Use of different protien for development of medical treatments

1. Chapter 4
4. Simple Proteins And Therapeutic Agents
4.1. Proteins as therapeutic agents

2.  Therapeutic Protein
 Proteins which are engineered in the laboratory for
pharmaceutical use are referred to as therapeutic
proteins.
 Proteins which are absent or lowin individuals with an
illness such as Cancer, Infectious diseases, Hemophilia,
Anemia, Multiple sclerosis, Hepatitis B/C, etc. Are
artificially synthesized on large scale through
genetically modifiedhost cells and delivered.
 Introduced in 1920’s Human insulin is considered to bethe first
therapeutic protein.

3.  Protein Therapeutics
 Our body naturally produces many of the proteins that are act
as therapeutics.
 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.
 Antibody baseddrugs, anticoagulants, blood factors, growth
factors, hormones, interferon, bone morphogenetic proteins,
interleukins and thrombolytic.

4. 4.2. Choice of gene expression systems and
optimizing gene expression of therapeutic proteins
 The various protein expression systems are bacteria, yeast, insect
or mammalian systems.
 The following factors determine the type of expression system
used to produce recombinant proteins:
 Time spent in expressing the protein
 Ease of handling the expression system
 Amount of protein needed
 Type of post-translational modifications, number of disulfide
bonds
 Destination of the expressed protein

5.  The process of expressing a recombinant
protein in an expression systemrequires
the following information/components.
 Identification of the gene that
encodes the protein of interest
 Generation of cDNA from the
respective mRNA
 Selection of suitable expression vector
to insert the gene sequence
 Selection of suitable systemthat can
express the vector
 Appropriate screening and scaling
up methods

6.  The core steps involved in producing the desired recombinant
protein are similar across the various expression systems

8. 1. Bacterial protein expression systems –
Escherichiacoli
 Bacteria act as rapid and simple systems of expressing recombinant
proteins due to the short doubling time.
 The media required to culture them are not expensive and the
methods adapted to scale-up bio production are straightforward.
 The most widely used host system is E. colisince there is ample
knowledge about its genetics, genome sequence and physiology.
 The genetic manipulation is easy and it also grows to high
densities and is suitable for large-scale fermentations.
 However, the cell wall of E. coli contains toxic pyrogens and the
expressed proteins may have to be extensivelytested before use.

9. Bacterial protein expression system – Escherichia coli

10. Expression of protein using E. coliinvolves the following steps:
1. Use of competent E. coli cells to take up DNA sequence of
interest
2. Integration of the DNA into bacterial genome or circularization
of the DNA sequence to exist as a plasmid
3. Selection of transformed E. coli using a selection marker
(antibiotic)
4. Expansion of selected E. coli to a higher scale in appropriate
culture media, such as classic LB options or EnPresso™ B
Growth Systems
5. Isolation and purification of intracellular/secreted proteins

11. Features
 Low cost culture methods
 Flexible system– can carry plasmids with multiple promoters,
tags and restriction sites
 Easy to scale up and produce higher yield of protein

12. 2. Yeast protein expression systems – Saccharomyces
cerevisiae
 The highly developed genetic system, ease of use, reducedtime input
and costs have made S. cerevisiae an attractive organismfor the
expression and production of recombinant proteins.
 Yeasts are able to carry specificallydesignedplasmids and this
ability is valuablein a recombinant protein expression system.
 The plasmid used consistsof restriction sites that can be used to
insert the gene sequence of interest.
 Transformation of yeasts with the plasmid produces the desired
protein and can be appropriately scaled up.

13. Yeast protein expression system– Saccharomyces cerevisiae

14. Expression of protein using S. cerevisiaeinvolves the following steps:
1. Use of competent E. coli cells to take up DNA sequence of interest
2. Integration of the DNA into bacterial genome or circularization of the
DNA sequence to exist as a plasmid
3. Selection of transformed E. coli using a selection marker (antibiotic)
4. Expansion of selected E. coli in appropriate culture media, such as
classic LB options or EnPresso™ Y Defined Media
5. Isolation of DNA or plasmid
6. Transformation into yeast
7. Screen the transformants for integration of DNA into yeast
chromosome
8. Selection and scaling-up of high expressing yeast clones in appropriate
culture media
9. Isolation and purification of intracellular/secreted proteins

15. Features
 Low cost culture methods
 Suitable for both intracellular and secreted proteins
 Provides eukaryotic post-translational glycosylation of proteins
although it results in high

16. 3. Insect cell expression systems – Sf9 and Sf21
 The cell lines derived from Spodopterafrugiperda, Sf9 and Sf21,
are frequently usedas recombinant protein expression system.
 Baculovirus is a lytic, dsDNA virus, routinely amplified in cells
of the insects belonging to Lepidoptera family.
 It is noninfectious in vertebrates and its promoters are inactive
in mammalian cells.

