Note: Descriptions are shown in the official language in which they were submitted.
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1
ELECTROPHORESIS APPARATUS AND METHOD
Technical Field
The present invention relates to an electrophoresis apparatus and method
s suitable for electrophoretically altering the original composition of a
mixture that
contains at least one ampholytic component.
Background Art
When ampholytic compounds, such as amino acids, peptides,
oligopeptides, proteins, and the like are present in a solution at a low
concentration, their charge-state of depends on the pH of their environment.
At a
certain characteristic pH value, the net charge - and consequently, the
electrophoretic mobility - of an ampholytic compound becomes zero. That pH
value is called the p1 value of the ampholytic compound. When two ampholytic
15 compounds have different p1 values, their net charge becomes zero at
different
pH values. Thus, if a pH gradient is established in an electric field, the~two
ampholytic species achieve zero net charge at different points of the pH
gradient
and can result in their separation. Such separations are called isoelectric
focusing (IEF) separations. IEF separations have been achieved in (i)
artificial
2o pH gradients created from non-amphoteric buffers either at constant or
spatially
varying temperatures, (ii) natural pH gradients created from carrier
ampholytes or
from the very components of the mixture to be separated (autofocusing), and
(iii)
immobilized pH gradients. IEF separations typically rely on anti-connective
means to preserve the stability of the pH gradient. The IEF principle has been
25 utilized for both analytical and preparative-scale separation of both
simple and
complex mixtures of ampholytic components. IEF separations have been
obtained in thin-layer format, column-format and in multi-compartment format,
in
both static and flowing media. In flowing media, separations have been
achieved
in both straight-through and recycling format. IEF separations often take
so considerable time because the electrophoretic mobility of each ampholytic
species becomes low as they approach the point in the pH gradient where they
become isoelectric. Therefore, there is a need for IEF separation schemes and
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equipment which (i) minimize the distance the components have to migrate
electrophoretically to achieve separation, (ii) maximize the electric field
strength
that brings about the electrophoretic separation without causing detrimental
heating effects, (iii) maximize the production rate that can be achieved in
unit
separation space and time, and (iv) minimize the use of auxiliary agents
needed
for the electrophoretic separation.
The present invention provides an apparatus and method which can
electrophoretically alter the original composition of a mixture that contains
at least
one ampholytic component.
Disclosure of Invention
In a first aspect, the present invention provides an electrophoresis
apparatus comprising:-
(a) a first electrolyte reservoir a,nd a second electrolyte reservoir;
(b) a first sample reservoir and a second sample reservoir;
(c) a separation unithaving a first electrolyte chamber in fluid connection
with
the first electrolyte reservoir, a second electrolyte chamber in fluid
connection
with the second electrolyte reservoir, a first sample chamber positioned
between
the first electrolyte chamber and the second electrolyte chamber, a second
2o sample chamber positioned adjacent to the first sample chamber and between
the first electrolyte chamber and the second electrolyte chamber, the first
sample
chamber being in fluid connection with the first sample reservoir, and the
second
sample chamber being in fluid connection with the second sample reservoir;
(d) a first ion-permeable barrier positioned between the first sample chamber
and the second sample chamber, the first ion-permeable barrier prevents
substantial convective mixing of contents of the first and second sample
chambers;
(e) a second ion-permeable barrier positioned between the first electrolyte
chamber and the first sample chamber, the second ion-permeable barrier
3o prevents substantial convective mixing of contents of the first electrolyte
chamber
and the first sample chamber;
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(f) a third ion-permeable barrier positioned between the second sample
chamber and the second electrolyte chamber, the third ion-permeable barrier
prevents substantial convective mixing of contents of.the second electrolyte
chamber and the second sample chamber;
(g) electrodes positioned in the first and second electrolyte chambers;
(h) means for supplying electrolyte from the first electrolyte reservoir to
the first
electrolyte chamber, and from the second electrolyte reservoir to the second
electrolyte chamber; and
(i) means for supplying sample or liquid from at least the first sample
reservoir
1o to the first sample chamber, or from the second sample reservoir to the
second
sample chamber.
In one preferred form, the first ion-permeable barrier is a membrane having
a characteristic average pore size and pore size distribution. In one form,
all the
ion-permeable barriers are membranes 'having a characteristic average pore
size
15 and pore size distribution. This configuration of the apparatus is suitable
for
separating compounds on the basis of charge and or size. .
In another preferred form, the first ion-permeable barrier is an isoelectric
membrane having a characteristic p1 value. Preferably, the isoelectric
membrane
has a p1 value in a range of about 2 to 12.
2o In another preferred form, the second and third ion-permeable barriers are
membranes having a characteristic average pore size and pore-size
distribution.
In another preferred form, at least one of the second or third ion-permeable
barriers is an isoelectric membrane having a characteristic p1 value.
Preferably,
the at least one isoelectric membrane has a p1 value in a range of about 2 to
12.
2s In another preferred form, both the second and third ion-permeable barriers
are isoelectric membranes each having a. characteristic p1 value. Preferably,
the
isoelectric membranes have a p1 value in a range of about 2 to 12. When both
the second and third ion-permeable barriers are isoelectric membranes, the
membranes can have the same or different characteristic p1 values.
so The isoelectric membranes are preferably Immobiline polyacrylamide
membranes. It will be appreciated, however, that other isoelectric membranes
would also be suitable for the present invention.
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Suitable isoelectric membranes can be produced by copolymerizing
acrylamide, N,N'-methylene bisacrylamide and appropriate acrylamido
derivatives
of weak electrolytes yielding isoelectric membranes with p1 values in the 2 to
12
range, and average pore sizes that either facilitate or substantially prevent
trans-
membrane transport of components of selected sizes.
In another preferred form, the apparatus comprises:
(h) ' means for circulating electrolyte from each of the first and second
electrolyte reservoirs through the respective first and second electrolyte
chambers forming first and second electrolyte streams in the respective
electrolyte chambers; and
(i) means for circulating contents from each of the first and second sample
reservoirs through the respective first and second sample chambers forming
first
and second sample streams in the respective sample chambers.
Preferably, means (h) and (i) are pump arrangements separately
15 controllable for independent movement of the electrolyte streams and the
sample
streams.
The apparatus may further include:
(j) means for removing and replacing sample in the first or second sample
reservoirs.
2o The apparatus may also further include:
(k) means to maintain temperature of electrolyte and sample solutions.
In another preferred form, the separation unit is provided as a cartridge or
cassette fluidly connected to the electrolyte reservoirs and the sample
reservoirs.
In one preferred form, the separation unit is provided as a cartridge or
25 cassette connected to the electrolyte reservoirs and the sample reservoirs.
The apparatus according to the present invention is suitable for
electrophoresis, isoelectric focusing and electrodialysis of molecules,
particularly
biomolecules including proteins, peptides, glycoproteins, nucleic acid
molecules,
recombinant molecules, metabolites, drugs, neutraceuticals, pharmaceuticals,
3o microorganisms including viruses, bacteria, fungi and yeasts, and prions.
