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ΒΙΟΣΥΝΘΕΤΙΚΗ ΠΑΡΑΓΩΓΗ ΙΝΣΟΥΛΙΝΗΣ. ΠΕΡΙΕΧΟΜΕΝΑ ΤΗΣ ΠΑΡΟΥΣΙΑΣΗΣ 1. Ορισμοί: Biopharmaceuticals - Biosimilars - Recombinant DNA (rDNA) - Plasmids - Discoveries.

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Παρουσίαση με θέμα: "ΒΙΟΣΥΝΘΕΤΙΚΗ ΠΑΡΑΓΩΓΗ ΙΝΣΟΥΛΙΝΗΣ. ΠΕΡΙΕΧΟΜΕΝΑ ΤΗΣ ΠΑΡΟΥΣΙΑΣΗΣ 1. Ορισμοί: Biopharmaceuticals - Biosimilars - Recombinant DNA (rDNA) - Plasmids - Discoveries."— Μεταγράφημα παρουσίασης:

1 ΒΙΟΣΥΝΘΕΤΙΚΗ ΠΑΡΑΓΩΓΗ ΙΝΣΟΥΛΙΝΗΣ

2 ΠΕΡΙΕΧΟΜΕΝΑ ΤΗΣ ΠΑΡΟΥΣΙΑΣΗΣ 1. Ορισμοί: Biopharmaceuticals - Biosimilars - Recombinant DNA (rDNA) - Plasmids - Discoveries led to the production of human recombinant insulin 2. Περί ινσουλίνης (discovery – structure – synthesis - μονάδες μέτρησης) 3. Παραγωγή βιοσυνθετικής ινσουλίνης (biosynthetic or recombinant or genetic engineered insulin): α. Insulin became the first commercial product of the biotech industry: Ιστορικά στοιχεία β. Production of insulin in bacteria/yeasts by using synthetic insulin genes: The “pre-proinsulin” method γ. Η παραγωγική διαδικασία για λήψη της βιοσυντιθέμενης ινσουλίνης σε καθαρή κρυσταλλική μορφή περιλαμβάνει πολύπλοκα επιμέρους στάδια 4. Προοπτικές του παρόντος εγχειρήματος

3 1α. Biopharmaceuticals: •Biological medicines (bio-medicine / bio-pharmaceutical substances): A medicine, whose active substance is made by a living organism. •Οι βιοφαρμακευτικές ουσίες (biopharmaceuticals), είναι φάρμακα που παράγονται με βιοτεχνολογικές μεθόδους. Έχουν προκαλέσει επανάσταση στην σύγχρονη Ιατρική και θεωρούνται ως οι σημαντικότεροι θεραπευτικοί παράγοντες του 21ου αιώνα. Σ’ αυτές συγκαταλέγονται: i)πρωτεϊνες που παράγονται με την τεχνολογία του ανασυνδιασμένου DNA (recombinant DNA technology, rDNAs). Χαρακτηριστικό παράδειγματα αποτελούν οι αυξητικοί παράγοντες και η ινσουλίνη στην δεκαετία του ii) μονοκλωνικά αντισώματα, ομοίως παραγόμενα με την τεχνολογία του ανασυνδιασμένου DNA (recombinant therapeutic monoclonal antibodies, mAbs). Χαρακτηριστικό παράδειγματα αποτελούν τα θεραπευτικά μονοκλωνικά αντισώματα και πρωτεϊνες με πολυπλοκότερη δομή στην δεκαετία του 1990 και πιο πρόσφατα.

4 1γ. Ανασυνδιασμένο DNA (recombinant DNA, r-DNA): •Defines a DNA sequence which has been artificially obtained by combining genetic material from different organisms, as is the case for a plasmid containing a gene of interest. The concept that DNA continues to function when it is transferred from one organism to another opened up infinite possibilities. Recombinant DNA heralded a new era in biology.

5 1β. Biosimilar medicines ( “ generic ” biological medicines): A medicine, that is similar to a biological medicine that has already been authorised (“γενόσημο” biopharmaceutical, in the broadest sense of the term) •For biosimilar medicines, the company needs to carry out studies to show that the medicine: i) is similar to the reference medicine; ii) does not have any meaningful differences from the reference medicine in terms of quality, safety or efficacy. •A generic medicine is a medicine that is developed to be the same as a medicine that has already been authorised, called the “reference medicine”.

