Downstream Processing

Downstream processing Biotech fourth semester Made by: Devender arora Roll no: 1229

Downstream Processing What is Downstream Processing? •

Downstream – ‘after the fermentation process’

•

Primary ‘unit operations’ of Downstream Processing ➢

Cell recovery/removal 

Centrifugatio, Dewatering, Ultra filtration , Precipitation, Spray drying

Downstream Processing •

Secondary ‘unit operations’ ➢

Protein purification: Adsorption chromatography, Gel permeation chromatography

➢

Protein processing: Immobilization ,Beading/Prilling

➢

Protein packaging: Sterilization , Bottling etc

Separation of cells and medium •

Recovery of cells and/or medium (clarification) ➢ For intracellular enzyme, the cell fraction is required ➢ For extracellular enzymes, the culture medium is required

•

On an industrial scale, cell/medium separation is almost always performed by centrifugation ➢

Industrial scale centrifuges may be batch, continuous, or continuous with desludging

Industrial centrifuges

Tubular bowl

Chamber

Disc

Properties of industrial centrifuges •

Tube: High centrifugal force, Good dewatering, Easy to clean, Limited solids capacity, Foams, Difficult to recover protein

•

Chamber: Large solids capacity, Good dewatering, Bowl cooling possible, No solids discharge, Cleaning difficult, Solids recovery difficult

•

Disc type: Solids discharge, No foaming, Bowl cooling possible, , Poor dewatering, Difficult to clean

Centrifugation properties of different cell types •

Bacteria :Small cell size, Resilient, High speed required, Low cell damage

•

Yeast cells: Large cells, Resilient, Lower speed required, Low cell damage

•

Filamentous fungi: Mycelia , Resilient, Lower speed required, High water retention in pellet

•

Cultured animal cells: Large cells, Very fragile, Very susceptible to damage

Dewatering •

Dewatering of whole cell fraction (use centrifugation)

•

Dewatering of culture medium or a lysed cell fraction (for recovery of a soluble protein fraction) ➢

Precipitation: Salting out – addition of a high concentration of a soluble salt (typically ammonium sulphate) causes proteins to aggregate and precipitate

➢ Ultra filtration : The solution is forced under pressure through a membrane with micropores, which allows water, salts and small molecules to pass but retains large molecules (e.g., proteins) ➢

Spray drying: Requires use of heat to evaporate water – unsuitable for most proteins

Cell disruption (for intracellular enzymes) •

Sonication : Use of high frequency sound waves to disrupt cell walls and membranes

•



Can be used as continuous lyses method



Better suited to small (lab-scale) operations



Can damage sensitive proteins

Pressure cells ➢ Apply apply high pressure to cells; cells fracture as pressure is abruptly released

•



Readily adapted to large-scale and continuous operations



Industry standard (Manton-Gaulin cell disruptor)

Enzymic lyses ➢

Certain enzymes lyses cell walls 

Lysozyme for bacteria; chitinase for fungi

Only useful on small laboratory scale

Protein purification •

Adsorption chromatography ➢ Ion exchange chromatography – binding and separation of proteins based on charge-charge interactions ➢

Proteins bind at low ionic strength, and are eluted at high ionic strength

…….. …….. …. Positively charge anionic ion exchange matrix

net negatively charged

cationic protein at selected ph Protein binds to matrix

Reminder about protein net charge, pI and pH •

All proteins have ionisable groups on the surface (N-terminal amino and carboxylate, Glu, Asp, His, Lys and Arg side chains)

•

These groups are charged or neutral depending on pH (e.g., -COO- + H+ D COOH)

•

The net charge on a protein changes at different pHs

•

Each protein has a pH where the net charge is zero (the pI: Isoelectric Point)

•

Useful rules: ➢

At pH > pI, protein net charge is negative

➢

At pH < pI, protein net charge is positive

➢ At pH = pI, protein net charge is zero

Typical ion exchange protein separation

Affinity chromatography •

•

Binding of a protein to a matrix via a protein-specific ligand ➢

Substrate or product analogue

➢

Antibody

➢

Inhibitor analogue

➢

Cofactor/coenzyme

Specific protein is eluted by adding reagent which competes with binding

Affinity chromatography Gel permeation chromatography (GPC) •

Also known as ‘size exclusion chromatography’ and ‘gel filtration chromatography’

•

Separates molecules on the basis of molecular size

•

Separation is based on the use of a porous matrix. Small molecules penetrate into the matrix more, and their path length of elution is longer.

•

Large molecules appear first, smaller molecules later

Downstream processing depends on product use

1. Enzyme preparations for animal feed supplementation (e.g., phytase) are not purified 2. Enzymes for industrial use may be partially purified (e.g., amylase for starch industry) 3. Enzymes for analytical use (e.g., glucose oxidase) and pharmaceutical proteins (e.g., TPA) are very highly purified