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Downstream Processing - Protein Extraction and Purification
bacterium Escherichia coli, and is completely free from the unwanted
Enzymes or proteins produced by microorganisms may be intracellular, periplasmic or secreted into the culture medium. For extracellular
enzymes, the degree of purification required is often minimal, as the final
product is intended for industrial use and does not have to be of high
purity. Such large-scale processes may yield tonnes of protein product.
Many other enzymes are produced in more complex intracellular
mixtures and represent a greater challenge to the protein purification
scientist. Those produced for therapeutic use must attain very high and
exacting standards of purity and to achieve this it may be necessary to
develop complex purification protocols. Again, recombinant DNA
technology has been able to help in this area. In the case of therapeutic
proteins, there are often advantages if the protein of interest can be
secreted, either into the periplasm or into the medium. This results in an
enormous reduction in the level of contaminating proteins and other
macromolecules, such that pure product can often be obtained in only
two or three steps of purification. Choice of a suitable expression system
can result in higher yields in the fermenter, thereby improving the specific
activity of the starting material. It is also possible to add groups to a
protein which aid its purification by conferring specific properties and
subsequently removing these groups when they are no longer required.
In designing a large-scale purification process the number of steps, and
the recovery of product at each step can have a major effect on the overall
yield of the product, as is shown in Figure 1. The typical recovery of a
chromatographic step is between 80% and 9070,so a complex purification requiring many steps may have an overall yield as low as 10% of the
starting material. This may not matter for a laboratory-scale purification, but for large-scale production it is important to optimize the overall
process from expression system or fermentation to final polishing step,
so as to minimize the number of purification steps required.
There are three main methods for the release of intracellular proteins
from microorganisms: enzymic, chemical or physical. Not all of the
techniques available are suitable for use on a large scale. Perhaps the
main example is sonication, which is frequently the method of choice for
the small-scale release of proteins. On a large scale it is difficult to
transmit the necessary power to a large volume of suspension and to
remove the heat generated.
Downstream Processing:Protein Extract ion and Pur iJicat ion
Recovery per step
Number of Steps
Figure 1 The eJtrect of recovery per step on overall recovery
Enzymic Methods of Cell Disruption
Lysozyme, an enzyme produced commercially from hen egg white,
hydrolyses beta- 1,4-glycosidic bonds in the mucopeptide of bacterial
cell walls. Gram-positive bacteria, which depend on cell wall mucopeptides for rigidity are most susceptible, but final rupture of the cell wall
often depends upon the osmotic effects of the suspending buffer once the
wall has been digested. In Gram-negative bacteria lysis is rarely achieved
by the use of lysozyme alone, but the addition of EDTA to chelate metal
ions will normally result in lysis. Although gentle, this technique is rarely
used for the large-scale extraction of bacterial enzymes, perhaps due to
the relatively high cost of lysozyme and the possibility of introducing
contaminants. It has been used for the large-scale release of an aryl
acylamidase from Pseudomonas fluorescens.
2.2 Chemical Methods of Cell Lysis
2.2.1 Alkali. Treatment with alkali has been used with considerable
success in small- and large-scale extraction of bacterial proteins. For
example, the therapeutic enzyme, L-asparaginase, can be released from
Erwinia chrysanthemi by exposing the cells to pH values between 11.0
' P. M.Hammond, C. P. Price, and M. D. Scawen, Eur. J . Biochem., 1983, 132,651.
