Lecture Notes in Pharmaceutical Biotechnology for Pharmacy Students..

1. Pharmaceutical Biotechnology/Lec. II
Dr. Fawaz A. Mustafa
PhD in Medical Physiology and Pharmacology
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2. Recombinant DNA Technologies
• Recombinant DNA (rDNA) molecules are prepared in
vitro via genetic recombination to merge more than one
source of the genetic material
• Therefore this new created sequences would not be exist
in the ordinary biological systems.

3. Recombinant DNA Technologies
• Genetic modification of organisms is done by Fusion of
any DNA fragment to DNA molecules able to maintain
themselves by autonomous replication. Such molecules
called replicons

4. Recombinant DNA

6. Recombinant DNA
• Thus Recombinant DNA technology or DNA cloning
technology:
(Application of plasmids in biotechnology)
• Fusing foreign DNA fragment to the isolated plasmid in
order to create a recombinant DNA molecule called
replicons
Replicons used as carriers for foreign DNA fragments are termed
vectors (include plasmids from bacteria or yeast, or DNA from
bactriovirus, animal virus or Plant virus).
Foreign DNA- isolated either from microbial, plant
or animal cell

7. Recombinant DNA Technologies
• Restriction enzyme used to cut DNA at a specific site
• Restriction enzymes have the remarkable property of being
able to recognize DNA on the basis of the specific host
marking
• The very selective action of the restriction enzymes is the
basis for their application in the recombinant DNA
technology
• A plasmid with only one recognition site for the restriction
enzyme EcoRI is treated with this enzyme
• The double-stranded DNA is then asymmetrically cut at the
recognition site, encompassing six bases, namely GAATTC
• This leads to linear DNA with typical short single-stranded
ends

8. Recombinant DNA Technologies
• If foreign DNA with recognition sites for the enzyme
EcoRI is also cut with this enzyme, fragments with
single-stranded ends characteristic for EcoRI are formed
• When the open vector and the foreign DNA fragments
are brought together, the various single-stranded ends
may recombine due to the presence of complementary
bases

9. Recombinant DNA Technologies
• Ligase enzyme used to close circular recombinant DNA.
• Introduction of recombinant DNA into host cell leads to
form (Transformant).
• Vector replicate in the host, thus all daughter cells will
inherit precise copy (a clone) of the recombinant DNA
molecule.

10. Monoclonal Antibodies
• Typically made by fusing myeloma cells with the spleen
from a mouse that has been immunized with the desired
antigen

12. Formulation of Biotech Products, Including
Biopharmaceutical Considerations

13. Microbial considerations
1. Sterility
• Most proteins are administered parentally and it should
be sterile (E.g.: Recombinant/purified protein vaccines
consist of protein antigens like Hepatitis B vaccine I.M.)
• (But are sensitive to heat and other sterilization
treatments) so cannot withstand [autoclaving, gas
sterilization, or sterilization by ionizing radiation]
• Then how to sterilize proteomic pharmaceuticals? What
about “aseptic conditions”?

14. Rules in the pharmaceutical industry for
aseptic manufacture
1. Equipment and excipients (autoclaved, or sterilization
by dry heat (>160oC), chemical treatment or gamma
radiation).
2. Filtration techniques used for the removal of micro-
bacterial contaminants .
A. Pre-filters (remove the bulk of the bio-burden and
other particulate materials).
B. The final 'sterilizing' step is filtration through 0.2 or
0.22 µm membrane filters.

17. Microbial considerations
2. Viral decontamination
A. Recombinant DNA products are grown in microorganisms, these
organisms should be tested for viral contaminants (like using
microscope) and appropriate measures should be taken if viral
contamination occurs:
i. To get rid of viral materials in the final product (by using
filtration, precipitation).
ii. Inactivation of viral contaminants in the final product (by using
heat, radiation).
B. Excipients with a certain risk factor (such as blood- derived
human serum albumin) should be carefully tested before use and
their presence in the formulation process should be minimized.

18. Microbial considerations
3. Pyrogen removal
Pyrogens are compounds that induce fever, that are of two
types endogenous pyrogens and exogenous pyrogens.
• Exogenous pyrogens (pyrogens introduced into the
body, not generated by the body itself) can be derived
from bacterial, viral or fungal sources.
• Bacterial pyrogens are mainly endotoxins shed from
gram negative bacteria (They are lipopolysaccharides
[cell wall from bacteria]) (stable under standard
autoclaving conditions! How to get rid of them?).

19. Fever inducing mechanism
Endotoxin pyrogen enters blood stream and binds to
lipopolysaccharide binding PTNs
Then bind to reticuloendothelial system (cells’ receptor
of circulate mononuclear and polynuclear cells: CD14
of macrophages)
Production and release of proinflammatory cytokines (IL-
1, IL-6, TNF-α, interferon, etc) of endogenous pyrogen
Inflammation and Fever (due to activation of arachidonic
acid pathway).

20. A general structure of endotoxin is shown in the following Figure
Responsible
for harmful
and useful
activities of
endotoxin

21. General notes on endotoxins
 They aggregate and form large units with M.wt. of over 106 in
water and sharing a general property of high negative electrical
charge.
 In addition to their tendency to adsorb to surfaces, thus
indicating that these compounds are amphipathic (hydrophilic
and lipophilic) in nature as they are lipopolysaccharides.
 Stable under autoclaving conditions, but break down when
heated in the dry state (thus equipment and containers are treated
at temperature above 160oC for prolonged periods (e.g. 30 minutes
dry heat at 250oC)).

22.  Pyrogen removal of recombinant products derived from
bacterial sources should be an integral part of preparation
process:
Ion exchange chromatographic procedures (utilizing its
negative charge) can effectively reduce endotoxins levels in
solution
 Excipients used in the protein formulation should be
essentially endotoxins-free
 For solutions, water for injection (compendia standards) is
(freshly) distilled or produced by reverse osmosis

23. Ion exchange chromatography

24.  The aggregated endotoxins cannot pass
through the reverse osmosis membrane.
 Removal of endotoxins immediately
prior to the filling of the final container
can be accomplished by using activated
charcoal or other materials with large
surfaces offering hydrophobic
interactions.
 Endotoxins can also be inactivated on
utensil surfaces by oxidation (e.g.
peroxide) or dry heating (e.g. 30
minutes dry heat at 250oC).