Medical Biotechnology Lecture Notes PDF

Medical biotechnology  Biopharmaceuticals are protein- based and may either be derived from genetically altered bacteria or fungi (also called biotech. drugs), Examples (Biopharmaceuticals) recombinant human insulin, human growth hormone and other products.  Firstly: The use of recom...

Medical biotechnology  Biopharmaceuticals are protein- based and may either be derived from genetically altered bacteria or fungi (also called biotech. drugs), Examples (Biopharmaceuticals) recombinant human insulin, human growth hormone and other products.  Firstly: The use of recombinant DNA technology to modify Escherichia coli bacteria to produce human insulin. The development of human insulin demonstrates the viability of using recombinant DNA technology to produce products with practical application. Human insulin (humulin) is the first therapeutic product produced by recombinant DNA technology. Insulin The hormone insulin is produced by the (J-cells of islets of Langerhans of pancreas. Human insulin contains 51 amino acids, arranged in two polypeptide chains. The chain A has 21 amino acids while B has 30 amino acids. Both are held together by disulfide bonds Diabetes mellitus: Diabetes mellitus affects about 2-3% of the general population. It is a genetically linked disease characterized by increased blood glucose concentration (hyperglycemia). The occurrence of diabetes is due to insufficient or inefficient (incompetent) insulin. Insulin facilitates the cellular uptake and utilization of glucose for the release of energy. In the absence of insulin, glucose accumulates in the blood stream at higher concentration, usually when the blood glucose concentration exceeds about 180 mg/dl, glucose is excreted into urine. The more serious complications of uncontrolled diabetes include kidney damage (nephropathy), eye damage (retinopathy), nerve diseases (neuropathy) and circulatory diseases (atherosclerosis).  Problems of insulin 1- In the early years, insulin isolated and purified from the pancreases of pigs and cows was used for the treatment of diabetics. There is a slight difference (by one to three amino acids) in the structure of animal insulin compared to human insulin. This resulted in allergy in some of the diabetics when animal insulin was administered. 2- Another problem with animal insulin is that large numbers of animals have to be sacrificed for extracting insulin from their pancreases. For instance, about 70 pigs (giving about 5 kg pancreatic tissue) have to be killed to get insulin for treating a single diabetic patient just for one year.  Raw Materials Raw materials are the most basic components to produce a product. In this section, some of the typical crude raw materials that lead to the industrial production of synthetic insulin will be discussed A- Raw materials used for preparation of recombinant plasmid 1-Desired Gene The human insulin gene isolated from the human DNA. However, often, mRNA encodes for insulin is used. Because the B cells of the pancreas make insulin, so make lots of mRNA molecules coding for insulin. This mRNA can be isolated from these cells and used to make cDNA of the insulin gene.  2- Vector Plasmid is the most preferred vector if bacterial host cells are used. The general features of plasmid in insulin production include 1- It usually replicates independently of bacterial chromosome to produce higher yield of products 2- Permits the reproduction of a foreign DNA by using the bacterial replicating system 3- Usually contains selective markers to eliminate undesired cells 4- Suitable to accommodate the size of the insulin gene It’s important that the vector you clone it into is an ‘expression vector’ – that is, a vector that includes a bacterial promoter sequence in front of your gene of interest. By including a bacterial promoter, you are giving the bacterium instructions to make a protein from your gene of interest – essentially, you are ‘tricking’ bacteria into producing a foreign protein  3- Specific enzymes Reverse transcriptase - synthesizes cDNA from the mRNA template that is required for the insertion into the vector Specific restriction enzymes – to cut DNA at specific sites, such as on the vector for the insertion of the desired insulin gene. Specific ligases – to join DNA fragments together after the insulin gene has been inserted into the vector so that the gene can be expressed in a host cell  B- Raw Materials used for the fermentation process 1- The host organism The bacterium Escherichia coli are commonly used as the host organism to produce the synthetic insulin. This is due to the fact that bacterial cells cannot do post-translational modification. After translation, post- translational conversion to insulin was carried out chemically. By contrast, yeast, as a eukaryote, is capable of post-translational modification, so this simplifies the production of human insulin.  2-Media LB Medium is used for the production. It is used as both seed-culture and the fermentation media, especially for the cultivation of Escherichia coli. The recipe of LB contains: 1) Bacto-tryptone, 2) Yeast extract, 3) Sodium chloride, 4) dH2O and 5) pH of 7.5. Other than the LB components, 2 more components – ampicillin and lactose are present in the media. These two unique components convert the medium into an enrichment liquid culture such that only the desired bacteria that contain the human insulin gene can grow in this type of media.  There are two methods of producing the insulin by genetic recombination: Method A: The 'A' and 'B' Insulin Chains (generating the chains individually and chemically combining them after) Method B: The Pro-insulin Method (creating a single-chain precursor, huma proinsulin, and cleave out a 35-amino acid peptide that joins the two chains) Obtaining of human insulin gene. In E. coli, β-galactosidase is the enzyme that controls the transcription of the genes.. The insulin gene and b-galactosidase gene are separated by a triplet codon for methionine. The cut plasmids are re-ligated by specific DNA ligases Media and equipment preparation The LB broth is prepared using the LB powder. It is autoclaved and ampicillin and lactose are added (after the sterilization to prevent denaturation or destruction). Inoculation is done by adding the transformed bacteria into the media In the Bioreactor (fermentation process) The fermentation broth contains two unique components - an antibiotic known as ampicillin and lactose. Bacterial cells that have sucessful transformation will contain the ampicillin resistance gene and the lac Z gene which encodes for β-galactosidase in the presence of lactose.  