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Insulin Production By Fermentation

Diabetes

Diabetes

For many years, insulin was obtained by purifying it from the pancreas of cows and pigs slaughtered for food. This was expensive, difficult and the insulin could cause allergic reactions. Once the structure of human insulin had been found, in 1955, the cow and pig insulin could be chemically modified to be the same as human insulin. It is now made by genetically-engineered microbes. They produce human insulin in a pure form that is less likely to cause allergic reactions. Human insulin is produced in a very controlled and clean environment. Genetically-engineered bacteria are grown in large stainless steel fermentation vessels. The vessel contains all the nutrients needed for growth. When the fermentation is complete, the mixture containing the bacteria is harvested. The bacteria are filtered off and broken open to release the insulin they have produced. It is then purified and packaged into bottles for distribution. All the equipment is kept sterile so that contamination cannot get into the medicine. Regular checks make sure that all the processes are working properly and the insulin meets the required quality. One of the possible future treatments for type 1 diabetes is gene therapy. Researchers have identified a faulty gene which makes people with the gene more likely to develop type 1 diabetes. In the future, this gene could be replaced by a fully-working version of the gene. This could prevent people from getting diabetes. In theory, gene therapy could even be used on embryos before they were implanted into the womb during in vitro fertilisation treatment. Is this a step too far for science or a great leap forward for medicine? What are the benefits and risks of developing gene therapy? How would you feel if you had a genetic disorder that could not be treated in a Continue reading >>

Industrial Fermentation

Industrial Fermentation

Industrial fermentation is the intentional use of fermentation by microorganisms such as bacteria and fungi as well as eukaryotic cells like CHO cells and insect cells, to make products useful to humans. Fermented products have applications as food as well as in general industry. Some commodity chemicals, such as acetic acid, citric acid, and ethanol are made by fermentation.[1] The rate of fermentation depends on the concentration of microorganisms, cells, cellular components, and enzymes as well as temperature, pH[2] and for aerobic fermentation[3] oxygen. Product recovery frequently involves the concentration of the dilute solution. Nearly all commercially produced enzymes, such as lipase, invertase and rennet, are made by fermentation with genetically modified microbes. In some cases, production of biomass itself is the objective, as in the case of baker's yeast and lactic acid bacteria starter cultures for cheesemaking. In general, fermentations can be divided into four types:[4] Production of biomass (viable cellular material) Production of extracellular metabolites (chemical compounds) Production of intracellular components (enzymes and other proteins) Transformation of substrate (in which the transformed substrate is itself the product) These types are not necessarily disjoint from each other, but provide a framework for understanding the differences in approach. The organisms used may be bacteria, yeasts, molds, algae, animal cells, or plant cells. Special considerations are required for the specific organisms used in the fermentation, such as the dissolved oxygen level, nutrient levels, and temperature. General process overview[edit] In most industrial fermentations, the organisms or eukaroyotic cells are submerged in a liquid medium; in others, such as the fe Continue reading >>

Cell Factories For Insulin Production

Cell Factories For Insulin Production

Abstract The rapid increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to escalate the demand for recombinant insulin in near future. Current manufacturing technologies would be unable to meet the growing demand of affordable insulin due to limitation in production capacity and high production cost. Manufacturing of therapeutic recombinant proteins require an appropriate host organism with efficient machinery for posttranslational modifications and protein refolding. Recombinant human insulin has been produced predominantly using E. coli and Saccharomyces cerevisiae for therapeutic use in human. We would focus in this review, on various approaches that can be exploited to increase the production of a biologically active insulin and its analogues in E. coli and yeast. Transgenic plants are also very attractive expression system, which can be exploited to produce insulin in large quantities for therapeutic use in human. Plant-based expression system hold tremendous potential for high-capacity production of insulin in very cost-effective manner. Very high level of expression of biologically active proinsulin in seeds or leaves with long-term stability, offers a low-cost technology for both injectable as well as oral delivery of proinsulin. Introduction The pioneering work of Stanley Cohen and Herbert Boyer, who invented the technique of DNA cloning, signaled the birth of genetic engineering, which allowed genes to transfer among different biological species with ease [1]. Their discovery led to the development of several recombinant proteins with therapeutic applications such as insulin and growth hormone. Genes encoding human insulin and growth hormo Continue reading >>