and 12.5 for 20 min.2 The success of this method relies on the alkali
stability of the desired product. The high pH may inactivate proteases,
and the method is of value for the combined inactivation and lysis of
2.2.2 Detergents. Detergents, either ionic, for example, sodium lauryl
sulphate, sodium cholate (anionic) and cetyl trimethyl ammonium
bromide (cationic), or non-ionic, for example Triton X-100 or X-450, or
Tween, have been used to aid cell lysis, often in combination with
lysozyme. Ionic detergents are more reactive than non-ionic detergents,
and can lead to the denaturation of many proteins. The presence of
detergents can also affect subsequent purification steps, in particular salt
precipitation. This can be overcome by the use of ion exchange
chromatography or ultrafiltration, but obviously introduces additional
2.3 Physical Methods of Cell Lysis
2.3.1 Osmotic Shock. Osmotic shock can be used for the release of
enzymes and proteins from the periplasmic space of a number of Gramnegative bacteria. The method involves washing the cells in buffer
solution to free them from growth medium, and then suspending them
in 20% buffered sucrose. After being allowed to equilibrate, the cells are
harvested and rapidly resuspended in water at about 4°C. Only about 48% of the total bacterial protein is released by osmotic shock, and if the
required enzyme is located in the periplasmic region it can produce a 14to 20-fold increase in purification compared with other extraction
techniques. A major disadvantage of osmotic shock is the large increase
in volume which occurs.
2.3.2 Grinding with Abrasives. Initially this technique was restricted
to the grinding of cell pastes in a mortar with an abrasive powder, such as
glass, alumina or kieselguhr. It has since been developed and mechanized
using machines originally developed for the wet grinding and dispersion
of pigments in the printing and paint industries. A typical product, the
Dynomill (W. A. Bachofen, Switzerland) can be used to release proteins
from a wide variety of microorganisms. It consists of a chamber containing glass beads and a number of fixed and rotating impeller discs. The cell
suspension is pumped through the chamber, and the rapid agitation is
sufficient to break even the toughest of bacteria. The disintegration
2T. Atkinson, B. J. Capel, and R. F. Sherwood, in ‘Safety in Industrial Microbiology and
Biotechnology.’, ed. C. H. Collins and A. J. Beale, Butterworth, Oxford, 1992, p.161.
Downstream Processing: Protein Extraction and PuriJication
chamber must be cooled to remove the heat which is generated. A
laboratory-scale model, with a 600 ml chamber can process up to 5 kg
bacteria per hour, and production scale models are available with
chambers of up to 250 litre capacity.
Many factors influence the rates of cell breakage, such as the size and
concentration of the glass beads, the type, concentration and age of the
cells, chemical pre-treatment, the agitator speed, the flow rate through
the chamber, the temperature, and the arrangement of the agitator discs,
and these have been investigated for yeasts3 and b a ~ t e r i a .This
~ - ~ type of
cell disrupter has the advantage that it can be readily mounted in an
enclosed cabinet when pathogenic or rDNA organisms are to be broken.
2.3.3 Solid Shear. Methods of cell disruption employing solid shear
have long been used on a small scale. It involves the extrusion of frozen
cell material through a narrow orifice at high pressure and an outlet
temperatures of about -20°C. It has found little application on an
industrial scale, due to limitations on the amount of material which can
2.3.4 Liquid Shear. Liquid shear is the principle choice for the largescale disruption of microbial cells, finding widespread application in
both industrial processes and in research. It is particularly useful for the
disruption of bacteria and yeast?
As with solid shear, the cells are passed through a restricted orifice
under high pressure, this time in a liquid suspension. For smaller scale
work, a French Press is used. Larger scale work usually employs a
homogenizer of the type developed for emulsification in the dairy
industry. A temperature increase of at least 10°C in a single pass is not
uncommon and it is necessary to pre-cool the cell suspension before
homogenization. The liquid shear homogenizer is normally operated at
wet cell concentrations of about 20%.
For large-scale work the Manton-Gaulin homogenizer (APV Ltd.,
Crawley, UK) is the most frequently used. It consists of a positive
displacement piston pump with a restricted outlet valve, which can be
adjusted to give the required operating pressure, up to 95 MPa. The
smallest Manton-Gaulin homogenizer, the 15M-8TA, has a throughput
of about 50 1 h-' at a pressure of 55 MPa. A larger version, the MC-4,
has a throughput of about 300 1 h-', again at a pressure of 55 MPa.
