Monoclonal antibodies

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• Monoclonal antibodies
• Anticancer therapy

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THE IMMUNE SYSTEM
DEFINITION - The integrated body system of
organs, tissues, cells cell products that
differentiates self from non self neutralizes
potentially pathogenic organisms. (The American
Heritage Stedman's Medical Dictionary)

• The Immune System consists of
• Innate Immunity
  Primary Response
• Acquired Immunity
  Secondary Response

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ANATOMY OF THE IMMUNE SYSTEM
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FUNCTIONING OF THE IMMUNE SYSTEM
HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE
CELL MEDIATED IMMUNE RESPONSE
ANTIGEN (1ST EXPOSURE)
ENGULFED BY
MACROPHAGE
ANTIGENS DISPLAYED BY INFECTED CELLS ACTIVATE
FREE ANTIGENS DIRECTLY ACTIVATE
BECOMES
APC
STIMULATES
HELPER T CELLS
CYTOTOXIC T CELL
B CELLS
STIMULATES
STIMULATES
MEMORY HELPER T CELLS
GIVES RISE TO
GIVES RISE TO
STIMULATES
STIMULATES
STIMULATES
ANTIGEN (2nd EXPOSURE)
ACTIVE CYTOTOXIC T CELL
MEMORY B CELLS
PLASMA CELLS
MEMORY T CELLS
STIMULATES
SECRETE ANTIBODIES
Defend against extracellular pathogens by binding
to antigens and making them easier targets for
phagocytes and complement
Defend against intracellular pathogens and cancer
by binding and lysing the infected cells or
cancer cells
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Lymphocytes

• Produce antibodies
• B-cells mature in bone marrow then concentrate in
  lymph nodes and spleen
• T-cells mature in thymus
• B and T cells mature then circulate in the blood
  and lymph
• Circulation ensures they come into contact with
  pathogens and each other

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B -Lymphocytes

• There are 10 million different B-lymphocytes,
  each of which make a different antibody.
• The huge variety is caused by genes coding for
  abs changing slightly during development.
• There are a small group of clones of each type of
  B-lymphocyte

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B -Lymphocytes

• At the clone stage antibodies do not leave the
  B-cells.
• The abs are embedded in the plasma membrane of
  the cell and are called antibody receptors.
• When the receptors in the membrane recognize the
  antigen on the surface of the pathogen the B-cell
  divides rapidly.
• The antigens are presented to the B-cells by
  macrophages

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B -Lymphocytes
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B -Lymphocytes

• Some activated B cells ? PLASMA CELLS these
  produce lots of antibodies, lt 1000/sec
• The antibodies travel to the blood, lymph, lining
  of gut and lungs.
• The number of plasma cells goes down after a few
  weeks
• Antibodies stay in the blood longer but
  eventually their numbers go down too.

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B -Lymphocytes

• Some activated B cells ? MEMORY CELLS.
• Memory cells divide rapidly as soon as the
  antigen is reintroduced.
• There are many more memory cells than there were
  clone cells.
• When the pathogen/infection infects again it is
  destroyed before any symptoms show.

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What are antibodies?

• An antibody is a protein used by the immune
  system to identify and neutralize foreign objects
  like bacteria and viruses. Each antibody
  recognizes a specific antigen unique to its
  target.
• Monoclonal antibodies (mAb) are antibodies that
  are identical because they were produced by one
  type of immune cell, all clones of a single
  parent cell.
• Polyclonal antibodies are antibodies that are
  derived from different cell lines.
• Isotypes
• According to differences in their heavy
  chain constant domains, immunoglobulins are
  grouped into five classes, or isotypes IgG, IgA,
  IgM, IgD, and IgE.
• IgG IgG1 (66), IgG2 (23), IgG3 (7) and IgG4
  (4) , blood and tissue liquid.
• IgAIgA1 (90) and IgA2 (10), stomach and
  intestines
• IgM normally pentamer, ocassionally hexamer,
  multiple immunoglobins linked with disulfide
  bonds
• IgD1 of proteins in the plasma membranes of
  B-lymphocytes, function unknown
• IgE on the surface of plasma membrane of mast
  cells, play a role in immediate hypersensitive
  and denfensive for parasite