Recombinant Dna Fermenter, Circa 1977

Recombinant Dna Fermenter, Circa 1977

Fermenters like this one used genetically-manipulated bacteria to produce the first human insulin in 1977 and the first human growth factor in 1979. Credit: © SSPL / Science Museum In 1972, Uni In 1972, University of California, San Francisco, biochemist Herbert Boyer met Stanford University geneticist Stanley Norman Cohen at a meeting in Hawaii. The two then kicked off a collaboration that eventually led to the creation of the first recombinant DNA, a landmark that ushered in the era of modern biotechnology. By combining Cohen's expertise with bacterial plasmids and Boyer's know-how about restriction enzymes, the two found that they could use bacteria as tiny factories for producing many human proteins. Boyer went on to found Genentech in 1976. In order to produce the proteins in mass quantities, the fledgling biotech company needed a way to grow transgenic bacteria on an industrial scale. To do that, they turned to the ancient art of fermentation. People had made wine, bread, and beer for thousands of years, yet it wasn't until World War I when tons of acetone and other explosive ingredients were needed that the process became industrialized, says Robert Bud, the principal curator of medicine at the Science Museum in London. Fermentation took another big leap in the 1950s and 1960s when scientists found new ways of growing large amounts of penicillin by continuously stirring air through the fermentation tank. The 750-liter fermenter depicted here—which was painted by Alan Stones to mark the 1986 opening of the chemical industry gallery at the Science Museum—was one of the first used by Genentech. Finely-tuned valves controlled the flow of air and other nutrients through the 3-meter-tall tank, which was critical to growing large batches of insulin without contamin Continue reading >>

Eli Lilly Humalog Manufacturing Facility, Carolina

Eli Lilly Humalog Manufacturing Facility, Carolina

Eli Lilly began production of the insulin product Humalog at its new bulk manufacturing facility in Carolina, Puerto Rico, in mid-2005. Scanning electron micrograph of E. coli as used for recombinant DNA techniques. Pro-insulin and human insulin, showing where the trypsin cleavage occurs. In April 2001 Eli Lilly announced it was constructing a new biotech bulk manufacturing facility in Carolina, Puerto Rico. Construction started in July 2002 and, following validation and commissioning, the plant was in production by mid-2006. This project was part of an overall $1bn investment into the company’s facilities in Puerto Rico, which was completed in August 2006. The 300,000ft² facility produces the rapid-acting insulin product Humalog, dispensed from the KwikPen (MirioPen), which is manufactured in Indianapolis. The production of this requires the use of recombinant DNA technology via insulin lispro [rDNA origin] injection. KwikPens became available for Humalog, Humalog Mix75/25 and Humalog Mix50/50 in February 2008. KwikPen is the third new insulin pen launched since 2007 to improve the daily management of diabetes. Previous models include the HumaPen MEMOIR, the world’s first digital insulin pen with memory; and HumaPen LUXURA HD, a reusable pen for patients who need insulin dosing in smaller increments. In February 2010, the plant was issued a warning letter by the US Food and Drug Administration (FDA) for deviations in cGMP API production. The FDA cited deviations during an inspection conducted in 2009. Deviations were found in 24 batches of Lyspro Insulin Zinc Crystals, the API in Humalog, released between December 2007 and March 2008. Production of the original Humalog pre-filled pens including the Humalog Mix75/25 and the Humalog Mix50/50 was discontinued Continue reading >>

Yeast Fermentation Processes For Insulin Production.

Yeast Fermentation Processes For Insulin Production.

Authors Diers IV , Rasmussen E , Larsen PH , Kjaersig IL MeSH Biotechnology Denmark Fermentation Genetic Vectors Government Insulin Recombinant Proteins Reproducibility of Results Saccharomyces cerevisiae Pub Type(s) Journal Article Review Language eng PubMed ID 1367289 Continue reading >>

