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Insulin Production By Fermentation

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 >>

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 >>

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 >>

How Insulin Is Made - Material, Manufacture, History, Used, Parts, Components, Structure, Steps, Product

How Insulin Is Made - Material, Manufacture, History, Used, Parts, Components, Structure, Steps, Product

Background Insulin is a hormone that regulates the amount of glucose (sugar) in the blood and is required for the body to function normally. Insulin is produced by cells in the pancreas, called the islets of Langerhans. These cells continuously release a small amount of insulin into the body, but they release surges of the hormone in response to a rise in the blood glucose level. Certain cells in the body change the food ingested into energy, or blood glucose, that cells can use. Every time a person eats, the blood glucose rises. Raised blood glucose triggers the cells in the islets of Langerhans to release the necessary amount of insulin. Insulin allows the blood glucose to be transported from the blood into the cells. Cells have an outer wall, called a membrane, that controls what enters and exits the cell. Researchers do not yet know exactly how insulin works, but they do know insulin binds to receptors on the cell's membrane. This activates a set of transport molecules so that glucose and proteins can enter the cell. The cells can then use the glucose as energy to carry out its functions. Once transported into the cell, the blood glucose level is returned to normal within hours. Without insulin, the blood glucose builds up in the blood and the cells are starved of their energy source. Some of the symptoms that may occur include fatigue, constant infections, blurred eye sight, numbness, tingling in the hands or legs, increased thirst, and slowed healing of bruises or cuts. The cells will begin to use fat, the energy source stored for emergencies. When this happens for too long a time the body produces ketones, chemicals produced by the liver. Ketones can poison and kill cells if they build up in the body over an extended period of time. This can lead to serious illne Continue reading >>

Fermentation Medias And Processes Thereof

Fermentation Medias And Processes Thereof

The present invention demonstrates the utility of carbonic acid amides such as urea or its derivatives, carbamates, carbodiimides & thiocarbamides as nitrogenous supplements in fermentation media for production of recombinant proteins to achieve enhanced bioconversion rates and peptides like insulin and insulin analogs, exendin and enzymes such as lipase using methanol inducible fungal expression systems such as Pichia. We claim: 1. A fermentation medium for production of a recombinant protein or a derivative or analog thereof or a secondary metabolite by improving phosphate uptake through fermentation using a microorganism, said medium characterized in that a residual concentration of urea, a derivative of urea, dimethylurea, diethylurea, N-acetylphenyl urea, isopropylpylideneurea, phenylurea or a combination thereof is maintained in a range of about 0.5 g/L to about 3 g/L, a methanol feeding rate range from about 6 g/L/h to about 20 g/L/h, basal salts per liter comprising: and 2. The fermentation medium according to claim 1, wherein the urea added in liquid, spray, powder or pellet form, and wherein the microorganism is selected from the group comprising E. coli, Streptomyces sp, Aspergillus sp, Rhizopus sp, Penillium sp, and Rhizomucor sp. 4. The fermentation medium according to claim 1, further comprising the recombinant protein, wherein the recombinant protein or a derivative or analog thereof yields a maximum product titre of above 0.5 g/L; and wherein the secondary metabolite effectuates bioconversion of compactin to pravastatin by at least 35%. 5. A process for production of a recombinant or non-recombinant protein product or a derivative or analog thereof or a secondary metabolite in the fermentation medium of claim 1, said process comprising steps of b) mainta 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 >>

How Did They Make Insulin From Recombinant Dna?

How Did They Make Insulin From Recombinant Dna?

