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Description  |
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BACKGROUND OF THE INVENTION
The invention relates to techniques for the separation, purification, or
both, of biolgical materials and, more specifically, to a method and
apparatus for isoelectric focusing and isotachophoresis.
Isoelectric focusing, isotachophoresis, and zone electrophoresis are
variants of electrophoretic techniques, differing in the buffer system
employed and mode of separation achieved. The theoretical distinction of
three methods has been described in some detail in Bier, 219 Science
1281-87 (1983).
Zone Electrophoresis ("ZE"), is the oldest of these techniques and most
commonly used. Separation is carried out in the presence of a background
of homogeneous buffer, and sample components separate according to their
mobilities in this buffer. No steady state is ever reached, but migration
continues with gradual broadening of sample zones due to diffusion and
other effects.
Isotachophoresis ("ITP") is a more recent variant of electrophoresis,
characterized by the fact that separation is carried out in a
discontinuous buffer system. Sample material to be separated is inserted
between a "leading electrolyte" and a "terminating electrolyte", the
characteristic of these two buffers being that the leader has to have ions
of net mobility higher than those of sample ions, while the terminator
must have ions of net mobilities lower than those of sample ions. In such
a system, sample components sort themselves according to decreasing
mobilities from leader to terminator, in a complex pattern governed by the
so-called Kohlrausch regulating function. The process has been described
repeatedly, as for instance, Bier and Allgyer, Electrokinetic Separation
Methods 443-69 (Elsevier/North-Holland 1979).
It is further characteristic of ITP that a steady state is eventually
reached, where all components migrate at same velocity (hence the name) in
sharply defined contiguous zones. Sample components can be separated in
such a contiguous train of components by insertion of "spacers" with
mobilities intermediary between those of the components one wishes to
separate.
Isoelectric focusing ("IEF"), also sometimes called electrofocusing, is a
powerful variant of electrophoresis. The principle of IEF is based on the
fact that proteins and peptides, as well as most biomaterials, are
amphoteric in nature, i.e., are positively charge in acid media and
negatively charged in basic media. At a particular pH value, called the
isoelectric point (PI), there is reversal of net charge polarity, the
biomaterials acquiring zero net charge.
If such amphoteric materials are exposed to a d.c. current of proper
polarity in a medium exhibiting a pH gradient, they will migrate, i.e.,
`focus` toward the pH region of their PI, where they become virtually
immobilized. Thus a stationary steady state is generated, where all
components of the mixture have focused to their respective PIs.
The pH gradient is mostly generated `naturally` i.e, through the electric
current itself. Appropriate buffer systems have been developed for this
purpose, containing amphoteric components which themselves focus to their
respective PI values, thereby buffering the pH of the medium.
Such buffer mixtures are known as `carrier ampholytes`, the best known
being "Ampholine", a trademark of the LKB Produkter AB, a Swedish company.
Other carrier ampholyte mixtures can be formulated by judicious mixing of
suitable ampholytes, as, for example, described in Bier, 211 J.
Chromatography 313-35 (1981).
The two variants, IEF and ITP, differ in that IEF attains a stationary
steady state whereas in ITP a migrating steady state is obtained. Thus, in
IEF a finite length of migrating channel is always sufficient. In ITP,
complete resolution may require longer migrating channels than is
practical. In such case, the migrating components can be virtually
immobilized by applying a counterflow of leading electrolyte, the rate of
counterflow being matched to the rate of frontal migration of the sample
ions. This is also known in the art.
IEF is most frequently carried out in polyacrylamide or agarose gels, where
all fluid flow disturbances are minimized. ITP is most often carried out
in capillaries. The sample is inserted at one end of the capillary, at the
interface between leader and terminator, and the migration of separated
components recorded by appropriate sensors at the other end of the
capillary. Both such systems are used mainly for analytical or
micro-preparative purposes.
The scaling up of any electrophoretic technique is difficult because of the
need to stabilize the fluid system against convection. The easiest fluid
stabilization is achieved in gels or with other supporting media, such as
granulated beds, etc. Unfortunately, such stabilized systems do not lend
themselves to separations involving flow of process fluid, yet such flow,
whether continuous or recycling, appears to be the best approach for
increasing the capacity of the techniques. Continuous flow is best carried
out in free fluids, unsupported by gels or granulated beds. Separations in
free fluids require stabilization against flow disturbances. These
disturbances could disrupt the orderly separation of sample components.
