Insulin and Erythropoietin Production

Insulin and Erythropoietin ProductionInsulin is a protein (polypeptide) discovered in 1921 by Banting with the pancreas being the site of its proceeds. It is do up of 51 amino acids, divided into 2 set up A and B, bonded by disulfide linkages. Chain A is do up of 21 amino acids with an intra-disulphide linkage, while chain B is made up of 30 amino acids (4).Why Insulin?Insulin is important in glucose metabolism, and is being utilize for the compensatement of Diabetes mellitus a metabolic disorder of glucose in the body. Initially, Insulin from animals was white plagued to treat this disorder however nowadays synthesized humanity Insulin is being used, this is because it is fast absorbed by the body, it has less allergic reactions, it contains less impurities, and it leavens good results (3).Recombinant process of producing InsulinSynthetic Insulin was basic produced in 1983 done genetic engine room, which involve extraction of the human DNA (1), once extracted, the gene for Insulin is isolated, and enzymes atomic number 18 used to expurgate it. The gene is then cut apply enzymes and put into the plasmid of a vector, where in most cases E. coli plasmid is used. Since Insulin contains two chains, two pieces of DNA argon extracted, and the genes for the two chains argon coupled to galactosidase enzyme of the bacteria. The plasmids formed are then inserted into a host electric cell E. coli and sealed using another enzyme called ligase. And the host on replicating produces the enzymes each containing one of the two chains each. Production is fol secondaryed by extracting and purifying the chains which are mixed in a reaction to reconstitute the disulphide bridges (1).ESCHERICHIA COLI AS RECOMBINANT INSULIN HOSTEntero-bacillus, gram-negative E. coli is to the advancedest degree 1 2m, it gutter survive in the presence/absence of oxygen, and it excessively grows in an optimum pH and temperature of 7.0 and 37oC respectively. It utilizes glucose a s its major carbon descent and post too use other carbon sources like pyruvate, glycerol, acetate, and other sugars. K-12 and B strains are mostly used in the laboratory (20)Reasons for choosing E. coliGenetic Engineering technologies were developed using E. coli as a role existence, and so, the genetics of E. coli are well known among other microorganisms, as such its the most used organism for the crossroadion of different proteins (14).Moreover E. coli has a well known safety and yield abilities, stable plasmid, controllable promoter, cheaper and easily assimilationd (6), E. coli also has fast harvest-home rate, its easy to handle, and has well known fermentation skills and the ability to produce last protein content (14). That is why most of the proteins licensed recently by FDA and EMEA, were produced in E. coli (5).With these, and the circumstance that Insulin is a simple polypeptide (protein) which does not require glycosylation for its bio activeness and stability, E . coli carrying the plasmids for production of insulin leave alone be used as the host for the production of InsulinStrain and plasmids BL21 strain containing the pMYW-A and pMYW-B plasmids and temperature repressor -c1857, impart be used for insulin production (21).Growth strategyThe various growth strategies that entrust be used to grow E. coli in order to make it keen and produce the desired product (11) includeMedium E. coli needs nutrients like carbon, nitrogen and others so a carbon source glycerol will be provided since its cheaper and more soluble than glucose (12), a source of nitrogen in the form of ammonium sulphate will also be provided. However such nutrients in large quantities contribute inhibit the growth of E. coli, as such a defined average that contain optimum concentrations 20gl-1 glycerol and 2gl-1 ammonium sulphate will be used (11). The medium will also consist of the following 3gl-1 KH2PO4, 1gl-1 MgSO4.7H2O, 0.8gl-1 citrate, and 6gl-1 K2HPO4 (23). Some trace elements will also be added to the medium. (23)Process and culture-strategies E. coli will be grown submerged in a sterile controlled stirred tank reactor, and fed- grasp will be used as the growth strategy so as to negate accumulation of acetate which can be inhibits its growth, and reduce the production of the insulin (18). The growth strategy will be divided into two initially batch mode will be used to initiate growth, after which the fed-batch exponential ply will be used to produce the insulin (21).After adapting the medium and alimentation method, oxygen transfer rates (OTRs) had to be increased through a suitable bioreactor design and over-head pressure (16). Large scale reactors usually reach high ORTs using air and normal aeration pressure, and so the oxygen partial pressure (pO2) will be increased by adding pure oxygen to the air-stream entering the reactor, thus increasing its oxygen transfer rates (16) DO will be maintained at 40% of air saturation and aeration rate at 1vvm. effervescing arising overdue to large number of cells and high aeration-rates will be understand by use of impellers for stirring simultaneously at 300rpm and the use of antifoam (ucolub N115) (16, 21). The process temperature and pH will be maintained at 30oC and 6.8 respectively so as to avoid partial proteolysis of the insulin protein.Bioreactor Design Bioreactor vessel is usually cylindrical and made up of stainless steel. It is composed of impeller for stirring, Air sparger is placed at the bottom of the vessel for introduction of air, it has close to inlets for introduction of acid/alkali for pH control and also for introduction of antifoams, nutrients and inoculum It is also has pH, DO and temperature probes for sensing (22), Microbial activity during fermentation usually produces heat, so the bioreactor design must throw in for removal of heat, and this