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Pressure BioSciences, Inc.
Pressure Cycling Technology (PCT):
A Novel, Enabling PlatformRevolutionizing Biomarker Discovery
BiotechnicaHannover, Germany
October 6, 2010
Forward Looking Statements
This presentation may contain forward looking statements that reflect management’s current views
and opinions as to the status of the Company’s products, technology and other future events and
operations. These statements are neither a promise nor guarantee, but involve risks and uncertainties that
could cause actual results to differ materially from those anticipated or indicated. Investors are
cautioned that any forward looking statements should be considered in light of such risks and uncertainties
including, without limitation, those detailed in the Company’s filings with the Securities and Exchange
Commission.
• Formed Sept 2004 - Sale of Boston Biomedica (BBI)
• NASDAQ CM: PBIO
• Started Operations in February 2005
• Began Instrument/Consumables Sales in Late 2007
• Fourteen (14) Employees
• Strong Management Team and Board of Directors
• 24 Issued Patents…Many More in Pipeline
• Focused on the Development and Commercialization of a Powerful, Proprietary, Enabling Platform
Pressure Cycling Technology (“PCT”)
Pressure BioSciences, Inc.
PCT is a Novel, Enabling Technology that
Uses Cycles of Hydrostatic Pressure
Between Atmospheric and Ultra-high
Levels (up to 35,000 psi and greater) to
Allow for the Precise Control of
Biomolecular Interactions
Pressure Cycling Technology (PCT)
History of High Pressure in Life Sciences
• 1623-1662: Blaise Pascal – described fundamental concepts of
pressure and vacuum
• 1895: H. Royer – pressure kills bacteria
• 1899: B.H. Hite et al. – pressure preserves milk
• 1914: P.W. Bridgman - pressure coagulates egg white
• 1989: High pressure processing of food products
• 2000: First International Conference on HPBB
• 2008: Fifth International Conference on HPBB in the USA
Understanding Hydrostatic Pressure
U.S. Navy Bathyscaphe Trieste (1958-1963)
Marianas Trench:38,713 ft (11,800m) deep
16,000 PSI (120MPa)
Significant portion of the Global Biosphere is subjected to high hydrostatic pressure!
Why Does PCT Work
• Pressure is a Thermodynamic Process
• Compressibility of Water
• Synergy of Pressure, Temperature and Chemistry
High Pressure Destabilizes Biological Membranes
Hydrostatic Pressure Applied
Hydrostatic Pressure Released
Lip
id b
ilaye
rs
Me
mbr
ane
Pro
tein
(Interdigitated bilayer,Hydrophobic hydration)
Current Extraction Methods• Mortar & Pestle• Dounce homogenizer (glass on glass)• Potter-Elvenhjem homogenizer (Teflon on
glass)• Enzymatic Digestion• Polytron shearing homogenizers• Blenders• Bead Beating• Sonication• Repeated Freeze/Thaw cycles• French Press (≤ 2000 PSI)
Barocycler™
NEP2320
PCT – Sample Preparation System
Barocycler™ NEP3229
The PCT Shredder
PCT Shredder (3rd GEN)• Long lasting lithium rechargeable batteries• Paddle for convenient pressure setting• Pressure levels -15, 30,45 lbs Force• PCT PULSE or Shredder Tubes• Heavy Duty and Robust Driver
PCT Shredder (1st GEN)• NiCad Batteries• Pressure set by pushing Driver• Pressure levels -15,30,45 lbs Force• PCT PULSE or Shredder Tubes
User-Adjustable Variables
• Pressure (up to 35 kpsi)
• Number of Cycles
• Cycle Profile
• Chemistry
• Temperature
Release of DNAwith the
PCT Sample Preparation System
(PCT SPS)
Genomics
• Samples : Tilapia and Goldfish were purchased
live, then frozen at -70 0C before processing. Total three samples were PCT processed for per each fish kind. Sample size is 0.4g ± 0.05.
• PCT conditions : 35 kpsi, 20 S up and 10 S down at 40C for 10 cycles.
• Buffer : Sat. Gdn/1% Chaps.
• Purification: Qiagen DNeasy Tissue Kit.
• PCR mitochondrial cytochrome b gene (as figure 4) by using a pair of universal primers which published in a paper by Paola Sebastio et al at J. Agric. Food Chem. 2001, 49, 1194-1199. Sequence of the primers used for PCR amplification: L 14841 (5’-AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA-3’) AND H15149 (5’AAA CTG CAG CCC CTC AGA ATG ATA TTT GTC CTC A-3’). About 380 bp amplicon was successfully amplified for all 6 DNA samples extracted by PCT from the two fish.