The rate of cell breakage and of protein release is dependent on a
F. Marffy and M. R. Kula, Biotechnol. Bioeng., 1974, 16,623.
J. R. Woodrow and A. V. Quirk, Enzyme Microb. Technol., 1982,24385.
5 S . T. Harrison, J. S. Dennis, and H. A. Chase, Biosepururion, 1991,2,95.
D. Foster, BiolTechnology, 1992, 10, 1539.
number of factors, including cell type, fermentation conditions, concentration and pre-treatment, such as freezing, as it is often observed that
microbial cells break more easily if they have first been frozen. It has also
been found that the presence of inclusion bodies makes E. coli cells more
easily b r ~ k e n . ~
The rate of protein release from yeast cells can be described by the
empirical first order rate equation:
Log ( R , / R , - R) = K n P"
Where: R, is the theoretical maximum amount of soluble protein to be
released, R is the actual amount of protein released, K is a temperature
dependent constant, n is the number of passes, P is the operational back
pressure, and a is a constant depending on the organism.
The value of the exponent a varies with the organism; for yeast it was
found to be 2.9.', and for E. coli it is about 2.0. A similar first order
equation can be used to describe the rate of release of proteins from other
organisms, although the value of the exponent varies.' There are many
examples of the use of the Manton-Gaulin homogenizers for the largescale disruption of microbial cells. Beta-galactosidase has been released
from E. coli,'o and carboxypeptidase from Pseudomonas spp." A large
number of enzymes have been isolated from the thermophilic bacterium
Bacillus stearothermophilus, including glycerokinase12 and a glucosespecific he~0kinase.l~
For a reliable process the conditions for cell
breakage must be carefully optimized, as variations in the degree of cell
breakage and protein release can have significant effects on subsequent
3 INITIAL PURIFICATION
3.1 Debris Removal
Following cell disruption, the first step in the purification of an
intracellular enzyme is the removal of cell debris. The separation of
solids from liquids is a key operation in enzyme isolation, and is
normally accomplished by centrifugation or filtration. Many protocols
'A. P. Middelberg, B. K. O'Neill, and D. L. Bogle, Biotechnol. Bioeng., 1991,38,363.
M. Follows, P. J. Hetherington, and M. Lilly, Biotechnol. Bioeng., 1971, 13,549.
C . R. Engler and C. W. Robinson, Biotechnol. Bioeng., 1981,23, 765,
J. J. Higgins, D. S. Lewis, W. Daly, F. G. Mosqueira, P. Dunnill, and M. D. Lilly, Biotechnol.
Bioeng., 1987, 20, 159.
F. Sherwood, R. G. Melton, S. M. Alwan, and P. Hughes, Eur. J. Biochem., 1985,148,447.
12P. M. Hammond, T. Atkinson, and M. D. Scawen, J. Chromatogr., 1986,366,79.
I3C. R. Goward, T. Atkinson, and M. D. Scawen, J. Chromatogr., 1986,369,235.
Downstream Processing: Protein Extraction and Purijication
also include the addition of small quantities of DNase at this point, to
break up long DNA chains which can cause the extract to become
3.2 Batch Centrifuges
Batch centrifuges are available with capacities ranging from less than
1 ml up to several litres, and capable of applying a relative centrifugal
force of up to 100000 x g (gravitational constant). However, for the
removal of bacterial cells, cell debris and protein precipitates, fields up to
20000 x g are adequate. Many centrifuges of this type, suitable for
intermediate-scale preparations, are available.
3.3 Continuous-flow Centrifugation
Because of the large volumes of liquid which need to be handled at the
beginning of a large-scale enzyme purification, it is preferable to use a
continuous flow centrifuge to remove particulate matter. Three main
types of centrifuge are available; the hollow bowl centrifuge, the disc or
multi-chamber bowl centrifuge, and the basket centrifuge. Hollow bowl
centrifuges have a tubular rotor which provides a long flow path for the
extract, which is pumped in at the bottom and flows upwards through
the bowl. Particulate matter is thrown to the side of the bowl, and the
clarified extract moves up and out of the bowl into a collecting vessel. As
centrifugation proceeds, the effective diameter of the bowl decreases, so
reducing the settling path and the centrifugal force which can be applied.