can be achieved by cooling with jackets and coils (16) Bioreactors must also be designed in a way that it can withstand high temperature and pressure and to allow cleaning-up and sterilizing (22).Growth analysisTemperature, pH, DO, foam, partial oxygen and carbon dioxide pressures, will be analysed on-line, other parameters like biomass, will be analysed by using optical compactness (OD600) and dry cell weight (offline). Cell viability will be analysed by using flow cytometry, the concentrations of substrates and metabolites by enzymatic methods while insulin will be analysed using electrophoresis methods like SDS-PAGE, and ELISA, while its purity will be check up ond by HPLC (8).Limitations/ProblemsThere are several problems that may arise during processing and can limit the use of this organism for Insulin production, these arePoor secretion because of the structure of its membrane (and tough cell wall), small amount of foldases, chaperones and increased concentrations of proteases, leading to low productivity (7).Solutions to this problem include all measures taken to increase lineament of secretion and production such asUse of secretion administrations like the system of -haemolysin (7)co-expression after co-cloning of foldases and chaperones (13)Improving the rates of gene-expression and using proteases deficient mutants like BL21 (18).use of E. coli mutants that are deficient of cell-wall (12)Limited post translational-modifications including disulfide-linkage formation, which is important for the insulin stability and biological activity (9).Solutions to this problem includeProduction of insulin with altered amino acid sequences through genetic engineering (9)Using E. coli mutants to enhance the formation of disulfide linkages e.g. Origami (15)iii. Exporting proteins into the periplasm which has disulphide bonding mechanisms (19).Codon biases due to large quantities of exact transfer-RNAs found in E. coli, the codons in the human-genes are often different from those that are found in this organism. This results in in good expression of some of these rare codons by the organism resulting in an unexpected protein synthesis termination or wrong incorporation of the amino acids (12).This problem can be solved by replacing codons that are rare in the desired gene by codons that are often found in the E. coli and by co-expressing the rare transfer-RNAs (15).ethanoate is usually formed as a by-product, and is inhibitory to growth of the cells (20).Solution is by using a fed-batch feeding method and by limiting DO level (11).Another problem is that large proteins are often obtained in an insoluble form (5) forming aggregates called inclusion bodies IBs (20).This can be solved by qualifying of temperature, increasing the strength of the promoter, adjusting the number of plasmids, concentrations of the inducer, and the composition of the media (9).Erythropoietin EPOEPO is a glycoprotein that is produced in the renal cortex of the kidney (10, 11). It has also being shown to be present in the humor, spleen, liver and the lungs (7, 17). I t is made up of 165 amino acids of ab come forward 18kDa (25), with a number of carbohydrates linked to the polypeptide through O and N glycosidic-bonds giving the glycoprotein a total weight of 34kDa.Two disulphide linkages kick in the molecule together (15) and the carbohydrates are responsible for the stability of the glycoprotein in-vivo,and increasing its half-life in the body (24).Why EPO?EPO functions to regulate the amount of red blood cells (RBC) in the blood by controlling the proliferation and differentiation of its immature cells to mature cells (1, 2, 22,). It is also involved in the growth and formation of blood vessels, and healing of wounds (6), it functions in the brain is not clear, but studies showed the glycoprotein to have some protective effects (18). Because of these functions EPO has being used in the treatment of anaemia caused by kidney failure and other causes (25).Recombinant production of EPODespite its importance, EPO in body is found in genuinely sma ll amounts and mostly in the urine (4), as such there is the requirement to produce EPO in large amounts, this leads to the work of isolating the glycoprotein from the urine (12, 21), and was used to identify its amino acid sequences, and synthesis of its DNA (9, 12), furthermore the human erythropoietin genes were cloned by Lin et al. (17), and so recombinant human EPO (rhuEPO) was produced in 1985 using CHO cells (14, 16).Chinese -Hamster- Ovary (CHO-Cells) as rhuEPO hostThese are epithelial cells derived from the ovary of Chinese hamster (a mammal). They grow well in culture and looks like cobble stones. The cells usually attach to a surface available but can be grown in suspension (20).CHO cells are grown best at 37oC and at pH 7.4 they are cultured in a suitable complex medium which can support their growth for many generations (20).CHO cell lines are now available from cell culture collections like the American type culture collection ATCC. Moreover human EPO expression plasm ids are now also commercially available, and are usually used for production of EPO using the CHO cells (27).Reasons for choosing CHO-cellsKarthik et al. (13) showed that CHO-cells are being used extensively in the industries for the production of many proteins, because they have demonstrated, to let some qualities likeThey can modify biological products post-translationally Proteins produce in CHO-cells have high glycosylation quality making them compatible and stable (13)Safety of the product Studies in 1989 have shown that most viruses do not multiply in CHO-cells (13)Ability to adapt easily and be grown in suspension (13).Products can now be purified to contain less contaminant (13).CHO cells have being used for a long time as such much entropy has being accumulated for