17. Insect cell expression systems – Sf9 andSf21

18. Expression of protein using baculovirus/insect cells involves
the following steps:
1. Use of competent E. coli cells to take up DNA sequence of interest
2. Integration of the DNA into bacterial genome or circularization of the DNA sequence to
exist as a plasmid
3. Selection of transformed E. coli using a selection marker (antibiotic)
4. Expansion of selected E. coli in appropriate culture media
5. Isolation of DNA or plasmid
6. Preparation of a secondplasmidcontaining viral genesrequiredfor multiplicationand
formation of virus particles
7. Co-transfection of the expression plasmid and the second plasmid into Sf9 or Sf21
insect cells
8. Purification of the recombinant viral stock
9. Amplification of the virus and additional plaque assays to increase the titer of the
recombinant viral stock
10. Infection of the insect cells with high-titer recombinant virus stock
11. Isolation and purification of intracellular/secreted proteins

19. Features
 Recombinant protein is highly expressed during the last phases of
lytic cycle before cell lysis
 Suitable to generate both cytoplasmic and secreted proteins
 Disulfide bonds in proteins are efficiently generated
 Provide majority of post-translational modifications found in
mammalian cells

20. 4. Mammalian cell expression systems – HEK293 and CHO
 The main challenge of using mammalian cells for expressing recombinant
proteins is the reduced efficiency and levelsof the protein expressed.
 However, cell lines such as HEK293 and CHO have been developed as efficient
transient and stableexpressionsystems, respectively.
 HEK293 cells are transiently transfected using liposomes, calciumphosphateor
PEGas transfection reagents.
 Though transient expression is relatively easy and simple, scaling up is
technically challenging.
 CHO cells are commonly used to stably express large quantities of recombinant
proteins.
 The process involves transfection of DHFR-deficient CHO cells with the gene of
interest and a DHFR selection cassette.
 The transfected cells are then screened in the presence of methotrexate to obtain
stably transfected cell pools.
 The selection and expansion process takes 2-3 months

21. Mammalian cell expression systems – HEK293 and CHO

22. Transient or stableprotein expression using mammalian cells involves
the following steps:
1. Use of competent E. coli cells to take up DNA sequence of interest
2. Integration of the DNA into bacterial genome or circularization of the DNA
sequence to exist as a plasmid
3. Selection of transformed E. coli using a selection marker (antibiotic)
4. Optional storage of clones in CloneStable™
5. Expansion of selected E. coli in appropriate culture media
6. Isolation of DNA or plasmid
7. Transfection of the expression plasmid to mammalian cells using X-
tremeGENE™ Transfection Reagents.
8. Selection of stable clones
9. Expansion of clones for transient batch expression OR expansion of clones
for 2-3 months for stable expression
10. Isolation and purification of intracellular/secreted proteins

23. Features
 Transient expression is easy and rapid
 Provide all the post-translational modifications found in
mammalian cells
 Stable transfection results in higher yield, scalability and
reproducible production

25. 4.3. Production of Recombinant Therapeutic
Proteins
 Recombinant DNA technology is widely used in the production of
therapeutic agents such as;
 hormones, cytokines, growth factors, antibiotics, vaccines, blood
products like albumin, thrombolytics, fibrynolytics, clotting
factors such as factor VII, factor IX, tissue plasminogen activator
and many more.
 All these therapeutic agents can be produced in a large quantity
and also more economically by using rDNA technology.

26. 1. Human Insulin Production
 The hormone insulin is essential for the control of blood sugar
levels.
 Diabetes mellitus is a disease in which some people cannot make
insulin themselves.
 This disease kills many people in the world every year.
 Insulin has been used in the treatment of diabetes mellitus since
1922 when Leonard Thompson became the first human to receive
an injection of man-made insulin.

