L6: Cell disruption

Question 1

What are the main differences between intracellular and extracellular products in bioprocessing?

Answer 1

Intracellular products require cell disruption and debris removal, while extracellular products are secreted and bypass these steps.

Question 2

Why is extracellular production generally preferred?

Answer 2

It reduces downstream processing complexity and cost.

Question 3

What are inclusion bodies (IBs), and why are they important?

Answer 3

Inclusion bodies are aggregates of insoluble proteins in recombinant bacteria, often containing the target product.

Question 4

What factors influence the choice of cell disruption method?

Answer 4

Cell wall structure, product stability, shear sensitivity, and impact on contaminants.

Question 5

What are the main types of organisms used in bioprocesses, and how do their cell wall structures differ?

Answer 5

Bacteria, yeast, fungi, mammalian cells, and plant cells; differences include thickness and complexity of the cell wall.

Question 6

How does the cell wall structure of Gram-positive bacteria differ from Gram-negative bacteria?

Answer 6

Gram-positive bacteria have a thicker peptidoglycan layer, while Gram-negative bacteria have an outer membrane with lipopolysaccharides

Question 7

Which cell types are easiest to disrupt mechanically?

Answer 7

Mammalian cells are easiest, followed by mycelia and Gram-negative rods

Question 8

What are the most common chemical cell disruption methods?

Answer 8

Osmotic shock, enzyme digestion, and solubilization.

Question 9

How does osmotic shock work to disrupt cells?

Answer 9

It bursts cells by drastically reducing extracellular solute concentrations.

Question 10

What enzymes are used to digest bacterial cell walls?

Answer 10

Lysozyme for Gram-positive bacteria and EDTA with enzymes for Gram-negative bacteria.

Question 11

Why are non-ionic detergents preferred over ionic detergents in solubilization?

Answer 11

Non-ionic detergents are less denaturing to proteins.

Question 12

What is the principle of mechanical disruption methods?

Answer 12

They physically break the cell wall using techniques like homogenization, grinding, ultrasonication, or bead beating

Question 13

What is a high-pressure homogenizer, and how does it work?

Answer 13

A device that forces cell suspensions through a small orifice under high pressure, causing shear and impact forces that break cells.

Question 14

What factors affect the efficiency of high-pressure homogenization?

Answer 14

Pressure, number of passes, temperature, cell age, and growth conditions.

Question 15

What are the main challenges of high-pressure homogenization?

Answer 15

Generation of heat, product damage, and variability in product release efficiency.

Question 16

How does bead beating disrupt cells?

Answer 16

Glass beads grind down cells in an agitated container, physically breaking them.

Question 17

What are the limitations of bead beating?

Answer 17

It generates heat quickly and may destroy sensitive products.

Question 18

How does ultrasonication disrupt cells?

Answer 18

A vibrating probe generates sound waves, creating cavitation that disrupts cell membranes.

Question 19

What are the key challenges of ultrasonication?

Answer 19

Heat generation and the need for cooling to prevent protein denaturation.

Question 20

What are some methods to evaluate the success of cell disruption?

Answer 20

Microscopy, laser particle sizing, and indirect assays like measuring intracellular protein release.

Question 21

What impact does freeze-thawing have on cell disruption?

Answer 21

It softens cells but makes them more resistant to mechanical disruption after repeated cycles.

Question 22

How do inclusion bodies affect the disruption process?

Answer 22

Their presence weakens the cell wall, making disruption easier, but the IBs themselves resist further breakdown.

Question 23

What are some key considerations for selecting a cell disruption method?

Answer 23

Compatibility with the product, cost, scalability, and impact on downstream processing.

Question 24

What role does modeling play in cell disruption?

Answer 24

It predicts protein release rates and helps optimize the number of passes or pressure settings.

Question 25

What is the summary of cell disruption in bioprocessing?

Answer 25

It is necessary for recovering intracellular compounds, involves various chemical and mechanical methods, and the choice depends on the product and cell type.