BACKGROUND

1. The Field of the Invention

The present invention relates to methods and apparatus for the isoelectric focusing of amphoteric substances. More particularly, the present invention is directed to the techniques for separating biological materials through the use of isoelectric focusing processes which enhance the separation characteristics of amphoteric biological substances and provide for efficient removal of heat generated during the isoelectric focusing process.

2. The Background of the Invention

Numerous areas of modern biology and genetic engineering depend on the availability of large quantities of high purity proteins. Currently available methods of protein purification include many kinds of chromatographic and electrophoretic techniques. Among these techniques, isoelectric focusing (hereinafter "IEF") has many attractive features.

The principle of IEF is based on the fact that certain biological materials (such as proteins, peptides, nucleic acids, and viruses) and even some living cells are amphoteric in nature — i.e.. they are positively charged in an acidic media and negatively charged in a basic media. At a particular pH value, called the isoelectric point (hereinafter "pi ¹¹ ) , these biomaterials will have a zero net charge.

Being charged in a pH gradient, the biomaterials migrate under the influence of an electric field until they reach the pH of their isoelectric point. At the isoelectric point, by virtue of their zero net charge, the biomaterials are not influenced by the electric field.

Diffusion of "focused" biomaterials away from their pi will cause them to once again become charged, whereby they will electrophoretically migrate back to their pi. Thus, the biomaterials focus into narrow zones (defined by the pH of the medium and the electric field applied) from which the biomaterials can be selectively separated.

In one known method of isoelectric focusing, the pH gradient is established by the introduction of carrier ampholytes into the electric field. "Carrier ampholytes" are defined as ampholytes of relatively low molecular weight having conductance as well as buffer capacity, in the isoelectric state. Mixtures of synthetic polyamino- polycarboxylic acids have been used as carrier ampholytes.

In order to establish suitable pH gradients for IEF, it is necessary to have access to a great number of carrier ampholytes with isoelectric points well distributed along the pH scale. A commercial mixture of such amphoteric substances (called "Ampholine") is available from LKB Produkter AB, a Swedish Company. Ampholine is thought to be principally composed of polyaminopolycarboxylic acid molecules made by reacting polyamines with acrylic acid.

By manipulating the pH range of the carrier ampholytes, isoelectric focusing has the potential for high resolving power. However, the potential of isoelectric focusing as a means for separating amphoteric substances has not been realized because of the time necessary and the quality of separation of prior art processes.

Since acids are attracted to the anode of the electric field and bases to the cathode during electrolysis, an increasing pH gradient from the anode to the cathode will develop in a convection free electrolytic conductor. The success of isoelectric focusing depends on the satisfaction of three conditions: (1) that the pH gradient is stable in time; (2) that an electrolyte deficit does not develop

within the field, thereby tending to quench the current and/or give rise to local overheating; and (3) that the pH gradient — d(pH)/dx — has a low value in the pH region of interest in the actual separation. Isoelectric focusing is most often practiced in small- scale batch instruments where the fluid is stabilized by either gels or density gradients established by a non- migrating solute such as sucrose. The capacity of such instruments for product separation is generally limited by the cross-sectional area of the apparatus. Because the apparatus cross-section is limited by the need to dissipate the heat generated by the electric field, larger scale preparative work has been proposed using continuous flow and recycling techniques. One known technique which comes close to combining high resolution with large quantitative capacity is the recycling isoelectric focusing method disclosed in United States Letters Patent No. 4,204,929 and No. 4,362,612.

Currently known recycling isoelectric focusing (hereinafter "RIEF") techniques involve dividing a fluid containing carrier ampholytes into a plurality of reservoirs and passing the contents of the reservoirs through an isoelectric focusing cell. The isoelectric focusing cell separates the fluids from adjacent reservoirs with ion non-selective permeable membranes which allow interchange of fluid constituents from channel to channel, but which inhibit bulk fluid flow. Electrodes establish an electrical potential transverse to the fluid flow thereby creating a pH gradient between successive channels. The fluid from each reservoir exiting the isoelectric focusing cell is pumped to the reservoir which feeds the isoelectric focusing cell. A heat exchanger cools the fluid within the reservoirs. As the fluid is pumped into the top of the reservoir, the fluid is directed from the

bottom of the reservoir back into the isoelectric focusing cell.

This technique, however, has serious design flaws which preclude its routine use for both research and industrial scale application. The principal design flaw in this technique is that liquid containing material that is semipurified during each circular passage is immediately remixed with the crude starting material in the cooling reservoirs. Thus, the whole process constitutes a continual dilution process in which original crude mixtures in the reservoirs are continually diluted so that final purity is never truly achieved.

The constant remixing of crude and semipurified material in the prior art greatly increases separation time and compromises the resolution of the subcomponents and final purity of the isolated materials. In effect, an asymptotic dilution of contaminants occurs in each separation channel, and therefore a zero contamination level can never be achieved using currently known RIEF techniques.

This constant remixing of semipurified and crude material not only requires very long periods of time for attaining satisfactory degrees of separation, but also requires long periods of time to prefocus the carrier ampholytes into the initial pH gradient. Hence, the overall time required for prefocusing and actual separation is quite, long.

