Apparatus for and method of isoelectric focussing

An apparatus for and a method for separating a mixture of proteins containing proteins of different isoelectric points, which method comprises flowing an aqueous feed stream containing the mixture of proteins along a thin-channel flow path established between first and second gas-permeable membranes, diffusing a gas through the first and second membranes and generally transversely across the flow path of the feed stream, the gas selected to establish a gradient in the concentration of the gas and to provide a gradient in pH across the flow path of the feed stream, and, after a defined time period, separating the feed stream into streams containing concentrated fractions of proteins having defined isoelectric points, by virtue of the difference in the direction of diffusion occurring of the proteins in the flow path.

REFERENCE TO PUBLICATION 
The analysis of steady-state transport of carbon dioxide in thin films of a 
protein solution was made and reported in "The Mechanism of Carbon Dioxide 
Transport in Protein Solution", Paper 96d, at the Annual Convention of the 
American Institute of Chemical Engineers, Los Angeles, Calif., Nov. 14-19, 
1982, hereby incorporated by reference. The bicarbonate formulation of 
carbon-dioxide transport was calculated to be small, but to be significant 
in solutions of hemoglobin, in agreement with literature data. 
Facilitation is shown to be minimized by diffusion potentials, the 
magnitudes of which also agree with reported measurements. It is shown how 
potential gradients engender uneven distributions of the proteins within 
the liquid films. The effect, analogous to isoelectric focusing, is most 
striking in cases of secondary, highly charged proteins which are much 
less concentrated than the primary protein mediating CO.sub.2 transfer. 
BACKGROUND OF THE INVENTION 
Where materials, such as biological materials like proteins, have an 
isoelectric point, the materials can be separated by isoelectric focusing, 
wherein a solution is passed through an electric-potential field and the 
materials in the solution are allowed to gravitate toward the isoelectric 
point of the material in the field. 
For example, when an electrical field is imposed upon a solution containing 
a mixture of proteins, the positively charged proteins migrate towards one 
electrode (the cathode) and the negatively charged proteins migrate in the 
opposite direction towards the other electrode (the anode). The 
characteristic charge of a particular protein, Z, is the net result of the 
hydrogen ion association/dissociation equilibria of the protein's numerous 
titratable groups; for example, --NH.sub.3.sup.+ =--NH.sub.2 +H.sup.+ and 
--COOH=--COO.sup.- +H.sup.+. Hence, Z is a function of pH, proteins are 
amphoteric and there is a characteristic pH value for each protein, pI, 
known as its isoelectric point, at which Z=0. Thus, if in addition to an 
electric field there is a gradient in the solution's pH, each protein can 
be made to migrate to a location between two electrodes at which the 
prevailing pH corresponds to that protein's pI. The vanishing of the 
protein's net charge at that point causes that particular protein species 
to cease migrating. 
In conventional isoelectric focusing technology, the necessary pH gradient 
is created by adding to the protein solution a concentrated mixture of 
relatively low molecular weight buffers known as ampholytes, the pI values 
of which span the range of the proteins to be separated. The solution is 
contained between two electrodes, for example, some 10 cm apart. 
Electrolysis of water caused by the applied voltage generates a pH 
gradient which is stabilized by the relatively rapid isoelectric focusing 
of the ampholytes. This is followed in turn by the isoelectric focusing of 
the slower moving proteins, the concentrations of which are sufficiently 
lower than those of the ampholytes as not to perturb radically the pH 
gradient. This methodology has been applied both as an analytical tool; 
for example, in order to determine a particular proteins's pI value, and 
as a means of separation, leading, with further treatment, to the 
isolation of purified proteins. 
Because of the low mobility of proteins, typically electrical fields of 100 
to 1000 volts/cm are employed, and, even so, long time periods of one or 
more days are often required, in order to accomplish desired degrees of 
separation. Usually, cooling must be provided to offset appreciable 
resistive heat whcih might otherwise denature the proteins. Oxygen 
generated at the anode also may cause denaturation. An inherent 
disadvantage of the conventional methodology is the contamination of the 
proteins by the ampholytes used in the process. 