These cells therefore can grow in the ampicillin environment and the transcription of the lac Z gene will in turn result in the transcription of the human insulin chain DNA. Bacterial cells that have failed the transformation do not contain the ampicillin resistance gene. As a result, the growth of these cells will be suppressed by ampicillin and will not replicate during the fermentation process. Moving on to the large scale, where transfected bacterial cells are transferred from the small flask and replicated under optimal conditions such as temperature, pH in fermentation tanks. This step involves process monitoring and control. The bacterial cell processes turn on the gene for human insulin chains and then insulin chains are produced in the cell.  (3) Downstream Processing Isolation of crude products Cells are removed from tanks and are lysed using different methods such as enzyme digestion, freezing and thawing and sonication. For enzyme digestion, lysosome enzyme is used to digest the outer layer of the bacterial cells and detergent mixture is subsequently added to separate the cell wall membrane.  Purification of crude product Centrifugation is conducted to help separate the cell components from the products. Stringent purification of the recombinant insulin chains must be taken to remove any impurities. This uses several chromatographic methods such as gel filtration and ion-exchange, along with additional steps which exploit differences in hydrophobicity. Obtaining of insulin chains The proteins isolated after lysis consists of the fusion of β-galactosidase and insulin chains due to the fact that there is no termination or disruption to the synthesis of these two proteins as the genes are linked together. Therefore, cyanogen bromide is used to split the protein chains at methionine residues, allowing the insulin chains to be obtained. Synthesis of active insulin Two chains (A and B) forms disulfide bonds using sodium dithionate and sodium sulphite, and the chains are joint through a reaction known as reduction-reoxidation under beta-mercaptoethanol and air oxidation, resulting in Humulin - synthetic human insulin.  PR-HPLC to obtain highly purified insulin Reverse-phase high performance liquid chromatography (PR-HPLC) is performed lastly to remove almost all the impurities, to produce highly purified insulin. The insulin then can be polished and packaged to be sold in the industires. The Proinsulin Process: another method to synthesize human insulin using the direct precursor to the insulin gene, proinsulin The proinsulin coding sequence is inserted into the non-pathogenic E. coli bacteria and the bacteria undergo fermentation where they replicate and produce proinsulin. The connecting sequence between the A and B chains is then spliced away with an enzyme and the resulting insulin is purified. A different downstream process is required for the Proinsulin process as compared to the Chain A and Chain B process. At the end of the both manufacturing processes, ingredients are added to insulin to prevent bacteria growth and maintain a neutral pH balance. Towards the end of the processes the ingredients to produce the desired duration type of insulin are also added. An example is adding zinc oxide to produce longer acting insulin.  Human Growth Hormone: Growth hormone is produced by the pituitary gland. It regulates the growth and development. Growth hormone stimulates overall body growth by increasing the cellular uptake of amino acids, and protein synthesis Insufficient human growth hormone (hGH) in young children results in retarded growth, clinically referred to as pituitary dwarfism Traditional treatment for dwarfism: The children of pituitary dwarfism were treated with regular injections of growth hormone extracted from the brains of deceased humans. It may be noted that only human growth hormone is effective for treatment of dwarfism. (This is in contrast to diabetes where animal insulin’s are employed Limitation in hGH production: The hGH is a protein comprised of 191 amino acids. During the course of its natural synthesis in the body, hGH is tagged with a single peptide (with 26 amino acids). The signal peptide is removed during secretion to release the active hGH for biological functions. The entire process of hGH synthesis goes on in an orderly fashion in the body. However, signal peptide interrupts hGH production by recombinant technology. The complementary DNA (cDNA) synthesized from the mRNA encoding hGH is inserted into the plasmid. The plasmid containing E. coli when cultured, produces full length hGH along with signal peptide. But E. coli cannot remove the signal peptide.  Further, it is also quite difficult to get rid of signal peptide by various other means. Unfortunately, there is no restriction endonuclease to do this job, hence this is not possible. A novel method for hGH production: Bio¬technologists have resolved the problem of signal peptide interruption by a novel metod The base sequence in cDNA encoding signal peptide (26 amino acids) plus the neighbouring 24 amino acids (i.e a. total 50 amino acids) is cut by restriction endonuclease ECoRI.  Now a gene (cDNA) for 24 amino acid sequence of hGH (that has been deleted) is freshly synthesized and ligated to the remaining hGH cDNA. The so constituted cDNA, attached to a vector, is inserted into a bacterium such as E. coli for culture and production of hGH. In this manner, the biologically functional hGH can be produced by DNA technology.