!n use, a sample to be treated is placed in the first and/or second sample
reservoirs and provided to, or circulated through, the first and/or second
chambers. Electrolyte is placed in the first.and second electrolyte reservoirs
and
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passed to, or circulated through, the respective first and second electrolyte
chambers without causing substantial mixing between the electrolyte in the two
electrolyte reservoirs. Electrolyte or other liquid can placed in first and/or
second
sample reservoirs if required. An electric potential is applied to the
electrodes
wherein one or more components in the first and/or second sample chamber are
caused to move through a diffusion barrier to the second and/or first sample
chamber, or to the first and/or second reservoir chambers. Treated sample or
product can be collected in the second and/or first sample reservoir.
In a second aspect, the present invention provides a method for separating
or removing at least one component from a sample by electrophoresis, the
method comprising the steps of:-
(a) providing an electrophoresis apparatus according to the first aspect of
the
present invention;
(b) , adding electrolytes to the first and second electrolyte reservoirs;
(c) , adding sample to at least one of the first and second sample reservoirs;
(d) optionally adding liquid to at least one of the first and second sample
reservoirs;
(e) providing electrolytes from the first and second electrolyte reservoirs to
the
first and second electrolyte chambers;
(f) providing sample or liquid in the first and second sample reservoirs to
the
first and second sample chambers;
(g) applying an electric potential between the electrodes causing at least one
component in the first or second sample chamber to move through the first ion-
permeable barrier into the other of the first or second sample chamber, or
through
at least one of the first and second electrolyte chambers, wherein no
substantial
connective mixing occurs between the electrolytes in the first and second
electrolyte chambers and the sample or liquid in the first and second sample
chambers.
Preferably, at least one sample component has a p1 value.
3o fn a preferred form, electrolyte from at least one of the first and second
electrolyte reservoirs is circulated through the first or second electrolyte
chamber
forming a first or second electrolyte stream.
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The choice of electrolye in the first and second electrolyte chambers will
depend on the p1 of compound or compounds to be treated, separated or
transferred from a sample chamber to the other sample chamber, or one or both
of the electrolye chambers, Similarly, the choice of the p1 of the isoelectric
membranes) will also depend on the p1 of compound or compounds to be
treated, separated or transferred form a given sample.
Electrolyes such as acetic acid or 5-amino caproic acid adjusted to desired
pH with HCI as the anolyte, and triethanol amine or morpholinopropanesulfonic
acid adjusted to desired pH with NaOH as the catholyte have been found to be
suitable for the separation of a. number of components from biological
samples.
Salt such as NaCI may also be added to the electrolyte to assist in the
transfer. It
will be appreciated, however, that other electrolytes would also be
applicable,
depending on the desired separation or treatment.
In another preferred form, electrolyte from both the first and second
~5 electrolyte reservoirs is circulated through the first and second
electrolyte
chambers forming first and second electrolyte streams.
In another preferred form, content of the first or second sample reservoir is
circulated through the first or second sample chamber forming a first or
second
sample stream through the first or second sample chamber.
2o In another preferred form, content of both the first and second sample
reservoirs are circulated through the first and second sample chambers forming
first and second sample streams through the first and second sample chambers.
In another preferred form, sample or liquid in the first or second sample
reservoir is removed and replaced with fresh sample or liquid.
25 Preferably, substantially all trans-barrier migration occurs upon the
application of the electric potential.
In another preferred form. step (g) is maintained until at least one desired
component reaches a desired purity level in the first or second sample chamber
or in the first or second sample reservoirs.
In a third aspect, the present invcention provides an electrophoresis
separation unit comprising:
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(a) a first electrolyte chamber;
(b) a second electrolyte chamber;
(c) a first sample chamber positioned between the first electrolyte chamber
and the second electrolyte chamber;
s (d) a second sample chamber positioned adjacent to the first sample chamber
and between the first electrolyte chamber and the second electrolyte chamber;
(e) an isoelectric membrane having a characteristic p1 value positioned
between the first and second sample chambers, the membrane prevents
substantial connective mixing of contents of the first and second sample
chambers;
(f) a first ion-permeable barrier positioned between the first electrolyte
chamber and the first sample chamber, the first ion-permeable barrier prevents
substantial connective mixing of contents of the first electrolyte chamber and
the
first sample chamber;
(g) a second ion-permeable barrier positioned between the second sample
chamber and the second electrolyte chamber, the second ion-permeable barrier
prevents substantial connective mixing of contents of the second electrolyte
chamber and the second sample chamber; and
(h) electrodes positioned in the first and second electrolyte chambers.
2o Preferably, the isoelectric membrane has a p1 value in a range of about 2
to 12.
In a preferred form, the first and second ion-permeable barriers are
membranes having a characteristic average pore sizes and pore-size
distributions.
2s In another preferred form, at least one of the first or second ion-
permeable
barriers is an isoelectric membrane having a characteristic p1 value.
Preferably,
the isoelectric membrane has a p1 value in a range of about 2 to 12.
In another preferred form, both the first and second ion-permeable barriers
are isoelectric membranes each having a characteristic p1 value. preferably,
3o each of the isoelectric membranes have a p1 value in a range of about 2 to
12.
Each isoelectric membrane can have the same or different characteristic p1
value.
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fn a fourth aspect, the present invention provides a method for separating
or removing at least one component from a sample by electrophoresis, the
method comprising the steps of:
(a) providing an electrophoresis unit according to the third aspect of the
present invention;
(b) adding electrolytes to the first and second electrolyte chambers;
(c) adding sample to at least one of the first and second sample chambers;
(d) optionally adding liquid to at least one of the first and second sample
chambers;
(e) applying an electric potential between the electrodes causing at least one
component in the first or second sample chamber to move through the
isoelectric
membrane into the other of the first or second sample chamber, or through at
least one of the ion-permeable barriers separating the first and second
electrolyte
chambers and first and second sample chambers, wherein no substantial
connective mixing occurs between electrolytes in the first and second
electrolyte
chambers and the sample or liquid in the first and second sample chambers.
Preferably, substantially all traps-membrane or traps-barrier migration
occurs upon the application of the electric potential.
Preferably, at least one sample component has a p1 value.
2o In a preferred form, step (e) is maintained until at least one desired
component reaches a desired purity level in the first or second sample
chamber.
In a fifth aspect, the present invention provides use of the apparatus
according the first aspect of the present invention to alter composition of a
sample containing at least one compound.
In a sixth aspect, the present invention provides a product obtained by the
method according to, the second aspect of the present invention.
In a seventh aspect, the present invention provides use of the
electrophoresis unit according the third aspect of the present invention to
alter
composition of the sample containing at least one compound.
in a eighth aspect, the present invention provides a product obtained by
the method according to the fourth aspect of the present invention.
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An advantage of the present invention is that the apparatus and method
can effectively and efficiently process and separate charged molecules and
other
components in samples.
Another advantage of the present invention is that the apparatus and
method have scale-up capabilities, increased separation speed, lower cost of
operation, increased safety considerations, lower power requirements, and
increased ease of use.
Yet another advantage of the present invention is that the apparatus and
method have improved yields of the separation component, minimal or no
inactivation of the separation component or other components in the sample,
and
improved purity of the separated component.
These and other advantages will be apparent to one skilled in the art upon
reading and understanding the specification.
Throughout this specification, unless the context requires otherwise, the
15 word "comprise", or variations such as "comprises" or "comprising", will be
understood to imply the.inclusion of a stated element, integer or step, or
group of
elements, integers or steps, but not. the exclusion of any other element,
integer or
step, or group of elements, integers or steps.