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The structure of antibodies

• http//www.path.cam.ac.uk/mrc7/igs/mikeimages.htm
  l

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Antibodies

• Also known as immunoglobulins
• Globular glycoproteins
• The heavy and light chains are polypeptides
• The chains are held together by disulphide
  bridges
• Each ab has 2 identical ag binding sites
  variable regions.
• The order of amino acids in the variable region
  determines the shape of the binding site

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ANTIBODIES
POLYCLONAL.
MONOCLONAL.
Derived from different B Lymphocytes cell lines
Derived from a single B cell clone
mAb offer Reproducible, Predictable Potentially
inexhaustible supply of Ab with exquisite
specificity
Batch to Batch variation affecting Ab reactivity
titer
Enable the development of secure immunoassay
systems.
NOT Powerful tools for clinical diagnostic tests
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How Abs work

• Some act as labels to identify
• antigens for phagocytes
• Some work as antitoxins i.e. they block toxins
  for e.g. those causing diphtheria and tetanus
• Some attach to bacterial flagella making them
  less active and easier for phagocytes to engulf
• Some cause agglutination (clumping together) of
  bacteria making them less likely to spread

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Different Immunoglobulins
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Type Number of ag binding sites Site of action Functions
IgG 2 Blood Tissue fluid CAN CROSS PLACENTA Increase macrophage activity Antitoxins Agglutination
IgM 10 Blood Tissue fluid Agglutination
IgA 2 or 4 Secretions (saliva, tears, small intestine, vaginal, prostate, nasal, breast milk) Stop bacteria adhering to host cells Prevents bacteria forming colonies on mucous membranes
IgE 2 Tissues Activate mast cells ? HISTAMINE Worm response
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History of Mab development

• 1890 Von Behring and Kitasato discovered the
  serum of vaccinated persons contained certain
  substances, termed antibodies
• 1900 Ehrlich proposed the side-chain theory
• 1955 Jerne postulated natural selection theory.
  Frank Macfarlane Burnet expended.
• Almost the same time, Porter isolated fragment of
  antigen binding (Fab) and fragment crystalline
  (Fc) from rabbit y-globulin.
• 1964 Littlefield developed a way to isolate
  hybrid cells from 2 parent cell lines using the
  hypoxanthine-aminopterin-thymidine (HAT)
  selection media.
• 1975 Kohler and Milstein provided the most
  outstanding proof of the clonal selection theory
  by fusion of normal and malignant cells
• 1990 Milstein produced the first monoclonal
  antibodies.

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The types of mAb designed

1. Murine source mAbs rodent mAbs with excellent
   affinities and specificities, generated using
   conventional hydrioma technology. Clinical
   efficacy compromised by HAMA(human anti murine
   antibody) response, which lead to allergic or
   immune complex herpersensitivities.
2. Chimeric mAbs chimers combine the human constant
   regions with the intact rodent variable regions.
   Affinity and specificity unchanged. Also cause
   human antichimeric antibody response (30 murine
   resource)
3. Humanized mAbs contained only the CDRs of the
   rodent variable region grafted onto human
   variable region framework

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Chemotherapy

• Shortcomings
• Nature of cytotoxin
• Lack of in vivo selectivity
• The mechanism of anti-proliferation on cells
  cycle, rather than specific toxicity directed
  towards particular cancer cell
• Host toxixity treatment discontinued, most of
  them had bad side-effects, such as no appetites,
  lose hair etc.

