cost-effectiveness : economically feasible basic biology...
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Biotechnology
What is Biotechnology? - Purposeful design and modification/assembly of bio-oriented materials (e.g.,
proteins/enzymes, microorganisms, plant/animal cells, tissues, stem cells etc..)
and unit processes to benefit humans or make a profit.
- Use and applications of biological system (cells, tissues etc..) or biomolecules (enzymes/proteins, antibodies, DNA/RNA) and key technologies to produce
valuable products at commercial scale and to treat diseases:
Cost-effectiveness : economically feasible
Basic Biology / Medical sciences - To discover and understand the underlying mechanisms of behaviors and
disorders in living organisms
Traditional Biotechnology (Before 1970) - Broad definition of Biotech : Using a biological system to make products
• Food processing : Fermented foods, Brewery, Dairy products, etc.
Biological process of brewing beer : conversion of starch to sugar followed
by fermentation by specific yeast
• Agriculture : Modifications of living plants for improved yield of crops via
artificial selection and hybridization: Breeding
ex) Crops with reduced vulnerability to frost, draught, and the cold
• Simple process
- Direct use of or isolation from original biological sources
- Fermentation: production of acetone using Clostridium acetobutylicum
Definition of Biotechnology based on the use of techniques/methods
• Use of recombinant DNA technology since 1973
- Cohen and Boyer : Gene manipulation techniques to cut and paste DNA
(using restriction enzymes and ligases) and transfer the new DNA into bacteria.
Revolutionize traditional biotechnology
• Combined use of different disciplines:
- Biology-based knowledge : Cell biology, genetics, molecular biology, etc
- Knowledge linked with practical applications :Biochemical Eng, Bioinformatics,
computational design, Organic chemistry etc.
• Use of genetically engineered microorganisms
- Enabling the production of existing medicines or products easily and cheaply
(ex: Insulin (51 amino acids) : discovered by Banting and Macleod from Univ. of Toronto, awarded Nobel Prize in 1923. Assistants : Charles Best (not Noble)
- First genetically engineered synthetic insulin (Humulin) by E. coli in 1982)
• Traditional Biotechnology industries : Adopts new approaches and modern techniques
to improve the quality and productivity of high value-added products
Modern Biotechnology (After 1970s)
Impact of recombinant DNA technology on the production of proteins
• Overcomes the problem of source availability : allows the manufacture of any proteins in whatever quantity it is required
• Overcomes the problem of product safety:
Transmission of blood-born pathogens such as hepatitis B, C, and HIV
via infected blood products
• Provides an alternative to direct extraction from inappropriate or dangerous source materials : Fertility hormones (FSH and hCG) from the urine of pregnant women; Urokinase from urines
• Facilitates the generation of newly designed proteins:
Therapeutic proteins or enzymes with desired property
• Development of therapeutics based on underlying mechanisms of diseases
- Development of new methods to cure diseases : Gene and cell (stem cells) therapies, therapeutic proteins
• Production of valuable products at commercial scale
Organic acids, Antibiotics, Amino acids, Proteins(enzymes), Biofuels, Vitamins,
Hormones, Alcohols, Fermented foods, Fine chemicals, etc..