The need for fluid stabilization is well recognized by practitioners of
the art and, to achieve it, a variety of principles have been utilized and
incorporated into diverse instruments.
One of the most common principles utilized for flow stabilization is
confinement of the process fluid, i.e. carrier buffer and sample solution,
to a narrow liquid film contained within a channel between two parallel
plates. Within the channel it is generally assumed that viscous forces
maintain fluid stability. Numerous such instruments have been designed and
patented for continuous flow electrophoresis.
In such continuous flow instruments the d.c. electric field is applied in a
direction perpendicular to buffer and sample flow. The critical feature of
such instruments seems to be the dimension of the gap between the two
parallel plates, i.e., the thickness of the fluid film. This is usually of
the order of 0.5 to 1.5 mm. The passage of the electric current generates
heat, and thus one or both of the parallel plates are cooled. The cooling
capacity of these plates sets the limit for power dissipation within the
apparatus. It is implicit in such continuous flow devices, whether applied
to ZE, IEF, or ITP, that separation of sample functions be obtained in a
single pass through the apparatus. This requires slow flow of buffer and
long residence time of the sample within the apparatus.
While such instruments are in reasonably wide use, their operation is
limited by several factors. Only very dilute solutions can be utilized, of
the order of 0.01 to 0.2% solute concentration, otherwise density
gradients between sample and carrier buffer may cause convective
disturbances. Three other factors disturb separation: (1) electroosmosis
causes a parabolic flow of liquid in the plane of the electric field,
electroosmosis being due to the electric charge at the inner surface of
the parallel plates; (2) the downward flow of the liquid through the
narrow gap also causes a parabolic flow velocity profile in a direction
perpendicular to that due to electroosmosis. Thus, the residence time of
the fluid in the center of the gap is much shorter than that of the fluid
close to the wall; (3) finally, as the fluid at the center of the gap is
warmer than at the walls, all electrophoretic parameters (conductivity,
viscosity, electrical field, mobility of ions, etc.) are affected. The
effects of the three factors are complex and cause the well known
`crescent phenomenon` (Strickler and Sacks, 209 Annals New York Acad. Sci.
497-514 (1973)), i.e., a crescent-like deformation of the migrating sample
zones. The crescent deformation is most pronounced closest to the walls of
the electrophoretic chamber. To minimize it, the sample stream is mostly
injected only into the center of the gap, thus seriously limiting the
throughput capacity of the apparatus. Of all these factors, gravity was
assumed to be the most important limitation on the performance of the
instruments. It is for this reason that McDonnell Douglas Astronautics Co.
has constructed and tested is well publicized continuous flow apparatus
specifically designed for operation in the reduced gravity of orbiting
spacecraft.
All present instruments of the continuous flow kind were designed and are
applied principally to ZE. The object of the present invention is to
demonstrate how these and other difficulties with current instruments can
be avoided. The invention is restricted only to IEF and ITP, where steady
states are achieved, and is not applicable to ZE. Thus, the apparatus and
method which are objects of the present invention may be considered as the
first ones specifically designed for IEF and ITP.
SUMMARY OF THE INVENTION
The present invention is directed to a method and apparatus for separation
and purification of proteins and other biological materials by isoelectric
focusing (IEF) or isotachophoresis (ITP). The invention is based on the
discovery that stabilization of fluid flow during preparation IEF or ITP
is achievable by rapid recirculation of process fluid through the narrow
channel of a continuous flow electrophoresis chamber of limited depth.
Thereby are eliminated the previously enumerated causes of fluid flow
disturbance due to temperature and density gradients, electroosmosis and
parabolic flow.
The apparatus suitable for implementation of the invention differs from
conventional continuous flow instruments in several respects: (1) there is
a matched set of in- and outflow ports at the opposite end of the
electrophoresis chamber; (2) means are provided for rapid recirculation of
the process fluid in closed external loops between each matched set of in-
and outflow ports; (3) these loops include individual refrigerated heat
exchangers.