• Lane identification : Figure 3 and 4: Lane 1 to 3 are from Goldfish and lane 4 to 6 are from Tilapia. Lane 7 in figure 4 is a PCR negative control.
Figure 2: Tilapia
1 2 3 4 5
Figure 1: Goldfish
Figure 3: Agarose gel showing total DNA
Figure 4: Bioanalyzer: PCR Products
DNA Extracted from Fish: Mitochondrial Cytochrome B Gene
DNA From Spinach Leaves
The PCT Shredder
PCT Alone PCT Plus Shredder Bead Beating
Release of RNAwith the
PCT Sample Preparation System
(PCT SPS)
Transcriptomics
Gene Expression Profiling
PCT +MW 1 2 3
PCT +MW 1 2 3
A. B. PCT C+ ControlPCT +
Sample: Rat Brain
PCT Condition: 4°C, 5 x 1 min cycles, 35 kpsiRNA Extraction Buffer: 1.1 ml 4M GTC/1% NP40
PCT Releases High Quality RNA for cDNA Microarray Analysis
microRNA Detection from Rat Tissue Samples
0
2
4
6
8
10
12
14
16
18
Liver Lung Brain Heart
Ct
M.P.
M.P.
PCT
PCT
y = 1.4486Ln(x) + 14.355R2 = 0.9982
10
15
20
25
30
35
40
1 10 100 1000 10000 100000 1000000
Template Dilution (fold)
Ct
• Both extraction methods yielded similar quality and quantitative RT/real-time PCR results
• PCT process is much easier to operate than M/P/H
• Excellent linearity on diluted real-time PCR templates were observed
Experimental Conditions:
PCT: 5 cycle, 35 kpsi, 4°C
miRNA Purified Using Ambion mirVana miRNA Kit
microRNA Assays Were Done with an hsa-miR-16 Probe Set on an ABI 9700 and 7500 Instruments
AgricultureImproved Extraction of DNA of Ca. Liberibacter Species from Plants and Cultivated CellsUsing Pressure Cycling Technology (PCT)Dr. Norman Schaad (FDWSRU, USDA-ARS, Fort Detrick, MD USA)APS Meeting 2090
Improved extraction of Rhizoctonia and Pythium DNA from wheat roots and soilsamples using pressure cycling technology Dr. Patricia Okubara (USDA, Pullman, WA)Can. J. Plant Pathol. Vol. 29, 2007
BioremediationAnalysis of Microorganisms in Oil Spills: Searching for Oil Eating BacteriaDr. Janet Jansson (Lawrence Berkeley Laboratories)Work in Progress
Counter-BioterrorismIntact Protein Liquid Chromatography Mass Spectromet ry for Bacteria StrainDifferentiation and Bacterial Toxin DetectionJohn H. Callahan (FDA/CFSAN, College Park, MD)
Use of Pressure Cycling Technology (PCT) in Sample Decontamination andBiomolecule Extraction for Analysis of the Anthrax Spore ProteomeBradford Powell, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USAManuscript Submitted
Example Applications
Proteins Under Pressure:
Applications of Pressure Cycling Technology in Proteomics and
Protein Biochemistry
Proteomics
Proteins Under Pressure
• Pressure promotes dissociation of oligomeric proteins
• Pressure promotes protein unfolding and re-folding
• Unfolding leads to hydration , i.e. volume reduction
• Pressure activates most hydrolytic enzymatic reactions
• Pressure leads to protein denaturation
• Pressure protects proteins from thermal denaturation
• Pressure may act in synergy with chemical denaturants
PCT-assisted Cell Lysis in Detergent-free Buffer
912
Grand Total: 1077
PCT-assisted243
“Conventional”165
669
834
HepG2 proteomes extracted either by PCT or by sonication in 50 mM AmBic
Analysis of Mouse Liver Lysates by 2DGE: Comparison of PCT, Sonication, and Ground Glass Tissue Grinder
Some Advanced ApplicationsDifferential Lysis: Exploiting The Pressure Profile
• Extract mitochondria
Systems Biology: Exploiting The Synergy of Pressure and Chemistry• ProteoSolveSB Kit
Pressure-dependant, detergent-free extraction of proteins, lipids and nucleic acids