Another serious drawback with the current RIEF techniques is the ability to dissipate the joule heat generated during the isoelectric focusing process. Current RIEF techniques cool the processed fluid within the reservoirs. Thus, extreme heating of the fluid sample may occur within the isoelectric focusing cell, irreparably

damaging the desired biological sample, before the fluid can be cooled in the reservoirs.

The use of dilute acids and bases for electrolyte solutions in the electrode chambers has been standard practice for virtually all isoelectric focusing systems. Typically the solutions used are 0.1 M sodium hydroxide (pH 12.5) for the catholyte and 0.1 M phosphoric acid (pH 2.3) for the anolyte. As the ampholytes focus, the subsequent pH gradient tends to be between these two pH extremes. Even when attempting to create shallow gradients with narrow range ampholytes, the extreme ends adjacent to the electrolyte solutions buffer to the electrolyte pH. This phenomenon is known as "spiking" or "acid notching".

In many isoelectric focusing applications spiking is not a major concern. However, in applications having a small number of separation channels or other limited area of separation, it is important to reduce or eliminate spiking to achieve high resolution separations. Otherwise the spiking tends to inhibit the formation of a narrow pH gradient which is important in achieving high resolution.

In summary, the burgeoning genetic engineering market in the United States and other Western nations has created an acute need for high resolution protein purification techniques. The manipulation of human and animal genes for numerous hormones, enzymes, and other protein molecules into bacteria, yeast, or mammalian cell liner and the subsequent large-scale production of these proteins has created an acute need for rapid high resolution methods for purification of proteins and other similar biological substances. Although other techniques are frequently used as first or even second step techniques in the purification of these molecules, preparative isoelectric focusing in a narrow pH gradient is an ideal environment in which to do either initial or final stage protein purification.

From the foregoing, it will be appreciated that what is needed in the art are apparatus and methods for isoelectric focusing of amphoteric substances which combine high resolution separation with large quantitative sample capacity.

Additionally, it would be a significant advancement in the art to provide apparatus and methods for recycling isoelectric focusing of amphoteric substances which do not mix semipurified sample with the crude sample. it would be another advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which efficiently remove heat generated during the process, thereby permitting increased power input. It would be a further advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which rapidly separate the desired substance from accompanying impurities.

It would be yet another advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which rapidly prefocus to establish a stable pH gradient.

Additionally, it would be a significant advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which inhibit spiking.

Another significant advancement in the art would be to provide apparatus and methods for isoelectric focusing of amphoteric substances which enable narrow pH gradients to be obtained and maintained without significant spiking in the separation channels adjacent the electrodes.

It would be still another advancement in the art to provide apparatus and methods for isoelectric focusing of amphoteric substances which provide high resolution separations.

Such methods and apparatus are disclosed and claimed herein.

BRIEF SUMMARY OF THE INVENTION The present invention includes novel apparatus and methods for isoelectric focusing of amphoteric substances within fluids containing carrier ampholytes. The present invention utilizes preparative isoelectric focusing techniques in order to process large volumes of fluid sample. However, fluid which passes through the isoelectric focusing cell is not mixed with the original crude fluid sample. Mixing is avoided by preferably using an alternating or dual reservoir system.

One reservoir of the dual reservoir system supplies the sample fluid to the isoelectric focusing cell and the other reservoir receives the fluid after passing through the focusing cell. After the supply reservoir empties, fluid flow into the isoelectric focusing cell is automatically diverted from the supply reservoir to the receiving reservoir. After appropriate time delay, fluid flow from the isoelectric focusing cell into the receiving reservoir is diverted into the supply reservoir. Fluid flow into and out of each reservoir is automatically alternated after each pass through the isoelectric focusing cell.

The apparatus within the scope of the present invention preferably includes an isoelectric focusing cell having a plurality of inlet ports and a plurality of corresponding outlet ports. The inlet and outlet ports permit fluid to flow into and out of the isoelectric focusing cell along discrete paths. Each inlet and outlet port is preferably coupled to a pair of reservoirs, and each reservoir has a reservoir entrance to allow fluid flow

into the reservoir and a reservoir exit to allow fluid flow out of the reservoir.

Fluid flow is preferably alternated into and out of each reservoir of each reservoir pair through use of reservoir inlet and outlet valves. The reservoir inlet and outlet valves preferably operate in response to the fluid level within the reservoir supplying fluid to the isoelectric focusing cell. When the reservoir empties, as measured by the fluid level, the corresponding reservoir outlet valve automatically engages to stop fluid flow from the empty reservoir and to start fluid flow from the full reservoir. After appropriate time delay, the reservoir inlet valve engages to stop fluid flow into the full reservoir and to start fluid flow into the empty reservoir. The apparatus within the scope of the present invention also preferably has a heat exchanger for removing heat generated by the isoelectric focusing cell. Heat is generated in the isoelectric focusing cell as a direct result of the power input to the isoelectric focusing cell by the electric potential across the focusing cell. The heat exchanger of the present invention may be coupled to the alternating reservoirs thereby cooling the fluid within the reservoirs. However, the heat exchanger is preferably coupled directly to the isoelectric focusing cell. Direct cooling of the isoelectric focusing cell permits greater power input to the focusing cell which results in rapid focusing of both the carrier ampholytes and the fluid sample.