Any discussion of documents, acts, materials, devices, articles or the like
2o which has been included in the present specification is solely for the
purpose of
providing a context for the present invention. It is not to be taken as an
admission that any or all of these matters form part of the prior art base or
were
common general knowledge in the field relevant to the present invention as it
existed in Australia before the priority date of each claim of this
application.
2s In order that the present invention may be more clearly understood,
preferred forms will be described with reference to the following drawings and
examples.
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Brief Description of the Drawings
Figure 1 is a schematic diagram of a separation unit for use in the present
invention.
.Figure 2 is a schematic diagram of an apparatus according to the present
5 invention utilizing the separation unit of Figure 1.
Figure 3 is an exploded view of a cartridge which may be used with the
separation unit of Figure 1.
Figure 4A is a plan view of a grid element which may be incorporated as a
component of a cartridge of a separation~unit.
Figure 4B is a reverse plan view of the grid element of Figure 4A.
Figure 5 is a cross-sectional view on the lines X-X of Figure 4A.
Figure 6 is a cross-sectional view on the lines XI-XI of Figure 4A.
Figure 7 is a cross-sectional view on the lines XII-XII of Figure 4A.
Figure 8 is a plan view of an alternative embodiment of a grid element
which may be incorporated as a component of a cartridge of a separation unit
. Figure 9 shows an apparatus utilizing the separation unit of Figure 1.
Figure 10 shows the image, at 280 nm, of the protein bands separated by
the iCE280 full-column-imaging capillary isoelectric focusing instrument, from
a
chicken egg-white sample, used as the starting material for the
electrophoretic
2o separation experiments described in Examples 1 to 3. The peaks labelled p1
3.52
and p1 9.61 correspond to dansyl phenylalanine and terbutaline, respectively,
used as isoelectric point markers. The egg-white sample was diluted 1:25 with
deionized water and filtered prior to analysis. Analysis conditions:
instrument:
iCE280 full-column imaging capillary IEF system, separation capillary: 5 cm
long,
100 micrometer I.D. fused silica, focusing medium: 8% carrier ampholytes for
pH
3 -10 in aqueous 0.1 % methylcellulose solution, focusing time: 5 minutes,
applied potential: 3,000 V.
Figure 11 shows the image, at 280 nm, of the protein bands separated by
the iCE280 full-column-imaging capillary isoelectric focusing instrument, from
so aliquots collected at the end of the experiment from the first sample
reservoir
(bottom panel) and second sample reservoir (top panel) of the electrophoretic
apparatus disclosed here and described in Example 1. Separation conditions:
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anolyte: 60 mL 80 mM acetic acid, pH = 2.9, catholyte: 60 mL 8 mM triethanol
amine, pH = 9.9, sample: 60 mL aqueous egg-white sample diluted 1:25 in
deionized water, separation time: 15 minutes, applied potential: 250 V, first
ion-
permeable barrier between the first electrolyte chamber and the first sample
chamber: p1 = 4.0 isoelectric membrane, second ion-permeable barrier between
the first sample chamber and the second sample chamber: p1 = 5.0 isoelectric
membrane, third ion-permeable barrier between the second sample chamber and
the second electrolyte chamber: p1 = 7.0 isoelectric membrane. The peaks
labelled p1 3.52 and p1 9.61 correspond to dansyl phenylalanine and
terbutaline,
1o respectively, used as isoelectric point markers. Analysis conditions:
instrument:
iCE280 full-column imaging IEF system, capillary: 5 cm long, 100 micrometer
I.D.
fused silica, focusing medium: 8% carrier ampholytes for pH 3 -10 in aqueous
0.1 % methylcellulose solution, focusing time: 5 minutes, applied potential:
3,000
V.
Figure 12 shows the image, at 280 nm, of the protein bands separated by
the iCE280 full-column-imaging capillary isoelectric focusing instrument, from
aliquots collected at the end of the experimenfi described in Example 2 from
the .
first sample reservoir (bottom panel) and the second sample reservoir (top
panel)
of the electrophoretic apparatus disclosed here. Separation conditions:
anolyte:
60 mL 2 mM acetic acid, catholyte: 60 mL 8 mM triethanol amine, sample: 60 mL
aqueous egg-white sample diluted 1:25 in distilled water, separation time: 15
minutes, applied potential: 250 V, first ion-permeable barrier between the
first
electrolyte chamber and the first sample chamber: polyacrylamide membrane
with a nominal molecular mass cut-off of 5,000 dalton, second ion-permeable
barrier between the first sample chamber and the second sample chamber: p1 =
5.0 isoelectric membrane, third ion-permeable barrier between the second
sample chamber and the second electrolyte chamber: polyacrylamide membrane
with a nominal molecular mass cut-off of 5,000 dalton. The peaks labelled p1
3.52 and p1 9.61 correspond to dansyl phenylalanine and terbutaline,
3o respectively, used as isoelectric point markers. Analysis conditions:
instrument:
iCE280 full-column imaging IEF system, capillary: 5 cm long, 100 micrometer
I.D.
fused silica, focusing medium: 8% carrier ampholytes for pH 3 -10 in aqueous
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0.1 % methylcellulose solution, focusing time: 5 minutes, applied potential:
3,000
V.
Figure 13 shows the image, at 280 nm, of the protein bands separated by
the iCE280 full-column-imaging capillary isoelectric focusing instrument, from
aliquots collected at the end of the experiment described in Example 3 from
the
first sample reservoir (bottom panel) and the second sample reservoir (top
panel)
of the electrophoretic apparatus disclosed here. Separation conditions:
anolyte:
60 mL 2 mM acetic acid, catholyte: 60 mL 8 mM triethanol amine, sample: 60 mL
aqueous egg-white sample diluted 1:25 in deionized water, separation time: 15
minutes, applied potential: 250 V, first ion-permeable barrier between the
first
electrolyte chamber and the first sample chamber: polyacrylamide membrane
with a nominal molecular mass cut-off of 1,000,000 dalton, second ion-
permeable
barrier between the first sample chamber and the second sample chamber: p1 =
5.0 isoelectric membrane, third ion-permeable barrier between the second
sample chamber and the second electrolyte chamber: polyacrylamide membrane
with a nominal molecular mass cut-off of 1,000,000 dalton. The peak labelled
p1
3.52 corresponds to dansyl phenylalanine, used as isoelectric point marker.
Analysis conditions: instrument: iCE280 full-column imaging IEF system,
capillary: 5 cm long, 100 micrometer I.D. fused silica, focusing medium: 8%
2o carrier ampholytes for pH 3 -10 in aqueous 0.1 % methylcellulose solution,
focusing time: 5 minutes, applied potential: 3,000 V.
Figure 14 shows the image of an SDS-PAGE gel used to analyze the
protein bands present in the aliquots collected from the first and second
sample
reservoirs of the electrophoretic apparatus disclosed here and described in
Example 4 during the isolation of IgG from human plasma. Molecular weight
markers (Sigma, St. Loius, MO, USA) were applied onto Lane 1, a
pharmaceutical-grade IgG preparation used as reference material onto Lane 2.