• Development of tools and methodology
Expression systems, Gene synthesis/Sequencing, Purification process, Formulation, Bioassays, Diagnosis, Delivery
Major focus of Biotechnology
• Multi-disciplinary field
- Integration of biological sciences with Engineering principles
cost-effectiveness
• Required disciplines
- Biology
- Physical, organic chemistry / Pharmacology, Electronics
- Biochemical engineering : Extension of chemical engineering principles to biological system Mass/Heat/Energy transfer, - Thermodynamics Bioreaction engineering, plant design, process control / optimization, and separations
Basic Biology
Biotechnology Bio-industry - Pharmaceutical - Biotech. company Engineering principles
Features of Biotechnology is a multi-disciplinary field
New paradigms in Biotechnology
• Massive and high-speed analysis system
- Genome and proteom-wide approach : Systemic approach
- Huge amounts of relevant knowledge
• Genomics (Gene chips) : Sequences of more than few hundreds genomes
- 1 million genes / chip
- Gene (mRNA) expression profiling in high throughput way
- Single nucleotide polymorphism (SNP)
• Proteomics (2-D gel, LC/MS, protein microarray)
- Functional genomics
- Bio-molecular interactions (Interactoms)
• Computational approach:
- Computational design of proteins, Bioinformatics
• Genome- and proteom-wide analyses: Global analysis • Integration of high-throughput analysis system
• Health care / Diagnostics :
- Therapeutics with high efficacy and low toxicity
- Gene therapy: correction of mutated genes - Immuno-therapy : use of immune system - Regenerative medicine: Replacement of damaged or defective organs - Diagnosis : Early detection and prevention of diseases POCT (Point of Care Testing), Genome sequence
• Agriculture : Crop production with high yield and quality ex) GMO issue
• Bio-based process: Environmental pollution, CO2 emission, Global warming, Climate change: Replacement of chemical processes
• Alternative energy (Bio-energy) : - Depletion of fossil fuels - Use of renewable sources : Corn, sugar cane, cellulose - C1 gas refinery : CH4, CO : conversion to transportation fuels or other compounds
Major application areas
• Protein engineering : Design of proteins/enzymes based on structural and mechanistic knowledge, molecular evolution, computational design
• Metabolic pathway engineering: Design of more efficient metabolic pathways:
high yield of target product, low by-product
• Computational modeling and optimization: Systems biology, Genome- and proteom-wide analyses, Design of proteins with desirable property
• Nano-biotechnology : Integration of nanotechnology
- Use of NPs for diagnosis, drug delivery, and imaging
- Nanomedicine
Key technologies and fields
• Cell culture engineering : Cultivation of microorganisms and mammalian cells
- Hybridoma technology : A technology of forming hybrid cell lines (called hybridoma) by fusing a specific antibody-producing B cell with a myeloma
(B cell cancer) cell that is selected for its ability to grow in culture media.
• Tissue engineering/Regenerative medicine : use of a combination of cells, engineering and materials/ methods, and suitable biochemical and physio-chemical factors to repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, skin, muscle etc,--> artificial organs )
- Organ-on-a chip: multi-channel 3-D microfluidic cell culture chip
artificial organ: in vitro multicellular human organisms
mimic an organ’s cellular physiological functions
• Synthetic biology : Creation of new bio-systems (Cells and biomolecules): Systematic, hierarchical design of artificial, bio-inspired system using robust, standardized and well-characterized building block
• Genome editing:
- DNA is inserted, deleted or replaced in the genome of an organism using
engineered nucleases, or "molecular scissors.
- The induced double-strand breaks are repaired through non-homologous
end-joining (NHEJ) or homologous recombination (HR), resulting in targeted
mutations ('edits').
- Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector-based
Nucleases (TALEN®), and the CRISPR/Cas 9 system
• Separation / purification technology : Recovery and purification of a target product
- Critical factor determining the economic feasibility of the bioprocess
Branches of Biotechnology
• Blue biotechnology : Marine and aquatic applications of biotechnology
• Green biotechnology : Agricultural applications
Plant biotechnology, transgenic plants
• Red biotechnology : Medical applications
- Pharmaceuticals, Nanomedicine, Regenerative medicine
• White biotechnology : Industrial applications
- Production of valuable compounds using enzymes and
microorganisms
Applications in four major industrial areas
Medical applications: treatment of diseases
• Proteins: Key biomolecules in metabolic and signaling processes • Control and regulate cellular function accurately and specifically • Diseases development: Abnormal regulation and control of signaling processes and dysfunction of proteins owing to mutations
Protein-based drugs • Small molecule-based drugs : Efficacy, side effect, safety
• Therapeutic proteins : High efficacy and safety, less toxicity
- Antibodies, proteins, enzymes, peptides etc.
ex) EPO, Interferon, Insulin, Avastin, Enbrel, Remicade, Herceptin,
EPO (Erythropoietin) : Stimulating the proliferation of red blood cells
Herceptin : Mab against EGFR2(Epidermal growth factor receptor 2) Avastin : Mab against VEGF (Vascular endothelial growth factor) Remicade, Humira: Mab against TNF-α (Tumor necrosis factor- α)
• World market
- EPO alone : ~ $ 11 billion per year
- Humira : ~ $ 9 billion per year
- $ 50 Billion (2007) $ 190 Billion (2015)
- Intensive investment in monoclonal antibodies
- Biosimilar
Therapeutic proteins will form the back-born of future biotech market
Rheumatoid Arthritis
• Autoimmune disease in which the normal immune response is directed against an individual's own tissue, including the joints, tendons, cartilages, and bones, resulting in inflammation and destruction of these tissues
• Affects between 0.5 and 1% of adults in the developed world with between 5 and 50 per 100,000 people newly developing the condition each year.