6 1γ. ….συνέχεια •Scientists associated with the initial development of recombinant DNA methods recognized that the potential existed for organisms containing recombinant DNA to have undesirable or dangerous properties. At the 1975 [Asilomar Conference on Recombinant DNA], these concerns were discussed and a voluntary moratorium on recombinant DNA research was initiated for experiments that were thought to be particularly risky. This moratorium was widely observed until the National Institutes of Health (USA) developed and issued formal guidelines for rDNA work.

7 1γ. ….συνέχεια •Today, recombinant DNA molecules and recombinant proteins are usually not regarded as dangerous. However, concerns remain about some organisms that express recombinant DNA, particularly when they leave the laboratory and are introduced into the environment or food chain.

8 1δ. Plasmids: •Plasmids are small circular molecules of extra-chromosomal DNA, present in nature in some bacteria. They contain a gene that confers antibiotic resistance to the bacterium, as well as sequences of origin and end of replication. Plasmid DNA can be modified by inserting fragments of foreign DNA into especially created insertion sites. Once introduced into bacteria, the plasmid (and thus the foreign DNA) is rapidly amplified thanks to the high frequency of replication of bacteria.

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10 1ε. Fundamental scientific discoveries of the past century, led to the production of human recombinant insulin and revolutionized the treatment of diabetes: •i) Restriction enzymes: In 1962 Werner Arber, a Swiss biochemist, and his colleagues proved the existence of what he called “molecular scissors”, i.e. proteins capable of cutting DNA. •They showed that the E. coli bacterium is equipped with an enzymatic system capable of recognizing and destroying exogenous DNA and called this group of proteins “restriction enzymes”. •They also showed that the activity of these enzymes is controlled: each enzyme targets a specific DNA sequence - called consensus or restriction site - which it recognizes and cuts. •In 1978 they received the Nobel Prize in Medicine for their discovery.

11 1ε. ….συνέχεια •The molecular tool used to cut DNA is a restriction enzyme (such as EcoR1). The enzyme has a precise shape that allows it to run along the groove of the double helix, scanning for the base letter sequence G A A T T C. EcoR1 then cuts the plasmid at this specific point......allowing a new piece of DNA to be inserted. •When it cuts, EcoR1 leaves a sticky end, which helps the new gene to attach. The joins are then stitched together by another enzyme called DNA ligase.

12 1ε….συνέχεια •ii) Ligases: Shortly after Arber’s discovery, Arthur Kornberg discovered that is was possible to join together fragments of DNA by using an enzyme that he called “DNA ligase”. •Kornberg was trying to construct an artificial viral DNA from fragments of viral DNA. After adding the ligase, he realized that the enzyme was capable of joining together fragments of DNA and re-establishing phosphodiester bonds between the nucleotides. •The artificial DNA was biological active, as it was able to replicate autonomously. Therefore, Kornberg was considered by the scientific community to be the scientist who was “able to generate life in a test tube”.

13 1ε….συνέχεια •iii)Transformation - Cloning: By the end of the 1960s, the techniques to cut and ligate DNA had been refined. However, scientists were still searching for a mechanism to copy DNA, in order to obtain sufficient amounts to work with. The discovery finally came in 1971, when scientists developed bacterial transformation. This technique consists in enhancing the introduction of plasmid DNA in bacteria - such as E. coli - a process which occurs in nature, albeit seldom. •Transformation is achieved by modifying some chemical- physical properties of the bacterial membrane using chemical substances (CaCl2) associated with rapid heat shock, or an electric shock at high voltage (electroporation). The bacteria become momentarily permeabilized to the exogenous plasmid DNA. •Conclusion: Restriction enzymes – Ligases – Cloning - Recombinant DNA technology: Scientists now understood how to cut, ligate and copy DNA.