Samples taken at 0, 10, 20, and 40 minutes respectively from the first sample
reservoir were applied onto Lanes 3, 4, 5, and 6. Samples taken at 0, 10, 20,
and
40 minutes respectively from the second sample reservoir were applied onto
Lanes 7; 8, 9, and 10. Separation conditions: anolyte: 2 L 2 mM 5-aminocaproic
acid adjusted to pH 4.8 with HCI, also containing 5 mM NaCI, catholyte: 2 L 2
mM
MOPSO adjusted to pH 6.8 with NaOH, also containing 5 mM NaCI, sample: 10
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93
mL human plasma sample diluted 1 to 3 with deionized.water, separation time:
40
minutes, applied potential: 250 V, first ion-permeable barrier between the
first
electrolyte chamber and the first sample chamber: polyacrylamide membrane
with a nominal molecular mass cut-off of 150,000 dalton, second ion-permeable
barrier between the first sample chamber and the second sample chamber: p1 =
5.8 isoelectric membrane, third ion-permeable barrier between the second
sample.chamber and the second electrolyte chamber: polyacrylamide membrane
with a nominal molecular mass cut-off of 150,000 dalton.
Modes for Carrying Out the Invention
Before describing the preferred embodiments in detail, the principal of
operation of the apparatus will first be described. An electric field or
potential
applied to ions in solution will cause the ions to move toward one of the
electrodes. If the ion has a positive charge, it will move toward the negative
~ 5 electrode (cathode). Conversely, a negatively-charged ion will move toward
the
positive electrode (anode).
In the apparatus of the present invention, ion-permeable barriers that
substantially prevent connective mixing between the adjacent chambers of the
apparatus or unit are placed in an electric field and components of the sample
are
2o selectively transported through the barriers. The particular ion-permeable
barriers used will vary for different applications and generally have
characteristic
average pore sizes and pore size distributions and/or isoelectric points
allowing
or substantially preventing passage of different components.
Having outlined some of the principles of operation of an apparatus in
25 accordance with the present invention, an apparatus itself will be
described.
Referring to Figure 1, a schematic representation of separation unit 2 is
shown for the purpose of illustrating the general functionality of a
separation
device utilizing the technology of the present invention. Separation unit 2
comprises first electrolyte inlet 4, and second electrolyte inlet 6, first
sample inlet
so 8, and second sample inlet 10, first electrolyte outlet 12, and second
electrolyte
outlet 14, and first sample outlet 16 and second sample outlet 18. Between
first
electrolyte inlet 4 and first outlet 12 is first electrolyte chamber 22.
Likewise,
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between second electrolyte inlet 6 and second electrolyte outlet 14 is second
electrolyte chamber 24. First sample and second sample inlets and outlets also
have connecting chambers. First sample chamber 26 running adjacent to first
electrolyte chamber 22 connects first sample inlet 8 to first sample outlet
16.
Similarly, second sample chamber 28 running adjacent to second electrolyte
chamber 24 connects second sample inlet 10 to second sample.outlet 18. lon-
permeable barriers 30 and 32 separate electrolyte chambers 22 and 24 from
first
sample and second sample~chambers 26 and 28, respectively. Between first
sample and second sample chambers 26 and 28 is ion-permeable barrier 34. In
one embodiment, when in use, first and second electrolyte 36 and 38 occupy
first
and second electrolyte chambers 22 and 24. It should be understood that during
operation, first and second electrolyte 36 and 38, as well as first and second
sample 56 and 66 may be stagnarit in, or flow through, the respective
chambers.
A schematic diagram of an apparatus utilizing separation unit 2 of Figure 1
, is shown in Figure 2 for the purpose of illustrating the general
functionality of.an
apparatus utilizing the technology of the present invention. In this purely
illustrative example, four chambers (first electrolyte chamber 22, second
electrolyte chamber 24, first sample chamber 26, and second sample chamber
28) are connected to four flow circuits. First electrolyte flow circuit 40
comprises
2o first electrolyte reservoir 42, electrolyte tubing 44, and electrolyte pump
46.
Second electrolyte flow circuit 41 comprises second electrolyte reservoir 43,
electrolyte tubing 45, and electrolyte pump 47. In the configuration shown in
Figure 2, electrolyte flow circuits 40 and 41 are running independently from
each
other so that the composition, temperature, flow rate and volume of first
electrolyte 36 and second electrolyte 38 can be suitably adjusted
independently
of one another. .
In the embodiment shown, first electrolyte 36 flows from first electrolyte
reservoir 42 through tubing 44 to pump 46 to first electrolyte chamber 22.
Second electrolyte 24 flows from second electrolyte reservoir 43 through
tubing
45 to pump 47 to second electrolyte chamber 24. First electrolyte 36 flows
through inlet 4 and second electrolyte 38 flows through inlet 6. First
electrolyte
36 exits separation unit 2 through outlet 12 and second electrolyte 38 exits
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separation unit 2 through outlet 14. After exiting separation unit 2,
electrolytes 36
and 38 flow through tubing 44 and 45 back into respective electrolyte
reservoirs
42 and 43. In one embodiment, electrolytes 36 and 38 are held stagnant in
electrolyte chambers 22 and 24 during separation. Electrolytes 36 and 38 can
also act as a cooling medium and help prevent a build up of gases generated
during electrophoresis.
First sample flow circuit 48 contains first sample reservoir 50, tubing 52
and pump 54. First sample 56 flows.from first sample reservoir 50 through
tubing.
52 to pump 54, then through inlet 8 info first sample chamber 26. In one
embodiment, the flow directions of first sample 56 and electrolytes 36 and 38
in
first sample chamber 26 are opposite. First sample 56 exits separation unit 2
at
outlet 16 and flows through tubing 52, then heat exchanger 68 that passes
through second electrolyte reservoir 43 before returning to first sample
reservoir
50 through tubing 52. In an alternative embodiment, heat exchanger 68 passes
15 through first electrolyte reservoir 42. In another embodiment, the flow
directions
of first sample 56 and electrolytes 36 and 38 in first sample chamber 26 are
the
same.
In addition to components of interest, first sample 56 may contain any
suitable electrolyte or additive known in the art~as demanded by the
procedure,
2o application, or separation being performed to substantially prevent or
cause
migration of selected components through the ion-permeable barriers. In a
preferred embodiment, sample from which constituents are removed is placed
into first sample reservoir 50. However, it is understood that in an
alternative
embodiment, sample from which constituents are removed is placed into second
sample reservoir 60.
Similarly, second sample flow circuit 58 contains second sample reservoir
60, tubing 62 and pump 64. Second sample 66 flows from second sample
reservoir 60 through tubing 62 to pump 64, then through inlet 10 into second
sample chamber 28. In one embodiment, the flow directions of second sample
66 and electrolytes 36 and 38 in second sample chamber 28 are opposite.
Second sample 66 exits separation unit 2 at outlet 18 and flows through tubing
62, then heat exchanger 70 that passes through second electrolyte reservoir 43
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before returning to second sample reservoir 60 through tubing 62. In an
alternative embodiment, heat exchanger 70 passes through first electrolyte
reservoir 43.
Second sample 66 may contain any suitable electrolyte or additive known
in the art as demanded by the procedure, application, or separation being
performed to substantially prevent or cause migration of selected components
through the ion-permeable barriers. In a preferred embodiment, sample from
which constituents are removed is placed into second sample reservoir 60.
However, it is understood that in an alternative embodiment, sample from which
constituents are removed is placed into first sample reservoir 50.