The ease with which the bowl can be changed, and the possibility of using
a liner to aid sediment recovery has contributed to the popularity of this
type of centrifuge. The flow rate must be determined empirically as it will
vary from one type of extract to another, but a rate of about 60 1 h-' is
generally satisfactory for the larger machines. Centrifuges of this type are
produced by Pennwalt Ltd. (Camberley, Surrey, UK) and by Carl
Padberg GmbH (Lahr, Germany).
Disc centrifuges provide an excellent means of clarifying crude
extracts, and in many cases the sediment may be discharged without
interrupting the centrifugation process. The bowl contains a series of
discs around a central cone. As the extract enters, particulate matter is
thrown outwards, impinging on the coned discs and sedimented matter
collects on the bowl wall. This provides a constant flow path, so there is
little loss of centrifugal efficiency during operation. A disadvantage of
these centrifuges is that some loss of product may be experienced during
the discharge process.
The rotors of instruments which do not have the facility to discharge
sediment during operation are tedious to clean and this again may result
in loss of product if the solids are required. These centrifuges achieve an
RCF (rotational centrifugal force) of about 8000 x g and have a
capacity of up to 20 kg of sediment. As with hollow bowl centrifuges,
the correct flow rate must be determined empirically. A variation of the
disc-type centrifuge is the multi-chambered bowl centrifuge, in which the
bowl is divided by vertically mounted cylinders into a number of
interconnected chambers. The feed passes through each chamber from
the centre outwards, before leaving the centrifuge. This type of arrangement also ensures a short and constant settling path as the bowl fills, and
is easier to dismantle and clean than the disc-type of centrifuge.
Typical centrifuges of these types are produced by De Lava1 Separator
Co. (New York, USA), and by Westfalia Separator Ltd. (Wolverton,
A problem suffered by all types of centrifugation when applied on an
industrial scale to enzyme recovery is that many homogenates produce
wet sloppy precipitates, which reduces the efficiency of centrifugation. l4
The degree of clarification achieved with an industrial continuous flow
centrifuge is never as great as that obtained with a laboratory centrifuge,
and subsequent steps may be necessary to achieve the desired degree of
clarification. One approach to alleviating this problem is to add a coarse
microgranular cellulose anion exchanger (cell debris remover (CDR),
Whatman) which binds cell debris and increases its density such that it is
removed more efficiently by low speed centrifugation.
3.4 Basket Centrifuges
These are designed to operate at much lower g forces, perhaps only 1000
rev min-I, and are basically centrifugal filters. The bowl is perforated
and is normally lined with a filter cloth. The main use of these centrifuges
is to collect large particulate material; in the context of enzyme purification this usually means ion exchange materials which have been used for
the batch adsorption of the desired protein. Examples of such centrifuges
are available from Carl Padberg GmbH (Lahr, Germany).
3.5 Membrane Filtration
Filtration is an alternative method of clarifying cell extracts. However,
microbial broths and extracts tend to be gelatinous in nature, and are
M.Hoate, P.Dunnill, and D. J. Bell, Ann. N . Y.Acad. Sci., 1983,413,254.
Downstream Processing: Protein Extraction and PuriJication
difficult to filter by traditional methods, unless very large filter areas are
This can be overcome by using tangential or cross-flow filtration. In
this method the extract flows at right-angles to the direction of filtration,
and the use of a high flow rate tends to reduce fouling by a self-scouring
action, although this action must be balanced against the possibility of
losses due to shear effects. Membranes with an asymmetric, anisotropic
pore structure are less prone to blockage than isotropic membranes, and
have been used for the large-scale recovery of L-asparaginase from
Erwinia chrysanthemi. A 1 m2 membrane assembly was used to harvest
the cells from 100 litre of culture fluid in 2.5 h, when the solids
concentration in the retentate increased from 0.55% to 22% dry weight.
This same membrane assembly was then used to clarify the extract
obtained by the alkali lysis of these bacteria. These data indicated that to
harvest the cells from 500 litre culture in 2.5 hours would require 7.5 m2
of membrane, and that the costs compared favourably with the costs of
centrifugation. Membranes suitable for cross-flow filtration are available as spirally wound cartridges, which offer the same surface area as
flat membranes, but in a more compact space.
