CLASS 1
Recurring Themes
Unmet healthcare needs from society Innovations from modern technology Commercial opportunities
Definition of Biotechnology
Integrated use of biochemistry, microbiology and engineering sciences in order to achieve technological (industrial) application of the capabilities of microorganisms,
cultures tissue cells and parts thereof
ABC in Molecular Biotechnology
DNA —transcription→ RNA —translation→ Protein
DNA discovery: Watsons and Crick 1953
CLASS 2 : Recombinant DNA Technology and the Birth of Genentech
What would happen if the error rate of DNA replication were zero?
- No evolution
- No life
- A self-sustaining chemical system capable of Darwinian evolution
In Central Dogma, does biological information always flow from DNA to RNA to Protein?
Reverse transcriptase is an exception.
64 triplet codons
254 quadruplet codons
Genetic Engineering Technology (Genentech)
- It is the foundation of modern biotechnology. Before the biotechnology revolution, drugs were either chemically synthesized/extracted from natural
sources/fermentation
- Genetic engineering opens a new ways to make drugs using natures machinery
- Synthetic Biology : each cell is a factory
- Recombinant DNA technology combines the genetic materials of more than one species to produce a new genetic template
- The expression of the recombinant DNA in cell cultures enables the production of protein products
Steps in Gene cloning and Expression
1. Obtain gene of interest
2. Insert the gene into host cells
3. Select cells that contain the desired gene
4. Induce the bacteria to make protein from the “foreign” gene
5. Collect and purify products
Plasmid Vector + DNA fragment to be cloned
↓ Enzymatically insert DNA into plasmid vector
Recombinant plasmid
↓ Mix E.Coli with plasmids in presence of CaCl2
Culture on nutrient agar plates containing ampicillin
Transformed E.Coli cell survives
Cells that no dot take up the plasmid die on ampicillin plates
↓ Independent plasmid replication
Cell Multiplication
Colony of cells each containing copies of the same recombinant
plasmid
Plasmids: Gene Carriers
1. Plasmid DNA and foreign DNA are both cut with the same restriction enzyme
2. Foreign DNA is inserted into the plasmid, where it inactivates the B-galactosidase gene
3. The recombinant plasmids introduce into the bacterium
4. All treated bacteria are spread on a nutrient agar plate and incubated
5. Colonies that grow on the plate contain a plasmic. Blue contains plasmids without inserts. White colonies contain recombinant plasmids. Cells that do not
take up any plasmids do not grow.
Restriction enzymes : Molecular Scissors
1. Restriction enzyme cuts double-stranded DNA at its particular recognition sited
2. These cuts produce a DNA fragment with two sticky ends
3. When two such fragments of DNA cut by the same restriction enzyme come together, they can join by base-pairing
4. The joined fragments will form either a linear molecule or a circular one. Other combinations of fragments can also occur
5. The enzyme DNA ligase is used to unite the backbones production a molecule of recombinant DNA
New Capital
Venture capital feeds the incubation stage providing the initial stage.
The first human protein was successfully made within 9 months
,The Genentech IPO
Buying the Genentech stocks in 1980s was an irrational act
The market indicates that it is willing to bet on a young inexperienced company, without any product, and revenues that are years away
- Genentech was the first biotech company to go public. The biotech landscape was changed on October 14, 1980, when Genentrach issued 1 million
shares at $35 each
- The market response was enthusiastic, with the stocks closed out at its first day at $70
Licensing Deals
1978: licensed insulin to Eli Lilly and Co.
