Quiz 3 - Pt. 4 (Sterilization) Flashcards
High temperature, short exposure time
Continuous Sterilization
Disrupts cell membrane. Mostly used at the laboratory scale.
Ultrasound
Extrude cell paste at high pressure
Pressing
Grind cells with glass, metal beads
Bead beating
_____ is a problem with all of these methods.
Heat dissipation
Salt differences to cause the membrane to rupture. Common.
Osmotic shock
Causes cell membrane to rupture. Common.
Freeze-thaw
Lysozyme attacks the cell wall.
Enzymatic
_______
* Separate inhibitory fermentation products from broth.
* Based on _____ for the compound of between the phases.
* Distribution coefficient: K_D = Y_L/X_N
* Y_L = concentration in the ____
* X_N = concentration in the ____
Liquid-Liquid Extraction
* solubility difference
* light phase
* heavy phase
New Product Candidate
⬇️
_____ - evaluation of product opportunities
⬇️
_____ - setting development objectives, preparation of budget
⬇️
Market Entry
⬇️
Commercial Products
Feasibility
Development Stage
_____________
* Salting out inorganic salts (NH4)2SO4 at high ionic strength
* Solubility _____ at low temperature (less than -5°C) by adding ______.
Precipitation
* reduction ; organic solvents
- Removal of solutes from aqueous phase to solid phase.
Adsorption
_______ is based on adsorption.
Chromatography
____________
- Membrane separation used to remove low molecular weight solutes.
- Used to remove salts from ________.
- Transport occurs due to a ________ driving force
Dialysis
- protein solutions
- concentration gradient
____________
- _____: Transport of water molecules from high to a low concentration pure water to salt water.
- In here, _____ is applied to salt phase causing water move against a ________.
- Salt phase becomes more ________.
Reverse Osmosis (RO)
- Osmosis
- pressure ; concentration gradient.
- concentrated
Ultrafiltration and Microfiltration
- Pressure driven molecular ____ to separate molecules of different size.
- ____________: retained components accumulate on the filter. Gel layer formed on the filter.
- _________: retained components flow tangentially across the filter.
- sieve
- Dead end filtration
- Cross flow filtration
- _____ filtration —–> bacteria, particulates, colloids
- _____ filtration ——> viruses, proteins/enzymes, polysaccharides
- _____ filtration —–> pyrogen, sugars, amino acids
- Reverse osmosis ——> salts
- (i2 ay nasa figure pls intindihin n’yo nalang)
- Micro-filtration
- Ultra-filtration
- Nano-filtration
_____________
- Separates mixtures into components by passing the mixture through a bed of absorbent particles.
- Solutes travels at _____ through the column resulting in the _____ of the solutes.
Chromatography
- different speeds;
- separation
- High specific interaction between a ligand on the particles and components in the mixture.
- Often based on antibodies.
Affinity Chromatography
Separation of molecules based on size and charge in an electric field.
Electrophoresis
Membrane separation to separate charged molecules from a solution.
Electrodialysis
Separation of Soluble Products
LAPADRUCCEE
- Liquid-liquid extraction
- Aqueous two phase extraction
- Precipitation
- Adsorption
- Dialysis
- Reverse Osmosis
- Ultrafiltration and microfiltration
- Cross-flow filtration and microfiltration
- Chromatography
- Electrophoresis
- Electrodialysis
General Approach
1. ______ of insoluble products or components.
2. ________ or concentration and removal of water.
3. ______ and removal of contaminated chemicals.
4. _____ preparation.
- Separation
- Primary isolation
- Purification
- Product
Figure: Major steps involved in the separation and purification of intracellular enzymes. (FCCRPUCSDL)
Fermentation
↓
Cell Removal and Concentration
↓
Cell disruption
↓
Removal of Cell Debris
↓
Protein precipitation or aqueous two phase extraction
↓
Ultrafiltration
↓
Chromatographic purification
↓
Solvent precipitation
↓
Dialysis
↓
Lyophilization
Factors that impact difficulty and cost of recovery
- Product can be biomass, _____ or ______ component.
- Fragile or ______.
- Concentration or titer in the ____.
- Typically _____and ____is more than 50% of total manufacturing costs
- intracellular or extracellular
- heat sensitive
- in the broth
- recovery and purification
Insoluble Products or Components
- Filtration
- Centrifugation
- Coagulation and Flocculation
- Most cost-effective, most common in industrial biotechnology.
Filtration
- Used to separate solids of solids of size 0.1 um to 100 um using centrifugal forces.
- Being replaced by ______.
Centrifugation
- Pretreatment to centrifugation, gravity settling or filtration to improve separation.
- Used wastewater treatment processes to improve clarification.
Coagulation and Flocculation
Formation of small flocs of cells using coagulating agents, electrolytes.
Coagulation
formation of agglomeration of flocs into settleable particles using flocculating agents, polyelectrolytes or CaCl2.
Flocculation
Cell Disruption - Intracellular Products
* Mechanical Methods
SBP
* Non-mechanical methods
OFE
Mechanical Methods
- Sonication
- Bead beating
- Pressing
Non-mechanical methods
- Osmotic shock
- Freeze-thaw
- Enzymatic
