Cell Disruption

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Cell Disruption
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What are we disrupting?
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Why disrupt cells?

• To get to the products we want!
• Biological products can be
• Extracellular (e.g. -OH, -COOH, AA, Antibiotics,
  Enzymes)
• Intracellular (e.g. Recombinant DNA products)
• Periplasmic (e.g. Recombinant DNA products)

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Methods of Disruption

• Mechanical
• Liquid Shear
• Ultrasound (sonication)
• Mechanical agitation (dyno-mill)
• Pressure (homogeniser)
• Solid Shear
• Grinding (ball mill)

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• Non-mechanical
• Desiccation (dehydration)
• Chemicals (Solvents, detergents, urea, basic
  conditions)
• Cell lysis
• Physical (osmotic, freeze/thaw)
• Chemical (detergent, chaotrope)
• Enzymatic (lysozyme, phage)

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Choice of Disruption Method

• The method selected for large scale cell
  disruption will be different in every case, but
  will depend on
• Susceptibility of cells to disruption
• Product stability
• Ease of extraction from cell debris
• Speed of method
• Cost of method

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Adverse Factors during Disruption

• Heat
• Shear
• Proteases
• Particle size
• DNA, RNA
• Chemical
• Foaming
• Heavy-metal toxicity

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Homogeniser

• Large scale, widely used
• Variables in design
• type of valve
• pressure
• single or two-stage
• surfactant type and content
• viscosity
• temperature
• Disruption occurs through turbulence and
  cavitation
• High pressure (400-600 bar)
• High solids (50 solids feed)
• High heat generation (1.5C/1000 psi)
• Throughput 4-21 kg dry yeast/hr

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Homogeniser

• Design equation
• k (release rate constant) is a function of
  temperature
• a is of order 1.5-3

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Bead Mill

• First Order
• k is a function of
• Rate of agitation (1500-2250 rpm)
• Cell concentration (30-60 wet solids)
• Bead diameter (0.2 -1.0 mm)
• Temperature

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Bead Mill Performance
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Osmotic Shock

• Dramatic change in the solute concentration of
  the liquid surrounding the microorganism can
  cause the cell to burst
• Pressure is calculated for dilute solutions from

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Sonication

• Highly effective at lab scale (15-300W)
• Poor at large scales
• High energy requirements
• Safety issues - noise
• Heat transfer problems
• Not continuous
• Protein lability

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Sensitivity of Cells to Disruption