The processing method differs also in a substantial manner from the
customary methods of operation of continuous flow electrophoresis
chambers: (1) rather than trying to achieve the desired degree of
separation in a single pass through the apparatus, only small shift
towards the final steady state is achieved in every pass through the
chamber; (2) rather than having slow flows and long residence times, of
the order of minutes to fractions of an hour, rapid recycling is
established, with residence times of the order of seconds. These and other
differences in apparatus and method of operation will become obvious from
further disclosure.
The stabilizing effect of rapid flow is so striking that cooling of the
electrophoresis chamber is no longer necessary. In most continuous flow
instruments, great emphasis is placed on uniformity of temperature within
the apparatus, to avoid convective disturbances. In the present invention,
the Joule heat generated by the applied electric current can be dissipated
in heat exchangers external to the apparatus. This is obviously a major
advantage, greatly simplifying the design of the apparatus. The Joule heat
is absorbed by the latent heat capacity of the process fluid during
transit through the electrophoresis chamber and released to the heat
exchanger. The allowable heating within the chamber is limited only by the
heat-sensitivity of the sample, rather than the stability of liquid flow.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a front view of a preferred embodiment of the invention;
FIG. 2 is a cross-sectional top view of the chamber shown in FIG. 1, taken
along plane II--II of FIG. 1;
FIG. 3 is a cross-sectional top view of an alternate embodiment of the
invention;
FIG. 4 is an overall schematic view of an apparatus as used for recycling
IEF according to the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Before considering the apparatus embodying the invention, understanding
will be facilitated by considering the principles underlying the device
and method. It is an essential feature of the present invention that fluid
flow disturbances inherent in continuous flow electrophoresis can be
largely overcome by recirculating the fluid rapidly through the chamber,
reducing the residence time to seconds only. This stabilizing effect was
an unexpected and fortuitous observation during the testing of the
apparatus: the faster the recirculating flow, the higher the power that
can be dissipated in the apparatus before fluid instabilities are
observed. The effect is very significant and not foreseeable on the basis
of prior art or literature. Careful analysis of the causes has revealed
that it is due to a synergistic contribution of several factors, no one of
which alone suffices to produce the observed effect.
Our analysis revealed the synergistic relation between the following major
factors:
1. The process is applicable only to electrophoretic methods resulting in a
steady state exhibiting self-stabilizing and self-sharpening sample
concentration boundaries, namely IEF and ITP. Thus, there exists the
necessity of using suitable buffer mixtures: appropriate carrier
ampholytes capable of generating pH gradients for IEF, and discontinuous
buffer systems for ITP. Random selection of homogeneous buffers used for
ZE will not result in effective fractionation.
2. To minimize gravitational effects it is necessary to maintain a short
residence time of fluids within the processing chamber. Density gradients
are unavoidable in all electrophoresis, but are particularly pronounced in
IEF and ITP, because of the sharpness of sample concentration boundaries.
Density gradients generate natural convection, but this requires a certain
time element to develop. In a situation such as continuous flow
electrophoresis, where both forced and natural convection can occur
simultaneously, the relative importance of the buoyancy can be determined
by the ratio of the Grashof and square of the Reynolds numbers:
##EQU1##
here g is the gravitational acceleration,.DELTA..rho. the density
difference, L the length, and U the through-flow velocity. The convection
effects will be negligible if above ratio is much smaller than 1. This
requires operation at relatively high Reynolds numbers, incompatible with
single pass operation and necessitating recirculation. This, in turn, is
compatible only with IEF and ITP modes of operation.
3. To further minimize gravitational effects, it is helpful to orient the
focused streams of flowing sample vertically, rather than horizontally. In
a horizontal orientation, the denser stream of focused sample would tend
to sediment in the course of transit through the chamber.
4. The most surprising discovery is the effectiveness of shear stress at
the walls of the chamber to minimize electroosmosis. To the best of our
knowledge, this has not been previously known, and the effect of shear
stress on electroosmosis has not been investigated. Shear stress, }, is
defined as the ratio of force, F, and surface, A, and is given by
##EQU2##
which is the well known Newton's law of viscosity, where .mu. is the
viscosity, U the fluid velocity at a distance y from the centerline of the
channel, and p the pressure. In laminar flow through a channel between
parallel plates, dp/dL is constant across the depth of the channel, as
otherwise the flow would not be laminar. Thus, .tau. varies linearly with
y, being at a maximum at the walls and zero at the center, where y=0. The
total discharge, Q, per unit width, W, of the channel is given by
##EQU3##
where b is the distance between the centerline and the wall of the
channel. Substituting for dp/dL, the shear stress can be ex expressed as a
function of Q/W by
##EQU4##
Thus, while the shear stress is increasing only linearly with Q, it is
proportional to the third power of 1/b. This explains the importance of
keeping the channel depth to the minimum consonant with reasonable
throughput and power dissipation. In practice, the wall to wall spacing of
the chamber 2b=D, of 0.05 to 0.1 cm was found best.