Production Of Genetic Engineered Insulin

Production Of Genetic Engineered Insulin

Pharmaceutics Human Insulin Production The hormone insulin is essential for the control of blood sugar levels. Diabetes mellitus is a disease in which some people cannot make insulin themselves. This disease kills many people in the world every year. Insulin has been used in the treatment of diabetes mellitus since 1922 when Leonard Thompson became the first human to receive an injection of man-made insulin. Production of Genetic Engineered Insulin 1. Human insulin is extracted from pancreas cells and an insulin-producing gene is isolated. 2. A plasmid DNA is extracted from a bacterium and cut with restriction enzyme, forming plasmid vector. 3. Insert human insulin-producing gene into the bacterial plasmid vector to form the recombinant DNA of human insulin-producing gene. 4. Introduce this recombinant DNA into a bacterial cell to form the recombinant bacterium. 5. The recombinant bacteria multiply in a fermentation tank and produce human insulin. 6. Insulin is extracted, purified and bottled. It is then ready to be injected into diabetic patients. Recombinant HB Vaccine Production Hepatitis B (HB) is one of the most common infectious diseases known to man. The World Health Organization estimates that there are as many as 285 million chronic carriers of this virus worldwide. Traditional vaccines use a weakened or killed form of a virus to force the body to develop antibodies that are strong enough to combat the virus. Traditional Vaccination for Hepatitis B Recombivax HB Recombivax HB was approved as a hepatitis B prevention vaccine in July 1986. Using recombinant DNA technology, Recombivax HB uses the surface antigen of the virus that stimulates the production of protective antibodies which combat the HB virus. Production of Recombivax HB 1. HB antigen producing gene i Continue reading >>

A Simplified And Efficient Process For Insulin Production In Pichia Pastoris.

A Simplified And Efficient Process For Insulin Production In Pichia Pastoris.

Abstract A significant barrier to insulin is affordability. In this manuscript we describe improvements to key steps in the insulin production process in Pichia pastoris that reduce cost and time. The strategy for recovery and processing of human insulin precursor has been streamlined to two steps from bioreactor to the transpeptidation reaction. In the first step the insulin precursor secreted during the methanol induction phase is recovered directly from the culture broth using Tangential Flow Filtration with a Prostak™ module eliminating the laborious and time-consuming multi-step clarification, including centrifugation. In the second step the protein is applied at very high loadings on a cation exchange resin and eluted in a mixture of water and ethanol to obtain a concentrated insulin precursor, suitable for use directly in the transpeptidation reaction. Overall the yield from insulin precursor to human insulin was 51% and consisted of three purification chromatography steps. In addition we describe a method for recovery of the excess of H-Thr(tBu)-OtBu from the transpeptidation reaction mixture, one of the more costly reagents in the process, along with its successful reuse. Continue reading >>

Enhanced Production Of Insulin-like Growth Factor I Fusion Protein In Escherichia Coli By Coexpression Of The Down-regulated Genes Identified By Transcriptome Profiling

Enhanced Production Of Insulin-like Growth Factor I Fusion Protein In Escherichia Coli By Coexpression Of The Down-regulated Genes Identified By Transcriptome Profiling

ABSTRACT The transcriptome profiles of recombinant Escherichia coli producing human insulin-like growth factor I fusion protein (IGF-If) during the high-cell-density fed-batch culture were analyzed using DNA microarrays. The expression levels of 529 genes were significantly altered after induction. About 200 genes were significantly down-regulated during the production of IGF-If after induction. Among these down-regulated genes, we rationally selected and coexpressed in E. coli producing IGF-If the prsA gene (encoding a phosphoribosyl pyrophosphate synthetase) and the glpF gene (encoding a glycerol transporter), which are involved in an early key step in the biosynthetic pathway of nucleotides and amino acids (Trp and His) and the first step in glycerol utilization, respectively. As a result, the production of IGF-If could be increased from 1.8 ± 0.13 (± standard deviation) to 4.3 ± 0.24 g/liter. The volumetric productivity was also increased from 0.36 ± 0.027 to 0.82 ± 0.048 g/liter/h. These results demonstrate that transcriptome profiling can provide invaluable information in designing engineered strains showing enhanced performance. Escherichia coli has been the workhorse for the production of recombinant proteins (7, 20). Various strategies have been employed for the development of E. coli strains which are able to efficiently produce recombinant proteins (14). Once a recombinant host strain is developed, a high level of production of recombinant proteins is generally achieved by high-cell-density fed-batch cultivation (13). It has been well known that the overproduction of recombinant proteins acts as a stress on the cells, resulting in induction of stress-responsive genes such as dnaK, groEL, ibpA, ibpB, and ompT (10, 17). Furthermore, the yield and specific Continue reading >>