Therefore, it is desirable to provide a new and improved method for the 
detection and/or separation of biological and other materials by 
isoelectric-focusing techniques, which overcome some of the disadvantages 
of the prior methods. 
SUMMARY OF THE INVENTION 
The invention relates to an apparatus for and a method of detecting and/or 
separating a mixture of materials having a difference in isoelectric 
points. In particular, the invention concerns an apparatus for and a 
method of concentrating particular proteinaceous material from a protein 
solution. 
It has been discovered that a feed stream, composed of a mixture of 
materials, such as an aqueous stream composed of a mixture of 
proteinaceous materials having different isoelectric points, may be 
separated into one or more concentrated streams containing the materials 
or proteins of defined isoelectric points, either for the purposes of 
detection or for separation. The method and apparatus of the invention 
avoid the difficulties associated with prior-art techniques for the 
detection and separation of proteins, such as the avoidance of electrical 
fields of high voltage, the denaturing of the proteins, the contamination 
of the proteins with ampholytes and other disadvantages associated with 
prior-art isoelectric-focusing and protein-separation techniques. The 
method of the invention comprises establishing a gradient in the 
concentration of a particular gas stream generally transversely across the 
path of flow of the feed stream to be separated, the gas stream selected 
to provide, with the feed stream, a gradient in pH conditions extending 
transversely across the flow path, and, thereafter, separating the 
material of different isoelectric points, as the different isoelectric 
material moves through the gradient in different directions into a 
concentrated stream of defined isoelectrical material, thereby providing a 
method for the separation and/or detection of the material so isolated in 
the gradient field. 
Typically, an aqueous solution containing a mixture of proteinaceous 
material having different isoelectric points is flowed through a defined 
flow path, such as a thin-film or other narrow flow channel. The flow path 
of the aqueous solution is defined between a pair of gas-permeable-type 
materials, and a gas stream composed of an acid or alkaline gas is 
disposed transversely to the direction of the flow path, by passing the 
gas stream through the membranes from one to the other side, typically 
with the use of an inert sweep gas on the opposing side, to establish a 
gradient in the concentration of the acid or alkaline gas across the flow 
path of the aqueous solution, as well as an electric potential gradient, 
and causes, by virtue of the directional diffusion of the different 
isoelectric-point material in the aqueous solution, the separation of the 
protein material, by virtue of such difference in pH and electrical 
potential across the flow path. 
Thus, the protein separation is based upon the difference in the diffusion 
direction of the proteins toward their isoelectric points in the aqueous 
solution; that is, the pH values at which particular proteins carry a net 
zero electrical charge. Where the pH in the solution is equal to the pI of 
a given protein, then that protein will not migrate further under the 
influence of an electrical field. In the method, if a mixture of proteins 
in an aqueous solution is contained in a flow path in which the pH varies 
continuously in a particular direction transverse to the flow, an 
electrical field arises as a diffusion potential or is externally imposed 
in the same direction, and the proteins will accumulate in the regions 
where the prevailing pH approaches their respective pI values. 
In the method of and apparatus of the invention, a gaseous concentration 
gradient across the flow path causes, typically by virtue of the chemical 
reaction of the gas with a solvent liquid carrier of the protein solution, 
a parallel gradient in the concentration of the reaction product for the 
gas solvent and an antiparallel gradient in pH. The gradient in 
concentration of reaction product causes that ionic species to diffuse 
across the flow path. The gradient in pH established across the flow path 
causes gradients in the concentrations of the charged protein species and, 
hence, their diffusion across the flow path. A diffusion potential arises 
because of the difference in the mobilities of reaction product and 
protein species. Where the pH in the flow path is equal to a particular 
protein's pI value, that protein's electrically driven migration is halted 
at that transverse position in the flow path. 
In the method of the invention, the proteins are subjected to a gas 
concentration gradient established by the gas diffusion across the flow 
path; thus generating electrical and concentration driving forces for 
their migration. A condition is approached in which the two driving forces 
balance one another, and an effective permanent degree of separation is 
obtained for the mixture of proteins into two fractions, each of which 
fractions has a defined isoelectric point. The basis of separation of the 
proteins in the invention is based on the direction in which the various 
proteins diffuse in the flow path, which diffusion direction depends upon 
the positive or negative charge of the protein and, hence, the particular 
isoelectric point relative to the prevailing pH in the flow stream, which 
distinguishes the protein and dictates into which fraction the protein 
will diffuse. 
