Lecture A Flashcards
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
What is Stoke’s Law?
the rate of sedimentation of spherical particles in a fluid of Newtonian viscosity characteristics is proportional to the square of the particle diameter
(as particle diameter increases by one order of magnitude, the settling velocity increases by two orders of magnitude)
What are the main designs of centrifugation?
- the tubular bowl
- the multi-chamber bowl (human
blood plasma) - the disc stack centrifuge
- the scroll centrifuge (sludge dewatering)
What is the structure of the disc stack centrifuge?
0 – 200 discs at an angle of 35 - 50º and kept 0.4 – 2 mm apart, dividing the bowl into separate settling zone
How does the disc stack centrifuge work?
culture broth fed into centrifuge continuously; solids settle on disc lower surface and migrate to bowl periphery; exit outside centrifuge and clarified extract through circular opening close to axis; typical force = 5,000-15,000xg; min. density difference = 0.01-0.03 kg/m3; min particle diameter = 5 μm
What is ‘polishing’?
supernatants are usually subjected to a final filtration for clarification purposes
What is broth conditioning?
A pre-treatment of a culture broth to improve filtration or centrifugation efficacy.
What does broth conditioning involve?
Changing particle size, altering interactions between particles, or modifying liquor viscosity.
What is the most commonly used pre-treatment in broth conditioning?
A filter aid (a slurry of inert, incompressible particles).
What size are the particles in a filter aid?
2–20 μm.
What is the purpose of a filter aid?
To form a permeable coating before the filter, preventing premature blocking.
What are two commonly used filter aid substances?
Diatomite (Dicalite®) and Perlite®.
What is diatomite derived from?
Fossilized diatoms.
What is Perlite® derived from?
Volcanic rock.
What is the structure of diatom skeletons made of?
Inert SiO₂ (silicon dioxide).
How much diatomite is produced annually in the US, and what percentage is used for filtration?
0.82 million metric tons per year; 75% is used for filtration.
What additional benefit does a cationic filter aid provide?
Reduces pyrogens, nucleic acids, and acidic proteins that may foul chromatography columns.
What are the three steps for using a filter aid?
(a) Apply a thin pre-coat layer to the filter.
(b) Mix the required amount of filter aid with broth and start filtration.
(c) For vacuum filtration, build a thick pre-coat on the drum surface before filtration.
What is an alternative broth conditioning strategy used in water treatment?
Flocculation facilitated by polycations.
What are common polycations used for flocculation?
Ammonium sulfate, sodium sulfate, and alum (aluminum potassium sulfate).
How do polycations aid in flocculation?
They remove water from protein surfaces, neutralize cell surface charges, and promote cell clumping.
Why do microbes normally resist clumping?
They have negatively charged surfaces, hydrophilic cell walls, bound water shells, and steric hindrance.
How do polycations promote aggregation?
They neutralize anionic charges, reduce hydrophilicity, and form high molecular weight polymer bridges.
What is the final result of flocculation?
Formation of large, loose particles that are easier to centrifuge.
