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Purification of recombinant human erythropoietin
Transcript of Purification of recombinant human erythropoietin
3. To promote an appropriate and suitable
environment for persons at work
4. To enable previous legislation to be replaced
by regulations and approved industry codes
of practice operating in combination with
the OSH Act 1994 Zahra Mohamed
Nur Farah Adibah Introduction The erythropoietin (Epo) is a hormone that sustains red blood cell mass by stimulates the rate of production of erythrocytic progenitor.
Distributing EPO start primarily from fibroblasts in the kidneys
EPO is mainly circulated by a liver cell meanwhile fetal state.
After birth, tiny blood vessel fibroblasts in the renal cortex become the main production site. The technique consist of using moderately filtering erythropoietin by transposing stage of high performance liquid chromatography to gain harmonized erythropoietin having a molecular weight about 34000 dalton on sodium dodecyl sulfate (SDS) and affecting a single peakon invert period HPLC (High-performance liquid chromatography). Scale up- bioreactor requirements The CHO cell line 6C2 is refined in fed batch line in 150 L bioreactor. In bioreactor cell culture medium is used containing of an enlightened amino acid postscript 50:50 DMEM/Hams F12 and consisting a 0.325% of a plant peptide,0.1% lutrol,6.4 g/L glucose,2.5 g/L NaHCO3,ethanolamine,ferric-citrate.ascorbic acid and sodium selenite and 0.6 g/L phosphate.
The cells are seeded about 5x105 cell/mL in 1250 m/L medium.
The PO2 is locate to 50% air dispersion, temperature to370 C the PH is set to 7.1 at the beginning. Throughout the path of the agitation it is decrease step wise to 6.9 Downstream processing Impurities present in the largest amounts should be removed first
Easiest to remove impurities should be removed first .
The most costly and difficult separations steps should be taken last.
Major differences in properties of the product and impurities should be the prime driving force when choosing processing steps.
Processes using different driving forces should be chosen and employed in the downstream processing. Downstream processing block diagram Primarily purification stages The broth, containing approximately 200-300mg of the rhEPO, is collected and put through centrifugation via disc-stacked centrifuge without any prior storage. This is the primary purification step because is involves removal of the host cells, which are classified as major impurities. The volume of the handling broth is thereby reduced as well.
The next unit operational step taken is in-depth filtration, added as a precautionary step for two reasons: to remove host cells overlooked by the centrifugation and to remove colloidal matter from the supernatant. Introduction (contd) Primarly purification stages (contd.) n-depth filtration is widely used for the clarification of cell culture harvests due to its relatively low economic costs. This step is also taken in order to separate the particles that would clog the polishing filtration membrane. Final purification stages In the final purification step, following the specific “rules of thumb”, impurities that have similar properties to those of the rhEPO are removed. The first unit operation in this process is AEX.
The next unit operation in the process of purification the rhEPO is ammonium sulfate precipitation.
The precipitate is usually separated by depth filtration (0.2µm filter Sartobran P or Duropore 2µm filter) (Alliger et al, 2006). Final purification stages (contd) The next purification step is reverse phase chromatography (RPC) which is very useful in this process. Firstly the different rhEPO isomers are well separated according to their carbohydrate backbones; secondly the rest of the host cell proteins are successfully removed. Thirdly this type of chromatography is known to be an effective step in the virus removal as well as virus inactivation by the usage of organic solvent. The final purification step is nano-filtration, which is a form of a dead-end filtration used as a sterilization step in order to remove potential viruses present. This filtration is carried out through a very specific membrane that is designed to remove particles as small as 15nm, such as Asahi’s Planova 15N. The filtrate acquired through nano-filtration step represents the final drug substance to be marketed (Alliger et al, 2006) Final purification stages (contd.) Economics and conclusion Economics and conclusion (contd.) In-depth filtration is widely used for the clarification of cell culture harvests due to its relatively low economic costs. This step is also taken in order to separate the particles that would clog the polishing filtration membrane. Primarily purification stages (Contd.) Economics analysis - Reasons for the rapidly increasing market value are naturally the increased number of demands in curing chronic disease and the need in large quantities.
- This requires an increase in manufacturing capacity.
- Also, a decrease in the expenses of manufacturing the bio- product.
Factors that are most critical in product manufacturing costs are the fermentation titer and the overall yield.
The fermentation titer depends mainly on the host cell expression system, the genetic stability of the host cell or cell line and the cell density.
The overall yield on the other hand is a result of the downstream process steps – of the number of the steps and the step yields.
The larger the scale the more significant the material costs.
The impact of the downstream processing costs on total manufacturing costs is high due to the steps used, the host used and the purification methods needed in order to get the proper result.
Conclusion A US study reported that the effectiveness for each $US1 spent on standard care can be achieved with $US0.81 spent on recombinant human erythropoetin therapy, i.e. treatment with recombinant human erythropoetin was about 23% more cost effective than standard care.
Based on both clinical and economic grounds, recombinant human erythropoietin should be considered a preferred alternative to transfusion, especially in patients who are likely to experience chemotherapy/radiotherapy-induced anaemia and those at higher risk of transfusion-related complications. References Alliger et al. 2006. Chromatographic purification of the recombinant human erythropoietin. United States Patent
Chiba et al. 1984. Process for producing erythropoietin. United States Patent.
Lai et al. 1985. Erythropoietin purification. United States Patent
Powel. 1997. Human erythropoietin gene: high level expression in stably transfected mammalian cells. United States Patent
Roger G.Harrison et al. 2003. Bioseparation science and engineering. New York. Oxford University Press