strains, Saccharomyces cerevisiae, Hansenulla polymorpha and Pichia pastoris are very commonly used for production of recombinant proteins . Like E. coli, they grow rapidly and are very easy to handle and amenable to various genetic manipulations. Besides, E.coli and yeast, mammalian cells, transgenic animals and plant expression systems are also employed as a host for large-scale production of recombinant insulin
  • 5. s Source E. coli E. coli S. cerevisiae P. pastoris Destination of product Cytoplasm Secreted Secreted Secreted Biomass cell dry weight (g/l) 80, in bioreactor with fed-batch culture 1.2, in shake flask with batch culture 5, in shake flask with batch culture 59, in bioreactor with fed-batch culture Typical spec. growth rate (1/h) 0.08– 0.12 not specified < 0.33 <0.03 Typical spec. production rate (mg/gh) 14.2 3.4 0.21 0.375 Product concentratio n (g/L) 4.34 0.009 0.075 3.075 Productivity (mg/l h) 1,085 4.01 1.04 17
  • 6. STEPS INVOLVED IN THE PRODUCTION OF INSULIN
  • 7. STEPS INVOLVED IN THE PRODUCTION OF INSULIN 1. One method of manufacturing insulin is to grow the two insulin chains separately. This will avoid manufacturing each of the specific enzymes needed. Manufacturers need the two mini-genes: one that produces the A chain and one for the B chain. 2. These two DNA molecules are then inserted into plasmids, small circular pieces of DNA that are more readily taken up by the host's DNA. 3. Manufacturers first insert the plasmids into a non-harmful type of the bacterium E. coli. They insert it next to the lacZ gene. LacZ encodes for 8-galactosidase. Next to this gene is the amino acid methionine, which starts the protein formation. 4. The recombinant, newly formed, plasmids are mixed up with the bacterial cells. Plasmids enter the bacteria in a process called transfection. Manufacturers can add to the cells DNA ligase, an enzyme that acts like glue to help the plasmid stick to the bacterium's DNA.
  • 8. 1. The bacteria synthesizing the insulin then undergo a fermentation process. They are grown at optimal temperatures in large tanks in manufacturing plants. The millions of bacteria replicate roughly every 20 minutes through cell mitosis, and each expresses the insulin gene. 2. After multiplying, the cells are taken out of the tanks and broken open to extract the DNA. The bacterium's DNA is then treated with cyanogen bromide, a reagent that splits protein chains at the methionine residues. This separates the insulin chains from the rest of the DNA. 3. The two chains are then mixed together and joined by disulfide bonds through the reduction-reoxidation reaction. An oxidizing agent is added. The batch is then placed in a centrifuge, a mechanical device that spins quickly to separate cell components by size and density. 4. The DNA mixture is then purified so that only the insulin chains remain. Procedures used include an ion-exchange column, reverse-phase high performance liquid chromatography, and a gel filtration chromatography column. Manufacturers can test insulin batches to ensure none of the bacteria's E. coli proteins are mixed in with the insulin.