Downstream Processing

workkinkajouBiotechnology

Dec 5, 2012 (4 years and 11 months ago)

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Downstream Processing

BTY323 Lectures 12, 13

What is
Downstream Processing
?


Downstream



‘after the fermentation process’


Primary ‘unit operations’ of
Downstream
Processing



Cell recovery/removal


Centrifugation



Dewatering


Ultrafiltration


Precipitation


Spray drying

Downstream Processing


Secondary ‘unit operations’



Protein purification


Adsorption chromatography


Gel permeation chromatography



Protein processing



Immobilisation



Beading/Prilling



Protein packaging


Sterilisation



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


Chamber


Large solids capacity


Good dewatering


Bowl cooling possible


Disc type


Solids discharge


No foaming


Bowl cooling possible




Limited solids capacity


Foams


Difficult to recover protein



No solids discharge


Cleaning difficult


Solids recovery difficult



Poor dewatering


Difficult to clean




Centrifugation properties of different cell
types


Bacteria


Small cell size


Resilient


Yeast cells


Large cells


Resilient


Filamentous fungi


Mycelial


Resilient


Cultured animal cells


Large cells


Very fragile



High speed required


Low cell damage



Lower speed required


Low cell damage



Lower speed required


High water retention in pellet



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



Ultrafiltration


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 lysis 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 lysis



Certain enzymes lyse 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 charged

(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
+



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

Loading starts

Loading ends,

Low salt wash begins

Protein absorbance

Peak of

unbound

protein

Salt gradient

0

1M

Salt gradient

begins

Salt gradient

ends

Eluted peaks of weakly bound (I),

moderately bound (II)

and tightly bound (III) proteins

II

III

I

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

Matrix Spacer arm

Affinity


ligand

+

Active
-
site
-
bound enzyme

1. Substrate analogue affinity chromatography

Matrix Spacer arm

Antibody


ligand

+

Antibody
-
bound enzyme

2. Immunoaffinity chromatography

Protein epitope

Enzyme

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

GPC in operation

Large protein Small protein

Short path length Longer path length

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



Fermentation

Culture supernatant

Centrifugation

to remove cells

Liquid preparation


to animal feed

market

Fermentation

Culture supernatant

Fermentation

Cell pellet

Intracellular fraction

Animal feed enzyme Analytical enzyme Therapeutic protein

Centrifugation

to remove cells

Centrifugation

to remove

medium

Protein

precipitation

Cell

lysis Centrifugation

Protein fraction

Protein

precipitation

Protein fraction

1 or 2 purification

steps

Semi
-
purified

protein

3
-
4 purification

steps

Homogeneous

protein

Sterile

bottling

To pharmaceuticals market

Lyophilisation

Bottling

To chemicals market

Operational diagram of large
-
scale fungal
batch fermentation system

Preculture Preparation of Fermentation Recovery of enzyme
-


inoculum containing medium