The content is about the general description of genetic material and further two techniques of biotechnology. The content includes two topics. Firstly with introduction to biotechnology it describe about DNA, recombinant DNA (rDNA) technology, history, goals, procedure of rDNA technology, tools, techniques, application, demerits and products of rDNA technology. Second portion entitiled as Hybridoma technology. this includes the basic principle, production of monoclonal antibodies, merits demerits and drugs from monoclonal antibody. New pharmaceuticals derived from biotechnology is covered in last. All the content is referred from books and internet sources.

1. RECOMBINANT DNA
TECHNOLOGY
&
HYBRIDOMA TECHNOLOGY
Presented To Ms. Gulnaz Fatima
By Sachin kumar Vishwakarma
-M.Pharm (Pharmaceutical Chemistry)
1

2. BIOTECHNOLOGY
■ Biotechnology is now defined as “Any technological application that
uses biological systems, living organisms or derivatives thereof to
make or modify products or processes for specific uses”.
■ Biotechnology includes following technologies :-
1. Bioprocessing Technology
2. Cell Culture
3. Recombinant DNA Technology
4. Cloning
5. Protein Engineering
6. Biosensors
7. Nano-biotechnology
2

3. DNA (DEOXYRIBOSE NUCLIC ACID)
■ Nucleic acid was first isolated by Johann Friedrich Miescher in 1868 from the pus
cells. He called it as “nuclein” because of its acidic nature.
■ DNA is a double-stranded molecule, made up of the two chains of nucleotides.
■ Each nucleotide of DNA consists of three parts:-
a. Nitrogenous Base (adenine, guanine, thymine, cytosine )
b. Pentose Sugar
c. Phosphate group
■ In 1953, F.H.C. Crick of Great Britain and J.D. Watson, a young American scientist,
published a brief paper describing how these three components might be arranged
in DNA.
■ In the Watson–Crick double helix model, DNA resembles a spiral ladder.
■ The functions of DNA is carrying huge amounts of information that determines all
biological activities of an organism, and which is transmitted from one generation
to the next.
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4. 4

5. RECOMBINANT DNA (rDNA) TECHNOLOGY
■ Recombinant DNA technology, which is also called gene cloning is an
umbrella term that encompasses a number of experiments protocols
leading to the transfer of genetic information (DNA) from one organism to
another.
OR
DNA molecules that are extracted from different sources and chemically
joined together; for example DNA comprising an animal gene may be
recombined with DNA from a bacterium.
■ Recombination of DNA occurs naturally in microbes. In the 1970s and
1980s, scientists developed artificial techniques for making recombinant
DNA.
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6. Discovery of rDNA technology
■ Discovery of DNA structure Watson & Crick in 1953
■ Isolation of DNA ligase in 1967
■ Isolation of REase in 1970
■ Paul Berg generated rDNA technology in 1972
■ Cohen & Boyer in 1973 produced first plasmid vector (In their
experiments, they successfully recombined two plasmids (pSC 101
and pSC 102) and cloned the new plasmid in E.coli.)
■ capable of being replicated within a bacterial host
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7. Goals of recombinant DNA technology
■ To isolate and characterize a gene
■ To make desired alterations in one or more isolated genes
■ To return altered genes to living cells
■ Artificially synthesize new gene
■ Alternating the genome of an organism
■ Understanding the hereditary diseases and their cure
■ Improving human genome
7

8. Procedure of making rDNA
Following 4 steps are involved in process of recombinant DNA technology:-
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9. Isolation of DNA
9

10. Cutting of DNA
■ DNA can be cut into large fragments by
mechanical shearing.
■ Restriction enzymes are the scissors of
molecular genetics and used to cut the
DNA in a specific sequence and so
called as sequence-specific enzyme.
■ Sequence-specific enzyme are found in
bacteria.
■ Restriction endonucleases (REases)
recognize DNA base sequence that are
palindrome.
■ REase make staggered cuts with
complementary base sequences for
easy circulization.
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11. Joining DNA
11

12. Amplifying the recombinant DNA
■ Transforming the recombinant DNA into a bacterial host strain.
■ The cells are treated with CaCl2
■ DNA is added
■ Cells are heat shocked at 42 C
■ DNA goes into cell by a somewhat unknown mechanism.
■ Once in a cell, the recombinant DNA will be replicated.
■ When the cell divides, the replicated recombinant molecules go to both
daughter cells which themselves will divide later. Thus, the DNA is
amplified.
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13. Amplifying the recombinant DNA(Cont.)
13

14. rDNA TECHNOLOGY (Complete process)
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15. 15

