10 - Pharmaceutical Biotechnology (PCS521) - Lecture Ten.
10 - Pharmaceutical Biotechnology (PCS521) - Lecture Ten.
10 - Pharmaceutical Biotechnology (PCS521) - Lecture Ten.
Biotechnology
(PCS521)
Lecture Ten
Dr. Asmaa Ramadan
Lecturer, Microbiology & Biotechnology Department – College of Pharmacy
Arab Academy for Science, Technology and Maritime Transport (AASTMT)
Therapeutic Applications of
Pharmaceutical Biotechnology:
Therapeutic Hormones
Hormones
- Hormones are defined as regulatory organic molecules synthesized and released
from a specific gland in the body that, regulate physiological activities by interacting
with a receptor present in/on a distant sensitive cell, evoking responses on specific
target organs or tissues that are adapted to react to minute quantities of them.
- Hormones are transmitted to their targets in the bloodstream after discharge from
the glands that secrete them.
- This mode of discharge (directly into the bloodstream) is called endocrine secretion.
- Human insulin produced by recombinant DNA technology was first approved for
general medical use in 1982.
- The initial approach to recombinant insulin production taken entailed inserting the
nucleotide sequence coding for the insulin A- and B-chains into two different E. coli cells
- These cells were then cultured separately in large-scale fermentation vessels, with
subsequent chromatographic purification of the insulin chains produced.
-The A- and B-chains were then incubated together under appropriate conditions in
order to promote interchain disulfide bond formation, forming 'human insulin crb'
- Crb: (chain, recombinant DNA, bacteria)
Chemical cleavage is
performed by addition
of Cyanogen Bromide
- An alternative method (developed in the Eli Lilly research laboratories), entails inserting
a nucleotide sequence coding for human proinsulin into recombinant E. coli.
- This is followed by purification of the expressed proinsulin and subsequent proteolytic
excision of the C peptide in vitro.
-This approach has become more popular, largely due to the requirement for a single
fermentation and subsequent purification scheme.
- Such preparations have been termed 'human insulin prb'.
- Any impurities present will be host microbial-cell-derived and, hence, are potentially
highly immunogenic. Therefore, stringent purification of the recombinant product must
thus be undertaken. This entails several chromatographic steps.
1.Addition of zinc which renders Zn-insulin longer to disassociate and, hence, longer
to leak into the blood from the injection depot site.
2.Addition of a protein to which the insulin will complex, and from which the insulin
will only be slowly released. The proteins normally used are protamines.
-'Insulin glargine’: differs from native human insulin in that the C-terminal
aspargine residue of the A-chain has been replaced by a glycine residue,
and the B-chain has been elongated (from its C-terminus) by two arginine
residues.
- Like insulin, it is synthesized as a high molecular mass from which the mature
hormone is released by selective proteolysis.
-The major biological actions of glucagon tend to oppose those of insulin, particularly
with regard to regulation of metabolism.
- Glucagon has an overall catabolic effect, stimulating the breakdown of glycogen, lipid
and protein.
(b) administration of insulin prior to a mealtime, but with subsequent omission of the
meal;
- In severe cases this can lead to loss of consciousness, and even death.
- Glucagon is then recovered and purified from the media by a series of further
precipitation and high-resolution chromatographic steps.
3-Human growth hormone (hGH):
• hGH (somatotrophin) is a polypeptide hormone
synthesized in the anterior pituitary gland.
- They directly and indirectly regulate reproductive function and the development of
secondary sexual characteristics.
- FSH and LH play critical roles in the development and maintenance of male
female reproductive functions.
- hCG, produced by pregnant women, plays a central role in maintaining support
systems for the developing embryo during early pregnancy.
- Insufficient endogenous production of any member of this family will adversely affect
reproductive function, which generally can be treated by administration of an
exogenous preparation of the hormone in question.
- Most gonadotrophins are synthesized by the pituitary, although some are made by
reproductive and associated tissues.
Follicle-stimulating hormone (FSH), luteinizing hormone (LH) and
human chorionic gonadotrophin (hCG)
• All three are heterodimeric glycoprotein hormones containing an identical α-
polypeptide subunit and a unique β-polypeptide subunit that confers biological
specificity to each gonadotrophin.
• α- and β-subunits are non-covalently linked.
• These oligomeric glycoprotein hormones are unusual because of the high
carbohydrate content
• In each case, both subunits of the mature proteins are glycosylated.
• The variable degree of glycosylation, especially of sialylation, creates a
spectrum of differences in charge, bioactivities, and elimination half-lives
A simple concept flowchart for
the preparation of recombinant
FSH:
• the critical nature of the carbohydrate on the human FSH. This restricts the
expression work to eukaryotic systems that are capable of making the required
post-translational modifications with high efficiency.
• Unlike other expression systems, mammalian cells are capable of proper
assembly of subunits and glycosylation.
• This cell line can be transfected readily and is fairly easy to grow.
• It can be maintained in essentially protein-free medium, facilitating product
purification and reducing the range of potential contaminants.
• Chinese hamster ovary cell-derived recombinant human FSH has been shown to
be identical to pituitary FSH in amino acid sequence, glycosylation sites, receptor
binding activity, and in vitro biologic activity