Individually adjustable flow rates of first sample, second sample, first
electrolyte and second electrolyte, when employed, can have a significant
. influence on the separation. Flow rates ranging from zero through several
milliliters per minute to several liters per minute are suitable depending on
the
~5 configuration of the apparatus and the composition, amount and volume of
sample processed. In a laboratory scale instrument, individually adjustable
flow
rates ranging from about 0 mlJminute to about 50,000 mL/minute are used, with
the preferred flow rates in the 0 mL/min to about 1,000 mL/minute range.
However, higher flow rates are also possible, depending on the pumping means
2o and size of the apparatus. Selection of the individually adjustable flow
rates is
dependent on the process, the component or components to be transferred,
efficiency of transfer, and coupling of the process with other, preceding or
following processes.
Preferably, all tubing 44, 52, and 62 is peristaltic tubing that is
25 autoclavable, chemically resistant, and biologically inert. One such tubing
is
Masterflex~ C-FLEX~ 50 A tubing. Also, pumps 46, 47, 54 and 64 are preferably
peristaltic pumps that are not in contact with electrolytes 36 and 38, and
samples
56 and 66. In the presently preferred embodiment, heat exchangers 68 and 70
are constructed from stainless steel, although other materials known in the
art are
3o suitably used. Preferably, heat exchangers 68 and 70 are autoclavable,
chemically resistant, biologically inert and capable of facilitating heat
exchange.
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Furthermore, it is preferable that first sample flow circuit 48, .second
sample flow circuit 58, first electrolyte flow circuit 40 and second
electrolyte flow
circuit 41 are completely enclosed to prevent contamination or cross-
contamination. In a preferred embodiment, reservoirs 50, 60, and 70 are
s completely and individually enclosed from the rest of the apparatus.
The separation unit further comprises electrodes 88a and 88b. Preferably,
the respective electrodes are located in the first and second electrolyte
chambers
and are separated from the first and second sample chambers by ion-permeable
barriers.
1o Electrodes 88a and 88b are suitably standard electrodes or preferably are
formed from platinum coated titanium expanded mesh, providing favourable
mechanical properkies, even distribution of the electric field, long service
life and
cost efficiency. Electrodes 88a and 88b are preferably located relatively
close to
ion-permeable barriers 30 and 32 providing better utilization of the applied
15 potential and diminished heat generation. A distance of about 0.1 to 6 mm
has
been found to be suitable for a laboratory scale apparatus. For scale up
versions, the distance will depend on the number and type of ion-permeable
barriers, and the size and volume of the electrolyte and sample chambers.
Preferred distances would be in the order of about 0.1 mm to about 10 mm.
2o Separation unit 2 also preferably comprises electrode connectors 78 that
are used for connecting separation unit 2 to power supply 72. Preferably,
power
supply 72 is external to separation unit 2, however, separation unit 2 is
configurable to accept internal power supply 72. Electrode connectors 78 are
preferably autoclavable.
25 Separation is achieved when an electric potential is applied to separation
unit 2. Selection of the electric field strength (potential) varies depending
on the
separation. Typically, the electric field strength varies between 1 V/cm to
about
5,000 V/cm, preferably between 10 V/cm to 2,000 V/cm and leads to currents of
up to about 1 A. It is preferable to maintain the total power consumption of
the
3o unit at the minimum, commensurable~with the desired separation and
production
rate.
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In one embodiment, the applied electric potential is periodically stopped
and reversed to cause movement of components that have entered the ion-
permeable barriers back into at least one of the fluid streams, while
substantially
not causing re-entry of any components that have entered other fluid streams.
In
s another embodiment, a resting period is utilized. Resting (a period during
which
fluid flows are.maintained but no electric potential is applied) is an
optional step
that suitably replaces or is included after an optional reversal of the
electric
potential. Resting is often used for protein-containing samples as an
alternative
to reversing the potential.
1o Separation unit 2 is suitably cooled by various methods known in the art
such as ice bricks or cooling coils (external apparatus) placed in one or both
electrolyte reservoirs 42 and 43, or any other suitable means capable of
controlling the temperature of electrolytes 36 and 38. Because both first
sample
flow circuit 48 and second sample flow circuit 58 pass through either
electrolyte
15 . reservoir 42 or 43, heat is exchanged between first and second samples
and one
or both of first and second electrolytes. Heat exchange tends to maintain the
temperature in first sample 56 and second sample 66 at the preferred, usually
low
levels.
In another form, there is provided an electrophoresis unit that comprises
2o four chambers (first electrolyte chamber 22, second electrolyte chamber 24,
first
sample 'chamber 26, and second sample chamber 28). Ion-permeable barriers
30 and 32 separate electrolyte chambers 22 and 24 from first sample and second
sample chambers 26 and 28, respectively. Between first sample and second
sample chambers 26 and 28 is ion-permeable barrier 34. Electrodes are housed
2s in the first and second electrolyte chambers and sample and/or fluid is
placed first
sample chamber 26 and second sample chamber 28. In use, an electric potential
is placed between the electrodes and one or more components in the first
sample
chamber 26 or second sample chamber 28 are caused to move to the other
sample chamber or to one of the electrolyte chambers.
30 ' Figure 3 is an exploded view of cartridge 100 which is preferably a
modular component of separation unit 2. When configured as a modular unit,
cartridge 100 preferably comprises housing 102 for holding in place or
encasing
the component parts of cartridge 100. In a presently preferred embodiment,
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cartridge 100 is generally elongated and has side walls 104 which are
generally
parallel to one another and the longitudinal axis A of cartridge 100. The
cartridge
is suitably generally octagonal, hexagonal, or ovular. In an octagonal
configuration, cartridge 100 has three end walls 106 on each side of side
walls
104 forming an octagon. However, two end walls on each side 106 are suitably
used to form a hexagon, or one curved end wall 106 on each side is suitably
used
to form a generally ovular shape. Furthermore, end walls 106 are suitably
either
straight or generally curved.
Extending around the base of side walls 104 and end walls 106 is a small
9o flange 108 that is generally perpendicular to side walls 104 and end walls
106
and projects inward toward the center of cartridge 100. Along the exterior of
either side walls 104 or end walls 106 is preferably a handle 110 to
facilitate
placement of cartridge 100~into separation unit 2. Flange 108 is preferably
configured to interact with lower gasket 112. In a preferred embodiment,.
lower .
gasket 112 is generally planar and configured to fit inside walls 104 and 106
of
cartridge 100. In a presently preferred embodiment, lower gasket 112.is made
from silicon rubber. Lower gasket 112 may be configured so that it has an .
aperture 114 extending in an elongated manner through the center of lower
gasket 112. Also extending through and adjacent each end of lower gasket 112
2o are alignment holes 116. fn a preferred embodiment, alignment holes 116 are
circular, forming generally cylindrical channels through lower gasket 112.
However, it is also contemplated that alignment holes 116 are suitably
triangular,
square, rectangular, hexagonal, octagonal, or similarly shaped.
Above lower gasket 112 is a generally planar lower ion-permeable barrier
z5 32. The external shape of ion-permeable barrier 32 is generally the same as
that
of lower gasket 112 and the interior of cartridge 100 so that ion-permeable
barrier
32 is configured to fit inside cartridge 100. Like lower gasket 112, ion-
permeable
barrier 32 preferably has two alignment holes of the same location and
configuration as alignment holes 116 in lower gasket 112. ion-permeable
barrier
30 32 substantially prevents convective mixing of the contents of first
electrolyte
chamber 22 and first sample chamber 26, while permits selective trans-barrier
transport of selected constituents upon application of the electric potential.