Because of the limitations of large-scale centrifugation the two
techniques are often combined to ensure that the extract is sufficiently
clear for subsequent chromatography.
4 AQUEOUS TWO-PHASE SEPARATION
An alternative to centrifugation or filtration is aqueous two-phase
separation. Aqueous two-phase systems are typically created by mixing
solutions of polyethylene glycol and dextran or polyethylene glycol and
salts such as potassium phosphate or ammonium sulphate to form two
immiscible phases. Proteins and cellular debris show differential solubility between the two phases, so that the technique can be used both for
the separation of proteins from cellular debris and for the partitioning of
enzymes during protein purification. The precise partitioning of a
protein depends on parameters such as its molecular weight and charge,
the concentration and molecular weight of the polymers, the temperature, pH and ionic strength of the mixture and the presence of polyvalent
salts such as phosphate or
The optimal conditions required
"M. S.Le and T. Atkinson, Process Biochem., 1985,20,26.
16H. Walter, D. E. Brooks, and D. Fisher, 'Theory, Methods, Uses and Applications to
Biotechnology: Partitioning in Aqueous Two Phase Systems', Academic Press, New York, 1985.
"N. L. Abbot, D. Blankschtein, and T. A. Hatton, Bioseparation, 1990,1, 191.
F. Tjerneld and G. Johansson, Bioseparation, 1990,1,255.
for a particular protein are found empirically. Although the conditions
required to achieve satisfactory separation can often be precisely defined,
the mechanism of partitioning is not fully understood.''
The phases can be separated in a settling tank, but a more efficient
and rapid separation can usually be achieved by centrifugation. Since
it is easier to separate liquids of different density than solids from
liquids on the large scale, this approach can be used to advantage in
large-scale enzyme purification. Although the relatively low cost of the
polyethylene glycol-salt system makes it attractive for large-scale use,
the more generally useful polyethylene glycol-dextran system can also
have economic advantages in comparison with other purification
methods, despite the high cost of purified dextran, providing the
total processing costs are evaluated.20 Its use is not restricted to
materials of microbial origin, and the method has been successfully
used to isolate materials from both plant2' and
including human alpha-L-antitrypsin expressed in transgenic milk. In
this case the relatively high starting purity of the protein meant that
after a single two phase separation the desired protein was 73%
Aqueous two phase separation can be adapted to offer biospecific
partitioning by attaching ligands to the polymers in order to alter the
partitioning of a
All phase-forming polymers can have
ligands attached covalently to them, and a wide range of such ligands
have been investigated. Because of their simple coupling chemistry, the
reactive dyes have frequently been used as l i g a n d ~ . *The
~ power of the
technique was demonstrated by the %-fold purification of yeast
phosphofructokinase that could be obtained in two steps using Cibacron Blue F-3GA immobilized on polyethylene
reactive dyes, a number of other ligands have been investigated. These
include cofactors, such as the pyridine nucleotides used successfully in
the affinity partitioning of a number of dehydrogenases.
On the process scale, affinity partition has been used for the purification of formate dehydrogenase from 10 kg quantities of the yeast
J. Huddleston, A. Veide, K . Kohler, J. Flanagan, S. Enfors, and A. Lyddiatt, Trends Biotechnol.,
2o K . H. Kroner, H. Hustedt, and M. R . Kula, Process Biochem., 1984, 19, 170.
2' H. Vilter, Bioseparation, 1990, 1,283.
22 M . J. Boland, Bioseparation, 1990, 1, 293.
23 D. P. Harris, A. T. Andrews, G. Wright, D. L. Pyle, and J. A. Asenjo, Biosepararion, 1997, 7,31.
24 G. Johansson and F. Tjerneld, in 'Highly Selective Separations in Biotechnology', ed. G. Street,
Blackie Academic and Professional, London, 1994, p.55.
2s G. Kopperschlager, Methods Enzymol., 1994,228, 121.