The feed stream to be separated typically comprises an aqueous solution or 
colloidal suspension of proteinaceous materials, the materials having a 
different isoelectric point. Such proteinaceous materials may comprise, 
but not be limited to, for example, enzymes, hormones, antibodies, amino 
acids, nucleic acids, albumin and hemoglobin, and other simple and complex 
proteins and other biological and biochemical materials. 
The particular selection of the gas employed to diffuse or to be passed 
into the flow path of the proteinaceous feed stream may vary, but 
typically comprises an acid or alkaline gas stream, which, on reaction 
with the solvent of the liquid carrier stream, such as the water, provides 
for the establishment of a pH gradient. The gases employed may, for 
example, be the acid anhydrides involving sulfur, carbon and nitrogen, 
such as, for example, sulfur dioxide and carbon dioxide, or their actual 
acids and bases, such as, for example, hydrogen sulfide and ammonia. The 
selected gases are employed to diffuse or otherwise to pass through and 
transversely across the flow path of the feed stream. Diffusion generally 
is established between two gas-permeable materials, and often includes an 
inert sweep gas employed on the one opposing side, so as to remove the 
diffused gas from the opposing side of the gas-permeable material, after 
passing across the solution flow path. A wide variety of inert sweep gases 
may be employed, which would include, but not be limited to, nitrogen, 
helium, argon, hydrogen and other inert gases which would not affect the 
establishment of the pH gradient, nor substantially react with the 
particular gas stream used for diffusion or the solution. 
Typically, the flow path, through which the proteinaceous feed stream is 
introduced, includes a gas-permeable membrane-type material which permits 
the introduction of the gas across the flow path of the feed stream. Such 
gas-permeable material may include various materials in various shapes, 
such as fibers, tubes and thin films, both supported and unsupported, 
typically spaced apart, to delimit a defined, narrow flow path, and may 
comprise, for example, gas-permeable cellulose acetate or cellulose 
triacetate-type membranes, as well as other polymeric membranes which may 
be gas-porous in nature or otherwise formed or treated, to permit the 
rapid diffusion of the selection gas therethrough and into the feed stream 
on the opposite side of the membrane. Such membranes would include, for 
example, a product known as Celgard, a polyolefin-type resin containing a 
plurality of micropores therein and being hydrophobic in nature, 
permitting the passage of gas from one side of the Celgard material, 
through the material and to the opposite side, wherein the feed stream is 
contained. The membrane material may be a silicone-type, gas-permable 
membrane; however, any membrane material selected must be compatible with 
the proteins, so that the surface of the membrane does not substantially 
denature the protein. 
If desired, the direction of diffusion and the diffusion rate may be 
enhanced further by the employment of various catalysts; for example, on 
the inner surface of the gas-permeable membrane, such as by immobilizing 
the particular catalyst on the surface thereof, to catalyze the gas 
reaction with feed stream solution, or by the employment of spaced-apart 
electrodes to enhance the potential difference across the flow path of the 
solution. The porous electrodes of opposite polarity may be placed between 
the flowing solution and the gas-permeable membrane. The degrees of 
purification of the recovered products and the degree of separation may be 
enhanced further by using multiple channels and a combination of a 
catalyst, multiple channels and electrodes. 
The invention will be described for the purpose of illustration only in 
connection with a particular embodiment; however, it is recognized that 
those persons skilled in the art may make various changes and 
modifications to the described embodiment, all falling within the spirit 
and scope of the invention.