• Most frequent during middle age, and women are affected 2.5 times as frequently as men
• Chronic disease
• Cause is not clear: Combination of genetic and environmental factors
- Possibilities of a foreign antigen, such as a virus or bacteria
• Description - Morning stiffness - Arthritis of 3 or more joints - Arthritis of hand joints - Symmetric arthritis - Rheumatoid nodules - Serum rheumatoid factor - Radiographic changes
Osteoclast : a type of bone cell that breaks down bone tissue Chondrocytes : cells found in healthy cartilage, and produce and maintain the cartilaginous matrix Cartilage : Resilient and smooth elastic tissue that covers and protects the ends of long bones at the joints
- Immune system ceases to recognize the body's normal constituents as "self,“ leading to production of pathological autoantibodies. - Autoantibodies attack the body's own healthy cells, tissues, organs, causing inflammation and damage.
• NSAIDs for stiffness • Corticosteroids for inflammation and to suppress the autoimmunity • Disease Modifying Anti rheumatic Drugs (DMARDs) - Methotrexate, Cyclosporine, Azathioprine, cyclophosphamide
Therapeutic drugs
Small molecule-based drugs
• Tumor Necrosis Factor (TNF)-α inhibitors: reduces inflammatory response
- Adalimumab (Humira): anti TNF-α Cost: $3,100 /M Global sales (2014): $13.0 billion Patent expired in 2016: First biosimilar in India: market at a price of $200
- Infliximab (Remicade): anti TNF-α
- Etanercept (Enbrel) : anti TNF-α
Fusion protein : TNF-α receptor fused to Fc of IgG1 antibody
• Tocilizumab (Actemra): anti IL-6 receptor
Biologics: Monoclonal antibodies or proteins
Disease Product name
Developer Sales ($ Millions)
Features 2004 2007
Gaucher’s
Ceredase® Genzyme 443 N/A Glucocerebrosidase (β-Glucosidase)
Purified from human placenta
Cerezyme® Genzyme 932
(2005) 1,048
Produced in CHO cells
3 Exoglycosidases process for Terminal Mannose
Fabry’s Fabrazyme® Genzyme 209 397 α-galactosidase
Mannose-6-phosphate for Glycotargeting Replagal TKT 57 168
MPS-1 Aldurazyme® Genzyme 12 204 α –L-iduronidase
Pompe Myozyme® Genzyme Approved
(2006) α-glucosidase
Therapeutic Enzymes : Enzyme replacement treatment
Treatment of Gaucher’s disease by Cerezyme costs up to $550,000 annually: Orphan drug and life-long treatment
Most of therapeutic enzymes : glycoproteins
β-Glucosidase
- Found by Phillipe Gaucher in 1882
- Biochemical basis for the disease in 1965 by Brady et al..
Glucosyl
CH2-CH-CH-CH=CH-(CH2)12-CH3
O=C-CH2-CH2-CH2-(CH2)n-CH3 N OH
Ceramide
OH-CH2-CH-CH-CH=CH-(CH2)12-CH3
O=C-CH2-CH2-CH2-(CH2)n-CH3 N OH
Glucose Ceramide
Gaucher’s Disease : Lysosomal Storage Disease
Autosomal recessive inheritance
- Caused by a recessive mutation in a gene located on chromosome 1, affecting both males and females
- Most common type of LSD
Glucocerebroside: Constituent of red and white blood cell membranes
Lysosomal storage diseases (LSDs): Lysosomal Enzymes
Lysosomes: Cellular organelles containing acid
hydrolase enzymes to break down waste materials and cellular debris
Cells’ garbage disposal system • Digestive organelle in the cell • Contains ~40 hydrolytic enzyme • Acidic pH (about pH4.8) within the
lysosome : the activity of lysosomal enzymes
(1) The ER and Golgi apparatus make a lysosome
(2) The lysosome fuses with a digestive vacuole
(3) Activated acid hydrolases digest the contents
(LSD) Lysosome
Nucleus
Mitochondria
Lysosome with substrate accumulation
(Normal cell) (LSD cell)
Normal cells Glucocerebrosides
Glucocerebrosides
Digestive vacuole
Gaucher cells
Digestive vacuole
Glucocerebrosidase
Incomplete digestion
Exocytosis
Residual vacuole
glucose ceramide +
Residual vacuole accumulated
No exocytosis
1/ 40,000~60,000 (Jew 1/~500) Swollen vacuoles Gaucher cells Accumulation in spleen, liver, kidney, brain Enlarged spleen and liver, liver malfunction, neurological complications etc..