Human Insulin Production Process & Requirement

Human Insulin Production Process & Requirement

1. Synthetic Insulin – R&D and Industrial level Manufacturing process & requirement (overall idea) Krishnasalini Gunanathan | Research Trainee| 2. Insulin • Insulin – • A hormone that regulates the amount of glucose in the blood • Produced by cells in pancreas- islets of Langerhans • types – Ultra short acting, short acting, intermediate acting, long acting • History of synthetic insulin • 1921 by Canadian sci Frederick G.Banting & Charles H.Best from Dog’s pancreas • 1982 Eli lilly corp., produced genetically engineered human insulin 3. Structure - Insulin • Insulin gene – a protein consist of two amino acid chains A above chain B held together with bonds • Chain A - 21 amino acid , Chain B – 30 amino acids • Formation of insulin – • Preproinsulin proinsulin single chain without signaling sequence Active protein insulin without link between A & B 4. Types Insulin Production • Type -1 • Two insulin chains are grown separately and inserted into plasmids and grown in E.coli • Two chains are linked by oxidation-reduction reaction using lysosomes and cyanogen bromide • Purified by chromatographic methods • Type- 2 • Known proinsulin process • Coded sequence are inserted to non-pathogenic E.coli bacteria & fermentation is carried out • Zn2+ (additives) are added to delay the absorption in the body • Type – 3 • Analog insulin is produced by changing its amino acid sequence & creating an analog, which clumps less & disperses more readily into the blood. Eg: Glargine insulin 5. Materials Required (R&D level) • Mother culture – Escherichia coli • UV-Chamber • Human protein with 20 amino acids which produce insulin • Enzymes, antibiotics and primer • PCR – cloning • Fermentation flasks – bacterial culture Continue reading >>

Genetic Engineering

Genetic Engineering

Genetic engineering involves the extraction of a gene from one living organism and inserting it into another organism, so that the receiving organism can express the product of the gene. A basic technique used is the genetic engineering of bacteria. It can be broken into the following key stages: Selection of characteristics. Identifying the gene from amongst all the others in the DNA of the donor organism. Isolation of the gene. Obtaining a copy of the required gene from the DNA of the donor organism and placing it in a vector. (A vector in biology refers to an organism that acts as a vehicle to transfer genetic material from a donor organism to a target cell in a recipient organism.) Insertion Use the vector to introduce the gene into the host cell. Replication Allow the host cell to multiply to make multiple clones of the genes. Example of Genetically Engineered Bacteria – Production of Human Insulin An example of genetically engineered bacteria is in the production of human insulin. Insulin is a protein hormone produced in the pancreas which has an important function in the regulation of blood sugar levels. Insulin facilitates the transport of glucose into cells. A deficiency in insulin is one of the causes of the disease diabetes mellitus or sugar diabetes in which the sugar levels in the blood become raised resulting in harmful consequences. At least 3% of the world’s population is affected by diabetes mellitus and sufferers of the disease require insulin injections to manage the disease. Pure Mouse Isotype Controls - IgG subclasses, IgA, IgM, IgE. Ad ICL - A trusted producer of high quality antibodies since 1977. icllab.com Learn more Before genetic engineering, insulin used for treatment was sourced from the pancreas of slaughtered pigs and cattle. This sour Continue reading >>

Biotechnology And Industry Microbiology Biopharmaceuticals From Microorganisms: From Production To Purification

Biotechnology And Industry Microbiology Biopharmaceuticals From Microorganisms: From Production To Purification

Introduction Biopharmaceuticals are mostly therapeutic recombinant proteins obtained by biotechnological processes. They are derived from biological sources such as organs and tissues, microorganisms, animal fluids, or genetically modified cells and organisms.1,2 Although several different expression systems may be employed including mammalian cell lines, insects, and plants, new technological advancements are continuously being made to improve microorganism production of biopharmaceuticals. This investment is justified by the well-characterized genomes, versatility of plasmid vectors, availability of different host strains, cost-effectiveness as compared with other expression systems.2,3 Bioprocessing is a crucial part of biotechnology. There is an anticipation that within the next 5 to 10 years, up to 50% of all drugs in development will be biopharmaceuticals. Examples include recombinant proteins obtained through microbial fermentation process.2,3 Bioprocessing for biopharmaceuticals production involves a wide range of techniques. In this review, we describe the main advances in microbial fermentation and purification process to obtain biopharmaceuticals. Biopharmaceuticals and the pharmaceutical industry Drug development is an extremely complex and expensive process. According to the Tufts Center for the Study of Drug Development4 (it may take approximately 15 years of intense research from the initial idea to the final product and development and costs usually exceed $2 billion. Low-molecular mass molecules are generically named as drugs while high-molecular mass drugs, which are represented by polymers of nucleotides (RNA or DNA) or amino acids (peptides and proteins), are called biopharmaceuticals.5 Biopharmaceuticals based in nucleic acids, such as small interfe Continue reading >>