DESCRIPTION OF THE EMBODIMENTS 
FIG. 1 is a schematic illustration of an apparatus for the separation of an 
aqueous protein solution, which apparatus 10 comprises a pair of 
spaced-apart, supported gas permeable membranes 2 and 4, typically 
cellulose acetate gas membranes permeable to carbon dioxide, which are 
stretched over porous plastic supports 12 and 14 to define with side walls 
11 and 13 a small, thin flow channel 20 of height H. The apparatus 10 has 
a length L and a wide W and includes C-shaped channel forming members 16 
and 18 on either side of the respective membrane supports 12 and 14, to 
define a channel 17 for the introduction, on one side of the gas-permeable 
membrane 4, of a carbon-dioxide gas at one end and the withdrawal of 
carbon-dioxide gas from the other end, and to define a channel 15 on one 
side of membrane 2, for the introduction of an inert nitrogen sweep gas 
into the inlet thereof and the withdrawal of the nitrogen gas together 
with diffused carbon-dioxide gas from the outlet, the carbon dioxide being 
that carbon-dioxide gas which is diffused from membrane 4 across the flow 
path 20 and through the gas-permeable membrane 2, to admix with the 
nitrogen sweep gas. The spaced-apart gas-permable membranes, which 
membranes need not be the same membranes, define a thin inlet 22 into the 
thin flow channel 20 at the one end, and, at the other end of the 
apparatus, there is defined a physical divider 24 projecting into the flow 
path 20, which is adapted to divide the protein mixture separated into a 
protein group I and a protein group II of defined isoelectric points. The 
protein effluent streams I and II are withdrawn through outlets 26 and 28 
at the one end of the apparatus 10. 
In operation, a pure carbon-dioxide-containing gas stream is introduced 
into the gas channel 17 between the member 18 and the one surface of 
gas-permeable membrane 4 at one end of the apparatus 10 and withdrawn from 
the other end, while an aqueous solution, containing, for example, a 
mixture of proteins, such as albumin (having a pI of 4.5 to 5.0) and 
hemoglobin (having a pI of 6.5 to 7.0), is introduced into the inlet 22 of 
the flow path 20 to pass from the one to the other end of the apparatus. A 
pure nitrogen inert sweep gas is introduced into the gas flow channel 15 
between the member 16 and the other surface of the gas-permeable membrane 
2, which membrane is permeable to the carbon dioxide. The carbon dioxide 
passes through the gas-permeable membrane 4 and into and transversely 
across the thin flow channel 20 of the protein mixture, to establish a 
gradient in the concentration of carbon dioxide transverse to the 
direction of flow. The carbon dioxide passing through the flow channel 20 
passes through gas-permeable membrane 2 and is removed with the nitrogen 
sweep gas. The aqueous solution of protein, prior to leaving the apparatus 
10, will have migrated into two groups, one group that has accumulated in 
that half of the flowing solution close to the carbon dioxide stream of 
greater carbon dioxide concentration, and the other group in the other 
half of the stream closer to the sweep gas. 
A CO.sub.2 gradient caused by the CO.sub.2 diffusion across the flow path 
provides, by virtue of the chemical reactions CO.sub.2 +H.sub.2 O=H.sub.2 
CO.sub.3 =HCO.sub.3.sup.- +H.sup.+, a parallel gradient in the 
concentration of bicarbonate ion, HCO.sub.3.sup.-, and an antiparallel 
gradient in pH. The bicarbonate gradient causes that species to diffuse 
from the CO.sub.2 to the N.sub.2 membrane side of the protein solution. 
The gradient in pH causes gradients in the concentrations of charged 
protein species and, hence, their diffusion. 
Since the HCO.sub.3.sup.- ion is much more mobile than proteins, the 
diffusion process perturbs the electrical charge balance throughout the 
solution, which brings about what is known as a diffusion potential. A 
relatively negative potential prevails on the N.sub.2 side of the 
solution. All charged species migrate under the influence of this 
potential, but where the pH is equal to a particular protein's pI value, 
that protein's electrically driven directional migration is halted. After 
a certain period of time (depending in part on the channel thickness (H)), 
the proteins in the solution will have distributed themselves, such that 
the electrical and concentration driving forces for their migration 
transverse to the direction of flow balance one another, and an 
effectively permanent degree of separation is attained at the other end of 
the apparatus. 