Recombinant DNA is a technology scientists developed that made it possible to insert a human gene into the genetic material of a common bacterium. This “recombinant” micro-organism could now produce the protein encoded by the human gene. 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 >>

Journal Of Microbiology And Biotechnology

Journal Of Microbiology And Biotechnology

Hae-Gwang Hwang, Kwang-Jin Kim, Se-Hoon Lee, Chang-Kyu Kim, Cheol-Ki Min, Jung-Mi Yun, Su Ui Lee, Young-Jin Son Department of Pharmacy, Sunchon National University, Suncheon 57922, Republic of Korea Recombinant Glargine Insulin Production Process Using Escherichia coli J. Microbiol. Biotechnol.2016 ; Vol.26-10 Abstract Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide glargine insulin when transformed into Escherichia coli JM109. The transformed E. coli cells were cultured by fed-batch fermentation. The final dry cell mass was 18 g/l. The prepeptide glargine insulin was 38.52% of the total protein. It was expressed as an inclusion body and then refolded to recover the biological activity. To convert the prepeptide into glargine insulin, citraconylation and trypsin cleavage were performed. Using citraconylation, the yield of enzymatic conversion for glargine insulin increased by 3.2-fold compared with that without citraconylation. After the enzyme reaction, active glargine insulin was purified by two types of chromatography (ion-exchange chromatography and reverse-phase chromatography). We obtained recombinant human glargine insulin at 98.11% purity and verified that it is equal to the standard of human glargine insulin, based on High-performance liquid chromatography analysis and Matrixassisted laser desorption/ionization Time-of-Flight Mass Spectrometry. We thus established a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. This established process for recombinant human glargine insulin may be a model process for the production of other human insulin analogs. diabetes, glargine, fed 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 >>

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 >>

A Simplified And Efficient Process For Insulin

A Simplified And Efficient Process For Insulin

RESEARCH ARTICLE Production in Pichia pastoris Sulena Polez1, Domenico Origi2, Sotir Zahariev1, Corrado Guarnaccia1, Sergio G. Tisminetzky1, NatasÌŒa Skoko1*, Marco Baralle1* 1 ICGEB, Trieste, Italy, 2 Biomanufacturing Sciences Network, Process Solutions, Merck SpA, Vimodrone (Milan), Italy * [email protected] (NS); [email protected] (MB) 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 pre- cursor secreted during the methanol induction phase is recovered directly from the culture broth using Tangential Flow Filtration with a Prostakâ„¢module eliminating the laborious and time-consuming multi-step clarification, including centrifugation. In the second step the pro- tein 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. Introduction Insulin is a relatively low-priced drug however, the chronic nature of Diabetes means the cost for insulin treatment is high, and together with an increasing number of patients, this financial burden challenges healthcare systems worldw Continue reading >>

Industrial Microbiology

Industrial Microbiology

Industrial Microorganisms There are various types of microorganisms that are used for large-scale production of industrial items. Learning Objectives Describe how microorganisms are used in industry to manufacture food or products in large quantities Key Takeaways The ability of specific microorganisms to produce specialized enzymes and proteins has been exploited for many purposes in industry. Industrial microorganisms are used to produce many things, including food, cosmetics, pharmaceuticals and construction materials. Microorganisms can be genetically modified or engineered to aid in large-scale production. exopolysaccharide: a type of sugar-composed polymer secreted by a microorganism into the external environment archaea: a taxonomic domain of single-celled organisms lacking nuclei that are fundamentally from bacteria. Industrial microbiology includes the use of microorganisms to manufacture food or industrial products in large quantities. Numerous microorganisms are used within industrial microbiology; these include naturally occurring organisms, laboratory selected mutants, or even genetically modified organisms (GMOs). Currently, the debate in the use of genetically modified organisms (GMOs) in food sources is gaining both momentum, with more and more supporters on both sides. However, the use of microorganisms at an industrial level is deeply rooted into today’s society. The following is a brief overview of the various microorganisms that have industrial uses, and of the roles they play. Archaea are specific types of prokaryotic microbes that exhibit the ability to sustain populations in unusual and typically harsh environments. Those suriving in the most hostile and extreme settings are known as extremophile archaea. The isolation and identification of vari 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 >>

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 >>

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