ProKyma Technologies Ltd Preparing cells and bacteria for analysis Spinout of Ploughshare Innovation

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ProKyma Technologies LtdPreparing cells and
bacteria for analysisSpin-out of Ploughshare
Innovations / Dstl out of research to develop
real-time automated detector for microbial
contamination

• March 2009
• Damian Bond, CEO

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What is the problem?

• Analysis of biological specimens is hampered by
  impurities in the sample.
• Most impurities are outside the cell.
• Antibody based assays matrix effects on avidity
• Fluorescent assays quenching on light
  transmission or emission
• Molecular assays inhibition of amplification
• Repeated washing steps required after the target
  has stuck to the impurity.
• If the impurities were removed first, then the
  problem is removed.

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Target Markets

• Antibody based cell measurement
• Blood grouping
• Fluorescent antibody staining (flow cytometry,
  FACs)
• Molecular assays
• Nucleic acid clean-up
• Molecular Diagnostics
• Clinical, eg. Sepsis from blood, TB from sputum
• Veterinary, e.g. Johnes disease from cattle
  faeces
• Environmental e.g. Legionella from water towers

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Product - KymaSep

• A flow-through system separating washing
  cells/bacteria from blood/soil/water/faeces
• Automated replacement for centrifugation
• Uses proprietary flow cell design
• reusable for some markets (e.g. blood grouping)
• disposable for others (e.g. PCR clean up)
• disposable cell costs 50p, sells for 4
• Instrument estimated at End User 10K

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What we set out to achieve

• Isolate intact cells from complex specimens e.g.
  blood or faeces
• Isolate all cells
• Small numbers of cells (10s) to large numbers
  107)
• Wash and concentrate
• Rapid automated
• Disposable

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What have we have achieved

• Indirect Coombs test at least 2x sensitivity of
  gel card system
• Removal of PCR inhibitors from bacterial culture
  in soil or blood on small volumes with
  quantifiable recovery
• Isolation of colony equivalent from blood culture
  to perform AST 24 hours earlier than plating
• Leukocyte isolation and fluorescent staining
  front end automation
• Microscopy
• CD4 FITC CD8 PE staining with flow cytometry
  detection
• Viability assay, e.g. T-Cell response to TB
  antigen
• Standardising system and electronics
• Miniturising of control systems suitable for
• Field or POC
• Large instrument processing 100s samples per day

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Limitations

• Scale efficient on 100 µL volume. After sample
  treatments and dilutions, this limits the
  sensitivity and therefore where the technology
  can be applied
• Efficiency on bacteria 30-50 of cells lost
  from the sample, limiting applications to large
  and heterogenous microbial populations. Can be
  solved
• Viscosity thick solutions not suitable. Sputum
  very difficult challenge.

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Current strategy

• Technical stepping stones
• Perfect instrumentation around large cells,
  leading to microbiology
• Blood grouping develops an engine to process
  100s of samples per day.
• Modules of 10 sample processors
• Standardise electronics, fluidics, software
  detection
• Pipe flow sample in, hold sample, wash sample -
  detector
• Module applied to low cost flow cytometry for POC
• Clean sample easier detection lower cost
  optics no sheathed flow
• PCR clean up of cells / bacteria

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Potential collaborations / research areas

• On existing development program (100 µL)
• Cell surface antigens, e.g. HIV therapy on
  CD4-CD8 ratios in low cost cytometry instrument
• Antibiotic sensitivity providing a purified
  colony equivalent 24 hrs earlier than culture
• MRSA, C. Diff purification on 103 / mL or above
  for existing detection methods
• Screening of soil micro-organisms for antibiotic
  production genes to identify novel variations
  (removal of soil inhibitors prior to gene chip
  analysis) (PHD Program?)
• Candida infection in blood

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Potential collaborations / research areas

• Currently outside our resources
• Scale-up to 1 mL development strategy
  understood.
• Improved molecular diagnostics in bacterial
  infections
• PCR inhibitor removal already demonstrated on
  small scale and can be proved on 1 mL
• Isolation of viable T-Cells would allow high
  throughput screening for latent TB infection
• Sputum treatments for active TB detection by
  improved acid fast stain (detect 103 104 / mL)
  or PCR (102 / mL)

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Potential collaborations / research areas

• Significant research program
• 10 mL sample processing
• Link to concentrator step (dielectrophoresis,
  covered in our patents or others)
• Rapid and reliable molecular detection of sepsis
  in whole blood
• 10 102 bacteria / mL by PCR
• Easier by mRNA or other target amplified reaction