10 - Pharmaceutical Biotechnology (PCS521) - Lecture Ten.

Pharmaceutical
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.
- Examples include insulin, glucagon, growth hormone and the gonadotrophins.

1- Insulin
- Insulin is a polypeptide hormone produced by the beta cells
of the pancreatic islets of Langerhans.
- It plays a central role in regulating blood glucose levels,
generally keeping it within narrow defined limits (3.5 - 8.0
mmol/L).
- It also has a profound effect on the metabolism of proteins
and lipids
- The most potent known stimulus of pancreatic insulin

release is an increase in blood glucose levels, often
occurring after meal times.
-Failure of the body to synthesize sufficient insulin
results in the development of insulin-dependent
diabetes mellitus (IDDM).
- This is also known as type-I diabetes or juvenile-

onset diabetes.
- IDDM may be controlled by parenteral
administration of exogenous insulin preparations,
usually by regular s.c. injection.
The Insulin molecule
23 aa 30 aa 34 aa 21 aa
- Although mature insulin is a dimeric structure, it is

synthesized as a single polypeptide precursor, i.e.
preproinsulin (108 amino acid polypeptide).
- It contains a 23 amino acid " signal sequence” at
its amino terminal end.
-This guides it through the endoplasmic reticulum
membrane, where the signal sequence is removed by a
specific peptidase, forming “Proinsulin”.
- Proinsulin is t h e n proteolytically processed, yielding
mature insulin and a 34-amino acid connecting
peptide (C peptide).
-The C peptide is further proteolytically modified by
removal of a dipeptide from each of its ends.
- Mature insulin consists of two polypeptide chains connectedby two interchain
disulfide linkages.
- The A-chain contains 21 amino acids, whereas the larger B-chain is composed of 30
residues.
- Insulins from various species conform to this basic structure, while varying slightly in
their amino acid sequence.
- Porcine insulin (5777 Da) varies from the human form (5807 Da) by a single amino
acid, whereas bovine insulin (5733 Da) differs by three residues.
Insulin production
Traditionally, commercial insulin preparations were produced by direct extraction from
pancreatic tissue of pigs (insulin emp: enzymatic modification of porcine insulin
(semisynthetic) ) and cattle, followed by multistep chromatographic purification.
However, the use of animal-derived product had potential disadvantages, including
immunogenicity and availability.
Immunogenicity:
- Bovine insulin differs from human insulin by three amino acids and it elicits an
immunological response in humans.
-Porcine insulin differs from human insulin by only a single amino acid (residue 30 of
the B-chain; threonine in humans, alanine in pigs) and is essentially non-immunogenic
in humans.
Availability:
Millions of people suffer from diabetes worldwide.
-The annual insulin requirement continues to grow, prompting concern of an insulin
shortfall from slaughterhouse sources.
Such issues and concerns underpinned the development of recombinant

human insulin products, now routinely used in the management of diabetes.
Production of human insulin by recombinant DNA technology
- 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.
-Prb: Proinsulin recombinant bacteria

Downstream processing of recombinant human insulin production from E.coli inclusion bodies
Formulation of insulin products
Insulin, whatever its source, may be formulated in a number of ways, generally in order
to alter its pharmacokinetic profile.
Fast (short)-acting insulins are those preparations that yield an elevated blood
insulin concentration relatively quickly after their administration (which is usually by s.c.
or, less commonly, by i.m. injection).
Slow-acting insulins, on the other hand, enter the circulation much more slowly from
the depot (injection) site. This is characterized by a slower onset of action, but one of
longer duration.
-N.B: Biphasic insulins include mixtures of short- and long-acting insulins, which
attempt to mimic normal insulin rhythms in the body.
- Recombinant Insulins:
In order to prolong the duration of insulin action, soluble insulin may be formulated to
generate insulin suspensions. This is generally achieved two ways:
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.
- the resulting long-acting insulins generated are termed protamine-Zn-insulin or

isophane insulin.
Recombinant Engineered (Modified) insulins
Short acting insulins:

