Production Of Human Insulin Research Articles

Metabolic Effects of Growth Hormone on Protein and Lipids

The development of techniques for large scale production of human growth hormone (GH) and insulin like growth factor-I (IGF-I) by recombinant deoxyribonucleic acid (DNA) technology has led to a resurgence in interest in their metabolic actions and clinical applications. The metabolic actions of GH can be divided into direct effects on carbohydrate, fat and protein, and indirect actions mediated primarily through IGF-I and insulin. IGF-I and insulin increase in response to GH treatment and the metabolic effects on carbohydrate, fat and protein of each are similar but not identical. Although the primary regulator of IGF-I is GH, nutritional effects on IGF-I may predominate during conditions of extreme undernutrition or overnutrition. Obesity is associated with impaired GH secretion, and total IGF-I concentrations in blood are reduced, assuming energy balance is maintained. GH treatment is associated with reduced percent body fat and increased lean body mass, although treatment of obesity with GH has been relatively ineffective, if combined with nutrient restriction.The direct effects of GH on glucose and fat metabolism are intimately related. GH-stimulated lipolysis leads to release of free fatty acids and inhibition of insulin-stimulated glucose uptake by skeletal muscle. This effect, combined with stimulation of glycogenolysis and gluconeogenesis, leads to hyperglycemia. The principal protein metabolic action of GH is a rapid reduction in amino acid oxidation. Within hours of exposure to GH, whole body protein synthesis (WBPS) increases, followed by an increase in the rate of skeletal muscle protein synthesis. This effect is associated with an increase in blood flow to muscle tissue, likely mediated by IGF-I and/or insulin. In summary, the effects of GH on body composition reflect a complex interaction between GH and its metabolic fuels in the short term, and the metabolic actions of IGF-I and insulin on carbohydrate, fat, and protein subsequently.

Computer‐aided process analysis and economic evaluation for biosynthetic human insulin production—A case study

Human insulin was the first mammalian protein produced in bacteria using recombinant DNA technology. Two technologies were developed; the first based on the separate expression of precursors of chains A and B of insulin, and the second based on the expression of a precursor of proinsulin as a Trp-E fusion protein. Both technologies utilized Escherichia coli as an expression system. Later, a third technology was developed based on a strain of yeast that can secrete a precursor of insulin. The second E. coli process, a variation of which has been commercialized by Eli Lilly and Co., is analyzed in this article from a process design and economic evaluation viewpoint. The objective of this work is to elucidate the technical complexity and high cost associated with the manufacturing of biopharmaceuticals. Human insulin is a good example of a protein-based biopharmaceutical produced in large quantities (a fex tons per year) that requires large scale equipment and presents a multitude of scale-up challenges. Based onthe analysis, a number of conclusions are drawn regarding the cost breakdown and cost dependency on process parameters. Recommendations are made for cost reduction and environmental impact minimization. This analysis was performed using a software tool for computer-aided bioprocess design. (c) 1995 John Wiley & Sons, Inc.

Production of human insulin in an E. coli system with Met-Lys-human proinsulin as the expressed precursor.

The construction of a gene encoding Lys-human proinsulin, its direct expression in E. coli, and the simple purification procedure are described here. The temperature inducible promotor was employed for induction in a very short time. The expression level could reach 20-30%. After simple downstream processing and only one step of Sephadex G50 purification, 150 mg recombinant Lys-human proinsulin with a purity of up to 90% could be obtained easily from 1 L of high density fermentation medium. The obtained product is in the form of Met-Lys-human proinsulin because of the failure of the bacterial host to remove the initiator methionine residue. The Lys-human proinsulin could be changed into human insulin by trypsin and carboxypeptidase B treatment in later steps. After separation with DEAE-Sephadex A25, human insulin with expected amino acid composition and full native biological activity could be obtained with a yield of 50 mg/L of fermentation medium.

Recombinant human insulin: III. High-performance liquid chromatography and high-performance capillary electrophoresis control in the analysis of step-by-step production of recombinant human insulin

The production of recombinant human insulin consists of five main stages, accompanied by considerable transformation of molecules, concerning size, secondary structure and the presence of charged groups. The application of different methods, i.e. size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE) (capillary zone electrophoresis and micellar electrokinetic capillary chromatography, to the analysis of insulin, insulin-related and non-insulin-related substances was studied. A combined HPLC-HPCE system for the step-by-step control of recombinant human insulin production technology is suggested. The advantages and shortcomings of these methods are discussed.