The isoelectric focusing cell preferably includes means for separating the fluid flow into a plurality of focusing cell passageways. Each focusing cell passageway is coupled to corresponding inlet and outlet ports. One proposed method of separating the fluid flow into the focusing cell passageways is with ion non-selective

permeable membranes arranged in a substantially parallel configuration which divides the focusing cell into the respective passageways.

Sensors may also be included to sense fluid properties at various locations in the apparatus. For example, pH and temperature sensors provide valuable data regarding status of the separation as well as information useful for efficient separation. Such data may be transmitted to a computer to control, monitor, and record the isoelectric focusing process.

Similarly, the power input to the isoelectric focusing cell may be monitored and may be adjusted by the computer in response to the temperature of the fluid within the focusing cell to maximize the efficiency of the focusing process. Such a technique would permit maximum power input to the isoelectric focusing cell which would produce an acceptable temperature rise. Likewise, the multichannel pump may be controlled by the computer to balance fluid flow through the apparatus with power input and fluid sample separation.

In addition, the present invention includes novel apparatus and methods for isoelectric focusing of amphoteric substances within fluids containing carrier ampholytes which inhibit the phenomenon known as spiking or acid notching. The apparatus preferably includes bipolar membranes to separate the electrode compartments from the focusing cell compartments. Although less preferred, anion and cation selective membranes combined may replace the single bipolar membrane. It has been found that the use of bipolar membranes to separate the electrode chambers significantly increases the resolution and separation characteristics of amphoteric biological substances.

Conventional isoelectric focusing cells use a single ion selective membrane to confine the anolyte and catholyte

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1 solutions within the electrode chambers. An anion permeable membrane used at the cathode (negatively charged electrode) repels positively charged ions. A cation permeable membrane used at the anode (positively charged 5 electrode) repels negatively charged ions. These membranes effectively block the passage of low pH molecules into the anolyte and high pH molecules into the catholyte. The ion selective membranes alone do not prevent the passage of cations generated within the anode compartment or anions

10 generated within the cathode compartment from entering the focusing cell.

It has been found that during the isoelectric focusing process, positively charged ions enter the focusing cell from the anolyte solution and negatively charged ions enter

15 the focusing cell from the catholyte solution. As the length of time increases during focusing, sufficient quantities of these charged ions enter the focusing cell altering the pH near the electrodes. The negative ions are trapped in the channels of the focusing apparatus near the

20 anode, thereby lowering the pH. Similarly, the positive ions are trapped in the channels of the focusing apparatus near the cathode, thereby raising the pH.

Experimental results suggest that if anions and cations can be prevented from passing into the focusing

25 cell channels from the electrode chambers, spiking in the end focusing cell channels is significantly reduced. This result is achieved according to the currently preferred embodiment of the present invention by providing a barrier between the electrode chambers and the focusing cell

30 channels which combines the characteristics of an anionic membrane and a cationic membrane. Such a barrier preferably comprises a single bipolar membrane; however, the use of an anion and a cation selective membrane in combination also accomplishes the same result. The use of

35

a suitable bipolar membrane to separate the electrode chambers from the focusing cell chambers permits very shallow pH gradients to be obtained thereby enhancing the resolution and separation characteristics of amphoteric biological substances.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of the recycling isoelectric focusing apparatus within the scope of the present invention.

Figure 2 is a partial cut-away perspective view of a reservoir housing for holding pairs of alternating reservoirs within the scope of the present invention.

Figure 3 is a partial cut-away perspective view of an isoelectric focusing cell within the scope of the present invention.

Figure 4 is an exploded perspective view of a cell separator within the scope of the present invention.

Figure 5 is an exploded perspective view of an electrode separator having dual ion-selective membranes within the scope of the present invention.

Figure 6 is an exploded perspective view of an electrode separator having bipolar membrane within the scope of the present invention. Figure 7 is a horizontal cross-sectional view of the isoelectric focusing cell of Figure 3 taken along line 7-7 of Figure 3.

Figure 7.1 is an enlarged view of Figure 7 showing specific details of an electrode separator within the scope of the present invention.

Figure 7.2 is an enlarged view of Figure 7 showing specific details of a cell separator within the scope of the present invention.

Figure 8 is a vertical cross-sectional view of the isoelectric focusing cell of Figure 3 taken along line 8-8 of Figure 3.

Figure 9 is an exploded perspective view of another isoelectric focusing cell within the scope of the present invention.

Figure 10 is a graph of pH verses channel number illustrating the results of Examples 5 and 6.

Figure 11 is a graph of pH verses channel number illustrating the results of Example 7.

Figure 12 is a graph of milliamperes verses time illustrating the results of Example 7.

Figure 13 is a graph of voltage verses time illustrating the results of Example 7. Figure 14 is a graph of conductivity verses channel number illustrating the results of Example 7.

Figure 15 is a graph of pH verses channel number illustrating the results of Example 8.

Figure 16 is a graph of milliamperes verses time illustrating the results of Example 8.

Figure 17 is a graph of voltage verses time illustrating the results of Example 8.

