Downstream processing Flashcards
What is Downstream Processing?
RECOVERY and PURIFICATION operations that follow chemical and biochemical reactions, especially fermentations, as well as animal cell culture or agricultural synthesis.
- Any treatment of the culture broth after fermentation is DSP;
- The main aim of DSP is to concentrate and purify the product;
- Because the products range is so wide, each recovery scheme will be different.
RIPP
DSP design: process decisions
1) What is the marketable price of the product? (i.e. very cheap, cheap, expensive, astronomic)
2) What is the level of the product in the fermentation broth?
3) What is the intended use of the product? (e.g. industrial enzyme, agricultural, therapeutic)
4) What is an acceptable product quality? (e.g. minimum purity required)
5) Where is the product? (e.g. intracellular, periplasmic, extracellular)
6) What are the physico-chemical properties of the product and the principal impurities? (needed for selection of appropriate separation techniques)
7) Is the product or the broth safe? (level of containment)
8) Are there any components in the broth likely to present problems in recovery? (e.g. antifoam)
Scale of fermentation and product concentration
Correlation between product concentration and price
Product concentration in the fermenter broth influences product selling price.
Product costs
- For more sophisticated products, the number of processing steps may easily be >10
- To obtain 50% overall yield in 10 steps the average yield for each unit operation should be well above 90%
- With a multi-step (>10) process there is a need to achieve high step yields (i.e. at least 90% & preferably >95%)
Give examples of different production yields
- Asparaginase production - 13 steps, 30% overall yield, equivalent 91% average step yield
- Penicillin acylase production - 14 steps, 52% overall yield, equivalent 94% average step yield
Biopharma product
- High price
- Stable
- Purity: <ppm></ppm>
</ppm>
Bioindustrial product
- Low price
- Stable
- purity: <10% to> 95% might contain several active components
- colour
Biopharma downstream process
Bioindustry-downstream process
General sequence of operations (RIPP)
R: Recovery /removal
Common first step in product recovery is removal of cells from fermentation broth; product may be either produced intracellularly or extracellularly (i.e. secreted in the liquid phase).
- Relatively little product concentration or improvement in quality occurs;
- Centrifugation & filtration are dominant operations in this segment;
- Typical large scale operations: settling /sedimentation /decanting; flotation; cell disruption; centrifugation; filtration
I: Isolation
- Wide variety of available techniques for isolation from cells or cell-free broth;
- These steps tend to be relatively non-selective;
- Significant increases in product concentration and quality by removing materials of widely divergent properties compared to the desired product;
- Typical large-scale operations: adsorption; liquid extraction; coagulation; flocculation, precipitation.
P: Purification
- These processes are highly selective for the soluble product & remove impurities of similar chemical functionality & physical properties.
- Typical large-scale operations: fractional precipitation; chromatography; ultrafiltration.
Examples: DSP flow for citric acid
Examples: DSP flow for Penicillin
Examples: DSP flow for Intracellular bacterial enzyme
DSP unit operations
1) Solid/liquid separations: centrifugation; sedimentation; filtration; microfiltration
2) Molecular filtration & concentration: ultrafiltration; reverse osmosis
3) Solids concentration: crystallisation; precipitation; extraction
4) Concentration of soluble products: adsorption & affinity chromatography
5) Purification: chromatography
What are the types of filtration?
- Membrane filtration
- Depth filtration
- Rotary vacuum filtration
- Tangential flow filtration (TFF)
Types of industrial centrifuges
- Tubular bowl
- Multi-chamber
- Disc
What is precipitation?
Precipitation is the formation of a solid in a solution during a chemical reaction. The solid is known as precipitate, while the remaining liquid is known as supernate.
- Proteins can be precipitated with the help of either ammonium or sodium sulphate or chilled ethanol or acetone;
- Organic solvents (e.g. methanol) are used to precipitate dextrans.
What are the 4 steps of HIC?
4 steps: equilibration, sample application, elution and regeneration.
- Equilibration: A buffer with a high ionic strength is initially applied to the column to ensure desired hydrophobic interactions between product molecules and the hydrophobic groups bound to the column.
- Elution: The isolated product molecules are eluted by decreasing the salt concentration.
- Regeneration: Ensures the release of any remaining bound molecules.
What are the main steps in affinity chromotograpgy?
3 steps: equilibration, sample application/wash and elution
- Sample application: the product of interest will bind to the resin containing the affinity ligand.
- By washing with binding buffer, the other proteins will pass through the column.
- Elution of the protein of interest will be done y increasing the pH of the elution buffer.
Describe Gel filtration chromatography / size exclusion chromatography (SEC)
SEC: separation based on size differences
- Uses a column packed with a porous gel
- Molecules with sizes bigger than the pores of the gel are unable to diffuse into the gel and are confined to the spaces between the beads.
- Molecules with smaller sizes can penetrate the pores within the gel to various degrees based on their size
What is Ion exchange chromatography (IEC)?
IEC uses resin that carries charged functional groups which interact with the oppositely charged groups on the product of interest.