Research, development, production, and safety of biosynthetic human insulin.

This paper provides some historical aspects on the research and development of Humulin (rDNA origin), the first human health-care product derived from rDNA technology more than a decade ago. Also referred to as biosynthetic human insulin, Humulin is currently produced via the human proinsulin route, using an Escherichia coli fermentation process. The authenticity, high purity, and safety of BHI has been investigated and verified by a complex battery of analytical and physicochemical methods. The daily treatment of more than two million diabetic patients worldwide with this rDNA human insulin not only demonstrates the value of rDNA technology in providing an important medical product, it is assurance that diabetic patients will have unlimited supplies of this vital hormone as well as potential analogue refinements.

Recombinant human insulin - II. Size-exclusion HPLC of biotechnological precursors. Factors influencing on retention and selectivity

Abstract

Stability and sterility of biosynthetic human insulin stored in plastic insulin syringes for 28 days

The stability and sterility of biosynthetic human insulin products stored at refrigerator and room temperatures in two types of plastic syringes and the stability of preservatives in the products were studied. Four types of biosynthetic human insulin were used: regular, isophane, combination, and extemporaneously prepared combination. Samples (0.4 mL) were withdrawn from multiple-dose vials into 39 polypropylene and 39 propylene-ethylene copolymer syringes. Three syringes of each type were analyzed immediately; the remaining syringes were stored in plastic bags, half at room temperature (23 degrees C) and half in the refrigerator (4 degrees C). A vial of each type of insulin was maintained under similar conditions. At days 1, 3, 7, 14, 21, and 28, samples from each syringe were analyzed by high-performance liquid chromatography for insulin potency and m-cresol and phenol concentrations. Samples of each product were also tested for sterility after 1, 2, and 4 weeks of storage at 4 degrees C and 23 degrees C. The potency of insulin in each of the biosynthetic human insulin products did not change significantly during the 28-day study in both types of plastic syringes and at both temperature settings. m-Cresol concentrations decreased in all samples; greater decreases occurred in samples stored at room temperature and in samples stored in polypropylene syringes. Phenol concentrations were less affected than m-cresol concentrations; greater decreases occurred in samples stored at room temperature. No significant decreases in insulin potency or m-cresol or phenol concentrations occurred in control samples stored in vials kept under similar conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction mechanism of trypsin-catalysed semisynthesis of human insulin studied by fast atom bombardment mass spectrometry

The production of semisynthetic human insulin for therapeutic purposes is of considerable importance. During trypsin-catalysed transformation of pig insulin into an ester of insulin of human sequence, the alanyl residue at position B30 is removed and replaced with an esterified residue of threonine. We have carried out this transformation in a medium enriched in 18OH2 and studied the product by MS. In contrast to a previous report, we find that incorporation of label into the B29 - B30 peptide bond occurs during the transformation with threonine methyl ester in aqueous N,N-dimethylacetamide. Quantitative data are presented and the implications of these findings are discussed.

紙パルプ分野で使われるバイオテクノロジーの用語について （ＩＩ） 遺伝子操作の方法と実際

Methods and practice of gene manupulation technique were explained for the production of human insulin. The DNA synthesizing and DNA sequencing were outlined with a few examples. Furthermore, peculiarity in studying gene technology was also described.

Enzymatic semisynthesis of human insulin: An update

Peptide bond formation can be enzymatically catalysed by the reverse reaction of proteases. Application is seen in the industrial production of human insulin. Human insulin derivative can be enzymatically prepared using either porcine insulin or the single chain B(1-29)-A(1-21) insulin precursor as the starting material. This is accomplished by either (1) digesting the starting material at Lys29 with Achromobacter lyticus protease I (Ach) and then coupling with Thr-X (X = blocking residue) (two-step reaction) or (2) subjecting Ala-B30 of porcine insulin or Gly-A1 of the single chain insulin precursor to transpeptidation with Thr-X (one-step reaction). Trypsin and Ach can be used for either type of reaction and, in the immobilized form, for the two-step reaction. Since the single chain insulin precursor can be produced by gene technology (yeast), use of immobilized trypsin or Ach and the two-step reaction using the single chain insulin precursor as the starting material ensures the continuous production of human insulin making it a feasible method for industrial manufacture.