Figure 18 is a graph of conductivity verses channel number illustrating the results of Example 8. Figure 19 is a graph of pH verses channel number illustrating the results of Example 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference is now made to the drawings wherein like parts are designated with like numerals throughout. As discussed generally above, the present invention is directed to apparatus and methods for isoelectric focusing of amphoteric substances, particularly biological materials. The apparatus of the present invention utilizes

preparative isoelectric focusing techniques which eliminate mixing of semipurified and crude amphoteric substances, which provide efficient removal of heat generated during the isoelectric focusing process, and which separate the electrolyte solutions from the separation channels with a barrier that minimizes the passage of both cations and anions into the separate channels.

A. Liquid Phase Isoelectric Focusing Apparatus

Referring first to Figure 1, a schematic view of the overall isoelectric focusing apparatus within the scope of the present invention is illustrated. An important concept of the present invention is eliminating the mixing of semipurified and crude amphoteric substances during recycling. Mixing is avoided by preferably using an alternating or dual reservoir system. Thus, a plurality of reservoirs 12, arranged in pairs, are provided. Reservoirs 12 are located in a reservoir housing 14 which is coupled to a coolant source 16 for cooling the reservoirs within reservoir housing 14.

One reservoir of each reservoir pair supplies the sample fluid to isoelectric focusing cell 20 and the other reservoir of each reservoir pair receives the fluid that has passed through isoelectric focusing cell 20. After the supply reservoir empties, fluid flow into isoelectric focusing cell 20 is automatically diverted from the supply reservoir to the receiving reservoir. Fluid flow from the isoelectric focusing cell into the receiving reservoir is then diverted into the supply reservoir. Thus, fluid flow into and out of each reservoir is automatically alternated after each pass through the isoelectric focusing cell.

Fluid flow is preferably alternated into and out of each reservoir pair through use of reservoir inlet valves 22 and reservoir outlet valves 24. The reservoir inlet and

outlet valves preferably operate in response to the fluid level within the reservoir supplying fluid to the isoelectric focusing cell. When the reservoir empties, as measured by fluid level sensors, the corresponding reservoir outlet valve automatically engages to stop fluid flow from the empty reservoir and to start fluid flow from the full reservoir. After appropriate time delay, the reservoir inlet valve engages to stop fluid flow into the full reservoir and to start fluid flow into the empty reservoir.

Also shown in Figure 1, the apparatus within the scope of the present invention preferably has coolant 16 coupled directly to isoelectric focusing cell 20, thereby providing direct removal of heat generated by the isoelectric focusing cell. The heat is generated by an electric potential across the isoelectric focusing cell. Power source 26 coupled to isoelectric focusing cell 20 provides the necessary electric potential. Direct cooling of the isoelectric focusing cell permits greater power input to the focusing cell which results in rapid focusing and separation of the fluid sample.

Sensors 28 may be provided to measure the temperature, pH or sample concentration in one or more of the outlet channels. Sensors 28 are preferably coupled to computer 80 which may control, monitor, and record the isoelectric focusing process. In addition, the power input to isoelectric focusing cell 20 from power source 26 may be monitored and adjusted by computer 30 in response to the temperature of the fluid within the focusing cell to maximize the efficiency of the focusing process. Similarly, multichannel pump 32 may be controlled by the computer to balance fluid flow through the apparatus with power input and fluid sample separation. Multichannel pump

32 returns the sample fluid exiting isoelectric focusing cell 20 to reservoirs 12.

Referring now to Figure 2, a reservoir housing 14 within the scope of the present invention is illustrated in a partial cut-away perspective view. Reservoir housing 14 is designed to accommodate a plurality of reservoirs arranged in pairs. Coolant is circulated around the reservoirs within reservoir housing 14 to remove heat generated during the isoelectric focusing process. Coolant enters reservoir housing 14 through reservoir coolant inlet 102 and exits reservoir housing 14 through reservoir coolant outlet 104.

Each pair of reservoirs, numbered generically as 106A and 106B, is coupled to an inlet channel 108 which directs fluid flow from the reservoir pair to the isoelectric focusing cell. Fluid flowing from the isoelectric focusing cell is directed to a corresponding reservoir pair through a plurality of outlet channels 110. Each outlet channel 110 is coupled to a corresponding reservoir pair. Fluid flow from each reservoir pair to the corresponding inlet channel 108 is controlled by a reservoir outlet valve. The reservoir outlet valve, as shown in Figure 2, includes a piston 112 having a series of gaskets 114 which direct fluid flowing from reservoir exit 116 into inlet channel 108. Piston 112 is operated by solenoids 118.

Reservoir inlet valves, which direct fluid flow from each outlet channel 110 into one reservoir of each reservoir pair, have a structure nearly identical to the reservoir outlet valves. Each reservoir inlet valve includes a piston 122 having a series of gaskets 124 which direct fluid flow from outlet channel 110 through reservoir entrance 126 and diffuser 128.

