Biopharmaceuticals are large molecule drugs made using cells or enzymes, often similar to natural biological compounds. Examples include proteins, peptides, nucleic acids, and gene therapy. The first approved biopharmaceuticals were recombinant human insulin in 1982 and recombinant tissue plasminogen activator in 1986. Today, biopharmaceutical sales reach over $200 billion annually and there are over 300 approved biopharmaceutical drugs on the market. Biopharmaceuticals are mainly produced using bacterial, mammalian, yeast, plant, or animal cells transfected with plasmids containing the gene for the target protein. Purification then isolates the target protein for clinical use.

1. Biopharmaceuticals –
proteins/peptides, nucleic acids,
gene therapy
Larry Baum
Hong Kong

2. Definition
Biopharmaceuticals: large molecule drugs
Often similar to natural biological compounds
Made using cells or enzymes
Examples:
» Proteins or peptides (antibodies, hormones)
» Nucleic acids (gene therapy)
» Cells (stem cells)

3. History
Insulin
» Recombinant human insulin, or Humulin
» Made by E. coli bacteria
» For diabetes
» Made by Genentech, licensed to Lilly
» First recombinant protein approved by FDA, in 1982
Tissue plasminogen activator
» Recombinant human tPA
» Made by CHO (Chinese hamster ovary) cells
» To reduce blood clotting in stroke or heart attack
» Made by Genentech
» Marketed from 1986

4. Market
Sales: ~US$200 billion (a quarter of all drugs)
Number: >300 biopharmaceuticals now
approved and on the market

5. Market growth

6. Top selling drugs
http://www.g-gts.com/en/business/market/

7. Biopharmaceutical companies
http://info.evaluategroup.com/rs/607-YGS-364/images/wp15.pdf

8. Examples: recombinant proteins
Blood factors (Factor VIII and Factor IX)
Thrombolytic agents (tissue plasminogen activator)
Hormones (insulin, glucagon, growth hormone, gonadotrophins)
Haematopoietic growth factors (Erythropoietin, colony stimulating
factors)
Interferons (Interferons-α, -β, -γ)
Interleukin-based products (Interleukin-2)
Vaccines (Hepatitis B surface antigen)
Monoclonal antibodies (Various)
Additional products (tumour necrosis factor, therapeutic
enzymes)

9. Monoclonal antibody: Rituxan
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10. Manufacture of protein
1. Choose cells
2. Transfect plasmid(s) of transgene and selector
3. Select transfected cell clones
4. Scale up
5. Purify protein
Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf

11. Manufacture of protein
1. Choose cells
» Bacteria
– E. Coli most popular
– Advantages: easy to handle; grow quickly
» Mammalian cells
– CHO cells most popular
– Advantages: proteins processed more like in humans, with post-
translational glycosylation, proper folding, and solubility
» Other transgenic sources: plants, yeast, animals
– Plants: cheap
– Yeast: easier and cheaper than mammalian cells, but more human-
like processing than bacteria
– Animals: cheaper than cells, but with same processing
Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf

12. Manufacture of protein
2. Transfect plasmid(s) of transgene and selector
» Use any non-viral transfection method
– Calcium phosphate precipitation, lipofection, electroporation, biolistic,
polymer-mediated
» Transgene:
1. Strong promoter
2. One intron, to increase cytoplasmic transport and translation efficiency
3. Transgene as cDNA
4. Change rare tRNA codons to common
» Selector: DHFR, GS
– Weak promoter, to divert expression to that of transgene
– DHFR: DHFR-deficient cells; MTX amplifies copies of plasmid
– GS: MSX selects clones with amplified copies of plasmid
Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf

13. Manufacture of protein
3. Select transfected cell clones
» Grow cell dilutions in presence of selector: MTX
(methotrexate) or MSX (methionine sulfoximine)
» Choose clones that survive selection
» Screen clones
– High and stable expression of intact recombinant protein
– Fast growth
– But growth and expression often inversely related
Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf

14. Manufacture of protein
4. Scale up
» Optimize growth of cells
– Can grow from 100,000 to 10 million cells/ml in 10 days
– Different medium for different growth phases
– Defined medium (serum-free): standardized, safer, cheaper
– Cells engineered for high growth: anti-apoptosis, growth factors
» Optimize protein yield
– Can reach 4 mg/ml in 20 days
– Batches (most common now) or continuous perfused culture
Reference: http://www.biomed.rutgers.edu/doc/papers/Wrum.pdf

15. Manufacture of protein
4. Purify protein
1. Capture: Ion exchange most common—by charge
2. Intermediate: Hydrophobic interaction most common—by hydrophobicity
3. Polishing: Gel filtration most common—by size
http://kirschner.med.harvard.edu/files/
protocols/GE_antibodypurification.pdf

