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.

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885.

9108

875.

1590

879.

8656

907.

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829.

8430

722.

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853.

8458

874.

1571

713.

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869.

8770

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9432

743.

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9177

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8836

721.

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8463

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877.

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920.

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8321

760.

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7156

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8130

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8259

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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.

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742.

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0

660.0 730.2 800.4 870.6 940.8 1011.0Mass (m/z)

755.0

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10

20

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100

% In

tens

ity

905.

7839

825.

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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

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750

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Mass (m/z)

1556.7

0

10

20

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40

50

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% In

tens

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855.

862

3

857.

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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