The Role of Chemical Engineering in the Era of Biotechnology

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BioChips
In-Soo Kim i.s.kim_at_lgchem.com LG Chem. Research
Park
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Contents

• Drug Discovery in Post Genomic Era
• BioChips Brief Introduction
• Components of BioChips
• Research on Biochips at LG

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Drug Discovery in Post Genomic Era
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Drug Discovery and Development Cycle
Clinical Research
Safety Metabolism
Lead Discovery and Optimization
Preclinical (1.5 - 3 Years)
Discovery (2 - 4 Years)
Clinical Trials (3 - 8 Years)
Approval (1 - 2 Years)
Lead Discovery
1 is approved
For every 5,000 discovery compounds...
5 candidates enter clinical trials...
FDA 1.5 Years
Phase I 1.5 Years
Phase II 2 Years
Phase III 4 Years
Phase IV
File NDA at FDA
File IND at FDA
Lead Optimization In Vitro Testing
Target Identification Validation Combinatorial
Chemistry HTS, Lead
Confirm effectiveness, monitor adverse reactions
from long-term use 1,000 to 5,000 patients
Approval to Market
Pharmacology Toxicology, PK/ADME Formulation
Evaluate effectiveness, look for side effects
100 to 500 patient volunteers
In Vivo Efficacy Testing
FDA required post marketing surveillance and
testing
Determine safety and dosage 20 to 100 healthy
volunteers
Review process and approval
300 - 500 million
0
A lengthy (7.5-15 years) and costly (300-500
million) process
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Paradigm shift in drug discovery
Predicted Growth Rate of me-too Market Only
7per year !
Genomics, HTS, Combichem
Labor intensive Time-consuming
Paradigm Shift
Genomics HTS Virtual Screening
Massive, Random Screening Search New Targets
Rational Drug Design Me too approach
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New Target Identification
Drug Targets
Family of Disease Gense (Total 923)
Gene Family
Total Gene
Receptor
Enzyme Modulator Protein Function Receptor Transcr
iption Factor Intracellular Matrix Extracellular
Matrix Transporter Ion Channel Unknown
32 14 10 9 7 6 6 4 7
Ion Channel, Transporter
Txn Factor
Natrue 409853(2001)
Secreted Proteins
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Genomics and Drug Targets
5000 Potential Targets

gt 2000 Validated Targets
500 Targets
Pre-genome
Post-genome
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BiochipsBrief Introduction
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Commercialization - New agricultural chemicals -
New drug compounds - New diagnostic test kits
Biosensor
HTS
Product Development - Combinatorial chemistry -
Diagnostic targets - High-throughput screenings -
Transgenics
BioMEMS
Increasing commercial value
Lab on a chip
Proteomics - Correlation of physiological
functions with protein - Protein-biomolecule
interactions
Protein chip
Functional genomics - Correlation of
physiological functions with genes -
Correlation level of tissue- related expression
Bioinformatics
DNA chip
Genomics - Primary sequence data -
Expressed-sequence tags - promoters, operons,
regulons - gene maps
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Overview

• Biochips(Microarrays)
• serve as core component of specialized
  genomic and proteomic analysis
  system
• drug discovery
• Epidemiological research
• in vitro diagnostic testing
• agricultural biotechnology
• 5 Major categories
• Biochips
• Instrumentation and equipment
• Reagents and other consumables
• analytical software
• custom services

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Biochips Demand by Type (M )
Item 1997 2001
2006 2011 Life science RD(B ) 32.6
50.6 74.6 104.2 biochip/000 RD
0.1 3.5 9.7
15.4 Biochips demand 4.2 176
720 1600 DNA chips
4.2 154 295 500 Laboratory
chips -- 14 180
460 Protein chips
-- 8 245 640
Biochip 8.6
31.4 45.0 48.5 Biochips Products
Service demands 48.7 560 1600
3300
Source The Freedonia Group, Inc.
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DNA Chips

• Applications
• Collection of gene expression data
• Disease targets for new drugs or diagnostics
  tests
• SNP identification
• Genotyping
• Pharmacogenomics
• Producers
• Affymetrics Genechip, Arrayer
• Agilent Microarray system. Plant
• Amersham Codelink system(Motorola)
• Genomic Solutions GeneMap microarrays
• Motorola Esensor Biochip
• Nanogen Nanochip
• Perkin Elmer Micromax
• Sequenom Spectrochip
• Spectral Genomics BAC DNA Chip ltBacterial
  Artificial chromosomegt