14 2. Περί ινσουλίνης •Insulin is a protein hormone. Its name is derived from the latin “insula” (island) as it is secreted by groups of endocrine cells called the islets of Langerhans in the pancreas. It enables glucose metabolism by activating glycolysis, it promotes the accumulation of glucose in the liver in the form of glycogen and favours the storage of fat. •Insulin is one of the best examples of how the progress - over the last century - in the scientific disciplines of molecular biology and genetic engineering has lead to successful diabetes mellitus therapy.

15 2α. Short history of the discovery of insulin: •1869: the young medical student Paul Langerhans identified under the microscope small areas of clear cells throughout the pancreas (islets of Langerhans). •1889: Oscar Minkowski and Joseph von Mering removed the pancreas from dogs and the animals developed diabetes. This meant that this gland and the islets of Langerhans were involved in regulating sugar levels in the blood. •1920: Frederick Banting working at the University of Toronto with the help of medical student Charles Best isolated the extraction from these cells and named it “isletin” (later insulin). When administered to dogs deprived of a pancreas, the extract lowered the glucose level in their blood.

16 2α. ….συνέχεια •1922: doses of purified insulin were successfully injected into diabetic children. In November of the same year, Eli Lilly started to produce purified insulin. •1951: insulin was the first protein to be sequenced by Prof. Frederick Sanger (Cambridge), marking an important milestone in molecular biology. •1961: παρασκευή του πρώτου ουδέτερου διαλύματος ινσουλίνης •1970s: Novo Nordisk started to produce “semi- synthetic” insulin. The company converted pig insulin into human one, by substituting an alanine residue on the B-chain with a threonine.

17 2α. ….συνέχεια •Some differences in the porcine / bovine insulin triggered an immune response in some patients. •The production of semi-synthetic insulin only partly solved the problem, as it still depended on the availability of animal insulin (difficult to obtain large amounts of pure insulin). •Therefore, the possibility to obtain unlimited quantities of human insulin made a strong impact on the pharmaceutical market.

18 2α. ….συνέχεια •1976: insulin became the first recombinant drug to be used for therapy in humans, by using newly developed genetic engineering techniques. •1982: η πρώτη παραγωγή βιοσυνθετικής ανθρώπινης ινσουλίνης με τεχνικές ανασυνδυασμένου DNA από την εταιρεία Eli Lilly. •1996: Lispro, το πρώτο βιοσυνθετικό ανάλογο ταχείας δράσης ινσουλίνης, από την εταιρία Eli Lilly. •Five researchers have earned Nobel Prizes for research related to insulin.

19 2β. Structure of insulin: •H ινσουλίνη αποτελείται απο δύο πολυπεπτιδικές αλυσίδες - την Α και την Β - μήκους 21 και 30 αμινοξέων, αντίστοιχα. Oι πλευρικές αλυσίδες τριών ζευγαριών κυστεϊνών συνδέονται μεταξύ τους με δισουλφιδικούς δεσμούς. Oι δύο απο τους τρεις δισουλφιδικούς δεσμούς συνδέουν μεταξύ τους τις δύο πολυπεπτιδικές αλυσίδες της ινσουλίνης, ενώ ο τρίτος απαντά στην Α αλυσίδα. Oι δισουλφιδικοί δεσμοί σταθεροποιούν τη δομή της ινσουλίνης. •Το γονίδιο που κωδικοποιεί την ινσουλίνη βρίσκεται στο 11 ο χρωμόσωμα του ανθρώπου (στην κορυφή του μικρού βραχίονα).

20 2β. ….συνέχεια

21 •The bovine hormone differs from the human one by only three amino acids, and the porcine one by a single amino acid. •Insulin tends to form dimers in solution. In the presence of zinc ions, insulin forms hexamers (groups of 6 molecules) shape. Insulin is stored in b-cells and secreted in the bloodstream as a hexamer. However, the active form is a monomer.

22 2γ. Synthesis of insulin: •In eukaryotic cells, insulin is initially synthesized as pre-proinsulin, a 110 amino acid polypeptide that contains additional sequences: • A “pre” amino-terminal sequence (signal peptide of 24 amino acids), which enables the secretion of the protein. • A central “pro” sequence (the C peptide of 35 amino acids) which determines the correct folding of the protein.

