Transcript Human Growth Hormone

Human Growth Hormone
CHEE450
By Leslie Davis
Human Growth Hormone
Simulates linear growth in humans
Effects on the growth plates of the long
bones
Increased cell growth
Increased protein, carbohydrate and lipid
metabolism
Human Growth Hormone
Generally used to treat those with GHdeficiencies
Beneficial effects: burns, bone fractures
and Turner’s syndrome
Shown promise in treating chemotherapy and
AIDS
Increases body mass
Human Growth Hormone
Polypeptide
191 amino acid residues
MW = 22125 daltons
Highly hydrophobic proteins
Historical Perspective
Isolated hGH in human pituitary 1956
Proof of effectiveness 1958
Obtained from human pituitary gland only
Urgent need to obtain human pituitary glands at
autopsy
High cost
Cloned from human gene in 1980s
Recombinant DNA Synthesis of hGH
Cloning of human DNA
Adapting of the cloned gene for
expression in Escherichia coli
E. coli did not have the biochemical
machinery
84 base pairs of ds DNA synthesized for direct
expression
Cloning of the gene
How it works
Chemical messenger
produced by the
pituitary gland
Pituitary Gland
Promotes growth during
childhood
Plays important
metabolic role
throughout adulthood
Production Process
Fermentation
Highly reproducible
Intracellular
E. coli W3110 with a pB322-derived plasmid
coding
Antibiotic resistance of genetically modified E.
coli strain
Typically tetracycline and ampicillin resistant
Production Process
Glucose and mineral salt medium
Exponentially growing cells in batch mode
Transferred from seed fermentor to production
bioreactor
10% of final volume
High cell density fermentation
Production Process
Fed-batch bioreactor
Glucose feed ~ 500-600 g/L
May use peptone for amino acid source
Temperature – near maximum tolerance of E.
coli
Agitation generally ranges 1000-1500 rpm
Slightly positive pressure
Production Process
Purification
Cell disruption
Microfiltration
Chromatographic methods
Genentech adapted anion exchange
chromatography and gel filtration technology
(early 80s)
Production Process
Issues with scale up
Mass transfer through high density cells
Oxygen
Glucose feed
Inhibitory ion formation
Acetate and formate
Final Product
White lyophilized powder
Low oral bioavailability (<1%)
Intended for intravenous, subcutaneous or
intramuscular administration
Sold in vials or cartridges containing
4 - 24 mg of somatropin
Preservatives
Water for injection
Final Product
Approximately 28 day shelf life
Oxygen Sensitive
Must be refrigerated
Improve the solubility, sodium dodecyl
sulfate (extremely surface active) or
denaturing agents - urea and guanidine
hydrochloride
Market
U.S. Manufacturers
Genotropin by Pharmacia & Upjohn Company,
Humatrope by Eli Lilly, Norditropin by Novo
Nordisk, Saizen and Serostim by Serono
Laboratories, Nutropin by Genentech
Market
Costs: $75 to $200 per week
World-wide sales of $1.6 billion
Approximately 20,000 U.S. children taking
drug
Prescribed for non-GH-deficient children if
(U.S.)
<3SD below mean for age category
<25% growth velocity
Discussion
Psychological issues
Cultural “heightism”
Increased metabolism,
reduced fat, anti-aging, gain
strength
Articles
References
 Human Growth Hormone, edited by Salcatore Raiti and
Robert A. Tolman, 1986
 Human Growth Hormone: Research and Clinical Practice,
edited by Roy G. Smith and Michael O. Thorner
 Influence of Scale-Up on the Quality of Recombinant
Human Growth Hormone, Bylund, Castan, Mikkola, Veide
and Larsson
 Modelling the effects of Glucose Feeding on a recombinant
E. coli Fermentation, Cockshott and Bogle
 Further Studies Related to the Scale-up of High Cell Density
Escherichia coli Fed-Batch Fermentations: The Additional
Effect of a Changing Microenvironment When Using
Aqueous Ammonia to Control pH
References
 http://pi.lilly.com/us/humatrope-pi.pdf
 http://www.hghnews.us/p/91.html
 http://www.hghhumangrowthhormone.com/hgh_human_growth
_hormone_sitemap/hgh_manufacturer.html
 http://www.novonordisk.com/therapy_areas/grow
th_hormone_therapy/default.asp
 http://patft.uspto.gov/netacgi/
 http://www.genotropin.com/patients/growth_disor
ders/index.asp