Pressure Cycling Technology

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Pressure Cycling Technology (PCT) Applications
in Life Sciences
Feng Tao, Ph.D. Pressure BioSciences, Inc.
At HPBB 2004 September 28, 2004

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As of Sept. 15, 2004

• Boston Biomedica, Inc. (BBI) is now
• Pressure BioSciences, Inc.
• A Company Develops Pressure Cycling Technology
  for Life Sciences Applications

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Objectives

• Introduce some Pressure Cycling Technology (PCT)
  applications in life sciences
• Biological Sample Preparation (Poster 44)
• Scope and examples
• Mechanisms
• Pathogen Inactivation
• Viral and bacterial inactivation
• Plasma protein activity retention
• Other PCT Applications

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What is PCT?
Pressure Cycling Technology An application of
high and low hydrostatic pressure cycles to
control biomolecular interactions
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• Sample Preparation

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Hard-to-Lyse Biological Samples
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Current Extraction Methods
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PCT Sample Processing

• Specially Designed PULSETM Tubes

BarocyclerTM NEP2017
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PULSE Tube
Specially designed multi-functional tube

• Single-Use
• Safer fully-contained
• Versatile, works with
• Standard and custom reagents
• Various sample types
• Range of sample sizes
• Convenient
• Efficient

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PULSE Tubes, Easy as 1, 2, 3 . . .
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(No Transcript)
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Corn Sprout DNA Extraction
PCT (MPa)
M 170 202 235 Neg
170 MPa 25 kpsi 202 MPa 30 kpsi 235 MPa 35
kpsi
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Evaluation of PCT Extraction

• PCT Procedure
• Pressure 35 kpsi (235MPa)
• 5 Cycles (30 s?, 30 s?)
• Temp 4o 37o C

• Positive Controls
• Mortar Pestle and/or Homogenizer
• Bead Beating
• Sonication
• Negative Control
• Expose to sample process buffer, but no physical
  treatment

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Extraction of DNA from Rat Liver with Different
Buffers
LS ATL GTC M
PCT MP PCT MP PCT MP N
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Gene Expression cDNA Microarray
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• Sample Preparation
• For Proteomics

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Extraction of Protein from Chicken Muscle with
Different Buffers
_____ B1 _______ B2_
_____ ____ B3_ ___ PCT
MP - PCT MP - PCT
MP - MW
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Rat Proteins Extracted by PCT SPS
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User Feedback
We obtained 37 higher protein yields using the
Barocycler relative to sonicationlysis buffer
together with the Barocycler effectively ruptured
the tough cuticle (of C. elegans)we found more
high molecular weight proteins more high basic
proteinsmembrane proteinsmore channel
proteins. H.A. Geiser et al.,
University of New Hampshire
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User Feedback
Our group has found that the PCT SPS
effectively extracts both estrogen and progestin
receptors of excellent qualitythe receptors
retain their biological activities... these are
exciting results, since receptor proteins are
inherently unstable, and current methods for
their extraction can be time consuming,
difficult, and inefficient. Dr. James
Wittliff, University of Louisville
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PCT Extraction Mechanisms Hypothesis
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Bubble Oscillation Dynamics Rayleigh-Plesset
Model
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Bubble Collapse Velocity (model)
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Freeze-thaw during Pressure Cycles
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Pressure Effects on Biomolecules P,T - Phase
Diagrams

• Water
• Nucleic Acid
• Lipid
• Molecular complexes
• Protein

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• Pathogens Inactivation
• and
• Preservation
• of Biological-active Proteins

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Inactivation of Pathogens by PCT

• Inactivation of broad range of Pathogens
• Enveloped and Non-enveloped Viruses, Bacteria,
  and Yeasts
• Retention of Therapeutic Protein Activity
• Immunoglobulins, Coagulation Factor, Blood
  Chemistries
• Short Pulses of High Pressure
• Optimum for Maintenance of Protein Function
• Sufficient to Inactivate Virus

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Rapid Inactivation of Bacteria by PCT
Enterococcus faecium
Escherichia coli 1
Bacillus cereus
1.E08
1.E08
1.E08
1.E06
1.E06
1.E06
Titer (cfu/ml)
1.E04
1.E04
1.E04
1.E02
1.E02
1.E02
0
100
200
300
400
0
100
200
300
400
0
100
200
300
400
Staphylococcus aureus
Escherichia coli 2
Pseudomonas aeruginosa
1.E09
1.E09
1.E08
1.E07
1.E07
1.E06
1.E05
1.E05
1.E03
1.E04
1.E03
1.E01
1.E01
1.E02
0
100
200
300
400
0
100
200
300
400
0
100
200
300
400
Pressure (MPa)
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Inactivation of HIV And HSV By PCT
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PPV Inactivation P, T
Atm control
Static Pressure
PCT
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HIV-1 Inactivation versus p24 Detection post PCT
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Effect of PCT on Serum Protein Activity
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Immunoglobulin Activity after PCT
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Stable Coagulation Factors Activity
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Protein C Activity in Plasma
235 MPa
300 MPa
335 MPa
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Inactivation of Pathogens by PCT

• Thermodynamic process
• No toxic or mutagenic chemicals to add or remove
• Rapid, well-controlled process
• Pressure transmitted at speed of sound
• No pressure gradients
• Safe
• Performed in closed container
• Scale-up potentials
• Accepted procedure in food processing industry
• Pressure chambers up to 24,000 L developed
• Mechanisms
• Micro-bubble collapse?
• Thermodynamic impact of viral proteins

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• Other Applications for PCT

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Protein Misfolding and Aggregation

• Misfolding and aggregation associated diseases,
  e.g. Alzheimers, Parkinsons, systematic
  amyloidosis/amyloid fibril formation and many
  types of cancer
• Diagnosis
• Amyloid proteins, small aggregates versus matured
  fibrils
• Proteomic analysis for clinical diagnostic assays
• Conversion of misfolded protein or protein
  aggregates using PCT

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Pressure Mediated Dissociation of Immuno-complexes

• Goal to release Factor IX from its
  antibody-derivatized resin using high pressure
  instead of high salt

• Longer pressurization, higher pressure, cycled
  pressure and temperature increase the amount of
  Factor IX released from its antibody-derivatized
  resin

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Pressure Effects on Ionization of Compounds

• Control of free-ion concentration, or pH
• Electrostriction
• Low barrier H-bond

• Hess Reinhard (1999) JACS, 121 (42), 9867-9870

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Summary

• Sample Preparation
• Extraction of biomolecules
• High precision extraction
• Integration of extraction and purification
• Pathogen Inactivation
• Pathogens in therapeutic products
• Preserving important proteins
• Protein folding
• Diagnose, converse, inactive misfolded proteins
• Solubilization, refolding of protein aggregates
• Protein binding
• Immuno-complexes diagnostics, affinity
  purification

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Static and Cyclic Pressure Effects

• Static Pressure
• Shearing
• Volume effect
• Covalent interactions
• Non-covalent interactions
• Solvent effects (hydrophobic interactions)
• Ionic interactions and hydrogen bonds
• Cyclic Pressure
• Kinetics of chemical reactions
• Fluid and air dynamics
• Micro-bubble collapse

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Pressure Cycling Technology

• Precise, uniform, rapid, scalable
• Powerful, suitable for combination use with other
  physical and chemical parameters, e.g. T,
  chemicals, electrical field, light
• Has not been widely used in life science
• Basic mechanisms need to be better understood