For example, when a protein solution containing albumin (pI 4.5 to 5.0) and 
hemoglobin (pI 6.5 to 7.0) is placed in the apparatus 10, the solution pH 
will approach albumin's pI near the CO.sub.2 stream and hemoglobin's pI 
near the N.sub.2 stream. In the bulk of solution, albumin will carry a 
negative charge and hemoglobin a positive charge, causing the albumin to 
accumulate near the CO.sub.2 stream as protein group II and hemoglobin 
near the N.sub.2 stream as protein group I. The electrical potential 
difference across the solution will approach 0.1 to 1.0 volt. 
The apparatus flow path H is made narrow, in order to minimize the time 
required for diffusion of the various protein species transverse to the 
direction of flow of the solution. The time for diffusion of a particular 
protein species is proportional to the square root of its molecular 
weight, as well as the square of the distance traveled, and inversely 
proportional to the voltage difference. The dimensions L and W of the 
apparatus are so selected, so that, with a given flow rate, the desired 
degree of protein separation is achieved before the liquid exits the 
apparatus 10. In the example cited, with H=2 mm, W=5 cm and L=50 cm, a 
flow of 10 to 100 ml/hr can be processed into streams of essentially pure 
hemoglobin (I) and albumin (II) solutions. 
Larger quantities of solution may be separated by immobilizing the enzyme 
carbonic anhydrase on the inner surfaces of the CO.sub.2 -permeable 
membranes, thereby catalyzing the CO.sub.2 hydration reaction and 
maximizing the bicarbonate, pH and electrical potential gradients. 
Larger quantities may also be separated by boosting the potential 
difference with porous electrodes 32, 34 placed between the flowing 
solution and the gas-permeable membranes 2, 4 and connected to a regulated 
potential source (not shown). The greater electrical driving force speeds 
up the transverse diffusion process, reducing the time to achieve 
separation and, thereby, allowing greater liquid velocity and volumetric 
flow through the same channel. 
In a further embodiment shown in FIG. 2, larger quantities are separated by 
a multi-channel system. In the use of multiple-channel systems, the 
volumetric flow rate through a single channel is as before, but the 
increase in number of liquid channels allows handling of more volume. As 
shown the apparatus 100 has gas channels 17a, 17b for CO.sub.2 and 15a, 
15b for N.sub.2. These channels are configured to have the gases enter and 
exit through side ports. Liquid channels 20a, 20b, 20c are provided and 
are configured to have liquid enter and exit through end ports. As in the 
embodiment of FIG. 1, gas channel 17a is formed from member 118 and porous 
membrane support 14a, gas channel 15b is formed from member 116 and porous 
membrane support 12c. The other channels are formed from the side walls of 
the apparatus 100 and porous membrane supports 14a, 14b, 14c and 12a, 12b, 
12c. Also included therein are corresponding membranes 4 a, 4b, 4c and 2a, 
2b, 2c and electrodes 32a, 34a, 32b, 34b and 32c, 34c. 
The divider 124 includes member 24a for dividing channel 20a, member 24b 
for dividing channel 20b and member 24c for dividing channel 20c. Divider 
124 also includes outlets 126 and 128 for protein groups I and II 
respectively. 
For refinements in the degrees of purification and separation of the 
proteins, additional stages, similar in design to that of FIGS. 1 and 2, 
may be added in series. Finally, with proteins of sufficiently high pI 
values, the solutions will be alkaline and then carbonate ion will play a 
role analogous to bicarbonate ion. 
EXAMPLE 
A representative application of the apparatus of FIG. 1 is used for the 
separation of the human blood proteins albumin (pI=4.8) and hemoglobin 
(pI=6.8) initially present in an aqueous solution at concentrations of 1 
millimole/liter. No other electrolytes are present at signficant 
concentrations, operation is at room temperature and pressure, and 100 
milliliters of feed solution are processed hourly. 
The dimensions of the apparatus's liquid channel are chosen as follows: 
(i) The height (H) influences strongly the magnitude of the electrical 
field (volts/cm) established between the gas channels which, in turn, 
determines the rates of protein migration into, respectively, albumin-rich 
and hemoglobin-rich portions. The height is chosen to be the narrowest 
possible without impairing the feasibility of splitting the exciting 
liquid stream into albumin-rich and hemoglobin-rich halves. Accordingly, 
the height is 2 mm. 