16. TOOLS OF rDNA TECHNOLOGY
Following tools are used in recombinant DNA technology:-
1. Restriction endonucleases
2. DNA ligases
3. Vectors
4. Others
a. Mutagens (physical and chemical agents hat produce mutations)
b. Use of Reverse Transcriptase to synthesize DNA
c. Synthetic nucleic acids
16

17. Restriction endonucleases
■ Bacterial enzymes that cut DNA molecules only at restriction sites.
■ Bacterial origin = enzymes that cleave foreign DNA
■ Named after the organism from which they were derived;
Examples:- EcoRI is from Escherichia (E) coli (co), strain Ry13 (R), and first
endonuclease (I) to be discovered.
■ Protect bacteria from bacteriophage infection (Restricts viral replication).
■ Type I –Cuts the DNA on both strands but at a non-specific location at varying
distances from the particular sequence that is recognized by the restriction
enzyme .
■ Type II –Cuts both strands of DNA within the particular sequence recognized
by the restriction enzyme.
17

18. DNA ligases (DNA joining enzymes)
■ The cut DNA fragments are covalently joined together by DNA ligases.
■ These enzymes were originally isolated from viruses. They also occur in E.coli and
eukaryotic cells. DNA ligases actively participate in cellular DNA repair process.
■ DNA ligase joins (seals) the DNA fragments by forming a phosphodiester bond between
the phosphate group of 5’-carbon of one deoxyribose with the hydroxyl group of 3’-carbon
of another deoxyribose.
18

19. Vectors (Carriers)
In genetic engineering, the vector (carrier) is the most widely used
method for the insertion of foreign, or passenger, genetic material into a
cell.
Types:-
1. Plasmid vectors
2. Lamda (λ) phase vectors
3. Cosmid vectors
4. Expression vectors
5. Yeast Artificial Chromosomes (YACS)
6. Bacterial Artificial Chromosomes (BACS)
19

20. Plasmid Vectors
■ Plasmids are extrachromosomal, double stranded,
circular, self-replicating DNA molecules.
■ They replicate independently of the bacterial
chromosome.
■ Useful for cloning DNA inserts less that 20 kb
(kilobase pairs).
■ Inserts larger than 20 kb are lost easily in the
bacterial cell.
■ Nomenclature of plasmids:- It is a common
practice to designate plasmid by a lower case p,
followed by the first letter(s) of researcher(s)
names and the numerical number given by
theworkers.
■ Example:- pUC19 is plasmid from University of
California and researchers designated as 19.
20

21. Techniques used in rDNA technology
Following techniques are used in rDNA technologies:-
■ PCR (Polymerase Chain Reaction )
■ Gel electrophoresis
■ Cloning libraries
■ Restriction enzyme mapping
■ Nucleic Acid Hybridization
■ DNA Microarrays
21

22. PCR (Polymerase Chain Reaction)
■ The polymerase chain reaction (PCR) is a technique by which small
samples of DNA can be quickly amplified, that is, increased to
quantities that are large enough for analysis.
■ Starting with just one gene-sized piece of DNA, PCR can be used to
make literally billions of copies in only a few hours.
■ Each strand of the target DNA will serve as a template for DNA
synthesis.
■ Short pieces of nucleic acid called primers are also added to help
start the reaction.
■ The primers are complementary to the ends of the target DNA.
■ After each cycle of synthesis, the DNA is heated to convert all the new
DNA int single strands. Each newly synthesized DNA strand serves in
turn as a template for more new DNA.
22

23. PolymeraseChainReaction
23

24. Applications of rDNA technology
■ Agriculture: growing crops of your choice (GM food), pesticide resistant
crops, fruits with attractive colors, all being grown in artificial conditions.
■ Pharmacology: artificial insulin production, drug delivery to target sites.
■ Medicine: gene therapy, antiviral therapy, vaccination, synthesizing
clotting factors.
■ Other uses:- fluorescent fishes, glowing plants etc.
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25. DEMERITS Of rDNA TECHNOLOGY
■ High cost process
■ Require sophisticated environment
■ More steps for a single products (eg.:- processing of the recombinant
protein)
■ Chemical instability of products (hydrolysis, deamidation,
racemization, oxidation)
■ Physical instability of proteins (denaturation, adsorption, aggregation,
precipitation)
■ immunogenicity
25

26. DRUG DISCOVERY rDNA
TECHNOLOGY
■ One of the most medically significant applications of
recombinant DNA technology is the modification of bacterial
cells to make substances useful to humans.
■ To make bacterial cells produce human proteins, a human
DNA gene with the information for synthesizing the protein is
inserted into the vector.
26

