Downstream Bioprocessing

Question 1

What is downstream bioprocessing?

Answer 1

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

Question 2

What’s the main aim of downstream processing?

Answer 2

Concentrate and purify products

Question 3

What does RIPP stand for in downstream processing?

Answer 3

R - recovery
I - isolation
P - purification
P - polish

Question 4

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)

Answer 4

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)

Question 5

What are the large medium and small scales of fermentation and the concentration of its products?

Answer 5

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

Question 6

How does product concentration in the fermentation broth influence product price?

Answer 6

As concentration decreases the price increases ????

Question 7

How should the number of steps affect the yield of a product in bioprocessing?

Answer 7

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%

Question 8

Give 2 examples of products stating their number of steps, overall yield and step yields?

Answer 8

Asparaginase production
13 steps - 30% overall yield - 91% average step yield

Penicillin production
14 steps - 52% overall yield - 94% average step yield

Question 9

State the differences between biotherapeutics and bioindustrial products

Answer 9

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

Question 10

Explain the downstream process for biopharma and bioindustrial downstream processes?

Answer 10

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

Question 11

Explain the Recovary/removal stage of RIPP

Answer 11

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

Question 12

Explain the isolation phase of RIPP

Answer 12

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

Question 13

Explain the purification stage of the RIPP process

Answer 13

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

Question 14

Explain the polishing stage of the RIPP process

Answer 14

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

Question 15

Explain the down stream process for citric acid

Answer 15

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

Question 16

Explain the downstream bioprocessing flow for penicillin

Answer 16

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

Question 17

Explain the down stream processing of intracellular bacterial enzymes

Answer 17

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

Question 18

What are the 4 down stream processing operations?

Answer 18

Centrifugation

Filtration

Precipitation

Chromatography

Question 19

Explain filtration

Answer 19

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

Question 20

Name 4 types of filtration

Answer 20

Membrane filtration

Depth filtration

Rotary vacuum filtration

Tangential flow filtration (TFF)

Question 21

Explain centrifugation

Answer 21

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

Question 22

Name 3 types of industrial centrifuges

Answer 22

Tubular bowl

Multi chamber

Disc

Question 23

Explain precipitation

Answer 23

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

Question 24

Explain chromatography

Answer 24

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

Question 25

Name 4 types of chromatography

Answer 25

Ion exchange chromatography

Affinity chromatography

Size exclusion chromatography

Hydrophobic interaction chromatography

Question 26

Explain hydrophobic interaction chromatography

Answer 26

Separation based on the reversible absorption of molecules based on their hydrophobicity

Question 27

What are the 4 steps in hydrophobic interaction chromatography?

Answer 27

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

Question 28

Explain Affinity chromatography

Answer 28

Separation based on a reversible absorption of bio molecules through bio-specific interactions with the ligand

Question 29

Explain the 3 steps of affinity chromatography

Answer 29

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

Question 30

Explain gel filtration chromatography/ size chromatography

Answer 30

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

Question 31

Explain ion exchange chromatography?

Answer 31

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

Question 32

Explain virus inactivation and filtration

Answer 32

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