Diffuser 128 causes fluid as it passes through reservoir entrance 126 to flow towards the periphery of each reservoir and descend downward along the reservoir periphery. Diffuser 128 includes a bore 130 which terminates at base 132 defined by two slits 134. As fluid flows through bore 130, it contacts base 132 causing fluid to flow through slits 134 towards the reservoir wall. The fluid then flows down the reservoir wall into the reservoir. Because the fluid flows along the reservoir wall periphery, heat exchange between the sample fluid within the reservoirs and coolant flowing through the reservoir housing is provided.

As discussed above, fluid flow is alternated into and out of each reservoir of each reservoir pair through use of reservoir inlet and outlet valves. The reservoir inlet and outlet valves operate in response to the fluid level within the reservoir supplying fluid to the corresponding inlet channel 108. When the reservoir empties, as measured by fluid level sensors 136, solenoids 118 engage causing piston 112 and gaskets 114 to shift stopping fluid flow from the empty reservoir and starting fluid flow from the full reservoir. After appropriate time delay, the corresponding reservoir inlet valve engages to stop fluid flow into the full reservoir and to start fluid flow into the empty reservoir.

Reservoir housing 14 of Figure 2 also preferably accommodates two electrolyte solution reservoirs 138. Electrolyte solution reservoirs 138 hold the anolyte and catholyte fluids which circulate through the compartment of the isoelectric focusing cell which houses the anode and cathode respectively. Electrolyte solution reservoirs 138 are not configured in pairs because there is no need to avoid mixing of the electrolyte solution.

B. Isoelectric Focusing Cell

Referring now to Figures 3-8, one isoelectric focusing cell 20 within the scope of the present invention is illustrated. Isoelectric focusing cell 20 includes a plurality of inlet ports 142 and a plurality of corresponding outlet ports 144. Isoelectric focusing cell 20 preferably includes means for separating the fluid flow into a plurality of focusing cell passageways 146. Each focusing cell passageway is coupled to a corresponding inlet port 142 and outlet port 144.

Electrodes 148 are located within the isoelectric focusing cell in order to create an electric potential across focusing cell passageways 146. Electrodes 148 may be constructed of platinum or any other suitable electrode material used in isoelectric focusing devices.

An electrolyte solution preferably surrounds each electrode 148. The electrolyte fluids associated with the anode and cathode are generally referred to as the anolyte and catholyte fluids, respectively. In conventional isoelectric focusing instruments, the anolyte is often a dilute solution of a strong acid, such as sulfuric or phosphoric acid, which flows in the anode or positive electrode compartment 150. The catholyte is often a dilute solution of a strong base, such as sodium hydroxide, which flows in the cathode or negative electrode compartment 152.

Other electrolyte solutions may also be used. For instance, narrow and stable pH gradients have been obtained using glycine, potassium nitrate (KN0 ₃ ) , and even water, as electrolyte solutions. Suitable results may be obtained by matching the anolyte and catholyte as closely as possible to the lower and upper pH of the pH gradient using 0.1 M glycine with the pH adjusted using either HCl or NaOH.

However, it has been found that the choice of electrolyte solutions has little effect on the formation of stable and

narrow pH gradients according to the principles of the present invention. In fact, experimental results suggest that the pH gradient formation in the present invention is strongly a function of ampholyte solution and not the electrolyte solution.

In one possible embodiment within the scope of the present invention each electrode compartment 150 and 152 is separated from focusing cell passageways 146 by both an anionic or anion selective membrane 153 and a cationic or cation selective membrane 155. The anionic and cationic membranes should preferably be ion-selective membranes of the type used in electrodialysis and ion exchange processes. Satisfactory ion-selective membranes are manufactured by Sybron Chemicals Inc. , Birmingham, New Jersey. Typical properties of ion-selective membranes which have been found suitable are summarized in Table 1.

Water Permeability

(ml/hr/ft ² /5psi) <30 <50 Membrane Thickness (mils) 16 16

The anionic and cationic membranes preferably form part of an electrode separator 154, shown best in Figures 3, 5, 6, 7 and 7.1. In Figures 3, 5, 7 and 7.1, each electrode separator includes an anionic membrane 153 and a cationic membrane 155 sandwiched between two supports 160 and two gaskets 162. In an alternative embodiment shown in Figure 6, the electrode separator includes a single bipolar membrane 157 sandwiched between two supports 160 and two gaskets 162. The bipolar membrane 157 possesses characteristics of both anionic and cationic selective membranes 153 and 155, respectively.

In fact, a single bipolar membrane is currently preferred over the use of dual anionic and cationic membranes. In some cases using dual anionic and cationic membranes fluid becomes trapped between the membranes. Without the capacity to circulate and cool the trapped fluid, the fluid may overheat and damage the membranes or otherwise limit the efficiency of the isoelectric focusing device. Thus, to avoid the possibility of fluid becoming trapped between the anionic and cationic membranes, use of a single bipolar membrane is currently preferred. The typical physical properties of one suitable bipolar membrane used within the scope of the present invention is approximated by the combined properties of the anionic and cationic membranes identified in Table 1. Supports 160 are generally of a material providing adequate support while being neutral in the electrical field and nonreactive with the electrolyte solutions and with the ampholytes. Supports 160 may be suitably constructed of ultra high molecular weight polyethylene. Gaskets 162 are preferably constructed of an elastic, nonreactive material such as neoprene. Other suitable materials with sealing capacity and neutrality, such as VITON ^® polymers manufactured by DuPont, may also be satisfactory under certain conditions. Isoelectric focusing cell passageways 146 are preferably separated by a plurality of cell separators 156. As shown more clearly in Figures 4, 7 and 7.2, each cell separator includes five components sandwiched together. Each cell separator includes a screen 158, two supports 160, and two gaskets 162.

