3. Contents
Introduction
Recombinant protein drugs
Importance of insulin
Recombinant insulin approaches
Human insulin production
Chain A & B method
Pro-insulin method
Applications
Conclusion
References
4. Introduction
“Protein which are engineered in the laboratory for
pharmaceutical use are referred to as therapeutic proteins”
Proteins which are absent or low in individuals with an
illness such as Cancer ,Infectious diseases, Hemophilia
BC, etc. are artificially synthesized on large scale through
genetically modified host cells and delivered
5. This therapeutic approach in treating diseases using
proteins and peptides is termed protein therapeutics
Protein therapy is similar to gene therapy, but unlike gene
therapy, protein therapy delivers protein to the body in
specific amounts (as would be ordinarily present), to help
repair illness, treat pain or remake structures
In 1920’s Human insulin is considered to be the first
therapeutic proteins
6. What is Recombinant protein?
• Recombinant protein are protein that are artificially made
through the recombinant DNA technology
• It provides a more efficient method to obtain large amount
of proteins
• Protein can be used in many areas such as diagnostic tools,
vaccines, therapeutics, detergents , cosmetics, food
production
9. Production of recombinant insulin
• Initial attempts for the production of recombinant
insulin started in the late 1970s
• First trial of human trial of recombinant insulin was
conducted in 1980
Insulin was the first recombinant DNA technology product
administrated to humans
Trial was successful and which lead to exploration of r DNA
technology for the pharmaceutical products
10. Insulin
Is a polypeptide hormone produced by the B cells of the
islets of Langerhans in the pancreas
Its main function is enabling the cells to take up glucose
(providing it with energy it needs)
Its required for normal glucose homeostasis
11.
12. Diabetes mellitus
DM is a genetically linked disease which is commonly found
in general population
Elevated blood glucose level is the characteristics of this
disease
Its due to the inefficient production of insulin
Lack of efficient insulin , glucose accumulate in blood
Elevated glucose levels, exceeding 180mg dL can result in
the excretion of glucose in urine
13. Importance of insulin
Without insulin, the blood glucose builds up in the blood
and the cells are starved of their energy source
Some of the symptoms that may occur include
14. The cells will begin to use fat ,the energy source stored for
emergencies
When this happens for too long a time the body produces
ketones, chemical produced by the liver
Ketones can poison and kill cells if they build up in the body
over an extended period of time. This can lead to serious
illness and coma
16. Recombinant insulin approaches
Cows & pigs Immunogenic
Yeast cells Costly
Plant cell Not fully developed
• E . Coli - Simple, well understood genetics
-Its very easy to manipulate
-Culturing cost is minimal
-Fermentation easy to scale up
-Ease of inclusion bodies purification
•
17. Industrial production of insulin
There two methods for the production of recombinant
human insulin from genetically modified bacterial culture
I. Two chain method (chain A & B method)
II. Pro insulin method
18. 1- Chain A & B Method
This method consists chemically synthesizing two
oligonucleotide which encode the 21 amino acid A chain 30
amino acid B chain individually in two different E. coli
21. Step 2: Insertion of cDNA of both chains into
plasmids
22.
23. Step 3: Transfection
Recombinant plasmids enter the bacteria in a process
known as transfection
Cacl2 treatment and electroporation can be used
These cells are later known as transformed cells
24. Step 4: Media and equipment preparation
The LB broth is prepared using LB powder
Its autoclaved and ampicillin and lactose are added
Inoculation is done by adding the transformed bacteria into
the media
Preparation of the bioreactor
25. Step 4- Fermentation:
This stage consists of small scaling( culture in shake flask)
to large scaling (fermentor)
Two chain are grown separately
The small scaling(early stage) uses shake flasks to do the
enrichment culture method for selecting the desired type of
E. coli for fermentation
26. The large scale where transfected bacterial cells are
transferred from small flask and replicated under optimal
condition such as temperature, pH in fermentation tank
Monitoring & control step
The bacterial cell process turn on the gene for human
insulin chain and then insulin chains are produced in the
cell
27. Down stream processing
When the products are subjected to series of processes
including separation and purification which are collectively
known as Downstream processing
• It is also known as product recovery
step 6: Isolation of crude product
Cells are removed from tank and are lysed using different
methods such as enzyme digestion, freezing etc.