regulatory reasons (13).They are easy to manipulate genetically (13).The isolation of cells deficient in Dihydrofolate-reductase enzymes leads to stable clones selection and genes amplification to increase prod uction (13).With all these, and the fact that EPO is a glycoprotein that requires glycosylation for its stability and activity, recombinant CHO cells are chosen to produce EPO.Cell lines and plasmids Cell lines which have the capability of glycosylating proteins (Pro-5), harboring the pGEX-HET-puro expression plasmid, will be used to produce the recombinant human erythropoietin (27).Growth strategyMedium Complex culture medium will be provided withGlucose as a source of carbon and energy, aminic acids as source of nitrogen,Salts will be included to make the solution isotonicVitamins and hormones will be added as co-factorsSerum is usually added to the culture medium to enhance the growth of the cell (20), but has the following disadvantagesIt chemicals are not defined and can cause cell growth inconsistency between batches (20)It is very expensive (20)The serum may contain proteins which can be difficult to separate and purify from the proteins secreted by the cells during downstrea m processing (20)It increases foaming and can be a source of contamination by viruses. (20)Therefore a serum-free (SF) media (16) will be used for the growth of the E. coli.Process and culture-strategies The cells will be grown aider on micro-carriers in a sterile controlled packed bed reactor, and perfusion method of production where some amounts of the medium is removed and replaced by fresh one and the cells are grown slowly will be used (28) because it was found to improve the glycosylation of the proteins more than fed-batch where there is fast growth of cells, (8). Before, many processes were run in a simple batch method, but nowadays, Perfusion or fed-batch methods are mostly employed and higher products are now realized (22). The production will be carried out in two stages the growth stage and the production stage. Normally stirring will be kept at 100 to 150 rpm, foaming will be avoided by adding Pluronic F68 (16).Temperature will be maintained at 37oC initially during gr owth and then reduced to 33oC during production, as was shown to increase the overall protein production, while maintaining the quality of the glycoprotein (3, 26). pH will be kept at 7.1 initially and then reduced to 6.8 (8, 26), by passing CO2 gas to the culture or by addition of concentrated sodium-bicarbonate solution in low quantities, because CO2 is also toxic to the cells and can also affect the production of EPO (20). In order to avoid the depletion of oxygen, the oxygen transfer rates (OTRs) will be increased above its utilization rate, with a constant supply of pure oxygen and air, while DO will be maintained at 20-50% of air saturation (20).Bioreactor DesignSince the cells are big and fragile, the design of the bioreactor has to be considered. Mammalian cell culture bioreactors are designed with bottoms that are round and are usually made up of glass/stainless steel (20). The impellers are usually marine or fling blade types fitted at the end of mechanical drives shafts so that both vertical and horizontal mixing are allowed at low stirring-rates (20). Temperature is controlled through coiled pipes or open ended fermenter jacket (20). pH, DO and temperature probes are used for sensing and have both air inlet and outlet for respiration.Growth AnalysisTemperature, pH and DO will be monitored on-line, because cells are immobilized, biomass formed cannot be measured directly therefore it will be monitored by measuring rate of glucose consumed day-after-day and the rate of lactate produced (28) Cell viability by flow cytometry, Glucose, glutamine, and lactate concentrations will be analysed using multi-parameter Bio-analytical system (26) while ammonia formed as fluff product of amino acid metabolism, will be analysed by colorimetric assay and by the use of detection-kit (26). EPO formed will be analysed using HPLC to determine its purity and its quality by Isoelectric focusing, SDS, and Bradford assay (26). The activity of EPO will be analysed by bio assay and by the use of protein assay-kit (27)Limitations/Problems.There are many limitations associated with CHO cells culture processes and they includeThey are fragile and highly sensitive to shear stress caused by agitation and bubble because the cells are large and have only cell membrane (20).This is usually solved using a suitable bioreactor-design and use of Pluronic F68 (20).They need a complex medium including serum which can cause problems in the downstream processing and is expensive (20).Solution to this is by using serum- free media (24, 25).Low yield of proteins have been produced from these cells, the productivity using the microbes being higher than the use of these cells. They also have slow growth rates (13).The problem of low productivity and slow growth rates can be solved through selecting cell lines that are better and optimizing cultural-strategies.Ammonia and lactate are generated during growth and can inhibit growth and also affect glycosylation (8).Solutio n is by optimizing the strategies of feeding and by monitoring (8).Glycosylation differences may arise from the EPO produced in the CHO-cells and the human EPO as seen in the way the two are sialylated terminally, as a result that the CHO-cells are not able to express an enzyme called alpha-2,6, sialyltransferase (27).Solution is by the use of CHO-cells harboring alpha-2, 6, sialyltransferase-cDNA expression-cassettes (27).REFERENCES1. Alcamo, I., DNA Technology the Awesome-Skill. Farming-dale. New York Academic Press. (2001).2. Banting Grolier Electronic publishing www.littletree.com.au/dna.htm accessed on 30/12/20103. 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