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Pressure Cycling Technology (PCT):A Novel, Enabling Platform

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Presentation at the 2010 Biotechnica Conference in Hannover, Germany by Nathan Lawrence Ph.D

Presentation at the 2010 Biotechnica Conference in Hannover, Germany by Nathan Lawrence Ph.D

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  • 1. Pressure BioSciences, Inc. Pressure Cycling Technology (PCT): A Novel, Enabling Platform Revolutionizing Biomarker Discovery Biotechnica Hannover, Germany October 6, 2010
  • 2. 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.
  • 3. • 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.
  • 4. 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)
  • 5. 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
  • 6. 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!
  • 7. Why Does PCT Work • Pressure is a Thermodynamic Process • Compressibility of Water • Synergy of Pressure, Temperature and Chemistry
  • 8. High Pressure Destabilizes Biological Membranes Hydrostatic Pressure Applied Hydrostatic Pressure Released Lipidbilayers Membrane Protein (Interdigitated bilayer, Hydrophobic hydration)
  • 9. 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)
  • 10. Barocycler™ NEP2320 PCT – Sample Preparation System Barocycler™ NEP3229
  • 11. 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
  • 12. User-Adjustable Variables • Pressure (up to 35 kpsi) • Number of Cycles • Cycle Profile • Chemistry • Temperature
  • 13. Release of DNA with the PCT Sample Preparation System (PCT SPS) Genomics
  • 14. • 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
  • 15. DNA From Spinach Leaves The PCT Shredder PCT Alone PCT Plus Shredder Bead Beating
  • 16. Release of RNA with the PCT Sample Preparation System (PCT SPS) Transcriptomics
  • 17. 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 kpsi RNA Extraction Buffer: 1.1 ml 4M GTC/1% NP40 PCT Releases High Quality RNA for cDNA Microarray Analysis
  • 18. 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) + 14.355 R2 = 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
  • 19. Agriculture Improved Extraction of DNA of Ca. Liberibacter Species from Plants and Cultivated Cells Using 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 soil samples using pressure cycling technology Dr. Patricia Okubara (USDA, Pullman, WA) Can. J. Plant Pathol. Vol. 29, 2007 Bioremediation Analysis of Microorganisms in Oil Spills: Searching for Oil Eating Bacteria Dr. Janet Jansson (Lawrence Berkeley Laboratories) Work in Progress Counter-Bioterrorism Intact Protein Liquid Chromatography Mass Spectrometry for Bacteria Strain Differentiation and Bacterial Toxin Detection John H. Callahan (FDA/CFSAN, College Park, MD) Use of Pressure Cycling Technology (PCT) in Sample Decontamination and Biomolecule Extraction for Analysis of the Anthrax Spore Proteome Bradford Powell, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA Manuscript Submitted Example Applications
  • 20. Proteins Under Pressure: Applications of Pressure Cycling Technology in Proteomics and Protein Biochemistry Proteomics
  • 21. 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
  • 22. PCT-assisted Cell Lysis in Detergent-free Buffer 912 Grand Total: 1077 PCT-assisted 243 “Conventional” 165 669 834 HepG2 proteomes extracted either by PCT or by sonication in 50 mM AmBic
  • 23. Analysis of Mouse Liver Lysates by 2DGE: Comparison of PCT, Sonication, and Ground Glass Tissue Grinder
  • 24. Some Advanced Applications Differential 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 Tissue (FFPE) • DNA, RNA and proteins
  • 25. “Live” Kidney Mitochondria Isolated by PCT
  • 26. ProteoSolve-SB Tissue Fractionation by Hydrostatic Pressure Cycling Technology: The Unified Sample Preparation Technique for Systems Biology Studies Vera Gross,1 Greta Carlson,1 Ada T Kwan,1 Gary Smejkal,1 Emily Freeman,2 Alexander R Ivanov,2 Alexander Lazarev1 1Pressure BioSciences, Inc., Woburn, MA; 2Harvard School of Public Health, Boston, MA Winner of The Journal of Biomolecular Techniques (JBT) Award for Outstanding Research Article
