ΒΙΟΣΥΝΘΕΤΙΚΗ ΠΑΡΑΓΩΓΗ
ΙΝΣΟΥΛΙΝΗΣ
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
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 1980.
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.
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.
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”.
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.
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.
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.
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.
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.
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”.
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 chemicalphysical 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.
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.
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.
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 “semisynthetic” insulin. The company converted pig
insulin into human one, by substituting an alanine
residue on the B-chain with a threonine.
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.
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.
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 11ο χρωμόσωμα του ανθρώπου (στην κορυφή
του μικρού βραχίονα).
2β. ….συνέχεια
2β. ….συνέχεια
• 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.
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.
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.
2δ. Μονάδες μέτρησης:
• MW: 5807.57 Da (σχετική μοριακή μάζα)
• 1 IU is the biological equivalent of about 34.7
μg / 0.0347 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 28.800 IU insulin
• 1 ml solution contains 100 IU insulin human,
equivalent to 3.47 mg.
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.
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.
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.
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.
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”.
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.
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.
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 1982.
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.
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.
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.
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).
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
3β. ….συνέχεια
Millions of diabetics now take human
insulin that can be mass-produced by
yeast, which becomes an insulinproducing
factory
through
genetic
engineering processes.
Recombinant
insulin
is
genetically
compatible with their bodies, just like the
produced naturally in our body.
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.
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, codonoptimized 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 highcell 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).
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.
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.
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 (10100 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.
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:
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)
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]
3γ) ….συνέχεια
Θ.
μονάδα
ταυτοποίησης
(χρωματογραφία μάζης)
της
ορμόνης
Ι. μονάδα ελέγχου σύμφωνα με τις αντίστοιχες
προδιαγραφές της Ευρωπαϊκής Φαρμακοποιίας (για
λήψη άδειας από τον Ευρωπαϊκό Οργανισμό
Φαρμάκων, EMA).
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.
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.
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.
4) ….συνέχεια
• Πρόκειται περί ενός φαρμάκου πρώτης γραμμής
για την υγεία των περίπου 450.000 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.
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 43.200 IU ή 432 ml διαλύματος
ινσουλίνης.
• εργαστηριακές τιμές της SIGMA για recombinant human insulin: 25mg/
122.50 ευρώ, 50mg/ 252.00 ευρώ, 250mg/ 971.00 ευρώ (τιμές Μαϊου
2013)