Effects Of Temperature Shift Strategies On Human Preproinsulin Production In The Fed-batch Fermentation Of Recombinantescherichia Coli

Effects Of Temperature Shift Strategies On Human Preproinsulin Production In The Fed-batch Fermentation Of Recombinantescherichia Coli

Abstract Preproinsulin is a well-known precursor of human insulin for the regulation of blood glucose levels. In this study, fed-batch fermentations of recombinantEscherichia coli JM109/pPT-MRpi were carried out for the overexpression of human preproinsulin. The expression of human preproinsulin was controlled by the temperature inducibleP2 promoter. The time-course profiles of fed-batch fermentation and SDS-PAGE analysis showed that human insulin expression was triggered by a culture temperature change from 30 to 37°C. Fermentation shift strategies, including the multi-step increase of temperature and the modulation of initiation time, were optimized to obtain high titers of cell mass and preproinsulin. The optimized fed-batch fermentation, consisting of a three-step shift of culture temperature from 30 to 37°C for 2 h, gave the best results of 43.1 g/L of dry cell weight and 33.3% preproinsulin content, which corresponded to 2.0- and 1.2-fold increases, respectively, as compared to those of fed-batch culture at a constant temperature of 37°C. Continue reading >>

Insulin Manufacturing

Insulin Manufacturing

The Insulin manufacturing facility is Asia’s largest Insulin production facility which utilizes Pichia pastoris as the expression system. The plant has a total installed capacity of about 130,000 litres of fermentation volume and is customized to suit the production of Insulin and Insulin analogs. The Purification suite is equipped with new-generation purification equipment of Industrial scale with advanced levels of control. The Preparative scale chromatography skids, which are the heart of the purification process, are one of the largest in the country to be utilized for purification of therapeutic proteins. The facility is staffed with well-trained and skilled technical supervisors to manage the high-tech production operations. The facility has been exposed to regulatory inspections from EU, USFDA and regulatory agencies from emerging countries and complies with their regulatory specifications. Continue reading >>

Recombinant Dna Technology In The Synthesis Of Human Insulin

Recombinant Dna Technology In The Synthesis Of Human Insulin

Recombinant DNA Technology in the Synthesis of Human Insulin The nature and purpose of synthesising human insulin. Since Banting and Best discovered the hormone, insulin in 1921.(1) diabetic patients, whose elevated sugar levels (see fig. 1) are due to impaired insulin production, have been treated with insulin derived from the pancreas glands of abattoir animals. The hormone, produced and secreted by the beta cells of the pancreas' islets of Langerhans,(2) regulates the use and storage of food, particularly carbohydrates. Fig. 1 Fluctuations in diabetic person's blood glucose levels, compared with healthy individuals. Source: Hillson,R. - Diabetes: A beyond basics guide, pg.16. Although bovine and porcine insulin are similar to human insulin, their composition is slightly different. Consequently, a number of patients' immune systems produce antibodies against it, neutralising its actions and resulting in inflammatory responses at injection sites. Added to these adverse effects of bovine and porcine insulin, were fears of long term complications ensuing from the regular injection of a foreign substance,(3) as well as a projected decline in the production of animal derived insulin.(4) These factors led researchers to consider synthesising Humulin by inserting the insulin gene into a suitable vector, the E. coli bacterial cell, to produce an insulin that is chemically identical to its naturally produced counterpart. This has been achieved using Recombinant DNA technology. This method (see fig. 2) is a more reliable and sustainable(5) method than extracting and purifying the abattoir by-product. Fig. 2 An overview of the recombination process. Source: Novo - Nordisk promotional brochure,pg 6. Understanding the genetics involved. The structure of insulin. Chemically, insuli Continue reading >>

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