One may also wish to define the residence time, R, of fluid in the electric
field, which is given by
##EQU5##
which allows to define the shear stress in terms of R and D Expressed in
these terms, the shear stress is proportional to L/R and inversely
proportional to the square of chamber thickness, being zero in the center
of the channel and maximal at the walls. Equation 6 has some interesting
implications, for instance, lengthening the chamber length L to increase
throughput Q, does not result in increased shear stress, as the ratio L/R
remains constant.
We are now in the position to give some typical values of one of our
apparatus and its mode of operation: L=30 cm; W=6 cm, D=0.075 cm, Q=3.5
cm3/sec, U=0.01 poise (gm/cm sec) then the calculated R=3.9 sec, .tau.=168
.times.y (gm/cm sec2), or at y=D/2 i.e., at the walls of the chamber,
.tau.max=6.3. Under such typical conditions, our apparatus tolerates a
continuous input of 200 watts d.c. electric power without fluid
disturbances. This power causes a temperature rise of approximately
12.degree. C. in a non-refrigerated chamber, easily tolerated by most
biological samples, provided the inflowing liquid was cooled in the
external heat exchanger to near freezing. Optional cooling of one of the
plates of the chamber avoids all temperature rise.
This should be contrasted to single pass conventional continuous flow
electrophoresis instruments where residence times for IEF are usually well
in excess of 600 sec, and the tolerated power is of the order of 10 watts
or less.
Our apparatus differs significantly from other electrophoretic apparatus
that utilizes shear for fluid stabilization. See Mattock, Aitchison and
Thomson, 9 Separation and Purification Methods 1-68 (1980). That apparatus
carries out separation in an annulus between two cylindrical electrodes,
an inner stationary one and an outer rotating one. Sample and carrier
buffer are introduced at the bottom of the annulus and the separated
fractions withdrawn at the top. The apparatus can be used only for ZE and
there is no possibility for fluid recycling. Moreover, liquid flows within
a channel with one stationary and one moving boundary, and dU/dy rather
than dp/dL is constant across the depth of the channel. Thus, the shear
stress is constant across the whole channel (see equation 1).
There are significant differences in the use of shear stress for fluid
stabilization in the two instruments: in the prior art apparatus, uniform
shear across the channel is used, while in the present invention, shear is
maximal at the wall of the chamber and zero at the channel center. This is
not a trivial difference: electroosmosis is exclusively a wall effect, and
that location is where maximal shear stress is needed. On the other hand,
biological materials are sensitive to shear, and thus the invention offers
the advantage of minimizing shear in the bulk of the liquid. Maximum flow,
of course, is in the center of a channel, where in our apparatus there is
zero stress. Furthermore, in the older instrument, rotationally induced
shear is in a direction perpendicular to the direction of liquid flow
through the annulus. In our apparatus, shear stress is in the direction of
the liquid flow.
As seen in FIGS. 1 and 2, the fluids to be processed are circulated through
a processing chamber in a first direction, and exposed to a d.c. current
in a second direction, roughly perpendicular to the first direction. The
chamber itself is constituted by two parallel plates 1 and 2, of an
electrically non-conducting material, such as plexiglass, glass, etc.,
these plates defining a relatively narrow channel for fluid flow. Multiple
parallel entry ports 8 for process fluid are provided at one end of the
chamber, matched at the opposite end of the chamber by outlet ports 7,
located at the bottom and top of the chamber, respectively. Electric
current is provided by electrodes 9 and 10, mounted on carriers fitted
with connectors 11 and 12. The electrodes are mounted in compartments,
usually lateral, of the chamber, as shown, and separated from the main
cavity of the chamber by electrically conducting but protein non-permeable
membranes 5 and 6. For IEF, these membranes may be either
ion-permselective or electrically neutral, while only the latter are
acceptable for ITP. Examples of such electrically neutral membranes are
the various types of dialyzing membranes commercially available, and
ion-permselective membranes may be the type used in electrodialysis. As is
known in the art, the electrode chambers are provided with ports 13 and 14
for circulation of the electrolytes.