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Monoclonal antibodies for cancer treatment

• Three mechanisms that could be responsible for
  the cancer treatment.
• mAbs act directly when binding to a cancer
  specific antigen and induce immunological
  response to cancer cells. Such as inducing cancer
  cell apoptosis, inhibiting growth, or interfering
  with a key function.
• mAbs was modified for delivery of a toxin,
  radioisotope, cytokine or other active
  conjugates.
• it is also possible to design bispecific
  antibodies that can bind with their Fab regions
  both to target antigen and to a conjugate or
  effector cell

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mAbs treatment for cancer cells
ADEPT, antibody directed enzyme prodrug therapy
ADCC, antibody dependent cell-mediated
cytotoxicity CDC, complement dependent
cytotoxicity MAb, monoclonal antibody scFv,
single-chain Fv fragment. Carter P Improving
the efficacy of antibody-based cancer therapies.
Nat Rev Cancer 20011118-129
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EVOLUTION OF MONOCLONAL ANTIBODY
1. TRANSGENIC DNA SPLICING / GENE KNOCK OUT
2. LIBRARIES a.BACTERIOPHAGE b. mRNA c. Cell
Surface
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Conventional production of mAbs

• The hybridoma technology
• spleen cells from immunized mice are fused
  with the murine myeloma cells.
• The several process had been developed at large
  scale.
• According to the different cell culture methods,
  it can calisifed into four fields
• Robottle cell culture process.
• Membrane binded cell culture process
• Microcarrier cell culture process
• Suspended cell culture process

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Applications of Monoclonal Antibodies

• Diagnostic ApplicationsBiosensors Microarrays
• Therapeutic ApplicationsTransplant rejection
  Muronomab-CD3Cardiovascular disease Abciximab
  Cancer RituximabInfectious Diseases
  PalivizumabInflammatory disease Infliximab
• Clinical ApplicationsPurification of drugs,
  Imaging the target
• Future Applications Fight against Bioterrorism

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• REPORT HIGHLIGHTS
• The global market for therapeutic monoclonal
  antibodies (mAbs) was estimated at 44.6 billion
  in 2011.  With the rollout of at least eight new
  therapeutic mAb products and expanded indications
  for existing products expected during the
  forecast period, the global mAb market is
  expected to rise at a compound annual growth rate
  (CAGR) of 5.3 to nearly 58 billion in 2016.
• The U.S. is projected to be the largest single
  market for therapeutic mAbs from 2011 to 2016.
  This particular market was nearly 19.8 billion
  in 2010 and reached 20.1 billion by 2011. BCC
  projects this market will grow to 27.4 billion
  by 2016, a CAGR of 6.4.
• Sales of mAbs in the rest of the world will
  remain higher than in the U.S.despite a higher
  CAGR in the U.S. versus the rest of the world.
  This market is expected to grow from 24.6
  billion in 2011 to 30.3 billion in 2016 at a
  CAGR of 4.3.

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Why should we be interested ?

• mAbs drive the development of multibillion dollar
  biotechnology industry.
• Many of the leading pharmaceutical companies have
  entered the mAb sector, attracted by quicker and
  less costly development, higher success rates,
  premium pricing, and a potentially reduced threat
  from generics
• The outlook for monoclonal antibody therapeutics
  is healthy. The ongoing success of existing
  products, combined with a bulging pipeline of new
  products awaiting approval and limited generic
  erosion, point towards robust growth in this
  segment

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• Anything other than Antibodies? Major
  application areas
• Health care / Diagnostics
• - Development of therapeutics efficacy,
  toxicity
• - Diagnosis early detection and prevention
  of diseases
• Agriculture Crop production with high yield and
  quality
• Bio-based process Pollution, CO2 emission,
• global warming
• Alternative energy (Bio-energy)
• - Depletion of fossil fuels
• - Use of renewable sources Corn, sugar cane,
  cellulose
• - Cost (?)