1978: licensed human growth hormone to AB Kabi
1980: licensed interferons to Hoffmann-La Roche
→ Genentech received upfront licensing fee and payments for reaching benchmarks
→ deals generated incomes, supported product development, and increased credibility
Evolution of a Biotech Startup
R&D house → Partnering with established companies (for clinical trials, late-state development, marketing) → Retaining more rights for next products → Retaining
all manufacturing and marketing rights
An Innovative Culture
Academic root Entrepreneurial Drive No dress up Fun Loves science Results driven Execs wear many hats
Intellectual Property
- In 1974, Cohen and Boyer on behalf of Stanford and University of California, filed a patent on their recombinant DNA technology
- In 1980, the US patent and Trademark Office granted the patent → the first significant parent to cover the new biotech
Patent protection continues to fuel innovation
Genentech Nowadays
- Market capitalization: 87.9 billion (17/10/08)
- Became wholly owned by Roche since 03/2009
- Product type: recombinant protein
- Product focus areas: oncology, immunology, metabolism, neuroscience, infectious disease
- Technology focus: recombinant DNA technology, genomic, bioinformatics, microarrays
- Examples: Zelboraf (small molecule), Avastin (proteins)
- In the pipeline: >25 candidates (2011)
Number of IPOs fell since the financial crisis
Innovation capital remained stagnant
, CLASS 3 : Putting Biology into a Bottle
Recombinant insulin starts a new chapter for biotechnology industry
- The first synthesis of Aspirin in 1899
- Penicillin, the first antibiotic discovered in 1928
- First recombinant product hit the market in 1982
Background : Diabetes is a serious and economic problem
- Diabetes affects more than 420 million people worldwide (2015)
- Diabetes is the leading cause of blindness in developed countries, a common cause amputation and kidney failure, and increases chances for developing
heart conditions
- The devastating complications of diabetes are imposing a huge burden on healthcare services. Estimatedly, diabetes account for 5%-10% of a nation's
health budget (US$612 billion in 2014)
Insulin: A standard treatment for diabetes since 1924
1921: First isolation of insulin from animals. (UofT) (Macleod and Banting awarded Nobel Prize in 1923 for discovery)
1922: Large scale production, commercialization (Eli Lilly, Novo Nordisk
Source of Insulin
The pancreas is an organ behind the stomach that is the main source of insulin in the body. Clusters of cells in the pancreas called islets produce the hormone and
determine the amount based on blood glucose levels in the body
Commercial Opportunities for the New Technology
1. Limitations of animal extraction (that can be solved by new technology)
a. Potential problems of allergic reactions
b. Slaughterhouse would not be able to provide enough supply of animal purchases
2. Market size of the proposed product
a. Increased diabetes patients
b. Insulin is the mainstream treatment
c. Lucrative markets
3. Regulatory environment
a. Animal insulin is already a mature product
b. Insulin is a relatively small and simple protein
Innovation
- Making insulin with recombinant DNA technology
Genetic Engineering for Insulin Productions
1. Obtain gene of interest
a. Insulin gene
2. Insert the gene into host cells
3. Select cells that contain the desired gene
4. Induce the bacteria to make protein from the “foreign” gene
5. Collect and purify products
a. Insulin protein
Obtaining the Insulin Gene
1. mRNA isolated bro b-cells in pancreas
2. cDNA produced by reverse transcriptase
3. mRNA destroyed
4. DNA strands produced by DNA polymerase
Why not obtain DNA directly from pancreas?
- Exons (<2% human genome)
- Introns (common in animals & plants, rare in bacteria)
Discovery in basic science → Biological Processing → Practical products, processes or systems
Bioprocess development is the link between basic science discovery and product manufacturing
Bioprocess Development for Large-scale production
- Eli Lilly the recombinant humans from Genentech in 1978.
- Its expertise in isolating antibiotics from fermentation was important is scaling the production of recombinant protein from bacterial cultures
What is Bioprocess Engineering About?