(ii) A suitable combination of liquid channel length (L) and width (W) is 
one which provides a total liquid channel volume (LCV) that ensures 
essentially complete protein separation by the end of the liquid residence 
time (LRT). Note that LCV=H.times.W.times.L, and that LRT=LCV/LFR, where 
LRF, the liquid flowrate, is 100 ml/hr. The minimum LRT in this case is 
calculated to be approximately 1000 seconds on the basis of 
(a) a potential difference, V, of 0.2 volt established between the upper 
and lower boundaries of the liquid channel, as result of the electrostatic 
interactions between bicarbonate and more slowly moving protein ions; 
(b) an average protein electrical charge of 5 equivalents/mole, negative 
for albumin and positive for hemoglobin; 
(c) protein diffusivities, D, of 5.times.10.sup.-7 cm.sup.2 /sec; 
(d) the law of electrical migration yielding the velocity of migration: 
EQU v=(zFD)/RT(V)/H 
where z is protein charge; F, Faraday's constant, is 96,500 
coulombs/equivalent; R, the ideal gas constant, is 8.314 
coulomb-volt/mole/.degree.K; and T, the absolute temperature, is 
295.degree. K. This yields an electrical migration velocity of 10.sup.-4 
cm/sec. Thus, the time for the proteins to travel an average distance of 
0.1 cm is 0.1/10.sup.-4 =1000 seconds. 
Given the liquid flowrate (LRF) of 100 ml/hr=0.028 cm.sup.3 /sec, then the 
liquid channel volume (LCV=L.times.W.times.H) must be at least 
1000.times.0.028=28 cm.sup.3, and thus the product L.times.W must be at 
least 28/H=140 cm.sup.2. To minimize the effect of protein back-diffusion 
towards the liquid entrance (which would detract from product purity), L 
should well exceed W. W is set at 5 cm and L is set at 50 cm to satisfy 
this criterion while providing nearly twice the necessary residence time. 
The gas flowrates are selected to maintain the necessary carbon dioxide 
gradient transverse to the liquid flow. Pure CO.sub.2 and N.sub.2 are fed 
to the respective lower and upper gas channels at slightly above 
atmospheric pressure. The electrical field is maintained high by 
minimizing the increase, along the flowpath, of the CO.sub.2 concentration 
in the initially pure N.sub.2 stream. The electrical field is rather less 
sensitive to the N.sub.2 buildup in the initially pure CO.sub.2 stream 
which, furthermore, is small because of the low N.sub.2 solubility in the 
liquid stream. 
An upper bound on the rate of CO.sub.2 permeation through the liquid film 
can be estimated from the CO.sub.2 permeability in pure water of 
approximately 1.5.times.10.sup.-5 (cm.sup.3 (STP)/(cm.sup.2 
-sec-(atm/cm)), and the area, L.times.W, of 250 cm.sup.2. With the 
CO.sub.2 partial pressure gradient maintained close to 1 atm/0.2 cm=5 
atm/cm, the steady state CO.sub.2 permeation rate will be no greather than 
0.02 cm.sup.3 (STP)/sec, or some 75 cm.sup.3 /hr. Thus, the CO.sub.2 
buildup in the N.sub.2 stream will be no greater than 0.01% of the total 
flow, if the N.sub.2 flow (which is effectively constant along the length 
of the apparatus) is 12.5 liters/min. Since N.sub.2 permeation through the 
liquid will be some 25 times slower than that of CO.sub.2, and the 
potential difference is comparatively insensitive to it, the CO.sub.2 flow 
is 0.1 liter/min. 
Since flow rate, pressure and purity are the only important aspects of the 
gas streams, convenience guides selection of gas channel dimensions. Their 
length and width are effectively constrained by the design to equal those 
of the liquid channel (50 cm and 5 cm, respectively). The height of the 
gas channel is also chosen, for simplicity, to equal that of the liquid 
channel, 2 mm. 
The gas and liquid channels are separated from one another by, 
polypropylene membranes (sold commercially as "Celgard"), the micropores 
of which exclude liquid water but offer no resistance to gases (including 
water vapor, whose loss from the solution may be prevented by 
prehumidfying the feed gases). The membranes are stretched over porous 
plastic supports situated on the gas side.