Lecture A Flashcards
(47 cards)
What is downstream processing?
A series of separation steps required to yield a metabolite or biopharmaceutical of interest
What is necessary for yielding a metabolite?
Sufficient purity
Cost effectiveness
What is a molecules value determined by?
Its dilution in a solvent: the more dilute, the higher the value (more dilute meaning lower concentration of solute - more rare/precious)
What knowledge is needed for separation and purification procedures to achieve a high yield of product?
- The attributes of the biomolecule of interest (size, shape, chemical nature, stability)
- The required purity
- The most cost efficient method for purification
Others include
- conc. of product in broth
- end-use of product
- biohazards in process
- specific impurities
- product price
- product market demand
Summary of downstream processing
Liquid volume quickly reduced with simultaneous purification while concentrating product
What are the four major steps in DSP?
- Solid-liquid separation
- Concentration
- Purification
- Formulation
What does solid-liquid separation include?
Separation of cell biomass from culture broth by either:
filtration or centrifugation
What are some primary considerations of solid-liquid separation?
- Chilled at 4ºC in harvest vessel (retards bioactivity)
- Work quickly
- Extra steps to stop bioactivity in broth such as protease activity or bacterial metabolism
- Low grade clean room (grade D in GMP)
Why is cellular location of biomolecules important?
Necessary to either retain extracellular supernatant or intracellular cells to later break open and recover protein (e.g. recombinant insulin in E.coli) - additional cost-time operation and major drawback
What determines whether filtration or centrifugation is used in solid-liquid separation?
Cell size and density
Glycocalyx presence
Formation of cellular aggregates/pellets
These influence efficacy/feasibility of separation techniques
How does filtration work?
Retains particles while allowing liquid to pass through a medium on the basis of size.
What is dead-end filtration flow dependant on?
Surface area of filter
Pore size
Applied pressure
Accumulated solids (cake)
What is the ‘cake’ in filtration?
Retention of particles on filter surface. Usually compressible and collapse (lose thickness) which blocks/blinds the filter
What is filter media usually made from?
Cellulose
Glass wool
Ceramics
Synthetic membranes
What do large scale process equipment use in filtration?
- vacuum filters like the rotary drum (0.5 – 3.0 m); filamentous fungi and yeast cells
- filter press
- disposable filters
What is this equipment used for?
used for the clarification of fermentation broths containing 10–40% solids by volume with particle sizes of 0.5 – 10 μm
How does the rotary drum work?
Partially submerged in the culture fluid and as it revolves in the trough it ‘sucks up’ liquid (0.1-2 rpm), leaving the cells as a cake on the surface of a porous fabric. It can operate continuously.
How does the filter press work?
Broth may be forced under pressure through variable number of cloth filters. Cheap system but operates in batch mode (and dismantling causes the filters to wear)
What is cross-flow microfiltration?
- particle size 0.2 μm – 10 μm
- uses pressures of 0.5 – 2 bar
- yields efficient separation (>99.9% cell retention)
- closed system (good containment)
- does not require the addition of a filter aid
What are the advantages of filtration?
- simple process
- lower power consumption relative to centrifugation
- separation independent of cell and media densities
How does centrifugation work at an industrial level?
usually low speed, continuous and of high capacity
What does centrifugation usually separate?
bacterial cells
How may deposited solids be removed?
intermittently or continuously
What does solid removal depend on?
- density difference between particles to be separated and the suspending fluid
- the viscosity of the fluid
- the size of the particles