Screen 158 is preferably constructed of a biocompatible mesh material such as nylon or teflon. It has been found that a pore size as large as 100 microns is

suitable for most amphoteric substances separated by isoelectric focusing.

Biocompatibility is an important characteristic for all components of the isoelectric focusing apparatus within the scope of the present invention. The term biocompatible material used in this specification includes materials which will not denature sensitive proteins being separated or react with or contaminate the proteins.

Generally, the amphoteric substances separated by isoelectric focusing have a molecular weight less than about 500,000. Higher molecular weight substances may be used, provided they remain in solution long enough to be separated effectively by isoelectric focusing. As a general rule, the larger the amphoteric substance, the lower the solubility. In many cases solubility can be increased by using suitable solubilizing agents. Thus, the limiting factor in determining suitable amphoteric substances is often the molecular weight of the amphoteric substance, and not the pore size of screen 60. it is currently preferred that the solubilizing agents be nonionic and not chemically reactive with either the protein or the ampholytes. Urea, for instance, at 1-5 Molar concentration has been used to enhance solubility without denaturing the proteins being separated. Non-ionic detergents such as Nonidet P-40 and low concentrations (5%- 10%) butanol have been used successfully. Basically, any nonionic substance can be added to the amphoteric substances to enhance solubility. For example, glycerol could be added to enhance the solubility of hydrophobic molecules.

An isoelectric focusing cell within the scope of the present invention may also include coolant chambers 164 for direct cooling of fluid flowing through the isoelectric focusing cell. Each coolant chamber 164 has a coolant

inlet 166 and a coolant outlet 168. Baffles (not shown) located within coolant chambers 164 create turbulent coolant flow which promotes heat transfer. Coolant flowing through coolant chambers 164 and reservoir housing 10 is preferably maintained around 4°C. One preferred coolant is a 20 percent solution of ethanol to prevent freezing in a refrigeration unit (not shown) .

Another isoelectric focusing cell within the scope of the present invention is illustrated in Figure 9. The isoelectric focusing cell of Figure 9 may be adjusted to increase or decrease the number of focusing cell passageways in the device. As shown in Figure 9, individual focusing cell elements 170 may be positioned between alternating focusing cell separators (illustrated in Figure 4) . Electrode separators 154 (illustrative embodiments of which are shown in Figures 5 and 6) may be used to separate the electrode compartments from the individual focusing cell elements 170.

Fluid flows into each focusing cell element through an inlet port 172 and exits each focusing cell element through a corresponding outlet port 174. Utilizing the embodiment illustrated in Figure 9, it is possible to customize the isoelectric focusing cell into the ideal number of focusing cell passageways required to effect the desired separation. A larger number of focusing cell passageways would be useful to separate two or more proteins which are very close to one another in pi (i.e. , pi of 6.4 and pi of 6.8) . Thus, in a narrow pH gradient made with narrow range ampholytes, a cell with many passageways would help separate the two proteins more efficiently.

Fewer focusing cell passageways would be needed to separate two proteins which have very different pi (i.e.. pi of 4 and pi of 8) . These would be separated in a wide

range gradient and only 3-4 passageways might be necessary for effective separation.

All materials used to construct those portions of the isoelectric focusing apparatus within the scope of the present invention which contact the fluid sample are preferably constructed of biocompatible materials. Biocompatible materials are preferred because most amphoteric substances to be separated are biomaterials such as proteins, peptides, nucleic acids, viruses, and even some living cells. Thus, it is important that all surfaces which contact the amphoteric substances are inert with respect to the amphoteric substances.

For example, upper and lower manifolds of reservoir housing 14 are preferably constructed of ultra-high molecular weight polymer (UHMW 1900, manufactured by High

Mowt) . Tubing used in the apparatus is preferably constructed of teflon. The isoelectric focusing cell is preferably constructed of acrylic. The reservoir tubes are preferably constructed of pyrex glass. In addition, manual valves (not shown) for filling and draining the isoelectric focusing system are preferably constructed of polycarbonate plastic. As discussed above, all materials used to construct the apparatus which will contact the amphoteric substances are preferably constructed of materials suitable for surgical or medical use.

In one possible mode of operation using the system described in conjunction with Figure 1, the solutions are gravity fed from each reservoir pair within reservoir housing 14 to isoelectric focusing cell 20. The isoelectric focusing cell is preferably vertically oriented, i.e.. focusing cell separators 156 are in the vertical direction.

In another possible mode of operation, the fluid is drawn through the isoelectric focusing cell against gravity

^■** - due to the suction forces of the pump. It has been observed that forcing the fluid through the focusing cell with pressure from the pump results in a more unstable flow pattern than when fluid is drawn through the focusing cell with suction forces from the pump.