Bacteria, Animal Tissue (Especially effective for lipid-rich tissues)
Standardization of Mass Spectrometry• Pressure-Enhanced Enzymatic Proteolysis
Extraction from Formalin Fixed Paraffin Embedded Ti ssue (FFPE)• DNA, RNA and proteins
“Live” Kidney Mitochondria Isolated by PCT
ProteoSolve-SB
Tissue Fractionation by Hydrostatic Pressure Cyclin g Technology:The Unified Sample Preparation Technique for System s Biology Studies
Vera Gross,1 Greta Carlson,1 Ada T Kwan,1 Gary Smejkal,1 EmilyFreeman,2 Alexander R Ivanov,2 Alexander Lazarev1
1Pressure BioSciences, Inc., Woburn, MA; 2Harvard School of PublicHealth, Boston, MA
Winner of The Journal of Biomolecular Techniques (JBT) Award for Outstanding Research Article
PCT-mediated Liquid-Liquid Extraction
1 5432
a
b
a
b
a
b
cc
a
b b
a
Pressure Applied
Patent Pending
ProteoSolve-SB
Protein, RNA and DNA Recovery From Rat Tissues
Br Ca Ad Li Kd Br Ca Ad Li KdBrain Cardiac Adipose Li ver Kidney MWS
EEE E EP P P P
Solvent removed by: E – evaporation; P - precipitation
ProteoSolve-SB
Rat Brain
Beef pericardial fat
673 737 801 865 929 993Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
699.
1371
883.
8964
698.
1401
855.
8623
909.
9142
857.
8772
911.
9307
744.
0679
885.
9108
875.
1590
879.
8656
907.
8981
829.
8430
722.
0797
853.
8458
874.
1571
713.
1217
869.
8770
913.
9432
743.
0856
767.
0527
899.
9177
801.
8069
905.
8836
721.
0809
841.
8463
895.
8985
877.
8512
942.
0816
786.
7094
920.
0858
851.
8321
760.
6949
810.
7156
825.
8130
782.
6794
735.
1141
673.
1355
952.
0440
815.
8259
930.
0551
725.
6609
773.
7536
711.
1127
677.
1275
889.
1473
707.
1061
694.240 695.938 697.636 699.334 701.032 702.730
Mass (m/z)
3726.4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
739.89029 741.70285 743.51541 745.32796 747.14052 748.95308
Mass (m/z)
1181.2
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
744.
067
9
743.
085
6
742.
103
0
660.0 730.2 800.4 870.6 940.8 1011.0Mass (m/z)
755.0
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
905.
7839
825.
7226
851.
7390
877.
7493
815.
7354
887.
8246
771.
6506
935.
8358
783.
6479
865.
7538
811.
6826
687.
5780
713.
5860
729.
6157
750.
6012
700.
5549
683.
5515
797.
6822
661.
5565
673.
5637
757.
6489
925.
8428
743.
6442
921.
8342
993.
8373
849.0 851.8 854.6 857.4 860.2 863.0Mass (m/z)
522.3
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
855.
766
4
857.
776
1
853.
750
4
851.
739
0
699.
137
1
698.
140
1
849.0 851.6 854.2 856.8 859.4 862.0
Mass (m/z)
1556.7
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
855.
862
3
857.
877
2
853.
845
8
851.
832
1
Phospholipids
Triacylglycerides
Direct Lipid Profiling by MALDI-TOF MS
Applications in Mass Spectrometry
Pressure-Enhanced Enzymatic Proteolysis
Some Pressure-Enhanced Enzymes
• Proteinase K• Lysozyme• Trypsin• Chymotrypsin• Lys C• PNGase F
Effect of High Pressure on Protein Activity
0
20
40
60
80
100
0 100 200 300 400
Pressure (MPa)
LDH AST
ALT
Amylase
LipaseAlk P’ase
Act
ivity
(%
of U
ntre
ated
Con
trol
)
Pressure (psi)
Pressure Impact on Lysozyme CatalysisDigestion of Fluorogenic Substrate at 50˚C
PCT-Enhanced Tryptic Digestion of BSA
.
Pacific Northwest National Laboratories:Application of Pressurized Solvents for Ultrafast T rypsin Hydrolysis in Proteomics: Proteomics on the Fly
Increase pressure can dramatically increase the rate of the enzymatic digestion.
PCT simplified sample preparation compared with other newer rapid digestion methods, such as MAPED and HIFU technologies .