The enzyme lysosome is used to digest the outer layer of the
bacterial cells
28. Step 7: purification of crude product
Centrifugation is conducted to helps to separate the
cell components
Stringent purification must be taken to remove any
impurities
Several chromatographic methods such as gel
filtration and ion exchanges are used
29. Step 8- Obtaining of insuin chain
The protein isolated after lysis consists of the fusion of B –
galactosidase and insulin chain
Its due to there is no termination or disruption
Cyanide bromide is used to split the protein chain at
methionine residues, allowing the insulin chains to be
obtained
31. Step 10 – PR- HPLC to obtain highly purified
insulin:
Its performed lastly to remove almost all the impurities to
produce highly purified insulin
The insulin then can be polished and packaged to be sold in
the industries
32.
33. Synthesis of the DNA containing the nucleotide sequences
of the A& B polypeptides chains of insulin
Plasmid + restriction endonuclease – insertion of the
insulin gene into plasmid ( circular DNA)
Restriction enzymes cut plasmidic DNA
DNA ligase agglutinates the insulin gene and the plasmidic
DNA .ie, plasmid + insulin gene
34. Introduction of recombinant plasmids into bacteria : E. coli
E.coli = factory for insulin production
Using E.coli- mutatants to avoid insulin degradation
Bacterium reproduces—the insulin gene replicates along
with plasmid E. Coli
35. Formed protein partly of a byproduct of A or B chain of
insulin
Extraction and purification of A and B chain
Connections of A and B chain by reaction forming disulfide
cross bridges results
pure synthetic human insulin
36. 2- Pro insulin method
Insulin is naturally synthesized as pre- pro insulinin the
pancrease
Its converted to proinsulin with the N- terminal signal
peptide enzymatically removed
The proinsulin coding sequence is inserted into the non –
pathogenic E . coli bacteria
Bacteria undergo fermentation , where they replicate and
produce proinsulin
37. The connecting sequence between the A &B chain is then
spliced away with an enzyme and resulting insulin is
prepared
The different downstream process is required for the
proinsulin process as compared to the chain A & chain B
process
Enzymatic proteolysis is a unique step for the proinsulin
production
38.
39.
40. Approval of recombinant insulin
The approval for using recombinant human insulin was
given in 1982
In 1986, Eli Lilly received approval for the manufacturing
and marketing of recombinant human insulin
The recombinant human insulin was marketed under the
trade name Humulin
41. APPLICATIONS
Several proteins are created from recombinant DNA
(recombinant proteins) and are used in medical
applications.
Hematopoietic growth factor.
Interferon's
Hormones
Recombinant protein vaccines
Tissue/bone growth factors and clotting factors
Biological response modifiers
Monoclonal/Diagnostic/Therapeutic antibodies
Recombinant proteins is extensively used in biotechnology,
medicine, and research
42. conclusion
Protein and peptide bases pharmaceuticals are rapidly
becoming a very important class of therapeutic agents and
are likely to replace many existing organic based
pharmaceuticals in the very future
Peptide and protein drugs will be produced on a large scale
by biotechnology processes and will become commercially
available for therapeutic use
43. References
1. Tripathi NK, Shrivastava A. Recent developments in
bioprocessing of recombinant proteins: expression hosts
and process development. Frontiers in Bioengineering
and Biotechnology. 2019;7.
2. Cheung AT, Dayanandan B, Lewis JT, Korbutt GS, Rajotte
RV, Bryer-Ash M, Boylan MO, Wolfe MM, Kieffer TJ.
Glucose-dependent insulin release from genetically
engineered K cells. Science. 2000 Dec 8;290(5498):1959-
62.
3. Glick BR, Patten CL. Molecular biotechnology: principles
and applications of recombinant DNA. John Wiley &
Sons; 2017 Jun 1.