16. Vaccines
Attenuated: Measles, Mumps, Tuberculosis
Killed or inactivated: Hepatitis A, Influenza, Pertussis
Toxoids: Tetanus, Diphtheria
Experimental types
» Subunit
» Vector
» DNA
» Peptide

17. Monoclonal antibodies

18. Monoclonal antibodies
Human antibody methods
» Fuse human B cells to myeloma cells
» Genetically alter mice to contain human antibody producing
genes
» Phage display: put human B cell antibody genes into
bacteriophage, infect E. coli, select clones producing desired
antibody
Conjugate antibodies: coupled to “payloads”
» Radioisotopes: Zevalin and Bexxar to treat lymphoma
» Cytokines: to generate anti-tumor response
» Toxins

19. Cytokines
Interferons
» α, β, and γ
» Fights viral, bacterial, or fungal infection
» To treat hepatitis, multiple sclerosis, cancer, leprosy
» Coupling with polyethylene glycol (PEGylation) prolongs absorption
Interleukins
» IL-2 induces tumor necrosis factor (TNF)
» To treat cancer
TNF
» α and β
» Activates T-cells and macrophages
» Block α to treat autoimmune diseases
– Crohn’s disease, arthritis, ankylosing spondylitis, psoriasis
– Antibody (Infliximab and Adalimumab) or soluble protein receptor for α (Etanercept)

20. Cytokines
Erythropoietin (EPO)
» Stimulates red blood cell production
» Treats anemia
» Made in mammalian cells
» Amgen won the race, with FDA approval in 1989
Granulocyte macrophage colony stimulating
factor (GM-CSF)
» Treats lack of blood cells in chemotherapy patients

21. Hormones
Insulin
Amylin
» Acts to slow glucose uptake during a meal
» To treat diabetes, along with insulin
Human growth hormone (hGH)
» Stimulates tissue growth
» To treat short height

22. Enzymes
Clotting factors
» Factor VIII and Factor IX
» To treat hemophilia
Glucocerebrosidase
» Processes gluococerebroside
» Mutated and deficient in Gaucher’s disease
» Very expensive: up to US$500,000/year

23. Gene therapy
Adding or changing a gene in a patient’s
cells to treat a disease
The disease may be genetic or acquired
Somatic or germ-line cells
Still experimental

24. Gene therapy methods
Virus: retrovirus, adenovirus, herpes
simplex (HSV), adeno-associated virus
(AAV), lentivirus (HIV)
Fusion: liposomes, DNA complexes
Physical: naked DNA, microprojectiles,
cationic peptide transduction domain
(PTD)

25. Example 1: ADA Deficiency
Adenosine deaminase deficiency
Mostly affects lymphocytes
Autologous T cells grown, ADA
retrovirus added, infused into patient
T cells survive for months in patients
Fewer infections
Leukemia in some patients after several
years

26. Retroviral Method
Producer cells
transgeneLTR LTR
Packaging cell line
MoMLV gag, pol, env genes
neo gene

27. Example 2: Cystic fibrosis
CFTR: chloride/water transport
Thick mucus causes lung infections
Adenovirus, AAV, liposomes into lungs
Adenovirus: <1% of cells, no chloride
secretion, inflammation
Liposomes: little chloride secretion, no
inflammation

28. AAV Method
transgeneAAV AAV
ITRITR
AAV rep, cap genesAd
ITR
Ad
ITR
helper adenovirus
AAV

29. Example 3: Brain tumor
Thymidine kinase/retrovirus producer
cells injected into tumor
Ganciclovir kills thymidine kinase cells
Bystander effect: gap junctions,
vascular endothelium, diffusion?
Tumor reduced in 25%, eliminated in
13% of patients

30. Brain gene therapy issues
Targeting
» Inject for small target area
» Whole brain
– Cross blood-brain barrier (bradykinin, mannitol)
– Migratory cells
– Replicating vector (HSV)
– Cerebrospinal fluid
Neuron transduction
» AAV, HIV, HSV
» Liposomes, PTD

31. Gene therapy obstacles
Low efficiency: few cells transduced
» Retrovirus: only dividing cells (liver, stem cells)
» Low viral titer (retrovirus, AAV)
» Physical barriers (solid tissue, absorbing tissue)
Transient expression
» Promoter shutdown (viral promoter)
» Cell turnover (lung epithelium, peripheral T cells)
Toxicity
» Immune response (adenoviral coat proteins, transgene itself)
» Replication-competent virus (HSV)

32. Popularity of gene therapy trials
>2000 trials so far
Most were for cancer
Some for acquired disease
Some for inherited disease

33. Future of gene therapy
More research
New vectors
New generations of current vectors
More trials
Occasional success

34. Stem cells
Bone marrow transplants
» For cancer treatment or genetic disease
» Common
» Requires life-long immunosupression
Other stem cells
» For diabetes, genetic diseases
» Research to find culture conditions and
growth factors to induce differentiation
» Still experimental

35. The EndThe End