27. RECOMBINANT DNA PRODUCTS
A. Hormones
B. Cytokines
C. Interferons
D. Interleukins
E. Enzymes
F. Vaccines
27

28. A. Hormones
a. Human insulin:- Human insulin was the first pharmacologically active
biological macromolecule to be produced through genetic
engineering. The FDA approved the drug in 1982 for treatment of
type1 (insulin-dependent) diabetes.
The insulin protein is a two-chain polypeptide containing 51 amino
acid residues. Chain A is composed of 21 amino acids, and chain B
contains 30.
Brand names:- HUMUIIN, NOVOLIN
b. Glucagon,
c. Human growth hormone,
d. Follicle-stimulating hormone
28

29. Production
of
recombinant
insulin
29

30. B. Cytokines
a. Erythropoietin Alfa:- It is a glycoprotein that stimulates red blood cell
production. Epoetin alfa (EPOGEN) is a 165 –amino acids
glycoprotein that is manufactured in mammalian cells by rDNA
technology.
Brand names:- EPOGEN, PROCRIT, EPOETIN ALFA
Epoetin is indicated to treat anemia.
b. Filgrastim
c. Sargramostim
d. Becapermin
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31. C. Interferons
 The interferons are family of small proteins or glycoproteins of
molecular masses ranging from 15,000 to 25,000 Da and 145 to 166
amino acids long.
 Their action is bimodal. The immediate effect is the recruitment of
natural killer cells to kill the host cell harbouring the virus. Interferons
then induce a viral resistance in cell in the immediate vicinity,
preventing spread of the virus.
a. Interferon Alfa-2a (recombinant)
b. Interferon Alfa-2b (recombinant)
c. Interferon Beta-2a (recombinant)
d. Interferon Beta-2b (recombinant)
31

32. D. Interleukins
a. Aldeslukin
b. Oprelvekin
c. Tumor necrotic Factor (recombinant):- The TNFs are members of a
family cytokinine.s that are produced primarily in the innate immunity
system by activated mononuclear phagocytes.
Brand name:- ETANERCEPT, ENBREL
32

33. E. Enzymes
a. Blood-Clotting Factors:-
• Tissue Plasminogen Activator
• Reteplase
• Tenecteplase
• Factor VIII
b. Anticoagulants:-
• Lepirudin recombinant:- It is an rDNA- derived protein produced in
yeast, has a molecular mass of approximately 7,000 Da.
33

34. F. Vaccines
 Vaccine production is a natural application of rDNA technology, aimed
at achieving highly pure and efficacious products.
 Current, there are four rDNA vaccines approved for human use.
a. Recombivax and Engerix-B:- For immunization against hepatitis B
virus. Both contain a 226-amino acid polypeptide chain.
b. LYMErix
c. Comvax:- It is a combination of Haemophilus influenza type b
conjugate and hepatitis B (recombinant).
34

35. HYBRIDOMA
TECHNOLOGY
35

36. HYBRIDOMA TECHNOLOGY
■ A hybridoma is a hybrid cell obtained by fusion of B lymphocyte with
usually a tumor cell of antibody forming system or B lymphocyte
(these are called myelomas).
■ In 1975, George Kohler and Cesar Milstein (Nobel Prize 1984) made
this dream a reality. They created hybrid cells that will make unlimited
quantities of antibodies with defined specificities, which are termed
as monoclonal antibodies (McAb). This discovery, often referred to as
hybridoma technology, has revolutionized methods for antibody
production.
■ The term hybridoma was coined by Leonard Herzenberg in 1975.
36

37. PRINCIPLE
■ This is based on the fusion between myeloma cells (malignant plasma
cells) and spleen cells from a suitably immunized animal. Spleen cells die
in a short period under ordinary tissue culture conditions while myeloma
cells are adopted to grow permanently in culture.
■ From the growing hybrids, individual clones can be chosen that secrete
the desired antibodies (monoclonal origin). The selected clones like
ordinary myeloma cells can be maintained indefinitely.
37

38. MONOCLONAL ANTIBODY
DEVELOPMENT PROCESS
■ Immunization of appropriate animals with antigen (need not be pure)
under study.
■ Fusion of suitable drug resistant myeloma cells with plasma cells,
obtained from the spleen of the immunized animal.
■ Selection and cloning of the hybrid cells that grow in culture and
produce antibody molecules of desired class and specificity against
the antigen of interest.
38

