Endocrine disorder

1. Chapter 10-Protein Therapeutics
•Pharmaceutical proteins and enzymes
•Monoclonal antibodies and recombinant antibodies

2. Table 10.1 Some recombinant proteins approved for
human use ($60 billion-2006)
Protein Company Disorder
Factor VIII Baxter, Bayer Hemophilia A
Factor IX Genetics Institute Hemophilia B
Tissue plasminogen
activator (TPA)
Genetech Acute myocardial
infarction
Insulin Eli Lilly, Novo Nordisk Diabetes mellitus
Human growth
hormone
Eli Lilly, Genetech,
Upjohn, Novo Nordisk
GH deficiency in
children (dwarfism)
Erythropoietin Amgen, Ortho Biotech Anemia
DNase I Genetech Cystic fibrosis
Various interferons
(IFN)
Schering, Biogen, Chiron,
Genetech
Hepatitis B and C,
multiple sclerosis

3. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 10
Protein Therapeutics
Table 10.1

4. Recombinant proteins-from http://en.wikipedia.org/wiki/List_of_recombinant_proteins -9/10/12
Human recombinants that largely replaced animal or harvested from human types
• Human growth hormone (rhGH) Humatrope from Lilly and Serostim from Serono replaced cadaver harvested
human growth hormone
• Human insulin (rhI) Humulin from Lilly and Novolin from Novo Nordisk among others; largely replaced
bovine and porcine insulin for human therapy. Some prefer to continue using the animal-sourced
preparations, as there is some evidence that synthetic insulin varieties are more likely to induce
hypoglycemia unawareness. Remaining manufacturers of highly-purified animal-sourced insulin include the
U.K.'s Wockhardt Ltd. (headquartered in India), Poland's Polfa Tarchomin S.A., Argentina's Laboratorios Beta
S.A., and China's Wanbang Biopharma Co.
• Follicle-stimulating hormone FSH replaced Serono's Pergonal which was previously isolated from post-
menopausal female urine
• Factor VIII Kogenate from Bayer replaced blood harvested factor VIII
Human recombinants with recombination as only source
• Erythropoietin (EPO) Epogen from Amgen
• Granulocyte colony-stimulating factor (G-CSF) filgrastim sold as Neupogen from Amgen; pegfilgrastim sold as
Neulasta
• alpha-galactosidase A Fabrazyme by Genzyme
• alpha-L-iduronidase (rhIDU; laronidase) Aldurazyme by BioMarin Pharmaceutical and Genzyme
• N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase) Naglazyme (TM) by BioMarin Pharmaceutical
• DNAse Pulmozyme by Genentech
• Tissue plasminogen activator (TPA) Activase by Genentech
• Glucocerebrosidase Ceredase by Genzyme
• Interferon (IF) Interferon-beta-1a as Avonex from Biogen Idec; Rebif from Serono; Interferon beta-1b as
Betaseron from Schering
• Insulin-like growth factor 1 (IGF-1)
Animal recombinants
• Bovine somatotropin (bST)
• Porcine somatotropin (pST)
• Bovine Chymosin

