Downstream Processing

Definitions • Bioprocessing

• Deals with the manufacture of biochemicals, biopharmaceuticals, foods, nutraceuticals, and agrochemicals.

• Bioseparations Engineering • Refers to the systematic study of the scientific and engineering

principles utilized for the large-scale purification of biological products.

• It is a broader term than the slightly dated downstream processing

• Downstream Processing • Specifically refers to the separation and purification segment of a

bioprocess which followed some form of biological reaction e.g. purification of an antibiotic following microbial fermentation.

Bioprocessing

Upstream processing

Biological reaction Bioseparation Biological

product

Upstream processing Bioseparation Biological product

What is separated in bioseparations?

What is separated in bioseparations?

Economics of Bioseparation

Nature of Bioseparation 1.Biological products are present in very low concentrations in

the starting material from which they are purified

2.Bioseparation has to be very selective in nature due to the presence of by-products and impurities with very similar chemical and physical properties.

3.There are tough quality requirements for products used for prophylactic, diagnostic and therapeutic purposes both in terms of active product content as well as in terms of the absence of specific impurities. • Injectable therapeutic products should be free from endotoxins

and pyrogens.

Nature of Bioseparation 4. Biological products are susceptible to denaturation and

other forms of degradation.

5. Many biological products are thermolabile and hence many bioseparation techniques are usually carried out at sub-ambient temperatures.

6. Bioseparation is frequently based on multi-technique

separation.

Nature of Bioseparations • Size: e.g. filtration, membrane separation, centrifugation

• Density: e.g. centrifugation, sedimentation, floatation

• Diffusivity: e.g. membrane separation

• Shape: e.g. centrifugation, filtration, sedimentation

• Polarity: e.g. extraction, chromatography, adsorption

• Solubility: e.g. extraction, precipitation, crystallization

• Electrostatic charge: e.g. adsorption, membrane separation,

electrophoresis

• Volatility: e.g. distillation, membrane distillation, pervaporation (membrane distillation with a phase change)

Physical forms separated in bioseparations • Particle-Liquid Separation

• F__ • M____ S___ • S____ • C____ • F___

• Particle-particle in liquid medium

• C_____ or Z____ C_____ • S____ E____ C____

Physical forms separated in bioseparations • Particle-Solute Separation in Liquid Medium

• Same as particle-liquid separation

• Solute-solvent separation • R____ O___ • N_____ • M____ S____ • A____ • P_____

Physical forms separated in bioseparations • Solute-Solute in Liquid Medium

• Selective A____ • E____ • D____ • M____ F____

• Liquid-Liquid separation

• (for immiscible liquids) D_______ or P_____ S_____ • D____

Bioseparation Techniques • Low resolution, high throughput

• Cell disruption • Precipitation • Centrifugation • Liquid-liquid extraction • Supercritical fluid extraction • Leaching • Microfiltration • Ultrafiltration • Adsorption • Filtration

Bioseparation Techniques • High resoltion, Low throughput

• Ultracentrifugation • Chromatography • Affinity separation • Electrophoresis

Considerations in developing a bioseparations process • The nature of starting material: e.g. a cell suspension, a crude

protein solution

• The initial location of the target product: e.g. intracellular, extracellular, embedded in solid material such as inclusion bodies

• The volume or flow-rate of the starting material

• The relative abundance of the product in the starting material, i.e. its concentration relative to impurities

Considerations in developing a bioseparations process • The susceptibility to degradation e.g. its pH stability,

sensitivity to high shear rates or exposure to organic solvents, and temperature effects.

• The desired physical form of the final product, e.g. lyophilized powder, sterile solution, suspension

• • The quality requirements, e.g. percentage purity, absence of

endotoxins or aggregates

• Process costing and economics

RIPP Scheme • Recovery • Isolation • Purification • Polishing

The main disadvantages of using the RIPP scheme are: • High capital cost

• Several techniques • Several instruments

• High operations cost • Too much solvents and material • Extensive labor • Prolonged time

• Lower recovery of product • Amount purified can be very small

PROPERTIES OF BIOLOGICAL MATERIALS

Size • Non-spherical molecules: Stokes-Einstein diameter

• Equivalent radius of a particle settling under gravity

Size • Example

• A suspension of kaolin (a type of clay used as adsorbent for biological material) in water became clear upon being allowed to stand undisturbed for 3 minutes at 20 °C. The height of the suspension in the vessel was 30 cm and the density of kaolin is known to be 2.6g/cm3. Estimate the diameter of the kaolin particles.

Molecular Weight

Diffusivity • Refers to random motion of molecules due to intermolecular

collisions • Net migration

• High conc. → low conc.

Diffusivity • Example

• The diffusivity of a protein having a Stokes-Einstein radius of 2 nanometers in a particular liquid is known to be 4.5x10-11 m2/s . Predict the diffusivity of another molecule having twice the Stokes-Einstein radius in the same liquid at the same temperature.

Sedimentation Coefficient • The tendency of macromolecules and particles to settle in a

liquid medium

Sedimentation Coefficient

Osmotic Pressure • Pressure differential generated across the membrane due to

tendency of water flowing from low to high solute concentration

• Osmotic difference across the membrane

Electrostatic Charge • Net negative: pH > pI • Net positive: pH < pI • No net charge: pI

Solubility • Salting in effect

• Low ionic strength • Addition of salt increases solubility

• Salting out effect

• High ionic strength • Addition of salt decreases solubility

Partition coefficient • Measure of how a compound distributes itself between two

liquid phases

Light Absorption • Chromophores – specific bonds or groups that absorb light at

a particular wavelenght

Fluorescence • Emission of light after absorption of energy (light)

Exercise problems • Two spherical molecules A and B were found to have

diffusivities of 4x10-10 m/s and 8x10-10m/s respectively in a particular medium. Which molecule has the larger diameter and by what percent is this diameter greater than that of the other?

• An ultrafiltration membrane separates two dilute myoglobin solutions: 0.01g/L and 0.05g/L respectively, both being maintained at 25°C .Calculate the osmotic pressure across the membrane.