23 2γ. ….συνέχεια •After pre-proinsulin is translated in the endoplasmic reticulum (ER), an enzyme cuts off the 24 amino-terminal amino acids (the “pre” sequence), leaving pro-insulin, which in turn folds and allows the formation of the disulphide bonds between cysteine residues. At this stage, the protein passes into the Golgi apparatus, where the C peptide is removed (the “pro” sequence), forming mature insulin, which is then stored in the Golgi vescicles.

24 2δ. Μονάδες μέτρησης: •MW: Da (σχετική μοριακή μάζα) •1 IU is the biological equivalent of about 34.7 μg / mg of human pure crystalline insulin (1/28.8 mg exactly) •1 mg of dry, pure, crystalline insulin περιέχει 28.8 IU •1 gr περιέχει IU insulin •1 ml solution contains 100 IU insulin human, equivalent to 3.47 mg.

25 3. Παραγωγή βιοσυνθετικής ινσουλίνης (biosynthetic or recombinant or genetic engineered insulin): 3α. Insulin became the first commercial product of the biotech industry: Ιστορικά στοιχεία •Sanger, in 1951, determined the sequence of insulin. And even then, it wasn't until 1979 that human insulin was able to be produced in large quantities. This was done by the use of recombinant DNA. The concept that DNA continues to function when it is transferred from one organism to another opened up infinite possibilities.

26 3α. ….συνέχεια •Recombinant DNA technology is based on the discovery - by Herbert W. Boyer and Stanley Cohen, at the Massachusetts Institute of Technology - that genes can be inserted into plasmids and then into bacteria, where they are continuously activated producing functional proteins. •In 1976, these scientists designed a single experiment to produce human insulin from a recombinant DNA.

27 3α. ….συνέχεια •However, the human DNA sequence for insulin was unknown and after the Asilomar conference, severe restrictions had been imposed on the production of human recombinant DNA. •Boyer and Cohen (the Genentech group) synthesized the human DNA insulin sequence based on its known amino acid sequence. Then, they inserted it into a plasmid and transformed into bacteria to produce insulin. •In fact, they assembled the human DNA sequence of insulin, without having to use "real" human DNA. They bypassed some of the restrictions on human recombinant DNA work resulting from the Asilomar conference.

28 3α. ….συνέχεια •This was the first insulin gene - artificially made in the laboratory and - capable of working in a living cell. •DNA biotechnology became a reality and has developed quickly.

29 3α. ….συνέχεια •INDIANAPOLIS, July 21, 1980 – Press release: Eli Lilly and company today announced that it has begun limited testing in healthy human volunteers of biosynthetic human insulin produced by recombinant DNA technology (followed careful animal studies and extensive laboratory testing). The company has started construction of the world’s first manufacturing facilities—at a cost of $40 million—to employ recombinant DNA technology to produce the biosynthetic human insulin. •“Biosynthetic human insulin is identical to the insulin naturally made inside the human body and it is expected to lessen chances of allergic and other reactions that some diabetics experience from current insulin, made from animal pancreas glands. Now we can believe that insulin supplies will always be adequate to meet public needs”.

30 3α. ….συνέχεια •The Asilomar conference Following the discovery of recombinant DNA, scientists questioned the safety of this new technology. Could the ability to join genes together have the same impact as the atomic bomb or the telescope? In 1975 at a conference in Asilomar, California, scientists, lawyers and the media came together for an open debate. They agreed to allow researchers to use only certain types of bacteria considered to be “safe” and restricted the use of mammalian DNA (measures equivalent to today’s restrictions on the use of the Ebola virus). Five years later these restrictions were amended, allowing considerable progress to be made in the field of mammalian research.

31 3α. ….συνέχεια •Washington News - October 29, 1982: •Eli Lilly & Co., which will market the insulin under the brand name of Humulin, told that the FDA federal scientists had reviewed test results and found the synthetic insulin safe and effective for diabetes treatment. •Lilly spokesman Ronald Culp in Indianapolis said the company has sent letters and mailgrams to 260,000 doctors across the nation advising them of the FDA approval.