All entry and exit ports are individually connected outside of the chamber
in a series of closed loops, so that a parallel and contiguous flow of
individual fluid streams is established through the chamber cavity. The
fact that the fluid loops outside the cavity are closed loops assures the
volumetric constancy of in- and outflow through each matched set of ports.
Ion transport between adjacent streams in the chamber takes place under
the influence of the electric field. Separate electrolyte circulation
paths are provided for each of the two electrode chambers.
For IEF, there is provided a matched number of entry and exit ports, the
number depending on the number of fractions desired. ITP requires
counterflow, thus necessitating inflow of buffer at one side of the
chamber and outflow at the other side. This flow can be accomplished
through inflow and outflow of excess fluid into the recirculation loops,
at opposite sides of the chamber, or by provision for separate
non-recirculating ports.
Although it has been found that the present invention eliminates the need
for cooling the chamber plates, cooling means may be provided if desired,
as shown in FIG. 3. Such means are most conveniently mounted on the
chamber back plate, as generally it is desirable to view that chamber
through a transparent covering over the front of the apparatus. Also,
sealing is facilitated by mounting the electrodes in the front plate. As
shown, a metallic plate 32, provided with channels 23 absorbs and
transfers heat. An insulating layer 22 electrically isolates the cooling
means.
Fluid addition or withdrawal during fractionation, if desired for any
reason, can be effectuated through any of the external fluid channels.
This is necessary for establishing counterflow in ITP, through the input
of leading electrolyte at one side of the chamber and withdrawal of excess
fluid so introduced at the outer side of the chamber. If desired, this
input or withdrawal could also be accomplished through additional ports in
the chamber.
A system for performing IEF according to the present invention is shown in
FIG. 4. There, a multichannel pump 500 provides fluid pressure in the
external loops, where constancy and uniformity of flow through each
channel are desirable. Preferably, the individual flows are also channeled
through a heat exchanger 1100, to dissipate the Joule heat generated by
the electric current. In addition, one may wish to introduce into all or
some selected fluid loops sensors 200 for pH, conductivity, electric
potential, optical density in the visible or ultraviolet range,
temperature, or other such fluid properties.
These sensors, along with data processor 1000 and collection valve 300,
comprise a monitoring means which, in addition to other detection and
control purposes, can locate the position of an isotachoporetic boundary
and regulate the counterflow to maintain that boundary. Finally, air and
pulse traps 700, reservoirs 600 to increase the volume capacity of the
system, and other attachments known to those in the art can be
incorporated into the external loops.
The apparatus of FIG. 4 may be constructed of glass, a variety of
machineable plastics or even ceramics. Cooling plates, if desired, can be
metallic, provided they are separated from the chamber's interior by
electrically insulating layers of suitable material, which could again be
either glass or plastic. The front side of the of the apparatus is
preferably transparent, to facilitate observation.
To initiate IEF fractionation, the apparatus is first filled with a
solution containing a suitable carrier ampholyte for the establishment of
the pH gradient. The sample to be fractionated can be premixed with this
solution, or can be added at a later stage. The apparatus is then purged
of air, which may require several reversals of flow direction through the
chamber and the external circuitry. Total fluid capacity is given by the
volume of the electrophoresis chamber and that of the external loops,
which can incorporate variable volume expanders. As a rule, chamber volume
is only a minimal faction of total volume. Circulation of suitable
electrolytes is then initiated through the electrode compartments. A
variety of electrolyte fluids are customarily used for this purpose, such
as dilute acids or bases. Finally, recirculation of process fluid is
initiated through the cooled heat exchanger and all other external
accessories.
