Cost-effectiveness : economically feasible Basic Biology ...bel.kaist.ac.kr/extfiles/lecture/2017spring/bs223/...proteins/enzymes, microorganisms, plant/animal cells, tissues, stem

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

1 2

3 3

2

1 CDRS

FR

VL VH

Structural and functional features of antibodies

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

Value chains from renewable sources

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.

Regenerative medicine

ESC: Embryonic stem cells iPSC: Induced pluripotent stem cells

- 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

Advances in CAR-T

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