Pharmaceutical microbial technology

1. • Mechanisms of action of antibiotics
• Structure and function of some antibiotics
• Drug resistance mechanisms
 Antifungal and antiviral drugs
 Industrial drug production methods
 Production of penicillin/chlorotetracyclin
 Use of recombinant technology in drug production
 Strategy for production of human insulin by E.coli or B. subtilis
The human genomic DNA library
 Transgenic animal technology
 Vaccine production
PHARMACEUTICAL MICROBIAL BIOTECHNOLOGY

2. PRODUCTION OF PENICILLIN
 Substrate: excess glucose leads to low yield, so
either supply slow steady input of glucose, or use
disaccharide lactose along with limiting N to enhance
drug production (stimulates production of 2° products
in idiophase).
 The medium is supplemented with phenylacetic acid,
which to add benzyl side chain to final product.
 The broth is removed from culture by filtration; crude
drug is extracted by adsorption, precipitation,
refinement by crystallization.

3. The kinetics of the penicillin
fermentation with Penicillium
chrysogenum.

4. Penicillin fermentation without addition of side-chain
precursors produces the natural penicillins (penicillin G)
To produce the most useful penicillins, those with
activity against gram-negative Bacteria, a combined
fermentation and chemical approach is used that leads to
the production of semi-synthetic penicillins.
Penicillin G can be chemically and or enzymatically
modified to make a variety of penicillin derivatives with
slightly different properties
These semi-synthetic penicillin derivative include
penicillin V, penicillin O, ampicillin and amoxycillin
PRODN. OF PENICILLIN AND ITS DERIVATIVES

5. PRODN. OF PENICILLIN AND ITS DERIVATIVES

6. PRODUCTION OF CHLORTETRACYCLINE
 Chlortetracycline is produced from Streptomyces
aureofaciens.
 Corn steep liquor and sucrose are used as carbon
source in large-scale production (glucose is avoided
because it causes catabolite repression of antibiotic
production).
 The production scheme for chlortetracycline is shown
in the next slide.

8. GEN. APPROACH FOR DESIGNING THE PRODN. OF
SYNTHETIC DRUGS
 Assumption: Drugs are small molecules which function by
interacting with specific enzymes/receptor.
 Goal: To develop a small molecule that specifically interacts with
active site of target enzyme/receptor.
 Strategies:
- Identify target protein and determine its 3-D structure.
- Hypothesize complementary small molecules.
- Chemically synthesize the proposed molecules.
- Test the biochemical function of synthetic molecules.
- Test the physiological function of synthetic molecules
- Modify molecules to optimize efficacy.

9. USE OF RECOMBINANT TECHNOLOGY IN DRUG
PRODUCTION
Introduction
 This technology takes advantage of the fact that MOs conduct
secondary metabolism leading to production of drugs.
 By growing large cultures in industrial setting, one can get the
MOs to produce the crude material from which the purified
product can be obtained.
 It is possible to manipulate MOs in culture and in particular,
manipulating high amounts of desired product to be produced.
 Recombinant technologies take advantage of industrial
microbiology by manufacturing microbes that can produce drugs
that are not naturally synthesized by microbes i.e, bacteria or
fungi that produce non-native metabolites.

10. USE OF RECOMBINANT TECHNOLOGY IN DRUG
PRODUCTION
 Several diseases are caused by a deficiency in production
of a protein or hormone.
Examples: Insulin deficiency leads to Diabetes; Factor VIII
deficiency causes hemophilia, etc.
 Traditionally, the medical approach has been to isolate
these components from other animals or cadavers, when
they are similar enough to also be active in humans (e.g.
porcine insulin from pancreas tissue, somatostatin from
human cadaver brain tissue, etc).
Problem: How might it be possible to produce a MO that can
synthesize a human protein like insulin? This would be an
advantage because it would open up the possibility for large scale

11. USE OF RECOMBINANT TECHNOLOGY IN DRUG
PRODUCTION
Advantages of MOs in recombinant technology
 Microorganisms are much cheaper, direct, unlimited source of
human drug products.
Example: 2 Liters of E coli can produce as much somatostatin as
that harvested from 100 sheep brains or cadavers!

12. THE APPLICATION OF GENETIC MANUPULATION TECHNIQUES
Defn. Genetic manipulation is the transfer of DNA between
different species using both in vivo and in vitro techniques, i.e.
genetic material derived from one species may be incorporated
into another species where it is expressed.
In vivo techniques: They make use of phage particles which pick
up genetic information from chromosomes of one species and
infect another species thus introducing the genetic information
from the first host. The information from the first host may then
be expressed in the second host.
In vitro techniques: It involves insertion of information into the
vector by in vitro manipulation followed by insertion of carrier
and its associated ‘extra’ DNA into the recipient cell.

