10 A Separation and Clarification Flashcards
What is the first step for an extracellular bioproduct?
- Remove / separate liquid material (supernatant)
What is the first step for an intracellular bioproduct?
- Remove / separate solid material (cell mass)
Separation
Means of separating solid and liquid material
ex:
- settling chambers
- centrifugation
- filtration
Settling Chamber
use gravity to remove heavy/large particles (from liquid or air)
Centrifugation
An instrument that generates centrifugal force.
Commonly used to separate particles in a liquid from the liquid
Clarification
- May also be referred to as Isolation
> precipitation
> filtration
Precipitation
Addition of chemical reagent that will result in a macromolecule precipitating out of solution
Filtration - Membrane
Separation of particles from liquid by applying pressure to the solution to force the it through a membrane/filter.
Membrane filtration systems are composed of four primary components:
- Feed/flow
- Pressure
- Membrane
- Filtrate
Normal Flow Filtration (NFF)
- Traps contaminants larger than the pore size on the top surface of the membrane
- Used for critical applications such as sterilizing and final filtration
Depth Filtration
Entraps contaminants both within the matrix and on the surface of the filter media
Tangential Flow Filtration
- Uses a cross flow (i.e. tangential flow) to reduce build up of retained components on the membrane surface
- Preferred for large scale operations
- Used in:
>Clarification/Purification
>Concentration
>Buffer Exchange
Filtration + Centrifugation
Tube with a built in membrane that uses centrifugal force to filter materials
Peristaltic Pump
Creates a gentle squeezing action to move fluid through flexible tubing
Cell Disruption
- Bacteria often express the protein into internal inclusion bodies
- Cell disruption is required when your expression system does not express the recombinant protein directly into the media
Cell lysis
Osmotic disruption of cell membrane
Enzyme digestion
Cell wall digested, leading to osmotic disruption
Chemical solubilization
Cell wall ( or membrane) partially solubilized chemically
Detergent
Solubilize proteins and disrupt lipids
Cell Disruption – Non Chemical Advantages
- Cheap operational costs
- Volume
- No addition of chemicals to bioproduct