A bioprocess includes an “upstream” process and a “downstream” process
1. Upstream: product production in cell cultures/fermentations
a. Engineering expression system
b. Maximize production of cell factory
c. Scale up and bioreactor design
2. Downstream: isolation of product and its formulation
a. Two major process
i. Solid liquid separation
ii. Purification
Upstream Process Development
1. Batch
a. Cell inoculum is added to a defined volume of medium in either fermenter or roller bottles, and allowed to grow and be harvested at
predetermined optimal time point
Recurring Themes
Unmet healthcare needs from society Innovations from modern technology Commercial opportunities
Definition of Biotechnology
Integrated use of biochemistry, microbiology and engineering sciences in order to achieve technological (industrial) application of the capabilities of microorganisms,
cultures tissue cells and parts thereof
ABC in Molecular Biotechnology
DNA —transcription→ RNA —translation→ Protein
DNA discovery: Watsons and Crick 1953
CLASS 2 : Recombinant DNA Technology and the Birth of Genentech
What would happen if the error rate of DNA replication were zero?
- No evolution
- No life
- A self-sustaining chemical system capable of Darwinian evolution
In Central Dogma, does biological information always flow from DNA to RNA to Protein?
Reverse transcriptase is an exception.
64 triplet codons
254 quadruplet codons
Genetic Engineering Technology (Genentech)
- It is the foundation of modern biotechnology. Before the biotechnology revolution, drugs were either chemically synthesized/extracted from natural
sources/fermentation
- Genetic engineering opens a new ways to make drugs using natures machinery
- Synthetic Biology : each cell is a factory
- Recombinant DNA technology combines the genetic materials of more than one species to produce a new genetic template
- The expression of the recombinant DNA in cell cultures enables the production of protein products
Steps in Gene cloning and Expression
1. Obtain gene of interest
2. Insert the gene into host cells
3. Select cells that contain the desired gene
4. Induce the bacteria to make protein from the “foreign” gene
5. Collect and purify products
Plasmid Vector + DNA fragment to be cloned
↓ Enzymatically insert DNA into plasmid vector
Recombinant plasmid
↓ Mix E.Coli with plasmids in presence of CaCl2
Culture on nutrient agar plates containing ampicillin
Transformed E.Coli cell survives
Cells that no dot take up the plasmid die on ampicillin plates
↓ Independent plasmid replication
Cell Multiplication
Colony of cells each containing copies of the same recombinant
plasmid
Plasmids: Gene Carriers
1. Plasmid DNA and foreign DNA are both cut with the same restriction enzyme
2. Foreign DNA is inserted into the plasmid, where it inactivates the B-galactosidase gene
3. The recombinant plasmids introduce into the bacterium
4. All treated bacteria are spread on a nutrient agar plate and incubated
5. Colonies that grow on the plate contain a plasmic. Blue contains plasmids without inserts. White colonies contain recombinant plasmids. Cells that do not
take up any plasmids do not grow.
Restriction enzymes : Molecular Scissors
1. Restriction enzyme cuts double-stranded DNA at its particular recognition sited
2. These cuts produce a DNA fragment with two sticky ends
3. When two such fragments of DNA cut by the same restriction enzyme come together, they can join by base-pairing
4. The joined fragments will form either a linear molecule or a circular one. Other combinations of fragments can also occur
5. The enzyme DNA ligase is used to unite the backbones production a molecule of recombinant DNA
New Capital
Venture capital feeds the incubation stage providing the initial stage.
The first human protein was successfully made within 9 months
,The Genentech IPO
Buying the Genentech stocks in 1980s was an irrational act
The market indicates that it is willing to bet on a young inexperienced company, without any product, and revenues that are years away
- Genentech was the first biotech company to go public. The biotech landscape was changed on October 14, 1980, when Genentrach issued 1 million
shares at $35 each
- The market response was enthusiastic, with the stocks closed out at its first day at $70
Licensing Deals
1978: licensed insulin to Eli Lilly and Co.