28. 2. Protropin (Human Growth Hormone) Production
 Human growth hormone is a polypeptide hormone synthesized in
the anterior pituitary.
 It promotes normal body growth and lactation and influences
various aspects of cellular metabolism.
 Dwarfism caused by insufficient production of HGH by the
pituitary gland.
 HGH can treat dwarfism – to help under sized children reach
their normal height and size
 Protropin was approved for treating human growth hormone
deficiency in children in May 1985.
 It has also been approved to be used in 67 countries where it is
marketed by licenses.

29. Protropin
 It is the first biotechnology-derived human growth hormone
treating thousands of children with GHI.
 Protropin (Synthetic versions by the trade names of Somatrem
and Somatropin etc.) is administered by injection during
childhood to stimulate and regulate body growth.

30. Productionof Genetic Engineered Protropin
1. A gene that produces growth hormone in humans is isolated.
2. The growth hormone production gene is inserted into the DNA
of E.Coli bacteria.
3. The bacteria recognise the inserted DNA as its own DNA and
begin to produce human growth hormone.
4. The bacteria multiply and produce the growth hormone in a
culturing media
5. Engineered E.Coli cell is allowed to multiply in the fermentor.
6. The growth hormone is extracted and purfied and is then ready
to be injected into children with GHI.

32. 3. Human Interferons-> to fight viral infections
 Scientists discovered an antiviral protein in 1957 that inhibited
growth of influenza virus in chicken embryos.
 It was named interferon because it interfered with the growth
of influenza virus.
 Anti viral proteins released by host cells (part of the immune
system)
 Interfere with viral multiplication
 Host cell specific but not virus specific
 Different types of cells in animals produce different interferons

33.  3 types of human interferon:
 alpha interferon (13 genes)
 beta interferon (2 genes)
 gamma interferon (1 gene)
 Alpha & beta usually produced early in viral infections
(viruses or viral RNA) and gamma appears later
 Presence of double-stranded RNA indicates cell is
infected
 Viral infected cells release alpha and beta interferons
Diffuse to neighboring cells -> Virus can’t replicate

34. Production Of Interferons By Genetic Engineering
 A DNA sequence coding for the product was synthesized and inserted
into E. coli.
 The recombinant product accumulates intracellularly as inclusion
bodies
 Large-scale manufacture entails an initial fermentation step.
 After harvest, the E. coli cells are homogenized and the inclusion bodies
recovered via centrifugation.
 After solubilization and refolding, the interferon is purified to
homogeneity by a combination of chromatographic steps.
 The final product is formulated in the presence of a phosphate buffer
and sodium chloride.
 It is resented as a 30 mg/ml solution in glass vials and displays a shelf-
life of 24 months when stored at 2–8°C`

35. 4. 4 Use Of Enzymes As Therapeutic Agents
 Recombinant enzymes can be used in various replacement
therapies.
a) DNase l
Is any enzyme that catalyzes the hydrolytic cleavage
of phosphodiester linkages in the DNA backbone, thus degrading
DNA.
Deoxyribonucleases are one type of nuclease, a generic term for
enzymes capable of hydrolyzing phosphodiester bonds that link
nucleotides.

36. Cont.,
 DNAse is an enzyme that cleans out the garbage DNA and other
cellular leftovers and dying cells.
 Recombinant Human DNAse reduces the viscosity of sputum in
Cystic fibrosis patients.
 Cystic fibrosis (CF) lung disease is characterized by a chronic
bacterial airway infection associated with a massive influx of
neutrophils.
 The rapid turnover of airway neutrophils leads to the
accumulation of large amounts of extracellular DNA which is
thought to hinder the clearance of respiratory mucus

37.  Cystic fibrosis (CF), also known as mucoviscidosis, affects most
critically the lungs, and also the pancreas, liver and intestine
 It is characterized by abnormal transport of chloride and
sodium across an epithelium, leading to thick, viscous
secretions i.e., Underlying cause is identified to the
mulfunction of ion transport
 Major clinical symptom is the production of viscous mucus in
the respiratory track

38. Cont,
Particularly affected are:
 The lungs, in which mucus compromises respiratory function.
 The pancreas, in which the mucus blocks its ducts in 85 percent
of cystic fibrosis patients, causing pancreatic insufficiency.
 This is chiefly characterized by secretion of greatly reduced levels
of digestive enzymes into the small intestine
 The reproductive tract, in which changes can render males, in
particular, subfertile or infertile