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Laboratory Chips

• Applications
• All life science investigations
• Drug discovery
• in vitro diagnostics
• Plant breeding
• Producers
• Caliper Agilent 2100 analyser, Caliper 250(HTS)
• Automated Workstation
• (Application Development System)
• Aclara eTAG system, LabCard chip
• Orchid Microfluidic System-layered flat chips
• Illumina Beadarray with Applera(PE Biosystems)

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Protein chips

• Applications
• Basic epidemiological research
• Secondary drug candidate screening
• in vitro diagnostic testing
• Pharmaceutical manufacturing
• Producers
• Ciphergen ProteinChip system(SELDI Technology)
• ltSurface Enhanced Laser
  Desorption/Ionizationgt
• Axon(Zyomyx) Protein chip scanning instruments
• Amersham Photolink photoactivated coating
  technology
• Perkin Elmer Hydrogel biochip and tyramide
  signal
• amplification(TSA)
  technology
• Prolinx Protein and peptide array under
  VersaLinx trademark

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Components of Biochips
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BioChip Integration of BT, NT and IT

• Substrate
• Surface Chemistry
• Contents
• Detection Mechanism
• Tagging Materials
• Detector

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Chip Components
Scanner
Detection Method
DNA, Protein etc. (contents)
Functional group
Base Plate
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Base Plate(substrate)
Polymer
Silicon
Glass

• Low cost material
• (disposable device)
• Material properties surface chemistries
• Poor solvent resistance
• Easy Processing

• Relatively high cost
• Well established processing
• (Wet and dry etching)
• Electrical conductivity
• Easy metallization

• Poor machinability
• Low background fluorescence
• Resistance against chemicals and UV
• Low electrical conductivity

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Surface Chemistry
Surface Attachment Advantage Disadvantage
PVDF Adsorption absorption No protein modification requirement, High protein binding capacity Non specific protein attachment, In random orientation
Nitrocellulose Adsorption Absorption No protein modification requirement, High protein binding capacity Non-specific binding, High background,Low density arrays
Poly-lysine coated adsorption No protein modification requirement Non-specific adsorption
Aldehyde-activated Covalent cross-linking High density and strong protein attachment High-resolution detection methods available Random orientation
Epoxy-activated Covalent cross-linking High density and strong protein attachment High-resolution detection methods available Random orientation
Avidin coated Affinity binding Strong, specific and high density protein attachment, low background Proteins have to be biotinylated
Ni-NTA coated Affinity binding Strong, specific and high density protein attachment, low background, uniform orientation Proteins have to be Hisx6 tagged
Gold coated silicon Covalent cross-linking Strong and high density protein attachment, low background, can be easily coupled with SPR and mass spectrometry Random orientation, tough to fabricate, not commercially available
PDMS nanowell Covalent cross-linking Strong and high density protein attachment, well suited for sophisticated biochemical analysis Random orientation
3D gel pad and agarose thin film Diffusion High protein binding capacity, no protein modification requirement Tough to fabricate, not commercially available
DNA/RNA coated Hybridization Strong, specific and high density protein attachment, low background, uniform orientation Sophistigated in vitro production of labeled proteins
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Contents
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Contents
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Classes of Capture Molecules
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Capture Molecules for Protein Arrays
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Detection Methods

• ELISA
• SPR
• - Ag-Ab complex formation results in refractive
  index change
• Non-contact AFM
• - surface topological change
• Planar waveguide
• - only the surface-bound flurophores are
  seletively excited
• Electro-chemical
• - conductivity or current change
• Scanner
• - confocal microscopy

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Research on Biochips at LG
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Sol-Gel Technology
Low Background Level High Sensitivity
Strong Intensity
Precursor Synthesis Biocompatible Porous
Structure at R.T Materials Formulation
Sol-Gel Materials
Adhesion Gelation Time Homogeneity
Plastic Substrate
Plastic Substrate
Detection
Microarraying
Scanning
1st,2nd antibody
Coating Materials Transparency Adhesion
Surface Properties Background Effect
Wash
Incubation
Incubation Time Temperature Buffer Condition
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Sol-Gel Technology morphology(AFM)
Spot ??
Nano-Scale image
Micro-Scale image
Spot ??