39. 39
ProductionofMonoclonal
Antibodies

40. MERITS OF HYBRIDOMA TECHNOLOGY
■ Hybridoma technology can make available highly specific antibodies in
abundant amounts.
■ The clones once developed are far cheaper than the traditionally
employed animals
■ (horses, rabbits) for producing antibodies.
■ The clones developed from the hybrids will also ensure constancy of the
quality of the product and will also avoid the batch to batch variation
inherent in the conventional methods.
40

41. DEMIRITS OF HYBRIDOMA TECHNOLOGY
■ Many patients develop immune response to monoclonal antibodies
produced by mice, as these are foreign proteins.
■ Hybridoma culture may be subjected to contamination.
■ System is only well developed for mouse and rat and not for the other
animals.
■ More than 99% of the cells do not survive during the fusion process-
reducing the range of useful antibodies that can be produced against
an antigen.
41

42. APPLICATIONS OF HYBRIDOMA
TECHNOLOGY
1. Production of Monoclonal antibodies
2. Early detection of Pregnancy
3. Diagnosis of HIV (detection of presence of HIV antibody)
4. Identification of types of Leukaemias (Monoclonal antibodies can
distinguish subsets of B cells and T cells)
5. Diagnosis and treatment of Cancer
42

43. MONOCLONAL ANTIBODY DRUGS
 RITUXIMAB
 GEMTUZUMAB OZOGAMICIN
 BASILIXIMAB
 DACLIZUMAB
 ABCIXIMAB
 INFLXIMAB
 ALEMTUZUMAB
43

44. NEW PHARMACEUTICALS
DERIVED FROM
BIOTECHNOLOGY
44

45. INTRODUCTION
■ Biotechnology encompasses any technique that uses living organisms
in the production or modification of products.
■ Thus, biotechnology products broadly refer to biopharmaceutical drugs
generated through researches in cell biology, genetics and
recombinant DNA technology.
45

46. PHARMACEUTICALS
DERIVED FROM
BIOTECHNOLOGY
46

47. A. rDNA Technology
a. Hormones
b. Cytokines
c. Interferons
d. Interleukins
e. Enzymes
f. Vaccines
47

48. B. Hybridoma technology
a. Antibody production
b. Diagnosis of diseases (ELISA, RIA)
c. Treatment of various diseases
d. Development of various drugs
48

49. C. Gene therapy
a. Treatment of Diseases on Genetic level
b. Modification in hereditary characteristic
c. Genetically modified plants (increase drug amount/potency )
d. Genetically modified animals (biological products and organs)
e. Transgenic animals
49

50. D. Fermentation
a. Production of wine
b. Production of Antibiotics
50

51. PRODUCTS OF BIOECHNOLOGY
 Anticoagulant drug –Lepirudin (Refludan®)
 Antisense drugs – Fomivirsen sodium injection
is approved for local treatment of cytomegalovirus
(CVM) retinitis in patients with AIDS
 Efavirenz (Sustiva®) – A non-nucleoside
reverse transcriptase inhibitor and the first anti-HIV
drug to be approved by FDA for once daily dosing in
combination with other anti-HIV drugs.
51

52. PRODUCTS OF BIOECHNOLOGY (Cont.)
 Clotting factors - Kogenate®, Recombinate®) –
Recombinant anti-hemophiliac factor indicated for
the treatment of classical hemophilia A in which there is a
demonstrated deficiency of clotting factor (Factor VIII )
 Growth factor – Becaplemin (regranex®)
 Systemic growth hormone - Somatropin recombinant
(Humatrope®)
52

53. PRODUCTS OF BIOECHNOLOGY (Cont.)
 Colony stimulating factors -
 Erythropoietin
 Interferon
 Vaccines – Genetically engineered vaccines use a
synthetic copy of the protein coat of a virus to “fool” the
body immune system into mounting a protective
response.
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54. REFERENCES
1. Tortora,G.J.; Funke,B.R.,“Microbiology”; 10th Edition; Benjamin Cummings;
San Francisco.
2. Satyanaryana,U.; Chakrapani,U.; “Biochemistry”; Elsevier, New Delhi.
3. Black,J.G.;”Microbiology”; 8th Edition; John Wiley and Sons.
4. Willey,J.M.; Sherweood,L.M.; “Prescott,Harley, and Klein’s Microbiology”;
7th Edition; Mc Graw Hill,Boston.
5. Beale,J.M.; Block,J.H.; “Wilson and Gisvold’s Textbook of Organic
Medicinal and Pharmaceutical Chemistry”; 12th Edition; Lippincott
Williams & Wilkins, New Delhi.
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55. THANK YOU FOR YOUR ATTENTION
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