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Key technologies and fields

• 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-wide analysis
• Nano-biotechnology Use of NPs for diagnosis
  and imaging
• Cell culture engineering Microorganisms and
  mammalian cells
• - Hybridoma technology A technology of
  forming hybrid cell lines (called hybridomas) by
  fusing a specific antibody-producing B cell with
  a myeloma (B cell cancer) cell that is selected
  for its ability to grow in tissue culture
  

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Branches of Biotechnology

• Separation technology Recovery and purification
  of a target product
• Synthetic biology Creation of new biosystems
  (Cells and biomolecules) Systematic,
  hierarchical design of artificial, bio-inspired
  system using robust, standardized and
  well-characterized building block

• Blue biotechnology Marine and aquatic
  applications of biotechnology
• Green biotechnology Agricultural applications
• Red biotechnology Medical applications
• White biotechnology Industrial applications

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Typical examples of Bio-Products
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
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General Bioprocess
Feedstock
Bioprocessing
Product
PRODUCT LINES
Cell culture Enzymatic
GAS
Biocatalyst
Bioreactor
Recovery product
LIQUID
SOLID
Feedstock
Bioprocessing
Product

• Gas
• Syn. Gas
• CO2
• Organic vapor
• Liquid
• Organic
• Sugar solution
• Solid
• Biomass
• Consumer Waste

• Immobilized Enzymes
• Ambient to Extreme
• Fermentation
• Immobilized
• Free cell
• Ambient to Extreme
• Bioreactors
• Continuous Systems
• Membrane
• Batch or Fed-batch

• Separation
• In situ
• Secondary
• Media
• Gaseous
• Aqueous
• Organic

• Pharmaceuticals
• Fine chemicals
• Specialty Chemicals
• Feedstock
• Bulk chemicals

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Bio-based economy Impact on global economy

• Shift from petroleum-based economy
• - Exhaustion and soaring price of petroleum
  (gt 100 /gallon)
• - Environmental issue
• Global warming (greenhouse gas, CO2 ,
  emission)
• Pollution
• Development of renewable source-based Bioprocess
• Replacement of chemical processes with Bio-based
  ones

White Biotechnology
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Value chains from renewable sources
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Key role of enzymes in Bio-based economy
Energy and Environmental issues - Depletion
of fossil fuels - Limitation to CO2
emission (Kyoto protocol)
Renewable source-based economy
Petrochemical-based economy
Chemical process
Bio-based process

Use of enzymes in Biofuel production from
renewable biomass such as starch and cellulose ?
amylase, cellulase etc.
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Enzymes

• Most proficient catalysts with high specificity
• Competitive and cost-effective processes

• Cleaning (Detergents)
• Textiles
• Starch Processing
• Brewing
• Leather
• Baking
• Pulp and Paper
• Food and Specialties
• Animal feeds
• Cosmetics

Industrial use for specialty chemicals

• Chiral drugs
• Chiral intermediates
• Semi-synthetic antibiotics
• Organic acids

Therapeutics

• Treatment of Gauchers disease

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Chemical company devoting to Biotechnology BASF
Emphasis on Bio-products mainly using Enzymes
Ecoflex
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Therapeutic proteins

• High specificity and less toxicity ? high safety
  and efficacy
• Therapeutic proteins
• - 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 Mab against TNF-a
  (Tumor necrosis factor- a)
• World market
• - EPO alone 11 Billion per year
• - 50 Billion (2007)? 190 Billion
  (2015)
• - Antibodies gt 50
• - Intensive investment in monoclonal antibodies
  Biosimilar

Therapeutic proteins will form the back-bone of
future medicinal therapy
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Structural and functional features of antibodies
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Blockbuster Therapeutic Antibodies
Approved Year Product Target Indication Company Market size(07) Antibody Type
1997 Rituxan CD20 Non-Hodgkin's lymphoma Genentech 4,603 Chimeric
1998 Herceptin Her2/neu Breast cancer Genentech 4,047 Humanized
1998 Synagis RSV RSV prophylaxis MedImmune 1,100 Humanized
1998 Remicade TNF- ? RA, Chrons disease JJ 5,234 Chiemric
2002 Humira TNF-? RA Abbott 3,064 Human
2003 Raptiva CD11a Psoriasis Genentech Xoma 211 Humanized
2004 Erbitux EGFR Colorectal cancer Imclone Bristol-Myers 1,336 Chiemric
2004 Avastin VEGF Colorectal cancer Genentech 3,335 Humanized
2006 Vectibix EGFR Colorectal cancer Amgen 170 Human
Million (Data Monitor Monoclonal 2008)
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Drawbacks of immunoglobulin antibodies

