Protein
Factor VIII
Factor IX
Tissue plasminogen activator (TPA)
Insulin
Human growth hormone
Erythropoietin
DNase I
Various interferons
(IFN)
Company
Baxter, Bayer
Genetics Institute
Genetech
Eli Lilly, Novo Nordisk
Eli Lilly, Genetech,
Upjohn, Novo Nordisk
Amgen, Ortho Biotech
Genetech
Schering, Biogen, Chiron,
Genetech
Disorder
Hemophilia A
Hemophilia B
Acute myocardial infarction
Diabetes mellitus
GH deficiency in children (dwarfism)
Anemia
Cystic fibrosis
Hepatitis B and C, multiple sclerosis

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

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 postmenopausal 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

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 virus produced 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

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.

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

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

• 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.

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

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

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

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