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In one embodiment, ion-permeable barrier 32 is formed from a membrane
with a characteristic average pore size and pore-size distribution. The
average
pore size and pore size distribution of the membrane is selected to facilitate
trans-membrane transport of certain constituents, while substantially
preventing
s trans-membrane transport of other constituents.
In another embodiment, ion-permeable barrier 32 is an isoelectric ion-
permeable barrier, such as an isoelectric membrane that substantially prevents
connective mixing of the contents of first electrolyte chamber 22 and first
sample
chamber 26, while permits selective trans-barrier transport of selected
1 o constituents upon application of the electric potential. Suitable
isoelectric .
membranes can be produced by copolymerizing acrylamide, N,N'-methylene
bisacrylamide and appropriate acrylamido derivatives of weak electrolytes
yielding isoelectric membranes with p1 values in the 2 to 12 range, and
average
pore sizes that either facilitate or substantially prevent trans-membrane
transport '
~5 of components of selected sizes.
Above lower ion-permeable barrier 32 is lower grid element 118 that is
generally planar and also shaped like lower gasket 112 and the interior of
cartridge 100 so that lower grid element 118 is configured to fit inside
cartridge
100. One of the functions of tower grid element 118 is to separate lower~ion-
2o permeable barrier 32 from ion-permeable barrier 34. Another function of
lower
grid element 118 is to provide a flow path for first sample 56. Like lower ion-
permeable barrier 32 and lower gasket 112, lower grid element 118 suitably
also
has alignment holes 116.
Above lower grid element 118 is generally planar ion-permeable barrier 34.
The external shape of ion-permeable barrier 34 is generally the same as that
of
lower gasket 112 and the interior of cartridge 100 so that ion-permeable
barrier
34 is configured to fit inside cartridge 100. Ion-permeable barrier 34
substantially
prevents connective mixing of the contents of first sample chamber 26 and .
second sample chamber 28, while permits selective trans-barrier transport of
3o selected constituents upon application of the electric potential.
In one embodiment, ion-permeable barrier 34 is formed from a membrane
with a characteristic average pore size and pore-size distribufiion. The
average
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21
pore size and pore size distribution of the membrane is selected to facilitate
trans-membrane transport of certain constituents, while substantially
preventing
trans-membrane transport of other constituents.
In another embodiment, ion-permeable barrier 34 is an isoelectric ion-
s permeable barrier, such as an isoelectric membrane that substantially
prevents
convective mixing of the contents of first sample chamber 26 and second sample
chamber 28, while permits selective trans-barrier transport of selected
constituents upon application of the electric potential. Suitable isoelectric
membranes can be produced by copolymerizing acrylamide, N,N'-methylene
1o bisacrylamide and appropriate acrylamido derivatives of weak electrolytes
. yielding isoelectric membranes with p1 values in the 2 to 12 range, and
average
pore sizes that either facilitate or substantially prevent trans-membrane
transport
of components of selected sizes.
Above ion-permeable barrier 34 are three upper components: upper grid
~5 element 120, upper ion-permeable barrier 38, and upper gasket 124. These
' three components are placed so that upper grid element 120 is immediately '
above ion-permeable barrier 34, ion-permeable barrier 38 is immediately above
upper grid element 120, and upper gasket 124 is immediately above ion-
permeable barrier 120. The configuration of the three upper components
suitably
2o mirrors that of the lower three components.
Components below ion-permeable barrier 34 having alignment holes 116
' may be connected together with a fastener, which.is any type of connector
configured to interact with alignment holes 116 and facilitate through flow of
first
sample 56. Similarly, components above ion-permeable barrier 34 having
25 alignment holes 116 may be connected together with a fastener, which is any
type of connector configured to interact with alignment holes 116 and
facilitate
through flow of second sample 66.
Components of cartridge 100 are suitably held in cartridge 100 by clip 126.
Clip 126 is suitably snap fitted or glued around the top of walls 104 and 106
of
3o cartridge 100.
Ion-permeable barrier 38 substantially prevents convective mixing of the
contents of second electrolyte chamber 24 and second sample chamber 28, while
.
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permits selective trans-barrier transport of selected constituents upon
application
of the electric potential.
In one embodiment, ion-permeable barrier 38 is formed from a membrane
with a characteristic average pore size and pore-size distribution. The
average
s pore size and pore size distribution of the membrane is selected to
facilitate
trans-membrane transport of certain constituents, while substantially
preventing
trans-membrane transport of other constituents.
In another embodiment, ion-permeable barrier 38 is an isoelectric ion-
permeable barrier, such as an isoelectric membrane that substantially prevents
1o convective mixing of the contents of second electrolyte chamber 24 and
second
sample chamber 28, while permits selective trans-barrier transport of selected
constituents upon application of the electric potential. Suitable isoelectric
membranes can be produced by copolymerizing acrylamide, N,N'-methylene
bisacrylamide and appropriate acrylamido derivatives of weak electrolytes
15 yielding isoelectric membranes with p1 values in the 2 to 12 range,
and.average
pore sizes that facilitate or substantially prevent trans-membrane transport
of
components of selected sizes.
Preferred grid elements 118 and 120 are shown in more detail in Figures 4
to 7. Figure 4A shows a plan view of a preferred grid element which is
2o incorporated as a component of cartridge 100 for separation unit 2. An
elongate
rectangular cut-out portion,128 which incorporates lattice 131 is defined in
the
center of the grid element. At each end of the grid element, an alignment hole
116 is suitably provided for alignment with the other components of cartridge
100.
Preferably, a triangular channel area 130 having sides and a base, extends and
25 diverges from each alignment hole 116 to cut-out portion 128. Upstanding
ribs
132, 134, and 136 (best shown in Figures 6 and 7) are defined in channel area
130. Liquid flowing through hole 116 thus passes along triangular channel area
130 between ribs 132, 134, and 136 and into lattice 131. Ribs 132, 134, and
136
direct the flow of liquid from hole 116 so that they help ensure that liquid
is evenly
so distributed along the cross-section of lattice 131. Ribs 132, 134, and 136
also
provide support to ion-permeable barrier 34 disposed above or below the grid
element. .
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Lattice 131 comprises a first array of spaced parallel members 138
extending at an angle to the longitudinal axis of the grid disposed above and
integrally formed with a second lower set of spaced parallel members 140
extending at approximately twice the angle of the first array of parallel
members
s 138.to the longitudinal axis of the grid. In the presently preferred
embodiment,
the first array of parallel members 138 extend at approximately a 45 degree
angle
from the longitudinal axis and the second array of parallel members 140 extend
at
approximately 90 degrees to the first array of parallel members 138, however,
other angles are also suitably used.
Referring to Figure 4B, the reverse side of the grid element is illustrated.
The reverse side is suitably relatively smooth and flat aside from cut-out
area 128
and alignment holes 116. The smooth, fiat surface fends to ensure sealing
between ion-permeable barriers 32 and grid element 118, and ion-permeable
barrier 38 and grid element 120, respectively.