39.  Patients are susceptible to frequent lung infections and some patients
develop antibiotic resistance bacteria and hence, bacteria accumulate
leading to a viscous mucous secretion, clogging the bronchia and
bronchioles.
 Thick mucous = alginate that is secreted by the bacteria + DNA
released when bacterial cells and degenerating leucocytes that
accumulate in response to infection are lysed
 The role of DNase I can hydrolyse long polymeric DNAchainsinto
shorteroligonucleotides and the purified enzyme can be delivered in an
aerosol mist to the lungs of CF patients to prevent respiratory distress.
 The enzyme could decrease the mucus viscosity in the lungs and allow
patients for easy breathing, thus reducing the severity and pain of the
patient.
 This enzyme was approved for use by the US FDA in 1994

40. cont.
 Initial in vitro studies proved encouraging: incubation of the
enzyme with sputum derived from a cystfibrosis patient resulted
in a significant reduction of the sputum’s viscosity.
 Genentech received marketing authorization for the product in
December 1993, under the trade name Pulmozyme.
 Pulmozyme is produced in an engineered CHO cell line
harbouring a nucleotide sequence coding for native human
DNase.

41. b) Alginate lyase
 Alginate, a polysaccharide polymer of β-Dmannuronate and α-L
glucoronate, form an elastic gel, which is related to its viscosity
and molecularweight.
 The excretion of alginate by mucoid strains of pseudomonas
aeruginosa may infect the lungs of cystic fibrosis patient
contributing significantly to the viscosity of the mucous.
 Hence treatment of cystic fibrosis depends on the DNase l therapy
and depolymeriztion of the alginate which would help to clear
blocked airways.
 Since the enzyme alginate lyase can liquefy viscous bacterial
alginate which in addition to DNase l is good therapeutic agent of
cystic fibrosis

43. Asparaginase
 Asparaginase is an enzyme capable of catalyzing the hydrolysis of L-
asparagine, yielding aspartic acid and ammonia
 Asparaginases are enzymes expressed and produced by microorganisms.
 They are used in food manufacture, and in medicine to treat some cancers.
 Asparaginase is used with or without other anticancer (chemotherapy) drugs
to treat acute lymphocytic leukemia (ALL).
 It works by starving tumor cells of needed nutrients and slowing tumor cell
growth.

44. Cont,
 The discovery and development of asparaginase as an anti-cancer drug began in
1953, when scientists first observed that lymphomas in rat and mice regressed
after treatment with guinea pig serum.
 Later it was found out that it is not the serum itself which provoke the tumour
regression, but rather the enzyme asparaginase.
 After researches comparing different kinds of asparaginases, the one derived
from Escherichia coli and Erwinia chrysanthemi turned out to have the best anti-
cancer ability.
 E. coli has thereby become the main source of asparaginase due to the factor that
it is also easy to produce in large amount.

45. Mechanism of action
 The rationale behind asparaginase is that it takes advantage of the fact that
acute lymphoblastic leukemia cells and some other suspected tumor cells are
unable to synthesize the non-essential amino acid asparagine.
 whereas normal cells are able to make their own asparagine; thus leukemic
cells require high amount of asparagine.
 These leukemic cells depend on circulating asparagine. Asparaginase,
however, catalyzes the conversion of L-asparagine to aspartic acid
and ammonia.
 This deprives the leukemic cell of circulating asparagine, which leads to cell
death.

46. Production of Asparaginase
 Asparaginase production by a recombinant Pichia pastoris strain
harbouring S. cerevisiae ASP3 gene.
 Escherichia coli and Erwinia sp. enzymes have been frequently used.
 The combination of recombinant DNA technology and large-scale
culture processes has enabled enzymes to be produced in much larger
quantities than what can be obtained from natural sources.

47. Cont.,
 Applying this technology to therapeutic asparaginase
production would mitigate the aforementioned problems.
 Fed-batch techniques for culturing E. coli have improved
productivity, thereby reducing the formation of toxic by-
products, enhancing the downstream processing, lowering
overall production costs and reducing the technical effort
required .

48. ♥ Asparaginase: enzyme used in therapy of
adult leukemia ,.

