L6: Cell disruption Flashcards
What are the main differences between intracellular and extracellular products in bioprocessing?
Intracellular products require cell disruption and debris removal, while extracellular products are secreted and bypass these steps.
Why is extracellular production generally preferred?
It reduces downstream processing complexity and cost.
What are inclusion bodies (IBs), and why are they important?
Inclusion bodies are aggregates of insoluble proteins in recombinant bacteria, often containing the target product.
What factors influence the choice of cell disruption method?
Cell wall structure, product stability, shear sensitivity, and impact on contaminants.
What are the main types of organisms used in bioprocesses, and how do their cell wall structures differ?
Bacteria, yeast, fungi, mammalian cells, and plant cells; differences include thickness and complexity of the cell wall.
How does the cell wall structure of Gram-positive bacteria differ from Gram-negative bacteria?
Gram-positive bacteria have a thicker peptidoglycan layer, while Gram-negative bacteria have an outer membrane with lipopolysaccharides
Which cell types are easiest to disrupt mechanically?
Mammalian cells are easiest, followed by mycelia and Gram-negative rods
What are the most common chemical cell disruption methods?
Osmotic shock, enzyme digestion, and solubilization.
How does osmotic shock work to disrupt cells?
It bursts cells by drastically reducing extracellular solute concentrations.
What enzymes are used to digest bacterial cell walls?
Lysozyme for Gram-positive bacteria and EDTA with enzymes for Gram-negative bacteria.
Why are non-ionic detergents preferred over ionic detergents in solubilization?
Non-ionic detergents are less denaturing to proteins.
What is the principle of mechanical disruption methods?
They physically break the cell wall using techniques like homogenization, grinding, ultrasonication, or bead beating
What is a high-pressure homogenizer, and how does it work?
A device that forces cell suspensions through a small orifice under high pressure, causing shear and impact forces that break cells.
What factors affect the efficiency of high-pressure homogenization?
Pressure, number of passes, temperature, cell age, and growth conditions.
What are the main challenges of high-pressure homogenization?
Generation of heat, product damage, and variability in product release efficiency.
How does bead beating disrupt cells?
Glass beads grind down cells in an agitated container, physically breaking them.
What are the limitations of bead beating?
It generates heat quickly and may destroy sensitive products.
How does ultrasonication disrupt cells?
A vibrating probe generates sound waves, creating cavitation that disrupts cell membranes.
What are the key challenges of ultrasonication?
Heat generation and the need for cooling to prevent protein denaturation.
What are some methods to evaluate the success of cell disruption?
Microscopy, laser particle sizing, and indirect assays like measuring intracellular protein release.
What impact does freeze-thawing have on cell disruption?
It softens cells but makes them more resistant to mechanical disruption after repeated cycles.
How do inclusion bodies affect the disruption process?
Their presence weakens the cell wall, making disruption easier, but the IBs themselves resist further breakdown.
What are some key considerations for selecting a cell disruption method?
Compatibility with the product, cost, scalability, and impact on downstream processing.
What role does modeling play in cell disruption?
It predicts protein release rates and helps optimize the number of passes or pressure settings.