The focusing process can best be visualized using colored samples, such as
red homoglobin, blue-stained albumin, or a suspension of green
chloroplasts from plant leaves. At the start, a uniform colored film of
flowing liquid is observed. Upon application of current, progressive
focusing is seen. The pH gradient originates at the electrodes and
progressively moves inwards. Thus, with a mixture of hemoglobin and blue
albumin, initial clearing of color from both sides of the chamber will be
seen. As the pI of the albumin is lower than that of hemoglobin, a faster
blue and slower red band will be seen at the cathodic side of the chamber,
the reverse being visible at the anodic side. Finally, the advancing bands
of the two proteins will merge at their respective pH's in the chamber. In
each pass through the chamber, the movement of the protein is
imperceptible, as there is only a small shift of protein distribution in
the short residence time. When final focusing is achieved, each protein
will be confined to a narrow band of colored material in continous
recirculation. Visually, the stability of the fluid is such that the bands
appear stationary and their flow is visible only by careful observation.
The reasons for the fluid stabilization have been given in a previous
section of the disclosure. Essentially, short residence time is needed to
minimize gravitational effects due to density gradients, and shear stress
at the walls minimizes electroosmosis. There is proportionality between
shear stress and total electrical power that can be dissipated in the
apparatus. This can be readily demonstrated if a colored protein sample,
such as hemoglobin or a stained protein, is focused. At any electrical
power, instability can be generated by lowering the flow rate, Q. This is
visualized by wavering of the colored protein bands, previously sharply
focused at higher power. The effect is even more dramatic if flow is
completely interrupted, while maintaining full power. This results in a
nearly instantaneous and dramatic disruption of the focused protein bands,
this being best described as feathering of the bands in lateral
directions. Rapid remixing of the contents of the electrophoresis chamber
is the result. If the electric power is first stopped and then the flow
interrupted, a different phenomenon is seen: the settling of the denser
protein bands to the bottom of the chamber as a result of gravity.
Finally, if power is stopped but recirculation maintained, there is no
immediate visual effect on the colored protein bands, diffusion being
quite slow.
Good results were obtained with three electrophoresis chambers, with the
following internal dimensions: Chamber A: 40 matched entry and exit ports,
chamber height 30 cm, width 6 cm, and variable depth (gap between front
and back plate) between 0.025 and 0.15 cm. Experiments with this chamber
have determined that optimal depth is of the order of 0.075 cm. Chamber B:
12 matched ports, height 20 cm, width 2.8 cm and depth 0.075 cm. Chamber
C: 48 matched ports, height 30 cm, width 6 cm and depth 0.075 cm. Only
chamber C had the external cooling, requiring frontal electrode
compartments, as illustrated in FIG. 3.
Most experiments were run at constant power, registering also current or
voltage. During focusing, the conductivity of the process fluid decreases
by nearly an order of magnitude, causing an increase in applied voltage
and decrease in amperage. Thus, the steady state is signaled by the
constancy of these two factors. To speed up the focusing process, the
power is kept at a maximum tolerated by heating and fluid stability, but
it was experimentally observed that reduction of power and flow rate, once
apparent steady state is reached, may marginally increase the sharpness of
resolution. The reasons for this are not clear, but can probably be
ascribed to increased irregularity of pump performance at high rate. The
pumps employed are peristaltic pumps with planetary gear drives, to
minimize wear and tear on the tubing and decrease pulsation.
Once steady state is reached, the fractions can be collected. A variety of
procedures has been employed, but in any of them, the first step is to cut
the power and recirculation. The fluid contained in the chamber is then
drained and rejected, as it rapidly remixes. Following this, the fractions
are collected by draining, pumping, or otherwise extracting the fluid from
each isolated external circulation loop. Several fraction collection
devices accomplishing the extraction of all channels simultaneously have
been constructed, but are not object of the present invention.
Utilization of the apparatus for ITP is more complicated, as it requires
the establishment of a counterflow. The chamber is filled with the leading
electrolyte, as is the appropriate electrode compartment -- the anodic for
separation of negatively charged species and the cathodic for positively
charged species. The remaining electrode is filled with the terminating
electrolyte. After priming the apparatus, the leader is replaced by the
terminator in a few (2-5) of the recirculating channels closest to the
terminator. The sample to be separated is then injected into one or more
of the circulating loops between leader and terminator and the power is
applied. This injection can be in a single bolus, or can be continued for
as long as it is desired during the run.
