Recombinant Pharmaceutics , concepts examples

Recombinant DNA Technology

1. Recombinant
Pharmaceuticals

2. What is recombinant
technology???

3. •Recombinant DNA technology involves
 using enzymes
and various laboratory techniques to manipulate and
isolate DNA segments of interest.
•This method can be used to combine (or splice) DNA
from different species or to create genes with new
functions

4. Pharmaceutical products of
DNA technology
•Recombinant DNA is a technology scientists
developed that made it possible to insert a human
gene into the genetic material of a common
bacterium. This “recombinant” micro-organism could
now produce the protein encoded by the human gene.

7. Recombinant Pharmaceuticals

8. Recombinant Pharmaceuticals
•A 
number of human disorders can be traced to the
absence or malfunction of a protein normally
synthesized in the body.
• Most of these disorders can be treated by supplying
the patient with the correct version of the protein
• Hence, modern pharmaceutical manufacturing
frequently relies upon recombinant drugs

9. Examples: Recombinant
Pharmaceuticals
•Human Insulin
• Human Growth Hormone
• Human blood clotting factors
• Vaccines
• Monoclonal Antibodies
• Interferons
• Antibiotics & other secondary metabolites

10. Human Insulin
•Earliest use of recombinant technology
• Modify E.coli cells to produce insulin; performed by Genentech in 1978
• Prior, bovine and porcine insulin used but induced immunogenic
reactions
• Also, there were many purification and contamination hassles.
• To overcome these problems, researchers inserted human insulin
genes into a suitable vector (E.coli)

11. Producing Recombinant Insulin
•First, scientists synthesized genes for the two insulin A & B
chains.
•• They were then inserted into plasmids along with a strong
lacZ promoter.
•• The genes were inserted in such a way that the insulin & B-
galactosidase residues would be separated by a methionine
residue.
•This is so that the insulin A & B chains can be separated easily
by adding cyanogen bromide

12. •The vector was then transformed into E.coli cells.
• Once inside the bacteria, the genes were "switched-on" by the
bacteria to translate the code into either the "A" chain or the "B"
chain proteins found in insulin
• The purified insulin A and B chains were then attached to each other
by disulphide bond formation under laboratory conditions
Producing Recombinant Insulin

14. Human Growth Hormones
•Somatostatin and Somatotrophin are two proteins that act in conjunction to
control growth processes in the human body,
•Their malfunction leading to painful and disabling disorders such as
Acromegaly (uncontrolled bone growth) and Dwarfism.
•Somatostatin was the first human protein to be synthesized in E. coli. Being
a very short protein, only 14 amino acids in length, it was ideally suited for
artificial gene synthesis.

15. •The strategy used was the same as described for
recombinant insulin,
•involving insertion of the artificial gene into a lacZ vector,
′
•synthesis of a fusion protein, and cleavage with cyanogen
bromide
Production of recombinant
Human Growth Hormones

16. Recombinant Blood clotting factors
•Human factor VIII is a protein that plays a central role in blood
clotting.
•The commonest form of haemophilia in humans results from an
inability to synthesize factor VIII
•The factor VIII gene is very large.
•The mRNA codes for a large polypeptide (2351 amino acids), which
undergoes a complex series of post-translational processing events,
eventually resulting in a dimeric protein consisting of a large subunit
and a small subunit.

17. •The two subunits contain a total of 17 disulphide bonds and a number of
glycosylated sites.
•As might be anticipated for such a large and complex protein, it has not been
possible to synthesize an active version in E. coli.
•Two separate fragments from the cDNA were used. Each fragment was
ligated into an expression vector along with Ag promoter (a hybrid between
the chicken b-actin and rabbit b-globin sequences) and a polyadenylation
signal from SV40 virus.
•The plasmid was introduced into a hamster cell line and recombinant protein
obtained.
Production of RBC factors

19. Production of RBC factors
•Alternative method- pharming
•The complete human cDNA has been attached to the
promoter for the whey acidic protein gene of pig,
leading to synthesis of human factor VIII in pig
mammary tissue and subsequent secretion of the
protein in the milk.
• The factor VIII produced in this way appears to be
exactly the same as the native protein

20. Recombinant Vaccines
•Two 
types:
(i) Recombinant protein vaccines: This is based on
production of recombinant DNA which is expressed to
release the specific protein used in vaccine preparation
(ii) DNA vaccines: Here the gene encoding for
immunogenic protein is isolated and used to produce
recombinant DNA which acts as vaccine to be injected
into the individual.