5. Expression systems are based on the insertion of a gene into a host cell for its translation and expression into protein. Host cells include :
Bacteria - e.g. Escherichia coli (E.coli), Bacillus subtilis (B. subtilis)
Yeast
Cultured insect cells
Cultured mammalian cells
The choice of cell type used depends upon the protein to be expressed. All require DNA to be cloned into the an appropriate vector.
Advantages of bacterial cells
simple physiology
short generation times, as bacteria grow and multiply rapidly
large yields of product - up to 10 % of mass (low cost)
With B. subtilis and some others, it is possible to induce secretion of a gene product into the surrounding medium. This method is in use in the
pharmaceutical industry in the production of hormones such as insulin and human growth hormone.
Disadvantages of bacterial cells
The expressed proteins often do not fold properly and so are biologically inactive.
The synthesised protein is often toxic to bacteria preventing the cell cultures from reaching high densities. A solution to this problem is to
incorporate an inducible promoter, which may be turned on to transcribe the inserted gene after the culture has been grown
Lack of enzymes responsible for post-translational modifications (effect on function of proteins), eg if the protein to be expressed is a
glycoprotein, there is not apparatus in the bacterium to 'stick on' the necessary sugar residues.
Advantages of yeast cells
Yeast is a simple eukaryote and performs many of the post-translational modifications required for human proteins
Can be induced to secrete certain proteins into the growth medium for harvesting- e.g. Hepatitis B virus (HBV) vaccine.
Disadvantages of yeast cells
Presence of active proteases that degrade foreign (expressed) proteins, thereby reducing their yield (a solution to this problem is the
construction of yeast strains from which the protease genes have been deleted).
Insect Cells-Expression of foreign proteins in insect cells through incorporation of their genes into baculovirus vectors
Advantages of insect cells
High level of expression
Correct folding
Post-translational modifications similar to those in mammalian cells
Cost, though more than for culturing bacteria and yeast, less than for mammalian cells e.g. potential vaccine for AIDS virusproduced by
expression of one of the HIV glycoproteins with this system
Disadvantages of insect cells
More difficult to work with
Expensive
Slow generation time
Not suitable for proteins with repetitive sequences
Cloning and expression of a foreign protein in a suitable host

6. Use of an appropriate expression vector and host
Example: A simple E. coli expression vector
utilizing the lac promoter. (a) The
expression vector plasmid contains a
fragment of the E. coli chromosome
containing the lac promoter and the
neighboring lacZ gene. In the presence of
the lactose analog IPTG, RNA polymerase
normally transcribes the lacZ gene,
producing lacZ mRNA, which is translated
into the encoded protein, b-galactosidase.
(b) The lacZ gene can be cut out of the
expression vector with restriction enzymes
and replaced by the Granulocyte-Colony
Stimulating Factor G-CSF cDNA. When the
resulting plasmid is transformed into E. coli
cells, addition of IPTG and subsequent
transcription from the lac promoter
produces G-CSF mRNA, which is translated
into G-CSF protein.

7. Table 10.3 Some therapeutic monoclonal antibodies
approved for human use
Type of antibody Company Therapeutic use
Mouse, Humanized Ortho Biotech, Protein
Design, Hoffmann-
LaRoche
Prevents kidney
transplant rejection
Chimeric Centocor Prevents blood clots
Chimeric Genetech, Hoffmann-
LaRoche
Non-Hodgkin
lymphoma
Humanized
(Herceptin)
Genetech HER2-positive breast
cancers
Humanized Am Home Prod, Celltech,
Schering, Millen. Pharm.
Certain leukemias
Humanized Genetech Asthma

8. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 10
Protein Therapeutics
Table 10.3

9. Antibody Structure
•Antibodies are immune system-related proteins
called immunoglobulins. Each antibody consists of
four polypeptides– two heavy chains and two light
chains joined to form a "Y" shaped molecule.
•The amino acid sequence in the tips of the "Y"
varies greatly among different antibodies. This
variable region, composed of 110-130 amino
acids, give the antibody its specificity for binding
antigen. The variable region includes the ends of
the light and heavy chains. Treating the antibody
with a protease can cleave this region, producing
Fab or fragment antigen binding that include the
variable ends of an antibody.
•The constant region determines the mechanism
used to destroy antigen. Antibodies are divided
into five major classes, IgM, IgG, IgA, IgD, and
IgE, based on their constant region structure and
immune function.

10. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 10
Protein Therapeutics
Figure 10.24 Structure of an antibody molecule
Complementarity-
determining regions (CDRs)

11. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 10
Protein Therapeutics
Figure 10.25 Genetically engineered chimeric antibody

12. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 10
Protein Therapeutics
Figure 10.26 Genetically engineered humanized antibody

13. Copyright © 2010 ASM Press
American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904
Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition
Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten
Chapter 10
Protein Therapeutics
Figure 10.40
Making monoclonal antibodies
even more effective therapeutic
agents: two ways