The selected buffer solution containing carrier ampholytes suitable for establishment of a stable pH gradient is loaded into one of the two reservoirs of each reservoir pair. A commercially available carrier ampholyte 0 solution is suitable. An electrolyte solution is loaded into those reservoirs coupled to electrode compartments 150 and 152. These electrode rinses are allowed to flow upward through isoelectric focusing cell 20 to permit venting of gases generated by electrolysis. 5 After equilibration of fluid flow and temperature, electric power is applied from power source 26. Typically, a gradient of about 200 volts to about 250 volts at a constant current is sufficient to cause rapid equilibration. Power input into the focusing cell is 0 maintained relatively constant throughout the separation process. Typical power input is in the range from about 30 watts to about 60 watts. It has been found that the width of the pH gradient significantly affects the time to establish equilibrium. Narrow pH gradients may take longer 5 to form than wide pH gradients.

As focusing of the carrier ampholytes occurs, the voltage may be increased up to about 500 to about 600 volts in order to maintain current flow. The maximum power input is limited by the allowable temperature rise in the 0 apparatus due to Joule heating. For this reason, continuous or periodic temperature monitoring is preferred. Typically, a reservoir temperature of 4 ^° C is maintained. The temperature may increase up to about 10°C without causing damage to most biomaterials. 5

In practice, the carrier ampholyte solution is circulated through the apparatus in order to establish a relatively stable pH gradient before the sample material is added. The sample material is then preferably added only to the channel or compartment having a pH relatively close to the pi of the one protein which is desired. It is possible to add the sample material to be focused at the beginning of the operation. Nevertheless, this will cause some of the material to be exposed to extremes of pH in the compartments adjacent to the electrodes. This may damage some pH sensitive biomaterials.

In addition, the protein of interest might be ionically altered by exposing it to the whole ampholyte mixture. For example, a protein with acidic pi would be brought into contact with the whole ampholyte mixture if mixed in with the beginning ampholytes prior to running. Some or all of the protein of interest could then bind to some basic ampholytes, thus creating artificial heterogeneity of charge. This might cause all or part of the protein of interest to focus at a different pH than it should. When added to the reservoir containing ampholytes at or very close to the pi of the protein, it never encounters extreme pH ampholytes, thus retaining its inherent charge and pi. A series of experiments were conducted to detect the difference in mass protein migration between a conventional recirculating isoelectric focusing device ("RIEF") which remixes semipurified fluid with the crude sample fluid and an isoelectric focusing device within the scope of the present invention which alternates fluid flow between pairs of reservoirs. The following examples are intended to be purely exemplary of the use of the invention and should not be considered limiting as to the scope of the present invention.

Example 1

In this Example, the difference in mass protein migration between a conventional RIEF device obtained from Ionics, Inc. of Watertown, Massachusetts, and corresponding to the device described in U.S. Patent Nos. 4,204,929 and 4,362,612 (hereinafter referred to as "mixing device") and an alternating isoelectric focusing device within the scope of the present invention (hereinafter referred to as "alternating device") was determined. Both the mixing and alternating devices were loaded with 1000 ml of solution containing 1 percent pH specific carrier ampholyte. The solution was prefocused for two hours to establish a pH gradient. Ten (10) milligrams of radioactive labeled Cytochrome C protein was introduced into channel 2 of both devices and allowed to circulate for 15 minutes. At that point, the mixing and alternating devices were turned off and all eight channels from each device were collected individually. A 0.5 ml sample was removed from each channel and counted for the presence of the radioactive labeled protein. The total volume of each channel was measured and the total presence of protein in each channel was calculated using the following formula: counts per minute (cpm)/0.5 ml x 2 x volume ^*■*** total counts per channel; counts per channel/total counts channel = fraction of total in a given channel.

The results of this Example show that after 15 minutes, 42% of the protein was still in channel 2 of the mixing device, while only 18% of the protein remained in channel 2 of the alternating device. Table 2 contains the experimental results for Example 1.

Table 2 Solution of 1000 ml 1% w/v ampholytes with 2 hour prefocus

10 g Cytochrome C + 500 μL 1-125 labeled Cytochrome C into channel 2 with a 15 minute run time.

Mixin Device of the Prior Art:

Example 2 In this Example the difference in mass protein migration between a mixing device and an alternating device was determined according to the procedure at Example 1 except that the devices were allowed to focus for 30 minutes after injecting the radio labeled protein before being turned off.

A 0.5 ml sample from each channel was taken and analyzed for the presence of radioactive protein. The results of the 30 minute run times show 13.7% of the protein activity still remaining in channel 2 of the mixing device. The alternating device showed only 3.7% of the protein activity remaining in channel 2. It is believed the 3.7% measured protein activity represents a reading of unbound radioactive iodine and not the presence of the Cytochrome C protein. Table 3 contains the experimental results for Example 2.

Table 3 Solution of 1000 ml 1% w/v ampholytes with 2 hour prefocus 10 mg. Cytochrome C + 500 μL 1-125 labeled Cytochrome C into channel 2 with a 30 minute run time.

2020 860 393

Example 3 In this Example, the difference in mass protein migration between a mixing device and an alternating device was determined according to the procedure of Example 1, except that the devices were allowed to focus for 45 minutes after injecting the radiolabeled protein before being turned off.