Successful in-solution digestions of single proteins and complex protein mixtures were achieved in 60 seconds
Data provided by Dr. Roger Biringer, Thermo Fischer Scientific
PCT-Enhanced Tryptic Digestion
A Comparison of PCT and CTRL for Post-Nuclear Membr ane Tryptic Digests
Barocycler Thermomixer
Unique Peptides 832 288
Unique Proteins 342 141
Integral Membrane Proteins 62 15
Barocycler Thermomixer
Unique Peptides 832 288
Unique Proteins 342 141
Integral Membrane Proteins 62 15
PCT enables a 2.5-fold increase in peptide identification compared to the conventional digestion procedure for post-nuclear membrane samples.
PCT is also more effective in the digestion of integral membrane proteins.
The Effect of Pressure Cycling on Proteolytic Cleav age Efficiency, Reaction Time and Protein Sequence CoverageEric Bonneil1; Roger Biringer2; Julian Saba2; Andre as Huhmer2; Pierre Thibault11Institute for Research in Immunology and Cancer, U niversité de Montréal, Montréal, Canada2Thermo Fisher Scientific, San Jose, CA
Lys-C Digestion of Monoclonal Antibodies
AMGEN: A Comparison of Methods for Efficient Digestion of Protein A Comparison of Methods for Efficient Digestion of Protein
TherapeuticsTherapeutics
Conclusion:This study demonstrated that pressure cycling provi ded the most effective method for digesting monoclonal anti bodies. Complete digestion can be obtained in a short perio d of time without inducing modifications such as methionine o xidation.While the microwave technique has established appli cability in a proteomics setting, the more stringent requiremen ts of the biopharmaceutical arena suggest limitations of the technique with respect for characterization of protein primar y structure
Deglycosylation of RNase B by PNGase F
Rapid Release of N-Linked Glycans from Glycoprotein s by Pressure-Cycling TechnologyZoltan Szabo, Andras Guttman, and Barry L. Karger*Barnett Institute, Northeastern University, Boston, Massachusetts 02115
Figure 1. The effect of the maximum pressure level of pressure cycling on PNGase F-mediated deglycosylation of the N-linked sugars from RNase B (Coomassie Brilliant Blue stained SDS-PAGE image). The bands at �15 and �18 kDa represent the deglycosylated and intact forms of RNase B, respectively. Deglycosylation of denatured RNase B was carried out at 37 °C for 5 min with 1:2 500 enzyme: substrate molar ratio in the presence of Triton X-100. Control: 5 min deglycosylation at atmospheric pressure and 37 °C. Pressure cycles:50 s pressure/1 0 s atmospheric. (Left lane) SDS-PAGE protein sizing standards: aprotinin (6 kDa), lysozyme (14 kDa), myoglobin (17 kDa),and carbonic anhydrase (28 kDa).
A Possible MechanismUntying the Gordian Knot
Newest Application
Extraction from Formalin-Fixed
Paraffin-Embedded Tissue (FFPE)
Pressure Cycling Technology (PCT) Significantly Improves Recovery of Proteins/Peptide s from
Formalin Fixed Paraffin-Embedded (FFPE) Tissue
4934 4806
3253
0
1000
2000
3000
4000
5000
6000
Fresh FFPE, 45,000 psi FFPE, no pressure
10932
7964
4737
0
2000
4000
6000
8000
10000
12000
Fresh FFPE, 45,000 psi FFPE, no pressure
Unique Proteins Identified Unique Peptides Identifie d
Data Courtesy of Dr. C. Fowler (AFIP)____________________________________________
"Our initial data show that for aorta samples, which are traditionally difficult to lyse, a greater amount of protein is recovered foll owing pressure-enhanced FFPE
extraction, compared to the standard non-pressure m ethod."
Dr. J. Van Eyk, Director, The Hopkins NHLBI Proteom ics Center, John Hopkins
Summary
Pressure Cycling Technology (PCT):
• Employs an orthogonal sample preparation technique
– Pressure, Temperature, Mechanical and Chemical Variables
• Has a wide variety of applications
– Genomics, Proteomics, Enzymology, etc.
• Tool for Discovery
Pressure BioSciences, Inc.
Pressure Cycling Technology (PCT)
A Novel, Enabling PlatformRevolutionizing Biomarker Discovery
Biotechnica, Hannover, GermanyOctober 6, 2010
Thank You