32 3α. ….συνέχεια About Humulin® It is the first therapeutic recombinant protein for human use that developed by Arthur Riggs, working with Genentech in 1978 and later acquired by Eli Lilly and Company. It was synthesized in a laboratory strain of Escherichia coli bacteria which had been genetically altered with recombinant DNA to produce biosynthetic human insulin. Eli Lilly and company arguably facilitated the approval by the FDA, in Prior to the development of synthesized human insulin, patients were dependent on animal insulin which was more costly, less accessible, and caused infections in some patients.

33 3α. ….συνέχεια The case for “Lispro” or “intelligent insulin: •Today there are different categories of insulin: rapid-acting, short-acting or regular, intermediate-acting, long-acting and premixed. Although the principle of action is the same, the rate at which they are absorbed are different. Monomers and dimers diffuse more rapidly in the blood compared to the hexameric form. Insulin preparations containing a majority of hexamers are absorbed more slowly. •Therefore, researchers aimed at obtaining molecules of recombinant insulin with reduced tendency to form dimers and hexamers. This is the case for “Lispro” or “intelligent insulin”, which is obtained by inverting lysine and proline residues at the C-terminal end of the B-chain. This modification allows a more rapid absorption of the hormone without altering the site for the receptor.

34 3β. Production of insulin in bacteria/yeasts by using synthetic insulin genes: The production of recombinant insulin can be carried out in different cellular systems: bacteria, yeast, insect cells and mammalian cells. Advantage of bacterial cells: •simple to handle •short replication time •produce high yields •low production costs.

35 3β. ….συνέχεια However, some eukaryotic proteins are processed after translation. This is the case for insulin, which is initially translated as pre-pro-insulin and through subsequent passages of maturation reaches the final sequence and conformation. In bacteria, such maturation processes do not take place. While E. coli was the original organism of choice for the production of insulin, it has somewhat fallen out of favor due to its inability to make post-translational modifications (the failure to modify that structure often means that the protein will be non-functional).

36 3β. ….συνέχεια Yeast cells: Mammalian, insect and yeast cells have all been studied as suitable replacements for bacteria, however, yeast cells are the most desirable. Yeast is an established industrial fermentation system ideally suited for large-scale production of recombinant eukaryotic proteins. Advantages of a yeast expression system : •the ease of genetic manipulation •rapid growth characteristics a of prokaryotic organism (can be grown quickly to very high cell densities, in defined medium) •translates into greater quantities of recombinant protein per fermentor volume •lower protein production cost •subcellular machinery for performing post-translational protein modification of eukaryotic cells

37 3β. ….συνέχεια Millions of diabetics now take human insulin that can be mass-produced by yeast, which becomes an insulin- producing factory through genetic engineering processes. Recombinant insulin is genetically compatible with their bodies, just like the produced naturally in our body.

38 3β i) The pre-pro-insulin method: “Application of simple fed-batch technique to high-level secretory production of insulin precursor, using a yeast strain with subsequent purification and conversion to human insulin” •Nowadays, human insulin is produced as recombinant fusion protein, using two major routes: •One route involves the production of an insulin precursor in the form of inclusion bodies, using bacteria as expression host with subsequent solubilization and refolding procedures. •The other route involves the utilization of yeast-based expression systems, leading to the secretion of a soluble insulin precursor (IP) into the culture supernatant. •Both routes are economically viable, however, most scientists favor the second route.

39 3β i). ….συνέχεια •The pre-pro-insulin method (PPM, περιγραφή μεθόδου): In order to simplify the purification process, scientists decided to devise a method in which insulin would be directly secreted in the culture media after its production inside the cell. Yeasts are given a signal to produce pre-pro-insulin, which is processed in the laboratory, normally releasing the mature protein. A synthetic insulin precursor (IP)-encoding gene, codon- optimized for expression in a yeast strain, is cloned in a suitable plasmid vector and integrated into the genome of the yeast strain. The selected recombinant yeast strain is grown to high- cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP is carried out at optimum methanol concentrations, which are kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant).