Gaucher’s disease : Occurrence and symptoms
Distended abdomen
Nucleases: Genome editing
• Nucleases or artificially engineered "molecular scissors."
- A type of gene engineering by which DNA is inserted, replaced, or
removed from a genome
• The nucleases create specific double-stranded break (DSBs) at desired locations in the genome, and harness the cell’s endogenous mechanisms to repair the induced break by natural processes of homogeneous recombination (HR) and nonhomogeneous end-joining (NHEJ).
• Applications: plants, livestock, human
• Four families of engineered nucleases
- Mega-nucleases: Endodeoxyribonucleases with a large recognition site
(double-stranded DNA sequences of 12 to 40 base pairs)
- Zinc finger nucleases (ZFNs)
- Transcription Activator-Like Effector Nucleases (TALENs)
- CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas system
• Ethical issues: Editing of human embryos - First approval in UK for human healthy embryos to alter genes after fertilization • Off-target effect : Non-specific mutations • Intellectual property issue
CRISPR/Cas 9 system
Bio-based economy: Impact on global economy
• Shift from petroleum-based economy
- Exhaustion and soaring price of petroleum (> $ 100 /gallon)
- Environmental issue:
Global warming (greenhouse gas, CO2 , emission), Pollution
• Development of renewable source-based Bioprocess
- Corn, starch, cellulose
- Conversion of C1 gas to transportation fuels and feedstocks
Methanotrophic bacteria : Methylococcus or Methylomonas species
• Replacement of chemical processes with bio-based ones
White Biotechnology
Company Products
BASF
Vitamin B-2 Methoxy isopropyl amine (chiral intermediate) Styrene oxide Amino acids
Eastman Chemical / Genencor Ascorbic acid
Degussa
Acrylamide Fatty acid – derived esters Polyglycerine ester Organo modified silicones and oleochemicals
Celanese / Diversa
Acetic acid Polyunsaturated fatty acids Non-digestible starch Polylactic acid (PLA)
Cargill Polylactic acid (PLA) (140,000 MT/yr)
DuPont / Genencor 1,3-Propanediol Terephthalic acid Adipic acid
Chevron / Maxygen Methanol
Typical chemical companies in bio-based production
Biomolecular Eng. Lab.
Enzymes
- Cleaning (Detergents) - Textiles - Starch Processing - Leather - Baking - Pulp and Paper - Food and Specialties - Cosmetics
• Most proficient catalysts with high specificity • Competitive and cost-effective processes
- Chiral drugs
- Chiral intermediates
- Semisynthetic antibiotics
- Organic acids
Synthesis of specialty chemicals
Use for biosciences
- DNA polymerase: Thermostability, fidelity
- Restriction enzymes: Specificity
- Alkaline phosphatase, Peroxidase
Use for daily life
Key role of enzymes in Bio-based economy
Energy and Environmental issues - Depletion of fossil fuels - Control of CO2 emission (Kyoto protocol)
Renewable source-based economy
Bio-based process
Enzymes
Petrochemical-based economy
Chemical process
Use of enzymes for biofuel and biochemicals from renewable biomass such as starch and cellulose amylase, cellulase etc.
- The immune system depends on multiple checkpoints to avoid over-activation of the immune system on healthy cells
- Tumor cells often take advantage of these checkpoints to escape detection by the immune system.
- CTLA-4 and PD-1 are checkpoints that have been studied as targets for cancer therapy: Checkpoint inhibitors
Immunotherapy for cancers
- T cells are removed from a patient and modified so that they express receptors specific to the particular form of cancer CAR(Chimeric antigen receptor) -T cell therapy
- The T cells are reintroduced into the patient. - The T cells can recognize and kill the cancer cells by secreting granzyme or perforin
Checkpoint inhibitors
CAR-T cell therapy
Diagnostics
• Diagnosis of disease as early as possible :
Best solution compared to treatments
• Prediction and treatment of diseases based on individual
genome sequence
- Personalized medicine
- Treatment with appropriate therapeutic agents
• Analysis / Detection of disease biomarkers:
- Invasive or non-invasive analysis
Biotechnology will have the greatest impact on humans
in the future in terms of health care, life-style, and economy.
- Therapeutic proteins
- Immuno-therapy
- Regenerative medicine
- Genome editing
- Bio-based economy : High-value compounds by bioprocess
- Diagnostics
Modern Biotechnology constitutes a variety of diverse areas
and technologies, requiring interdisciplinary collaborations.
Perspectives