• 3D Structure Multi-Porous Structure

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Sol-Gel Technology morphology
Signal distribution CLSM image

• Microspot ??? Signal? ??? ??

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Sol-Gel Technology characteristics
Antigen Protein chip? ??? LOD ??
Negative Control
p24 50pg/spot
p24 250pg/spot
p24 500pg/spot
200ng/ml
2ng/ml
2pg/ml
2fg/ml
20ag/ml

• ??? Sensitivity, Activity
• 3D Nanoporous Capsule ?? ? ??? Protein ?? ??, ??
• Pore Size Control? ??? ??? ?? ?? ??
• Good Spot Morphology? Uniformity ???? ??? ??

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Introduction of TB

• The worlds largest infectious disease
• Infects one third of the worlds population
• 8 million new clinical cases per year
• Kills gt 3 million people per year
• MDR gt10 in China, Russia
• Co-infection with NTM
• HIV is re-activating latent TB
• HIV patients are very susceptible to TB
• In the poorest nations, TB kills the majority of
  HIV patients
• TB/HIV patients are difficult to diagnose and
  treat

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Introduction of TB Diagnosis

• Smear - insensitive, 104 bacteria/ml of
• sputum required
• X-ray - highly skilled, cant differentiate
• active previous infection
• Sputum culture slow, requires expensive
• equipment and skilled lab staff.
• Tuberculin skin test - poor specificity
• Serodiagnosis - detects IgG fast, non-specific
• PCR can detect dead or contaminated TB

TB is one of most difficult bacterial diseases
to diagnose treat
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• PNA Chip for Mycobacterial genotyping

Conventional Concept
LG Concept
DNA
PNA
vs.
Dry process (Epoxy)
Wet Process
Glass/ Silicon
Plastic (Polystyrene)

• Higher stability using PNA probes instead DNA
• Efficient surface chemistry using dry
  process(plasma treatment)
• Using cost-effective and flexible plastic
  substrate

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Genome Structure of Mycobacteria
Protein Gene Distribution Map (Mycobacterium
tuberculosis)
MT1385 MT1703
Between 16S and 23S rDNA

• Example Mycobacterium tuberculosis H37Rv
• Total Bases 4,411,532 base pairs
• Target region of M-chip Inter Transcriptional
  Spacer( ITS)
• - sequence No. 1,473,383 1,473,657

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PNA vs. DNA
Advantages

• No salt dependency
• Higher hybridization forces
• High Sensitivity
• Resistant to Nuclease
• ? Stable Structure

Poly amide backbones Electrically neutral str.
Phosphodiester backbones Negative charge
Disadvantages

• Expensive
• Hydrophobic

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Plastic substrate
Optical Resins and its Selection Guide for
Plastics Healthcare Applications.

• Bio-compatibility
• Optical properties
• Surface property
• Mechanical stability
• Chemical stability
• Rheological property

PMMA Polymethylmethacrylate PS Polystyrene PC
Polycarbonate COC Cyclic Olefin Copolymer
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Plasma Treatment
? ???? ??
Step 1. ???(??? ????)
? ???? ??(PS)
Step 2. ??? ??(???)
Step 3. ??? ??(??)
? ??? ? Gases (Ally glycidyl ether)
(Ar, O2, H2)
???? ??? ??? ????? ????(?? ? ???? ???? ??)
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Plasma Treatment
Step 1 Injection Molding

• Low auto-fluorescence resin LG GPPS 15NFI
• Optical lens quality mold nano scale roughness
• Optimum polymer processing condition

Advantage of plastics slide vs. glass slide
Design Free ? Lower cost for 3D or complex
design
Step 2 Plasma Surface Treatment
Epoxide Surface Clear Polystyrene Slide
Auto-fluorescence by 532 nm laser 700

• Monomer Allyl Glycidyl Ether
• Process Activation?Grafting?Radical
  Termination at low pressure(lt100 Pa), at low
  temperature(lt 50 ?)