16] Use of immobilized Achromobacter protease I for semisynthesis of human insulin

This chapter explains the immobilization of Achromobacter protease I as well as the use of the immobilized enzyme in the enzyme-assisted semisynthesis of human insulin. The protected insulin derivative obtained from the enzyme-catalyzed reactions is subsequently deblocked in a one-step chemical procedure, which yields human insulin. For large-scale production, Achromobacter protease I seems to be the ideal enzyme to use in enzyme-assisted semisynthesis of human insulin. The enzyme exhibits several characteristics that make it suitable to use for synthetic purposes. For example, the rate of condensation of 30 B -dealanine-porcine insulin (DAI) to L-threonine tert-butyl ester catalyzed by Achromobacter protease I is at least 10 times higher than that observed for the trypsin-catalyzed condensation reaction. The experiments discussed in the chapter are helpful in solving some of the principal problems in the semisynthesis of human insulin from porcine insulin. Although they were done on a semipreparative scale, the obtained results indicate that application of immobilized Achromobacter protease I in the semisynthesis of human insulin has great potential as a tool for the large-scale production of human insulin.

Diabetes I: insulins.

Sixty years after the first therapeutic use of insulin treatment of insulin dependent diabetes continues to be with subcutaneous insulin injections. Nevertheless, this does not imply lack of progress. Recent years have seen important developments in insulin purification, the commercial production of human insulin, the development of new techniques of insulin adminis? tration, and the growing acceptance that good control reduces diabetic complications. The present change to U100 (100 units/ ml) insulin has resulted in important changes in the range of insulins marketed?notably, only highly purified insulins will be available in the new strength.

Human Insulin

Recently an improved process for the production of human insulin from porcine insulin has been described. After being characterized, this insulin has been tested and shown to be safe and effective in diabetics in a short-term, double-blind, cross-over study. The availability of sufficient quantities of this human insulin now permits short- and long-term clinical investigations of the possible benefits of human insulin in the treatment of diabetics.

Bacterial production of human insulin.

Bacterial production of human insulin.

Insulin resistance: Definition and treatment

Two cases of insulin resistance are described, and recent developments in the pathogenesis and treatment of insulin resistance are reviewed. Both immune and nonimmune types of insulin resistance have been described. Immune resistance is related to the presence of circulating antibodies directed against exogenous insulin or the insulin receptor sites. Nonimmune resistance is associated with obesity, ketoacidosis, infection, or endocrinopathies. Treatment of insulin-resistant diabetics can include proper diet and weight control; use of insulin in large quantities; selection of less antigenic forms of insulin, such as pork, fish, or sulfated insulin; oral hypoglycemics such as tolbutamide; and immunosuppressive therapy with corticosteroids. The production of human insulin by recombinant DNA technology promises benefits to patients with high levels of antibodies directed against insulin from animal sources. True insulin resistance is a rare phenomenon, which must be documented adequately before vigorous treatment is considered.

Bacteria directed to produce insulin in test application of genetic code

Putting the genetic code to the test, California researchers have synthesized genes that "instruct" an enfeebled laboratory strain of<i>Escherichia coli</i>(EK-1) to produce the two amino acid chains of insulin. When these peptide chains are separated from the enzyme to which they are attached, purified, and mixed together, the result is a chemical structure identical to human insulin. Making the announcement are scientists from City of Hope National Medical Center at Duarte, Calif, near Los Angeles, and the research and development laboratories of Genentech, Inc, in South San Francisco. Genentech funded the work. A long-range development program now is beginning, "aimed at the eventual commercial production of human insulin" by this means. Because the precise sequence of amino acids in insulin is known, the researchers said, they were able to select nucleotides that code for each of these amino acids and "design the necessary genes on paper." Then, using

Bacteria directed to produce insulin in test application of genetic code

Putting the genetic code to the test, California researchers have synthesized genes that "instruct" an enfeebled laboratory strain of<i>Escherichia coli</i>(EK-1) to produce the two amino acid chains of insulin. When these peptide chains are separated from the enzyme to which they are attached, purified, and mixed together, the result is a chemical structure identical to human insulin. Making the announcement are scientists from City of Hope National Medical Center at Duarte, Calif, near Los Angeles, and the research and development laboratories of Genentech, Inc, in South San Francisco. Genentech funded the work. A long-range development program now is beginning, "aimed at the eventual commercial production of human insulin" by this means. Because the precise sequence of amino acids in insulin is known, the researchers said, they were able to select nucleotides that code for each of these amino acids and "design the necessary genes on paper." Then, using