  • 27. PCT-mediated Liquid-Liquid Extraction 1 5432 a b a b a b c c a b b a Pressure Applied Patent Pending ProteoSolve-SB
  • 28. Protein, RNA and DNA Recovery From Rat Tissues Br Ca Ad Li Kd Br Ca Ad Li KdBrain Cardiac Adipose Liver Kidney MWS EEE E EP P P P Solvent removed by: E – evaporation; P - precipitation ProteoSolve-SB
  • 29. Rat Brain Beef pericardial fat 673 737 801 865 929 993 Mass (m/z) 0 10 20 30 40 50 60 70 80 90 100 %Intensity 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 %Intensity 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 %Intensity 744.0679 743.0856 742.1030 660.0 730.2 800.4 870.6 940.8 1011.0 Mass (m/z) 755.0 0 10 20 30 40 50 60 70 80 90 100 %Intensity 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 849.0 851.8 854.6 857.4 860.2 863.0 Mass (m/z) 522.3 0 10 20 30 40 50 60 70 80 90 100 %Intensity 855.7664 857.7761 853.7504 851.7390 699.1371 698.1401 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 %Intensity 855.8623 857.8772 853.8458 851.8321 Phospholipids Triacylglycerides Direct Lipid Profiling by MALDI-TOF MS
  • 30. Applications in Mass Spectrometry Pressure-Enhanced Enzymatic Proteolysis
  • 31. Some Pressure-Enhanced Enzymes • Proteinase K • Lysozyme • Trypsin • Chymotrypsin • Lys C • PNGase F
  • 32. Effect of High Pressure on Protein Activity 0 20 40 60 80 100 0 100 200 300 400 Pressure (MPa) LDH AST ALT Amylase Lipase Alk P’ase Activity (%ofUntreatedControl)
  • 33. Pressure (psi) Pressure Impact on Lysozyme Catalysis Digestion of Fluorogenic Substrate at 50˚C
  • 34. PCT-Enhanced Tryptic Digestion of BSA . Pacific Northwest National Laboratories: Application of Pressurized Solvents for Ultrafast Trypsin 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
  • 35. Data provided by Dr. Roger Biringer, Thermo Fischer Scientific PCT-Enhanced Tryptic Digestion
  • 36. A Comparison of PCT and CTRL for Post-Nuclear Membrane 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 Cleavage Efficiency, Reaction Time and Protein Sequence Coverage Eric Bonneil1; Roger Biringer2; Julian Saba2; Andreas Huhmer2; Pierre Thibault1 1Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Canada 2Thermo Fisher Scientific, San Jose, CA
  • 37. Lys-C Digestion of Monoclonal Antibodies AMGEN: A Comparison of Methods for Efficient Digestion of ProteinA Comparison of Methods for Efficient Digestion of Protein TherapeuticsTherapeutics Conclusion: This study demonstrated that pressure cycling provided the most effective method for digesting monoclonal antibodies. Complete digestion can be obtained in a short period of time without inducing modifications such as methionine oxidation. While the microwave technique has established applicability in a proteomics setting, the more stringent requirements of the biopharmaceutical arena suggest limitations of the technique with respect for characterization of protein primary structure
  • 38. Deglycosylation of RNase B by PNGase F Rapid Release of N-Linked Glycans from Glycoproteins by Pressure-Cycling Technology Zoltan 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).
  • 39. A Possible Mechanism Untying the Gordian Knot
  • 40. Newest Application Extraction from Formalin-Fixed Paraffin-Embedded Tissue (FFPE)
  • 41. Pressure Cycling Technology (PCT) Significantly Improves Recovery of Proteins/Peptides 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 Identified 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 following pressure-enhanced FFPE extraction, compared to the standard non-pressure method." Dr. J. Van Eyk, Director, The Hopkins NHLBI Proteomics Center, John Hopkins
  • 42. 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
  • 43. Pressure BioSciences, Inc. Pressure Cycling Technology (PCT) A Novel, Enabling Platform Revolutionizing Biomarker Discovery Biotechnica, Hannover, Germany October 6, 2010 Thank You

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