Referring to Figure 5, the upper and lower surfaces of first and second
parallel members 138 and 140 are preferably rounded. When parallel members
138 and 140 are rounded, the absence of any sharp edges help prevent damage
to ion-permeable barrier 34 and provide extra support. Lattice 131 evenly
distributes the flow of liquid over the surface of ion-permeable barrier 34.
The
2o use of a first set of members 138 disposed above a second set of members
140
tends to ensure that the liquid in a stream is forced to move up and down,
changing direction frequently, which helps to encourage mixing of the liquid
and
tends to inhibit static flow zones.
The thickness of the.grid element is preferably relatively small. In one
2s presently preferred embodiment, exterior areas 144 of the element are 0.8
mm
thick. Sealing rib or ridge 142 (also shown in Figures 4A and 4B) extends
around
the periphery of lattice 131 to improve sealing. Ridge 142 is preferably
approximately 1.2 mm thick measured from one side of the grid element to the
other. The distance between the opposite peaks of lattice elements 138 and 140
so measured from one side of the grid to the other is preferably approximately
1 mm.
The relatively small thickness of the grid provides several advantages. First,
it
results in a more even distribution of liquid over ion-permeable barrier 34
and
assists in inhibiting its fouling by macromolecules.
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Also, the volume of liquid required is decreased by the use of a relatively
thin grid which enables relatively small sample volumes to be used for
laboratory-
scale separations, a significant advantage over prior art separation devices.
Finally, if the electric field strength is maintained constant, the use of a
relatively thinner grid element enables less electrical power to be deposited
into
the liquid.. If less heat is transferred into the liquid, the temperature of
the liquid
remains lower. This is advantageous since high temperatures may destroy both
the sample and the desired product.
Figure 8 illustrates grid element 144 for an alternative embodiment of the
1o present invention. Grid element 144 utilizes an ion-permeable barrier
having a
much larger surface area than that of grid elements 118 and 120. The principal
.
operation of grid element 144 is suitably generally the same as that of the
smaller
grid elements although holes 146 through which first sample 56 or second
sample 66 are fed are located in two opposite corners of grid 144 and there
are
many more channels 148 feeding streams from holes 146 to central portion 150
of grid 144. The cartridge, cartridge casing, and other components are
increased
in size and shape so as_to match that of grid 144.
Figure 9 is a diagram of a presently preferred embodiment of a separation
apparatus 200 for use in accordance with the present invention. The separation
20 . apparatus comprises separation unit 2 configured to accept cartridge 100
and
clamp 86. Clamp 86 is used to fix separation unit 2 in place once a component
cartridge is placed into separation unit 2. In the presently preferred
embodiment,
clamp 86 is constructed from aluminium and is~preferably anodized. Clamp 86 is
preferably a simple screw clamp unit so that a screw-operated knob may be used
2s to open and close clamp 86. The separation apparatus shows first sample
reservoir 50, second sample reservoir 60, and first and second electrolyte
reservoirs 42 and 43 in electrolyte compartment 202.
In order that the present invention may be more clearly understood,
examples of separation methodology is described with reference to the
preferred
3o forms of the separation technology as described.
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Example 1
An apparatus according fio the present invention, shown in Figure 9, was
used to separate the proteins present in chicken egg-white into two fractions.
An
electrophoresis separation cartridge, shown in Figures 3 to 7, was adapted to
be
used in the apparatus. The first ion-permeable barrier placed between the
first
electrolyte chamber and the first sample chamber was a p1 = 4.0 isoelectric
membrane prepared from Immobiline chemicals (Pharmacia, Sweden),
acrylamide and N-N'-methylene bis-acrylamide. The second ion-permeable
barrier placed between the first sample chamber and the second sample
10 chamber was a p1 = 5.0 isoelectric membrane prepared from Immobiline
chemicals (Pharmacia, Sweden); acrylamide and N-N'-methylene bis-acrylamide.
The third ion-permeable barrier placed between the second sample chamber and
the second electrolyte chamber was a p1 = 7.0 isoelectric membrane prepared
from Immobiline chemicals (Pharmacia, Sweden), acrylamide and N-N'-
~ 5 methylene bis-acrylamide. _
The first electrolyte reservoir was filled with 60 mL of an 2 mM acetic acid
solution, pH 2.9. The second electrolyte reservoir was filled with 60 mL of an
8
mM triethanol amine solution, pH 9.9. The first and second sample reservoirs
were filled with 30 mL each of a filtered chicken egg-white solution, diluted
with
2o deionized water at a rate of 1 to 25. The anode was placed into the first
electrolyte chamber, the cathode into the econd. electrolyfie chamber. The
applied potential was 250 V, the separation time was 15 minutes. Aliquots were
taken for analysis from the sample reservoir chambers before separation and at
the end of the separation.
25 Full-column-imaging capillary isoelectric focusing on an iCE280 instrument
(Convergent Bioscience, Toronto, Canada) was used to analyze the egg-white
samples. The fused silica separation capillary was 5 cm long, its internal
diameter was 100 micrometer. The focusing medium contained 8% carrier .
ampholytes to cover the pH 3 -10 range, in an aqueous, 0.1 % methylcellulose
~ solution. Seventy-five microliter of the sample to be analyzed was mixed
with
150 microliter of the focusing medium, filled into the capillary and focused
for 5
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26
minutes at 3,000 V. Dansyl phenylalanine (p1 = 3.52) and terbutaline (p1 =
9.61)
were used as p1 markers.
Figure 10 shows the results for the egg-white feed sample. The peaks
between pixels 650 and 850 correspond to ovalbumin isoforms, those between
pixels~1250 and 1350 correspond to ovotransferrin isoforms.
As a result of the electrophoretic separation, proteins with p1 values lower
than .5.0, such as ovalbumin (p1 = 4.7), accumulated in the first sample
reservoir,
on the anodic side of the p1 = 5.0 isoelectric membrane (bottom panel in
Figure
11). Proteins with p1 values greater than 5.0, such as ovotransferrin (p1 =
6.1)
accumulated in the second sample reservoir, on the cathodic side of the
isoelectric membrane (top panel in Figure 11 ).
Example 2
The same apparatus as in Example 1 was used to separate the proteins
~5 present in chicken egg-white into two fractions. An electrophoresis
separation
cartridge, shown in Figures 3 to 7, was adapted to be used in the apparatus. '
The
first ion-permeable barrier placed between the first electrolyte chamber and
the
first sample chamber was a polyacrylamide membrane with a nominal molecular
mass cut-off of 5,000 dalton. The ion-permeable barrier between the first
sample '
2o chamber and the second sample chamber was a p1 5.0 isoelectric membrane
prepared from Immobiline chemicals (Pharmacia, Sweden), acrylamide and N-N'-
methylene bis-acrylamide as in Example 1. The third ion-permeable barrier
placed between the second sample chamber and the second electrolyte chamber
was a polyacrylamide membrane with a nominal molecular mass cut-off of 5,000
25 dalton.
The~first electrolyte reservoir was filled with 60 mL of a 2 mM acetic acid
solution, pH 3.8. The second electrolyte reservoir was filled with 60 mL of an
8
mM triethanol amine solution, pH 9.9. The first and second sample reservoirs
were filled with 30 mL each of a filtered chicken egg-white solution, diluted
with
3o deionized water at a rate of 1 to 25. The anode was placed into the first
electrolyte chamber, the cathode into the second electrolyte chamber. The
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27
applied potential was 250 V, the separation time was 15 minutes. Aliquots were
taken for analysis from the sample reservoir chambers at the end of the
separation.