49. Use of asparginase will
decrease aspargine level so
inhibiting tumor growth (depress
the tumor vitality).
Leukemic cells have little or no ability
to synthesize asparagine and must
scavenge it from blood to synthesize
their proteins

50. Side effect
 The main side effect is an allergic or hypersensitivity reaction; anaphylaxis is
a possibility.
 Additionally, it can also be associated with a coagulopathy as it decreases
protein synthesis, including synthesis of coagulation factors (e.g. progressive
isolated decrease of fibrinogen) and anticoagulant factor
(generally antithrombin III; so leading to bleeding or thrombotic events such
as stroke.
 Bone marrow suppression is common but only mild to moderate, rarely
reaches clinical significance and therapeutic consequences are rarely
required.

51. Debriding agents
 Debridement refers to the process of cleaning a wound by removal of foreign material and dead
tissue.
 Although debridement may be undertaken by physical means (e.g. cutting away dead tissue,
washing/cleaning the wound), proteolytic enzymes are also often used to facilitate this process.
 The enzyme is formulated in an aqueous-based cream, and in others it is impregnated into special
bandages.
Different debriding agents used;
 Surgical Debridement
 Autolyticdebridement
 Enzymatic Debridement

52. Enzymatic Debridement
 Highly selective method of wound debridement
 Uses naturally occurring proteolytic enzymes that are
manufactured by the pharmaceutical and healthcare
industry specifically for wound debridement.
 These exogenously applied enzymes work alongside the
endogenous enzymes in the wound.

53. Cont.,
 Several enzyme debriding agents have been developed
including bacterial collagenase, papain/urea,
fibrinolysin/DNAse, trypsin, streptokinase-streptodornase
combination, and subtilisin.
 Only the first three products are widely available
commercially in those markets where they are registered,
although availability varies geographically.

54. Enzymatic Debriders
 Trypsin
 Collagenase-BasedProducts
 Papain-BasedProducts
 Papain-Urea-Chlorophyllin Copper Complex

55. Trypsin
 Trypsin is a 24 kDa proteolytic enzyme synthesized by the
mammalian pancreas in an inactive zymogen form: trypsinogen.
 Upon its release into the small intestine, it is proteolytically converted
into trypsin by an enteropeptidase.
 Active trypsin plays a digestive role, hydrolyzing peptide bonds.
o Trypsin used medically is generally obtained by the enzymatic
activation of trypsinogen, extracted from the pancreatic tissue of
slaughterhouse animals.

56. Collagenase-Based Products
 Collagenase, is an enzymatic debriding agent derived from Clostridium histolyticum belonging
to the metallopeptidase family.
 It specifically hydrolyzes peptide bonds and digests all triple helical collagen and will not degrade
any other proteins lacking the triple helix.
 This is a unique feature of bacterial collagenase; since none of the other available proteases can
digest collagen.
 The enzyme liquefies necrotic tissue without damaging granulation tissue.
 Collagenase digests the lower portion of an eschar working from the bottom up giving the
appearance of working more slowly.
 Collagenase has been shown to be gentle to viable cells and might promote angiogenesis and
epithelialization.

57. Papain-Based Products
 Papain is a nonspecific proteolytic enzyme derived from the fruit of
the papaya tree (Carica papaya).
 Papain breaks down fibrinous material in necrotic tissue and
requires the presence of sulfhydryl groups, such as cysteine, for its
activity
 It does not digest collagen, and it requires specific activators that
are present in necrotic tissue in order to be stimulated.
 Urea is combined with papain because urea is able to expose the
activators of papain in necrotic tissue.
 Urea also denatures proteins, making them more susceptible to
proteolysis by papain.

58. Cont,
 The combination of papain and urea is approximately twice as effective at
digesting protein compared with papain alone.
 Papain-urea products should be applied daily with a moisture retentive dressing.
 Papain-urea preparations produce more exudate digesting eschar from the top
which may irritate the surrounding skin.
 Papain use is known to produce an inflammatory response and possibly as a
result of this, considerable pain is often experienced with the use of this method
 Hydrogen peroxide solution may inactivate papain as well as salts of heavy
metals such as lead, silver, and mercury have been shown to inactivate papain.

59. Papain-Urea-Chlorophyllin Copper Complex
 Chlorophyllin, an anti-agglutinin, has been added to
preparations of papain/urea in an attempt to reduce the
pain.
 In summery there are favorable clinical results that
reveal papain-urea chlorophyllin copper complex’s
proteolytic action thoroughly cleanses lesions of all
necrotic tissue debris and then maintains optimal
circulation so that affected tissue will benefit from both
hematological and nutritive elements.