40 3β i). ….συνέχεια Using immobilized metal ion affinity chromatography (IMAC) for IP purification, 95% of the secreted product is recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP is trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth. Important remarks: •Today, the insulin gene can be isolated from the human genome using the technique called Polymerase Chain Reaction (PCR). This extremely precise and rapid method to select and amplify a DNA segment was invented by Kary Mullis, in 1985, who was awarded the Nobel Prize for Chemistry in 1993 for his groundbreaking invention. PCR enables the amplification of nucleic acid fragments, provided that the initial and terminal nucleotide sequences of these fragments are known.

41 3β i). ….συνέχεια •Once isolated, the insulin gene can be modified at its ends by adding on sequences recognized by a restriction enzyme. The same enzyme also recognizes a specific sequence on the plasmid. The restriction enzyme cuts both the plasmid and the insulin gene ends in a specific and asymmetric manner, producing sticky ends that facilitate the insertion of the insulin gene to be fixed into the plasmid by a DNA ligase. •Since only some of the bacteria will take up the plasmid, a gene encoding an enzyme which breaks down a certain antibiotic is also included in the plasmid, which allows bacteria with the plasmid to grow on a plate containing the antibiotic while the other bacteria die. These bacteria are then allowed to grow and replicate, which allows the plasmid and the insulin gene to replicate millions of times.

42 3β i). ….συνέχεια •The selection of high-producing clones can dramatically enhance the whole production process, along with significant decrease in production costs. •The high-level protein production can be reached by taking care of the following factors: Adequate copies of vector ( copies per cell) and suitable promoters (the segment of DNA located immediately in front of each gene. The promoter regulates when, how much and how often the gene is transcribed). •Protein purification is a cost-saving area and pure protein is recovered with higher yield and lower cost. •Yeast strains grow on a simple mineral media and does not secrete high amounts of endogenous protein. Therefore the heterologous protein secreted into the culture is relatively pure and purification is easier to accomplish.

43 3γ) Η παραγωγική διαδικασία της βιοσυντιθέμενης ινσουλίνης : •Η διαδικασία λήψης ανασυνδιασμένης ινσουλίνης σε καθαρά κρυσταλλική μορφή περιλαμβάνει πολύπλοκα επιμέρους στάδια, η λειτουργία των οποίων παρακολουθείται με την εφαρμογή του κατάλληλου λογισμικού. •Θα χρησιμοποιηθεί σύγχρονος εξοπλισμός για την επάνδρωση και λειτουργία δύο εργαστηρίων – ενός Βιοτεχνολογικού και ενός Αναλυτικής Χημείας. •Αναλυτικά, η παραγωγική διαδικασία θα περιλαμβάνει τα εξής επιμέρους στάδια:

44 3γ) ….συνέχεια Α. Δημιουργία του συνθετικού γονιδίου ινσουλίνης Β. Δημιουργία του ανασυνδιασμένου πλασμιδίου - φορέα του γονιδίου Γ. ενσωμάτωση (transformation) των πλασμιδίων με το γονίδιο της ινσουλίνης στα προς καλλιέργεια κύτταρα μυκήτων Δ. επιλογή του καταλλήλου κυτταρικού στελέχους - κλώνου (να περιέχει όσο πιο πολλά αντίγραφα του πλασμιδιακού γονιδιακού φορέα) Ε. μονάδα πρωτογενούς κυτταροκαλλιέργειας των ανασυνδιασμένων μυκήτων (προσθήκη 95 g αλκοόλης Α, σ’ ένα λίτρο θρεπτικού υλικού καλλιέργειας)

45 3γ) ….συνέχεια ΣΤ. μονάδα ολοκληρωμένης ζύμωσης των ανασυνδιασμένων μυκήτων (ελεγχόμενη συγκέντρωση αλκοόλης Β, 2 g σ’ ένα λίτρο θρεπτικού υλικού) Ζ. συλλογή κυττάρων με συνεχόμενη φυγοκέντρηση Η. μονάδα απομόνωσης και καθαρισμού της προδρόμου ινσουλίνης που εκκρίνεται στο υγρό υλικό της καλλιέργειας / μετατροπή σε ινσουλίνη [χρωματογραφία – βιομετατροπή (transpeptidation, deprotection) – κρυσταλλοποίηση]

46 3γ) ….συνέχεια Θ. μονάδα ταυτοποίησης της ορμόνης (χρωματογραφία μάζης) Ι. μονάδα ελέγχου σύμφωνα με τις αντίστοιχες προδιαγραφές της Ευρωπαϊκής Φαρμακοποιίας (για λήψη άδειας από τον Ευρωπαϊκό Οργανισμό Φαρμάκων, EMA).