• Complicated process for selecting cell lines and
• the production using mammalian cells ? very
  expensive
• Intellectual property barriers
• Tend to aggregate due to large size ( 150 KDa)
• Difficult to penetrate inside the cells
• Limited binding affinity due to confined binding
  surface

Ideal scaffold for alternative therapeutics

• High-level soluble expression in bacteria
• High stability (thermodynamic, pH)
• Easy design of binders with high affinity for a
  target
• Low immunogenicity and cytotoxicity

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Therapeutics based on non-antibody scaffold
New paradigm in therapeutic proteins

• Development of new therapeutics with high
  efficacy and
• low side effect from non-antibody protein
  scaffold
• Designer therapeutic proteins
• IP issue and cost-effectiveness

GlaxoSmithKline, Amgen Bristol-Myers-Squibb,
Boehringer Ingelheim Eli Lilly, Roche, Avidia,
Ammunex. Affibody, Ablynex, Adnexus Therapeutics
.
Strategic alliance or merger between big pharma
and biotech companies
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Non-antibody scaffolds
Human lipocalin
Ankyrin
Human fibronectin
Z domain of Staphylococcal protein A
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Therapeutic Enzymes
Disease Product Developer Sales (USMillions) Sales (USMillions) Features
Disease Product Developer 2004 2007 Features
Gauchers Ceredase Genzyme 443 N/A Glucocerebrosidase Purified from human placenta
Gauchers Cerezyme Genzyme 932 (2005) 1,048 Produced in CHO cells 3 Exoglycosidases process for Terminal Mannose
Fabrys Fabrazyme Genzyme 209 397 alpha-galactosidase Mannose-6-phosphate for Glycotargeting
Fabrys Replagal TKT 57 168 alpha-galactosidase Mannose-6-phosphate for Glycotargeting
MPS-1 Aldurazyme Genzyme 12 204 alpha L-iduronidase
Pompe Myozyme Genzyme Approved (2006) Approved (2006) alfa-glucosidase
Treatment of Gauchers disease by Cerezyme costs
up to 550,000 annually
Most of therapeutic enzymes glycoproteins
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Gauchers Disease Lysosomal Storage Disease
Caused by a recessive mutation in a gene located
on chromosome 1, affecting both males and females.
- Found by Phillipe Gaucher in 1882 - Biochemical
basis for the disease in 1965 by Brady et al..
Glucosyl
Ceramide
Glucocerebrosidase (b-glucosidase)
Autosomal recessive inheritance
Ceramide
Glucose
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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 is
  required for lysosomal enzymes to be active

(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 with substrate accumulation
Lysosome
Nucleus
Mitochondria
(Normal cell)
(LSD cell)
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Gauchers disease Occurrence and symptoms

• 1/ 40,00060,000 (Jew 1/500)
• Swollen vacuoles ? Gaucher cells
• Accumulation in spleen, liver, kidney, brain
• Enlarged spleen and liver, liver malfunction,
• neurological complications etc..

Distended abdomen
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Diagnostics

• Diagnosis of disease as early as possible
• Best solution compared to treatments
• Prediction and treatment of diseases based on
  individual genomes
• - personalized medicine
• - treatment with appropriate therapeutic
  agents
• Analysis / Detection of disease biomarkers
• - Invasive or non-invasive analysis

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Perspectives

• Biotechnology will have the greatest impact on
  humans in the future in terms of health,
  life-style, and economy.
• - Therapeutic proteins
• - Bio-based economy Bioprocess and
  Bio-Energy
• - Diagnostics
• Modern Biotechnology constitutes a variety of
  diverse areas and technologies, requiring
  interdisciplinary collaborations.