Full-column-imaging capillary isoelectric focusing on an iCE280 instrument
s (Convergent Bioscience, Toronto, Canada) was used-to analyze the egg-white
samples. The fused silica separation capillary was 5 cm long, its internal
diameter was 100 micrometer. The focusing medium contained 8% carrier
ampholytes to cover the pH 3-10 range, in an aqueous, 0.1 % methylcellulose
solution. Seventy-five microliter of the sample to be analyzed was mixed with
150 microliter of the focusing medium, filled into the capillary and focused
for 5
minutes at 3,000 V. Dansyl phenylalanine (p1 = 3.52) and terbutaline (p1 =
9.61 )
were used as p1 markers.
As a result of the electrophoretic separation, proteins with p1 values lower
than 5.0, such as ovalbumin (p1 = 4.7), accumulated in the first sample
reservoir,
15 on the anodic side of the p1 = 5.0 isoelectric membrane (bottom panel in
Figure
12). Proteins with p1 values greater than 5.0, such as ovotransferrin (p1 =
6.1)
accumulated in the second sample reservoir, on the cathodic side of the p1 =
5.0
isoelectric membrane (top panel in Figure 12). Neither ovalbumin nor
ovotransferrin were lost into the first or second electrolyte chambers despite
the
2o fact that the first and third ion-permeable barriers were not isoelectric
membranes
as in Example 1. At the end of the separation, the solution pH in the first
and
second sample reservoirs was 4.7 and 6.7, respectively.
Example 3
25 The same apparatus as in Example 1 was used to separate the proteins
present in chicken egg-white into two fractions. The first ion-permeable
barrier
placed between the first electrolyte chamber and the first sample chamber was
a
polyacrylamide membrane with a nominal molecular mass cut-off of 1,000,000
dalton. The ion-permeable barrier between the first sample chamber and the
so second sample chamber was a p1 5.0 isoelectric membrane prepared from
Immobiline chemicals (Pharmacia, Sweden), acrylamide and. N-N'-methylene bis-
acrylamide as in Example 1. The third ion-permeable barrier placed between the
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28
second sample chamber and the second electrolyte chamber was a
polyacrylamide membrane with a nominal rholecular'mass cut-off of "1,000,000
dalton.
The first electrolyte reservoir was filled with 60 mL of a 2 mM acetic acid
solution, pH .3.8. The second electrolyte reservoir was filled with 60 mL of
an 8
mM triethanol amine solution, pH 9.9. The first and second sample reservoirs
were filled with 30 mL each of a filtered chicken egg-white solution, diluted
with
deionized water at a rate of 1 to 25. The anode was placed into the first
electrolyte chamber, the cathode into the second electrolyte chamber. The
applied potential was 250 V, the separation time was 15 minutes. Aliquots were
taken for analysis from the sample reservoir chambers at the end of the
separation, and analyzed by full-column-imaging capillary isoelectric focusing
on
an iCE280 instrument.
As a result of the electrophoretic separation, proteins with p1 valu,es lower
~5 than 5.0, such as ovalbumin (p1 = 4.7), accumulated in the first sample
reservoir,
on the anodic side of the p1 = 5.0 isoelectric membrane (bottom panel in
Figure
13). Proteins with p1 values greater than 5.0, such as ovotransferrin (p1 =
6.1)
accumulated in the second sample reservoir, on the cathodic side of the p1 =
5.0
isoelectric membrane (top panel of Figure 13). Neither ovalbumin nor
20 ovotransferrin were lost into the first or second electrolyte chambers
despite the
fact that the average pore size of the first and third ion-permeable barriers
was
large enough to permit their passage through these barriers. At the end of the
separation, the solution pH in the first and second sample reservoirs was 4.7
and
6.2, respectively. r
Example 4
The same apparatus as in Example 1 was used to purify immunoglobulin
G (IgG) from human plasma. The first ion-permeable barrier placed between the
first electrolyte chamber and the first sample chamber was a polyacrylamide
3o membrane with a nominal molecular mass cut-off of 150,000 dalton. The ion-
permeable barrier between the first sample chamber and the second sample
chamber was a p1 5.8 isoelectric membrane prepared from Immobiline chemicals
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29
(Pharmacia, Sweden), acrylamide and N-N'-methylene bis-acrylamide. The third
ion-permeable barrier placed between the second sample chamber and the
second electrolyte chamber was a polyacrylamide membrane with a nominal
molecular mass cut-off of 150,000 dalton.
s The first electrolyte reservoir was filled with 2 L of a 2 mM 5-amino
caproic
acid solution that also contained 5 mM NaCI, its pH was adjusted to 4.8 with
HCI.
The second electrolyte reservoir was filled with 2 L of a 2 mM MOPSO solution
that also contained 5 mM NaCI, its pH was adjusted to 6.8 with NaOH. The
anode was placed into the first electrolyte chamber, the cathode into the
second
electrolyte chamber. The applied potential was 250 V. Initially, both sample
reservoirs were filled with deionized water. Potential was applied for 2
minutes to
remove any unpolymerized material from the membranes. After 2 minutes, all
reservoirs were emptied, the electrolyte reservoirs were refilled with fresh
electrolytes, the sample reservoirs were filled with 15 mL each of human
plasma
~5 diluted 1 to 3 with deionized water. Potential was applied for 40 minutes.
Aliquots were taken for analysis from the sample reservoir chambers at 0, 10,
20,
and 40 minutes, respectively.
Figure 14 shows the image of an SDS-PAGE gel used to analyze the
protein bands present in the aliquots collected from the first and second
sample
2o reservoirs ,of the preparative-scale isoelectric focusing apparatus during
the
isolation of IgG from human plasma. Molecular weight markers (Sigma, St.
Loins, MO, USA) were applied onto Lane 1, a pharmaceutical-grade IgG
preparation used as reference material onto Lane 2. Samples taken at 0, 10,
20,
and 40 minutes respectively from the first sample reservoir were applied onto
25 Lanes 3, 4, 5, and 6. Samples taken at 0, 10, 20, and 40 minutes
respectively
from the second sample reservoir were applied onto Lanes 7, 8, 9, and 10. IgG
was purified within 40 minutes.
These examples indicate that remarkably rapid separation of ampholytic
components can be achieved using the apparatus and method disclosed here.
30 ~ The high production rates are attributed to the short electrophoretic
migration
distances, high electric field strength and good heat dissipation
characteristics of
the system.
CA 02421450 2003-03-06
WO 02/24314 PCT/AU01/01193
The invention has been described herein by way of example only. It will
be appreciated by persons skilled in the art that numerous variations and/or
modifications may be made to the invention as shown in the specific
embodiments without departing from the spirit or scope of the invention as
broadly described. The present embodiments are, therefore, to be considered in
all respects as illustrative and not restrictive. Other features and aspects
of this
invention will be appreciated by those skilled in the art upon reading and
comprehending this disclosure. Such features, aspects, and expected variations
and modifications of the reported results and examples are clearly within the
scope of the invention where the invention is limited solely by the scope of
the
following claims.
