Downstream Bioprocessing Flashcards
What is downstream bioprocessing?
Downstream processing refers to recovery and purification operations that follow chemical and biochemical reactions, especially fermentations, as well as animal cell culture or agriculture synthesis
- any treatment of the culture after fermentation is downstream processing
- each recovery scheme different as product range is large
What’s the main aim of downstream processing?
Concentrate and purify products
What does RIPP stand for in downstream processing?
R - recovery
I - isolation
P - purification
P - polish
What are the process decisions in downstream processing design? (Questioned asked about the product like quantity quality use just to name a few, 8 questions)
1) what is the marketable price of the product (very cheap, cheap, expensive, astronomical)
2) what is the level of the product in the fermentation broth
3)what is the intended use of the product (industrial enzyme, agricultural, therapeutic)
4) what is an acceptable product quality (minimum purity required)
5) where is the product (intracellular, periplasmic, extracellular)
6) what are the physio-chemical properties of the product and the principal properties (needed selection of appropriate separation techniques)
7) is the product or the broth safe ( level of contamination)
8) are there elements in broth likely to cause issues in recovery?(antifoam)
What are the large medium and small scales of fermentation and the concentration of its products?
Large scale (200,000L or more )
Ethanol 7-12% concentration
Citric acid 10% conc
Lactic acid 5% conc
Penicillin 3-5% conc
Amino acids 0.2-10% conc
Medium scale (50,000 - 200,000L)
Cephalosporin 3% conc
Streptomycin 1% conc
Extracellular enzymes 5-10% conc
Small scale (less that 50,000L)
Riboflavin. 0.01-0.7% conc
Vitamin b12. 0.006-0.2% conc
Monoclonal antibodies 0.1 % conc
How does product concentration in the fermentation broth influence product price?
As concentration decreases the price increases ????
How should the number of steps affect the yield of a product in bioprocessing?
More sophisticated products number of steps can be greater than 10
To obtain 50% overall yield in 10 steps the average yield for each unit operation should be above 90%
I other multi step processes need to achieve high step yields at least 90% or more than 95%
Give 2 examples of products stating their number of steps, overall yield and step yields?
Asparaginase production
13 steps - 30% overall yield - 91% average step yield
Penicillin production
14 steps - 52% overall yield - 94% average step yield
State the differences between biotherapeutics and bioindustrial products
Biopharma products
High price
Stable
Purity - less than 1ppp host proteins - less than 10 of DNA/dose pyrogens
Sterile
Product aggregation
Bioindustrial products
Low price
Stable
Purity - between 10-95% - might contain active components - colour
Explain the downstream process for biopharma and bioindustrial downstream processes?
Biopharma
- process at kg scale
- ultra high purification factor
- gentle to product
- must be validated
- virus removal/inactivation
- contained process
- reasonable process yield
- can be very expensive
Bio industry
- Processing at ton Scale
- necessary purification factor
- low processing
- high process yield
- green process
- low investment cost
- contained process - working environment
Explain the Recovary/removal stage of RIPP
Common first step in product recovery is removal of cells from fermentation broth
Product may be either produced intracellularly or extracellularly (secreted in liquid phase)
- relatively little product concentration or improvement in quality occurs
- centrifugation and filtration are dominant operations in this segment
- typical large scale operations: setting/sedimentation/ decanting / flotation / cell distribution / centrifugation / filtration
Explain the isolation phase of RIPP
Wild variety of available techniques for isolation from cells or cell-free broth
These steps tend to be relatively non selective
Significant increase in product concentration and quality by removing materials of widely divergent properties compared to desired product
Typical large scale operations; absorption / liquid extraction / coagulation/ flocculation / precipitation
Explain the purification stage of the RIPP process
These processes are highly selective for soluable product and remove impurities of similar chemical functional and physical properties
Typical large scale operations : fractional precipitation / chromotography / ultrafiltration
Explain the polishing stage of the RIPP process
Final processing steps which end with product packaging in a form that is stable, easily transportable and convenient
Final purity depends on product application
The end use of produce dictates final sequence of operation utilised