According to the principles well known to the practitioners of the art of
ITP, the samples will migrate, displacing the leader, and will be followed
by the terminator. At the same time, sample components will sort
themselves according to their respective electrophoretic mobilities in the
chosen electrolyte system. There is usually also significant concentration
of sample zones, proteins often reaching a concentration of several
percent. The advancing band of the fastest sample can be monitored in a
variety of ways: color (if visible), absorption in the ultraviolet,
conductivity, or electric potential gradient. The potential is uniform
within the leader zone, but has a sharp stepwise increase at every
interface between successive zones, such as leader-sample, sample-sample,
or sample-terminator interface. Monitoring of potentials is as a rule most
effective, because universally applicable. A colored sample is very
helpful, however, for the initial setting of approximate parameters of
flow and electric power.
Once the leading boundary of sample zone has sufficiently advanced, it has
to be virtually immobilized by counterflow of leading electrolyte. The
excess fluid so introduced has to be withdrawn from the opposite side of
the chamber, occupied by the terminator. This procedure is well known to
the practitioners of the art. Typically, the inflow of the leader will be
carried out through the input ports close to the leader electrode and the
terminator withdrawn from the ports close to the terminator electrode.
Thus, quite large supplies of leader and terminator have to be available,
to avoid depleting the system.
The balancing of the input flow of the leader can be adjusted either
manually, to maintain constancy of the position of the leader-sample
interface, or can be automated. Electronic circuitry has been constructed
which senses the electric potential between each successive pair of the
exit ports of the chamber, thus monitoring the whole process. When the
chosen position of the critical interface is neared, counterflow is
initiated. At that time the circuitry can be employed in two alternate
modes: it either controls the applied power at constant leader
counterflow, or adjusts the counterflow at constant power. Obviously, both
have to be preset manually at approximately proper relation for the system
to work effectively. Under construction is an array of sensors monitoring
the ultraviolet absorption in all recirculating channels. The logic of the
operation is controlled by a data processor 1000 (FIG. 4), such as a
personal computer.
EXAMPLE 1
The following description summarizes a series of experiments which have
been used to test the apparatus as they were being developed. All three
previously described chambers were used. The objective was the isoelectric
focusing separation of carbon monoxide treated human hemoglobin, a red
protein, and human serum albumin, stained blue with Bromphenol Blue dye.
Typically, a 1% solution (w/v) of Ampholine (Trademark of LKB Produkter,
AB of Sweden), broad pH range of 3.5 to 10, was used as the carrier
ampholyte, the two proteins having been used at varying concentrations,
ranging from 0.2 to 5 mg/ml. The apparatus is primed with this solution
and run at constant power input until separation is visually completed.
"Once focusing is obtained, various combinations of pumping rates and
power can be used to try to determine conditions of optimal resolution."
This is easily determined visually, by observing the distribution of the
colored proteins in the recirculating flow loops.
Experience has shown that the process is very forgiving, being compatible
with a wide range of conditions of flow and power. Initially, there is
advantage of maximizing power, to shorten the focusing time. Routinely,
constant power of 200 watts was applied with residence time of liquid in
the chamber of about 3-6 seconds. Before final collection, there appears
to be some minor advantage in reducing the power to about 80-120 watts and
prolonging the residence time to about 10 seconds.
At the end of an experiment, all the fractions are collected and analyzed
for pH and protein concentration. A typical result illustrates the sharp
focusing of the proteins at their respective PI values:
______________________________________
hemoglobin
albumin
tube # pH (mg/ml) (mg/ml)
______________________________________
10 7.95 0.11 0
11 7.78 0.23 0
12 7.62 0.71 0
13 7.47 2.95 0
14 7.21 3.25 0
15 6.99 0.47 0
16 6.79 0.20 0
29 5.09 0 0.23
30 4.97 0 1.38
31 4.84 0 3.14
32 4.76 0 5.78
33 4.69 0 6.96
34 4.58 0 3.18
35 4.48 0 0.38
______________________________________
EXAMPLE II
This example illustrates the ability to purify by isoelectric focusing
monoclonal antibodies obtained from mouse ascites fluid. It also documents
the ability of the apparatus to handle proteins which precipitate at their
isoelectric point, a common problem. Mouse ascites fluid was diluted to
1/3 in a solution containing 1% Ampholine, pH range 5-9 and 3 molar
concentration of urea. | | |