- 'Insulin lispro' (tradename Humalog®): differs from
native human insulin in that the amino acid proline and Lysine
at positions B28 and B29 are switched in places.
- 'Insulin Aspart': differs from native human insulin in that the
"proline" residue at B28 has been replaced by “aspartic
acid".
These simple amino acid substitutions decrease the propensity

of individual molecules to self-associate (dimerize), ensuring
that they begin to enter the bloodstream from the site of injection
immediately upon administration.
Long-acting insulins:
-'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.
- The overall product appears to

precipitate in the subcutaneous tissue, it
resolubilizes very slowly and, hence, a
greatly prolonged duration of release into
the bloodstream is noted
- (Administered as a single daily dose)

‘Insulin detemir‘: is an alternative engineered long-acting insulin product.
- This differs from native insulin in that it is devoid of the

threonine B30 residue and contains a 14-carbon fatty acid
residue covalently attached to the side chain of lysine residue
B29.
-This allows the insulin to bind reversibly to albumin, both at the

site of injection and in plasma. This, in turn, ensures constant and
prolonged release of free insulin (prolonged duration of action of
up to 24 hr).
2- Glucagon:
-Glucagon is a single-chain polypeptide of 29 a.a residues. It is synthesized by the
alpha-cells of the islets of Langerhans.
- 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.
-A prominent metabolic effect is to increase blood glucose levels (i.e. it is a

hyperglycaemic hormone).
- Indeed, the major physiological function of glucagon is to prevent
hypoglycaemia which remains the most frequent complication of insulin
administration to diabetics. It usually occurs due to:
(a) administration of an excessive amount of insulin;
(b) administration of insulin prior to a mealtime, but with subsequent omission of the
meal;
(c)due to increased physical activity.
- In severe cases this can lead to loss of consciousness, and even death.
- Although it may be treated by oral or i.v. administration of glucose, insulin-

induced hypoglycaemia is sometimes treated by administration of glucagon.
- Glucagon preparations produced via recombinant means using an engineered
S. cerevisiae strain (Novo Nordisk) or E . coli strain (Eli Lilly).
- Upstream processing (aerobic batch-fed fermentation) is followed by an adjustment

of media pH in order to dissolve precipitated product (glucagon is insoluble in
aqueous-based media in the pH range 3-9.5).
- This facilitates subsequent removal of the yeast by centrifugation.
- 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.
• Biological effects of growth hormone: it primarily

displays anabolic activity. It partially stimulates
the growth of bone, muscle and cartilage cells
directly.
- GH has a potentially wide range of therapeutic
uses however, its major application has been for
the treatment of short stature.
- rhGH was first produced in E. coli in the early

1980s.
4-The gonadotrophins:
- The gonadotrophins are a family of glycoprotein hormones for which the gonads
represent their primary target.
- 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 first step is gene isolation

& forming mRNA
• reverse transcriptase to form
cDNA, DNA polymerase to form
dsDNA
• This is followed by plasmid
production,
• transfection into Chinese
hamster ovary (CHO) cell line,
which produces crude protein.
• The FSH is then isolated from
the medium and purified to the
final product.
The Chinese hamster ovary (CHO) cell line is the best chosen to
express recombinant human FSH & LH for clinical use because of:
• 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
N.B: The purification step is performed by immunochromatography using specific

anti-FSH monoclonal antibodies.

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Pharmaceutical

- Examples include insulin, glucagon, growth hormone and the gonadotrophins.

- The most potent known stimulus of pancreatic insulin

- This is also known as type-I diabetes or juvenile-

- Although mature insulin is a dimeric structure, it is

Such issues and concerns underpinned the development of recombinant

-Prb: Proinsulin recombinant bacteria

- the resulting long-acting insulins generated are termed protamine-Zn-insulin or

Short acting insulins:

These simple amino acid substitutions decrease the propensity

- The overall product appears to

- (Administered as a single daily dose)

- This differs from native insulin in that it is devoid of the

-This allows the insulin to bind reversibly to albumin, both at the

alpha-cells of the islets of Langerhans.

-A prominent metabolic effect is to increase blood glucose levels (i.e. it is a

(a) administration of an excessive amount of insulin;

(c)due to increased physical activity.

- Although it may be treated by oral or i.v. administration of glucose, insulin-

- Upstream processing (aerobic batch-fed fermentation) is followed by an adjustment

- This facilitates subsequent removal of the yeast by centrifugation.

• Biological effects of growth hormone: it primarily

- rhGH was first produced in E. coli in the early

• The first step is gene isolation

N.B: The purification step is performed by immunochromatography using specific

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