1978: licensed human growth hormone to AB Kabi
1980: licensed interferons to Hoffmann-La Roche
→ Genentech received upfront licensing fee and payments for reaching benchmarks
→ deals generated incomes, supported product development, and increased credibility
Evolution of a Biotech Startup
R&D house → Partnering with established companies (for clinical trials, late-state development, marketing) → Retaining more rights for next products → Retaining
all manufacturing and marketing rights
An Innovative Culture
Academic root Entrepreneurial Drive No dress up Fun Loves science Results driven Execs wear many hats
Intellectual Property
- In 1974, Cohen and Boyer on behalf of Stanford and University of California, filed a patent on their recombinant DNA technology
- In 1980, the US patent and Trademark Office granted the patent → the first significant parent to cover the new biotech
Patent protection continues to fuel innovation
Genentech Nowadays
- Market capitalization: 87.9 billion (17/10/08)
- Became wholly owned by Roche since 03/2009
- Product type: recombinant protein
- Product focus areas: oncology, immunology, metabolism, neuroscience, infectious disease
- Technology focus: recombinant DNA technology, genomic, bioinformatics, microarrays
- Examples: Zelboraf (small molecule), Avastin (proteins)
- In the pipeline: >25 candidates (2011)
Number of IPOs fell since the financial crisis
Innovation capital remained stagnant
, CLASS 3 : Putting Biology into a Bottle
Recombinant insulin starts a new chapter for biotechnology industry
- The first synthesis of Aspirin in 1899
- Penicillin, the first antibiotic discovered in 1928
- First recombinant product hit the market in 1982
Background : Diabetes is a serious and economic problem
- Diabetes affects more than 420 million people worldwide (2015)
- Diabetes is the leading cause of blindness in developed countries, a common cause amputation and kidney failure, and increases chances for developing
heart conditions
- The devastating complications of diabetes are imposing a huge burden on healthcare services. Estimatedly, diabetes account for 5%-10% of a nation's
health budget (US$612 billion in 2014)
Insulin: A standard treatment for diabetes since 1924
1921: First isolation of insulin from animals. (UofT) (Macleod and Banting awarded Nobel Prize in 1923 for discovery)
1922: Large scale production, commercialization (Eli Lilly, Novo Nordisk
Source of Insulin
The pancreas is an organ behind the stomach that is the main source of insulin in the body. Clusters of cells in the pancreas called islets produce the hormone and
determine the amount based on blood glucose levels in the body
Commercial Opportunities for the New Technology
1. Limitations of animal extraction (that can be solved by new technology)
a. Potential problems of allergic reactions
b. Slaughterhouse would not be able to provide enough supply of animal purchases
2. Market size of the proposed product
a. Increased diabetes patients
b. Insulin is the mainstream treatment
c. Lucrative markets
3. Regulatory environment
a. Animal insulin is already a mature product
b. Insulin is a relatively small and simple protein
Innovation
- Making insulin with recombinant DNA technology
Genetic Engineering for Insulin Productions
1. Obtain gene of interest
a. Insulin gene
2. Insert the gene into host cells
3. Select cells that contain the desired gene
4. Induce the bacteria to make protein from the “foreign” gene
5. Collect and purify products
a. Insulin protein
Obtaining the Insulin Gene
1. mRNA isolated bro b-cells in pancreas
2. cDNA produced by reverse transcriptase
3. mRNA destroyed
4. DNA strands produced by DNA polymerase
Why not obtain DNA directly from pancreas?
- Exons (<2% human genome)
- Introns (common in animals & plants, rare in bacteria)
Discovery in basic science → Biological Processing → Practical products, processes or systems
Bioprocess development is the link between basic science discovery and product manufacturing
Bioprocess Development for Large-scale production
- Eli Lilly the recombinant humans from Genentech in 1978.
- Its expertise in isolating antibiotics from fermentation was important is scaling the production of recombinant protein from bacterial cultures
What is Bioprocess Engineering About?
A bioprocess includes an “upstream” process and a “downstream” process
1. Upstream: product production in cell cultures/fermentations
a. Engineering expression system
b. Maximize production of cell factory
c. Scale up and bioreactor design
2. Downstream: isolation of product and its formulation
a. Two major process
i. Solid liquid separation
ii. Purification
Upstream Process Development
1. Batch
a. Cell inoculum is added to a defined volume of medium in either fermenter or roller bottles, and allowed to grow and be harvested at
predetermined optimal time point