13. BASIC REQUIREMENTS FOR THE IN VITRO TRANSFER AND EXPRESSION
OF FOREIGN DNA IN A HOST BACTERIUM
 A ‘vector’ DNA (*plasmid or **bacteriophage ) molecule
capable self incoparation into a host cell.
 Method of splicing (joining/uniting) foreign genetic information
into the vector. Use of restriction endonuclease and ligase
enzymes.
 Introduction of recombinant DNA into the host cell and selecting
for their presence. Use of drug resistance markers
 Assessment of the ‘foreign’ gene’s product from the population.
Achieved by testing the host cell for selected characteristics e.g.
testing if the host can confer drug resistance, etc.
*Plasmid is an extrachromosomal genetic element (DNA) found
among various strains of E. coli and other bacteria.
**Bacteriophage is the virus that infect bacterial cells.

14. BASIC REQUIREMENTS FOR THE IN VITRO TRANSFER AND
EXPRESSION OF FOREIGN DNA IN A HOST BACTERIUM

15. RESTRICTION ENZYME RECOGNITION SEQUENCE AND ENZYME
ACTION
 Restriction enzymes show
specificity for certain
substrates (DNA).
 They bind and digest DNA
within specific sequence of
nucleotides (restriction site
or recognition sequence)
 They are commonly referred
to as 4-base pair cutters or
6-base pair cutters ie
recognize the restriction
sites with a sequence of 4 or
6 nucleotides.
 Digestion of DNA by
enzyme EcoRI or KpnI
produces DNA fragments
with cohesive ends.

16. THE ERA OF GENETIC ENGINEERING/RECOMBINANT DNA
TECHNOL. IN DRUG PRODN.
Introduction
 Because of the almost universal nature of the genetic
language (genetic information is encoded in linear
sequences of A, T, G, and C in a DNA strand), it is
possible to transfer human genes into bacterial cells, then
those cells should be able to synthesize human gene
products.
 In practice, genes can be easily shuttled into microbeby
the use of vectors such as plasmids or viruses.
 The technology takes advantage of natural routes for
genetic transmission. The bacteria will automatically
secrete the newly synthesized products.

17. 1. Isolate human insulin gene
2. Subclone into plasmid vector
3. Transform E coli with recombined plasmid
4. Select transformants that contain plasmid (antibiotic
resistance)
5. Screen transformants for ones producing the human
protein
6. Grow up large amount of the insulin-producing strain,
harvest insulin from growth medium – secreted as a
secondary metabolite.
7. Purify, test quality (absence of bacterial toxins).
STRATEGY FOR PRODUCTION OF HUMAN INSULIN BY E.
COLI OR B. SUBTILIS

18. STRATEGY FOR PRODUCTION OF HUMAN INSULIN BY E.
COLI OR B. SUBTILIS (CONT’D)
NB:
 This is called a recombinant protein because it is a human protein
that is synthesized in a non-human species. In this case, an
eukaryotic protein is synthesized by a prokaryotic organism!
 In some cases, the protein product is not correctly processed, so
after purification, it must be chemically modified to activate it.
 Currently, S. cerevisiae (eukaryotic ) is also used in the production
of human proteins like insulin. These yeasts are also able to be
transformed by similar means to secrete desired products.

19. HUMAN GENOMIC DNA LIBRARY
• Chromosomal DNA from the
human tissue (e.g. pancreas for
insulin) is isolated and then
digested with restriction enzymes.
• A plasmid is digested with the
same enzyme and DNA ligase is
used to fuse the genonic DNA
pieces and the plasmid DNA
randomly.
• Recombinant plasmids are then
used to transform bacteria (E.
Coli), and each E. coli clone will
contain a plasmid with a human
genomic DNA fragment.
• Each clone is considered a “book”
in this “library” of DNA
fragments.

21. TRANSGENIC ANIMAL TECHNOLOGY
Some current examples
• Human monoclonal antibodies (MAbs) produced by transgenic
mouse.
• Human MAbs produced by corn & soybean.
• Human Hb synthesized in transgenic pigs a blood substitute.
• Human tissue plasminogen activator (anticoagulant) produced in
transgenic goats.
• With progress in cloning technologies ( e.g. dolly the sheep), it
may soon be possible to produce herds of transgenic cattle, all
producing a human protein of interest.
• Human protein production in cows: If the gene is modified so
that it is only expressed in mammary tissue, then potentially,
human product could be isolated simply from milk.