Advantage of plasma process vs. wet process
Various characteristics with same monomer
Micro scale patterning with different functions
? Auto-fluorescence measurement GenePix 4000B,
100 Power, 600 PMT level ? Plasma is the forth
state of matter(solid, liquid, gas, and plasma)
consisted of ions, electrons, free radicals, and
other neutral species.
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Format of M-Chip
No
Species
No
Species
1
12
1
M. tuberculosis
12
M. terrae
2
13
2
M. avium-intracellulare
13
M. flavescens
3
14
3
M. fortuitum
14
M. smegmatis
4
15
4
M. chelonae
15
M. malmoense
5
16
5
M. abscessus
16
M. simiae
6
M. kansasii
17
M. marinum- ulcerans
6
17
7
M. gordonae
18
M. gastri
7
18
8
M. scrofulaceum
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M. avium
8
19
9
M. szulgai
20
M. intracellulare
9
20
10
M. vaccae
21
M. genavense
10
21
11
M. xenopi
22
M. lentiflavum
11
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Positive control
Negative control
Position marker/Hybridization control
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M-chip process
Plasma treatment
PNA linkage
PS
Micro-Arraying
PS
Epoxy-Modified PS slide
PNA
PNA
PNA
PNA
DNA
Hybridization
PS
PS
NC mis- match match
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M-Chip protocol
Step 1 Sample Preparation

• Heat Extraction of Genomic DNA of Mycobacteria
  with Chelex resin

Step 2 Amplification of Target

• Polymerase Chain Reaction One-tube Nested
  Asymmetric
• PTC200 Cycler MJResearch

Step 3 Arraying and Post-process

• Piezorray Perkin Elmer, Non-contact
• Epoxy coated Plastic slide of Lg chem, Epoxy
  coated glass slide of corning and nunc
• Incubation for Immobilization of DNA/PNA probe
  55?, 100 relative humidity, 16 hrs
• Post-process washing with SDS solution and
  distilled water

Step 4 Hybridization / Washing and Detection

• Eliminate Prehybridization of microarray slide
  and Denaturation of PCR product
• Hybridization Mixture 100 ul of Hybridization
  Buffer 5 ul of PCR products
• Hybridization reaction 55?, 1hrs
• Washing SSC solution / 2 min, Distilled Water
  / 1 min
• Detection GenePix4000B

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Results
?? ?? OK
 
??? ?? 1 ?? ???? PCR ??
?? ???? ?? 1 ?? 2 ?? 3 ?? 4
?? 1.3 1.8 5.3 6.3 7.2
? ???? Corning ? Epoxide ????(??) ? ???? ????
?? ??(?? ??)
???? OK
??? ?? 2 PCR ?? 3? ? ??
?? ?? B100 k copy ?? C 100 k copy ?? D 100 k copy ?? D 100 copy
???? 84 16 67 33
???? 20 9 24 6
???? OK
? ???? Corning ? Epoxide ????(??)
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Results
Cel (Glass, Aldehyde)
Nunc (Glass, Epoxy)
Corning (Glass, Epoxy)
LG1 (Activation time 30sec)
LG2 (Activation time 60sec)
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Results
Cel (Glass, Aldehyde)
Nunc (Glass, Epoxy)
Corning (Glass, Epoxy)
LG1 (Activation time 30sec)
LG2 (Activation time 60sec)
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Results
TB
Position Marker
Positive control
Multiple infection
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Manufacture system
Required technologies for manufacturing of
Diagnostic chip
Nucleic acid extraction
PCR
DNA/PNA chip

• Sampling
• Extraction method
• Sample contamination
• Purity of DNA/RNA

• Reaction composition
• Reaction volume
• Reaction control
• Sensitivity
• Specificity
• Cross contamination
• Electrophoresis

• Spotting condition
• Post-spotting management
• Hybridization composition
• Hybridization volume
• Hybridization condition
• Sensitivity
• Specificity
• Stability
• Contamination
• Non-specific signal
• Reaction control system
• Data analysis program

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QC Flow chart for manufacturing
Check point for QC
Kit QC
Spot QC
Probe QC
Check probes
Check the slide
Check purity
Check the product
Check spots
Check the spot reaction
Check the program
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Thank you!