1. Human Growth Hormone
CHEE450
By Leslie Davis

2. 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
Clinical studies have generally shown that the drug is effective for approximately 80% of its users

3. Human Growth Hormone
Generally used to treat those with GH-deficiencies
Beneficial effects: burns, bone fractures and Turner’s syndrome
Shown promise in treating chemotherapy and AIDS
Increases body mass
Growth hormone deficiency may occur when the pituitary gland does not secrete sufficient amounts of endogenous GH
HIV patients with wasting or cachexia to increase lean body mass and body weight, and improve physical endurance

4. Human Growth Hormone Polypeptide 191 amino acid residues
MW = daltons
Highly hydrophobic proteins

5. 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
In 1956, researchers established that the human pituitary produced human growth hormone
It was proved effective in 1958
The only source of the hormone were human pituitaries. This resulted in numerous efforts to collect human pituitaries at autopsy so that the hormone could be extracted and given to children.

6. 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

7. Cloning of the gene

8. How it works Chemical messenger produced by the pituitary gland
Promotes growth during childhood
Plays important metabolic role throughout adulthood
Pituitary Gland

9. 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

10. 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

11. 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 rpm
Slightly positive pressure

12. Production Process Purification Cell disruption Microfiltration
Chromatographic methods
Genentech adapted anion exchange chromatography and gel filtration technology (early 80s)

13. Production Process Issues with scale up Inhibitory ion formation
Mass transfer through high density cells
Oxygen
Glucose feed
Inhibitory ion formation
Acetate and formate

14. Final Product White lyophilized powder
Low oral bioavailability (<1%)
Intended for intravenous, subcutaneous or intramuscular administration
Sold in vials or cartridges containing
mg of somatropin
Preservatives
Water for injection

15. 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

16. 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

17. 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

18. Discussion Psychological issues
Cultural “heightism”
Increased metabolism, reduced fat, anti-aging, gain strength
Articles
Taller college graduates make more money, and most (80%) presidents have been the taller candidate
Being teased or bullied, having poor self-esteem, feeling athletically incompetent, and being treated as younger than their chronological age by older ppl are frequent concerns of short children or their parents

19. 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

20. References http://pi.lilly.com/us/humatrope-pi.pdf