26G.Johansson, G. Koperschlager, and P. A. Albertsson, Eur. J . Biochem., 1983, 131, 589.
Downstream Processing: Protein Extract ion and Pur ifica t ioli
Candida bodinii, using the triazine dye, Procion Red HE-3B, immobilized
Although aqueous two-phase separation is a method which can easily
be scaled up to a manufacturing level, it does not appear to be often used
for industrial-scale purifications.
5.1 Ammonium Sulfate
Salting out of proteins has been employed for many years, and fulfils the
dual purposes of purification and concentration. The most commonly
used salt is ammonium sulfate, because of its high solubility, lack of
toxicity towards most enzymes, and low cost.
The precipitation of a protein by salt depends on a number of factors:
pH, temperature, protein concentration, and the salt used.28The protein
concentration is particularly important when scaling-up, because most
large-scale purifications are carried out at higher protein concentrations
than laboratory-scale purifications. This can have a dramatic effect on
the concentration of salt needed to precipitate a given protein.
5.2 Organic Solvents
The addition of organic solvents to aqueous solutions reduces the
solubility of proteins by reducing the dielectric constant of the medium.
Various organic solvents have been used for the precipitation of proteins,
with ethanol, acetone, and propan-2-01 being the most important.
Because proteins are denatured by organic solvents it is necessary to
work at temperatures below 0°C.
Because of their flammable nature, requiring flameproof equipment to
be used, and high cost, coupled with a low selectivity, organic solvents
are not often used in large-scale enzyme purification. The one notable
exception is in the blood processing field, where ethanol precipitation is
the major method for the purification of albumin; indeed it has been
developed into a highly automated, computer-controlled system.29
’’M.R. Kula, in ‘Extraction and Purification of Enzymes. Applied Biochehmistry and Bioengineer-
ing’, ed. L. B. Wingard, E. Katchalski-Katzir, and L. Goldstein, Academic Press, New York,
28 M. C. Dixon and E. C. Webb, ‘The Enzymes’, Longmans, London, 1979.
29 P. Foster and J. G. Watt, in ‘Methods of Plasma Fractionation’, ed. J. Curling, Academic Press,
New York, 1980, p.17.
5.3 High Molecular Weight Polymers
Other organic precipitants which can be used for the fractionation of
proteins are water-soluble polymers like polyethylene glycol. This has
the advantage of being non-toxic, non-flammable and not denaturing to
proteins. It is mainly used in the blood processing field.
5.4 Heat Precipitation
When a protein is sufficiently robust, heat treatment can provide a high
degree of purification as an initial step. In a large-scale example, 55% of
unwanted proteins were removed in a single step by heating an E. coli
extract containing recombinant Staphylococcal Protein A at 80" for
10 m h 3 ' If the recombinant protein to be purified is from a thermophilic
organism the results of an initial heat treatment can be even more
dramatic: when an E. coli extract containing recombinant malate
dehydrogenase from Thermus aquaticus is heated to 80" for 20 min the
enzyme in the supernatant is about 90% homogene~us.~'
The purification of proteins by chromatography has been a standard
laboratory practice for many years. These same chromatographic
techniques can equally well be applied to the isolation of much larger
quantities of protein, although the order in which they are used must be
For the purification of high value/low volume products, typically
therapeutic or diagnostic proteins, chromatography is the most widely
used method. Chromatography is the only method with the required
selectivity to purify a single protein from a complex mixture of proteins
to a final purity of greater than 95%.
6.1 Scale-up and Quality Management
In analytical chromatography, as well as in many laboratory-scale
applications, the quantity of sample to be applied is small, and the
overall aim is to achieve the maximum number of peaks or to produce a
small amount of highly purified protein. The flow rates used are low, as
'OK.A. Philip, P. M. Hammond, and G. W. Jack, Ann. N . Y. Acad. Sci., 1990,613,863.
3 1 R. M. Alldread, D. J. Nicholls, T. K. Sundaram, M.D. Scawen, and T. Atkinson, Gene, 1992,114,