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49 4. Προοπτικές του παρόντος εγχειρήματος •The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries. India is the world’s largest population of diabetics. India has 30 million diabetes sufferers.

50 4) ….συνέχεια •Η βιοσυνθετική ινσουλίνη παράγεται σε καθαρή ενέσιμη εμπορική μορφή σε τρείς Ευρωπαϊκές χώρες (Γαλλία, Ελβετία, Δανία). Επίσης, στην Ινδία, στην Κίνα και στις Ηνωμένες Πολιτείες της Αμερικής. •Η χρησιμοποιούμενη βιοτεχνολογική μέθοδος παραγωγής συνιστά τεχνολογική καινοτομία για την χώρα μας. Bασιζόμενη στην τεχνολογία του ανασυνδιασμένου DNA και την χρησιμοποίηση του "κυτταρικού εργαστηρίου" μυκήτων, αυξάνει αποδεδειγμένα σε σημαντικό βαθμό την παραγόμενη ινσουλίνη και με πολύ χαμηλό κόστος πρώτων υλών, γεγονός που θα μειώσει την μονάδα κόστους του παραγομένου προϊόντος.

51 4) ….συνέχεια •Αναμένεται ότι η πτώση της τιμής της θα την καταστήσει πιο προσιτή στους ολοένα αυξανόμενους ινσουλινο-εξαρτώμενους ασθενείς αλλά προπάντων στις οικονομικά ασθενέστερες πληθυσμιακές ομάδες (εντός και εκτός Ελλάδος). •Μετά την απομόνωση της ινσουλίνης σε καθαρή μορφή - πλην της χρήσης για ερευνητικούς σκοπούς - θα επιδιωχθεί η παραγωγή του πρώτου "made in Greece" βιοϊσοδύναμου (biosimilar) κλινικού ενεσίμου σκευάσματος ινσουλίνης.

52 4) ….συνέχεια •Πρόκειται περί ενός φαρμάκου πρώτης γραμμής για την υγεία των περίπου Ελλήνων διαβητικών, κάτι που λείπει παντελώς από την χώρα μας αλλά και πολλές άλλες χώρες. Για τον λόγο αυτό εισάγεται από τις μεγάλες πολυεθνικές φαρμακευτικές εταιρίες του εξωτερικού (Novo Nordisk Δανίας, Elli - Lilly Αμερικής, Sanofi Γαλλίας) με ό,τι οικονομικές απώλειες συνεπάγεται για την χώρα μας στην αντιμετώπιση μιας ισόβιας νόσου, όπως είναι ο σακχαρώδης διαβήτης.

53 4) ….συνέχεια •Η αρχική άδεια κυκλοφορίας δεν χορηγείται από τον ΕΟΦ αλλά από την ΕΜΑ (European Medicines Agency), ισχύει για συνολικά 34 χώρες της ΕΕ και της ΕΟΚ και ο χρόνος έγκρισης ανέρχεται σε 210 ημέρες. •Απόδοση της μεθόδου: ~1.5 g of 99% pure recombinant human insulin per liter of culture broth, δηλαδή IU ή 432 ml διαλύματος ινσουλίνης. •εργαστηριακές τιμές της SIGMA για recombinant human insulin: 25mg/ ευρώ, 50mg/ ευρώ, 250mg/ ευρώ (τιμές Μαϊου 2013)


Κατέβασμα ppt "ΒΙΟΣΥΝΘΕΤΙΚΗ ΠΑΡΑΓΩΓΗ ΙΝΣΟΥΛΙΝΗΣ. ΠΕΡΙΕΧΟΜΕΝΑ ΤΗΣ ΠΑΡΟΥΣΙΑΣΗΣ 1. Ορισμοί: Biopharmaceuticals - Biosimilars - Recombinant DNA (rDNA) - Plasmids - Discoveries."

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