21. Recombinant Protein Vaccines
• A pathogen produces its proteins in the body which elicit an immune
response from the infected body.
•The gene encoding such a protein is isolated from the causative
organism
•This DNA is expressed in another host organism, like genetically
engineered microbes; animal cells; plant cells; insect larvae etc,
resulting in the release of appropriate proteins.
•These when injected into the body, causes immunogenic response
against the corresponding disease providing immunity.

22. DNA Vaccines
•Refers to the recombinant vaccines in which the DNA is used
as a vaccine
•The gene responsible for the immunogenic protein is
identified, isolated and cloned with corresponding expression
vector.
•Upon introduction into the individuals to be immunized, it
produces a recombinant DNA.
•This DNA when expressed triggers an immune response and
the person becomes successfully vaccinated.

23. The mode of delivery of DNA vaccines
•The mode of delivery of DNA vaccines include:
•direct injection into muscle;
•use of vectors like adenovirus, retrovirus etc;
•In-vitro transfer of the gene into autologous cells and
reimplantation of the same and particle gun delivery
of the DNA

24. DNA Vaccines
•In certain cases, the responsible gene is integrated into live
vectors which are introduced into individuals as vaccines.
• This is known as live recombinant vaccines. e.g. vaccinia
virus.
•Live vaccinia virus vaccine (VV vaccine) with genes
corresponding to several diseases, when introduced into
the body elicit an immune response but does not actually
cause the diseases

25. Recombinant Antibodies
•An immunoglobulin which produced because of the introduction of
an antigen into the body, and which possesses the ability to
recognize the antigen.
•Recombinant antibody has significant advantages compared with
the conventional antibody and therefore its use becoming more
popular now days.
•The fact that no animals are needed in the manufacturing procedure
of the recombinant antibodies, in addition, the manufacturing time
is relatively short compared with the conventional method.
Moreover, the quality of the final product is higher

26. Production of Recombinant
Antibodies
•The production of non-animal recombinant antibodies can be broken
down into five steps:
(1)creation of an antibody gene library
(2) display of the library on phage coats or cell surfaces

(3) isolation of antibodies against an antigen of interest
(4) modification of the isolated antibodies and
(5) scaled up production of selected antibodies in a cell culture expression
system

27. Interferons
•IFNs are a group of signaling proteins made and
released by host cells in response to the presence of
pathogens, such as viruses, bacteria, parasites, or
tumor cells.
•In a typical scenario, a virus-infected cell will release
Interferons causing nearby cells to heighten their
anti-viral defenses.

28. Production of Recombinant IFNs
•Recombinant DNA technology has proved the most
satisfactory route to the large scale production of human
interferons.
•The genes of all three types of HuIFN have been cloned in
micro-organisms and expression obtained.
•HuIFNβ and γ produced in this manner lack the
glycosylation present in the naturally occurring substances
but this does not affect their specific activity.

29. Production of Recombinant IFNs
•Greatly improved methods of purification, including
immuno-adsorption chromatography on monoclonal
antibody columns, are now available
•so there should be no difficulty in supplying adequate
amounts of pure interferon of all three types although, up
till now, only HuIFNα has been readily available.

30. Recombinant Secondary Metabolites
•The importance of antibiotics to medicine has led to
much research into their discovery and production.
•GM micro-organisms are used to increase production.
•Another technique used to increase yields is gene
amplification, where copies of genes coding for
enzymes involved in the antibiotic production can be
inserted back into a cell, via vectors such as plasmids.

31. Production of Recombinant Plant SM
•PSM can also be produced by rDNA technology in plant suspension
cultures, micro-organism cultures and hairy root cultures
•A. rhizogenes mediated transformation which can transfer foreign
genes into the transformed hairy root.
•E .g.: 6-hydroxylase gene of Egyptian henbane which was
introduced to Deadly nightshade using A. rhizogenes.
•Engineered roots showed an increased amount of enzyme activity
and a five-fold higher concentration of scopolamine.