Crystallisation is often key
Typical large scale operations : size exclusion chromatography / reverse phase chromatography / crystallisation / desiccation / lyophilisation / spray drying
Explain the down stream process for citric acid
Segment 1
- after fermentation filtration to remove solids
Segment 2
- adding lime precipitates the calcium salt of citric acid isolating product from soluble impurities
Segment 3
The citrate is then purified by conversion back to acid form and by filtration to remove calcium sulphate
Segment 4
Polishing is achieved by crystallisation
Explain the downstream bioprocessing flow for penicillin
Segment 1
Involves filtration to remove solids
Segment 2
The filtered broth is acidified and extracted with an organic solvent
Extract is then stripped with buffer to isolate a concentrate
Segment 3
PH of concentrate is adjusted with acid and product is purified by second extraction into organic solvent
Segment 4
Final polishing consists of vacuum concentration, crystallisation and drying of crystalline penicillin
Explain the down stream processing of intracellular bacterial enzymes
Segment 1
Cells concentration, enzymes are released and cell debris removed
Segment 2
Isolation ( total precipitation) fractional precipitation, undesired products removed
Segment 3
Enzyme purification by ultrafiltration and chromatography
Segment 4
Final polishing - precipitation, centrifugation and lyophilisation
What are the 4 down stream processing operations?
Centrifugation
Filtration
Precipitation
Chromatography
Explain filtration
Filtration is a mechanical operation for separation of solids from liquids or gases by interposing a medium to a porous membrane
The fluid can pass through the membrane but solids are retained
Name 4 types of filtration
Membrane filtration
Depth filtration
Rotary vacuum filtration
Tangential flow filtration (TFF)
Explain centrifugation
Cetrifigayion is used to delegate particles from liquid by gravitational forces
Dependant on
- particle size
- density difference between the cells and the broth
- brith viscosity
More dense components migrate to away from axis of centrifuge
Less dense component migrate towards axis
Name 3 types of industrial centrifuges
Tubular bowl
Multi chamber
Disc
Explain precipitation
Precipitation is the formation of a solid in a solution during a chemical reaction
The solid is known as a 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 ( methanol) are used to precipitate dextrans
Explain chromatography
Chromatography is a highly specialised and sensitive technique used for separation of mixtures by passing them through a stationary phase (e.g resin beads) which separates the analyse of interest from other molecules in mixture and allows isolation
Relies on differences such as:
- size
- charge or interaction with water
Name 4 types of chromatography
Ion exchange chromatography
Affinity chromatography
Size exclusion chromatography
Hydrophobic interaction chromatography
Explain hydrophobic interaction chromatography
Separation based on the reversible absorption of molecules based on their hydrophobicity
What are the 4 steps in hydrophobic interaction chromatography?
4 steps : equilibration, sample application, elution and regeneration
Equilibrium
- 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
Explain Affinity chromatography
Separation based on a reversible absorption of bio molecules through bio-specific interactions with the ligand
Explain the 3 steps of affinity chromatography
Equilibration, sample application/ wash and elution
Sample application
- the product of interest will bind to resin containing affinity ligand
- by washing with binding buffer , the other proteins will pass through the column
Elution of the proteins of interest will be done by increasing the ph of the elution buffer
Explain gel filtration chromatography/ size chromatography
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
Explain ion exchange chromatography?
Uses resin that carried charged functional groups which interact with the oppositely charged groups on the product of interest
Positively charged proteins bind to negatively charged beads
- negatively charged proteins flow through
Explain virus inactivation and filtration
Inactivation
- low ph chemically inactivated viruses ( typically done after the protein a affinity chromatography steps)
- other chemicals can be used to destroy viruses like urea but all inactivation steps mushy take care to minimise product damage
Filtration
- nano filtration is typically done after product capture and polishing stages, prior to formulation of the antibody as a drug product
IS AN FDA REQUIREMENT
Viruses are the smalles example
Of a microbial contaminant, so validating their removal also validated the removal of larger microbe contaminants like bacteria or fungi