22. MONOCLONAL ANTIBODIES (MABS)
Monoclonal antibody= An antibody produced from a
single clone of cells. It has uniform structure and
specificity.
Some terminologies
B lymphocite: a cell of the immune system that
differentiates into antibody-producing cell.
Myeloma cells: Malignant tumor of antibody-producing
cells (that no longer produces antibody of its own).
Hybridoma: a fusion of an immortal (tumor) cell with a
lymphocyte to produce an immortal lymphocyte.

23. HOW TO USE HYBRIDOMA TECHN. TO PRODUCE
MONOCLONAL ANTIBODIES
1. A mouse is immunized with the antigen of interest and left for
weeks to produce B lymphocytes.
2. The spleen tissue (rich in B lymphocytes) is removed and fused
with myeloma cells to make hybridomas.
3. The hybidomas are grown in in vitro culture containing
hypoxanthine, aminopterin and thymidine compounds ( so-
called HAT medium).
4. Fused hybrids are selected for antibody production.
5. Positive antibody-producing cells are cloned.
6. Desired clones are cultured and frozen.
7. Monoclonal antibodies are purified.
Nb. Hybridoma tumors can be kept alive in mouse

25. MONOCLONAL ANTIBODIES (CONT’D)
DIFFERENCES BTW MONOCLONAL AND POLYCLONAL ANTIBODIES
Monoclonal antibodies Polyclonal antibodies
Contains single antibody
recognizing only a single
determinant on an antigen.
Contains many antibodies
recognizing many determinants
on an antigen.
Single class of antibody is
produced.
Various classes of antibodies are
produced.
Can make a specific antibody
using an impure antigen.
Can make a specific antibody
using only a highly purified
antigen.
Highly reproducible Reproducibility and
standardization is difficult.

26. VACCINE PRODUCTION
 Vaccine is a biological molecule/preparation that
provides active acquired immunity to a
particular disease
Earliest strategies relied on:
 Production of weakened or killed form of the pathogen -
but still able to elicit an immune response ( e.g. polio
virus)
 Availability of a non-virulent strain or similar non-
pathogenic strain that could be used to elicit a cross-
reactive immune response ( e.g. cowpox, smallpox).
 Limitation of this strategy: non-virulent strains have not
been identified for most infectious viruses and bacteria,
and attenuation procedures are not always 100%
complete, leading to the possibility of introducing live
pathogenic organisms via vaccine delivery itself.

27. NEW APPROACHES FOR PRODUCING VACCINE
1. Creating recombinant viral particles to serve as vaccine
vectors e.g. vaccinia virus.
 Attenuated forms of vaccinia virus (which is responsible
for cowpox and smallpox) have existed for many years.
 This is an enveloped, DNA virus similar to many other
animal viruses (enveloped instead of naked like many
bacteriophages).
 It is possible to grow this virus in culture: grow up
mammalian cell line, then infect it with vaccinia virus,
culture produces large amounts of viral particles, which
bud from the surface of the cultured cells.

28. NEW APPROACHES FOR PRODUCING VACCINE
(CONT’D)
2. Overexpressed viral proteins or combinations of viral
proteins in bacteria or yeast are isolated and used as
immunogen in vaccine mixtures.
 Isolated proteins may not present the same traits as in
the native viral particles.
 It is possible to genetically modify the viral genome so
that non-vaccinia proteins are produced.
 Using genetic recombination it is possible to produce
protein antigens for use in synthesizing vaccines. (see
diagram below)
Imunogen Antigen that is capable of inducing immune
response.

29. CLONING OF FOREIGN DNA INTO VACCINIA VIRUS
• Gene for different viral
surface protein can be
inserted and will then be
expressed on the surface of
infected cells and ultimately
incorporated into the
envelope of the new viral
particles.
• Modified viral particles are
harvested and used, as
vaccine – essentially these
particles present the new viral
antigen to the immune system
so that it can respond with a
primary response.
• Gen. Modified vaccinia is
used as vaccine: it is safer,
more reproducible and can be

30. OTHER VACCINES UNDER INVESTIGATION/ DEVELOPMENT
 Hepatitis B – recombinant protein used as immunogen.
 Malaria vaccine – Plasmodium falciparum surface
protein expressed in recombinant yeast cultures, trial
conducted in the Gambia.
 HIV vaccine –1st attempts were made in early 1990s
using recombinant gp120 protein produced in
mammalian cells. The trials proved ineffective.
 AIDS vaccine (VAX BB) trials - currently underway in
US and Thailand – includes a mixture of different
gp120 variants.
 HIV1 env/gag/pol genes - inserted into vaccinia viral
genome under vaccinia viral gene control.
 gp120/CD4 complex- under lab investigation as
potential immunogen vaccine.