The results of the 45 minute run times show that both devices were virtually void of protein in channel 2. Residual amounts of radiation were detected, which were believed to represent readings of unbound free iodine. Table III contains the experimental results for Example 3.

Table 4

Solution of 1000 ml 1% w/v ampholytes with 2 hour prefocus 10 mg. Cytochrome C + 500 μL 1-125 labeled Cytochrome C into channel 2, with a 45 minute run time.

Mixing Device: Channel

1

2

3 4

5

6

7

8

The foregoing experimental results demonstrate that the alternating isoelectric focusing device within the scope of the present invention provides significantly more rapid mass migration of protein out of a given pH channel as opposed to conventional recycling isoelectric focusing devices. This is particularly important because protein migration out of a channel is of prime importance when normal operation protocol is observed.

Under normal separation procedure using equipment within the scope of the present invention, the ampholytes are prefocused to establish a pH gradient across the focusing cell passageways. The sample introduced into the prefocused machine is usually a mixture of contaminants and the desired protein. The sample is preferably injected into a channel nearest the isoelectric point of the desired protein. In that channel the desired protein will not migrate, but remain at that point in the pH gradient. The contaminant evacuation from the channel containing the protein of interest is of greatest importance in rapidly obtaining the purified protein. The foregoing examples support the proposition that greater levels of purity can

be achieved in a shorter period of time by not remixing the content of a channel.

A series of additional experiments were conducted to determine the effect the pH of the electrolyte solutions had on the final pH gradient in the isoelectric focusing apparatus. These experiments were also intended to understand how to control spiking or acid notching so that high resolution separations could be more easily realized.

Example 4

In this example, the effect of electrolyte solution pH on the ultimate pH gradient was determined using an eight channel isoelectric focusing device having a single anion selective membrane separating the catholyte solution from the ampholytes and a single cation selective membrane separating the anolyte solution from the ampholytes. The catholyte solution was 0.1 M sodium hydroxide having a pH of about 12.5. The anolyte solution of 0.1 M phosphoric acid having a pH of about 2.3. Narrow range ampholytes designed to produce a pH gradient from pH 7 to pH 9 were used. Upon focusing the apparatus, spiking was observed in those channels adjacent to the electrolyte solutions.

Example 5 In this example, the effect of electrolyte solution pH on the ultimate pH gradient was determined using an eight channel isoelectric focusing device having a single anion selective membrane separating the catholyte solution from the ampholytes and a single cation selective membrane separating the anolyte solution from the ampholytes. The anolyte solution was glycine buffered to a pH of 6.0. The catholyte solution was glycine buffered to a pH of 8.0. Narrow range ampholytes designed to produce a pH gradient from pH 7 to pH 9 were used. Upon focusing the apparatus,

spiking was observed in those channels adjacent to the electrolyte solutions, but to a lesser degree than that of Example 4. The results of this Example are illustrated in Figure 10.

Example 6 In this example, the effect of electrolyte solution pH on the ultimate pH gradient was determined using an eight channel isoelectric focusing device having both anion and cation selective membranes separating the anolyte and catholyte solutions from the ampholytes. The anolyte solution was glycine buffered to a pH of 6.0. The catholyte solution was glycine buffered to a pH of 8.0. Narrow range ampholytes designed to produce a pH gradient from pH 7 to pH 9 were used. Upon focusing the apparatus, almost no spiking was observed in those channels adjacent to the electrolyte solutions.

Figure 10 provides a graphical comparison of the results from Examples 5 and 6. The results suggest that using a dual ion-selective membrane and appropriately buffered electrolyte solutions that very narrow pH gradients can be successfully achieved, thereby permitting high resolution separation of amphoteric substances.

Example 7

In this example, the effect of electrolyte solution on the ultimate pH gradient was determined using an eight channel isoelectric focusing device having bipolar membranes separating the anolyte and catholyte solutions from the ampholytes. The anolyte and catholyte solutions were both 0.1 M glycine. Upon focusing the apparatus, almost no spiking was observed in those channels adjacent to the electrolyte solutions. The procedure was followed three (3) times to establish baseline operation.

Repeatable pH gradients were generated when using 0.1 M unadjusted glycine. The results are shown graphically in Figures 11-14.

Example 8

In this example, the effect of different electrolyte solutions on the ultimate pH gradient was determined using an eight channel isoelectric focusing device having bipolar membranes separating the anolyte and catholyte solutions from the ampholytes. The following anolyte and catholyte solutions were tested: 0.1 M glycine (anolyte and catholyte); 0.1 M KN0 ₃ (anolyte and catholyte); 0.1 M H ₃ P0 ₄ (anolyte)/0.1 M NaOH (catholyte); and water (anolyte and catholyte) . Upon focusing the apparatus, almost no spiking was observed in those channels adjacent to the electrolyte solutions. The results are shown graphically in Figures 15-18.

The pH gradient and conductivity gradient from all runs using the different electrolyte solutions are virtually identical. This suggests that the pH gradient is a function of the ampholyte solution. Outside variables appear to have no substantial effect on the formation of a stable, reproducible pH gradient. The volts and milliamperes follow the same pattern, but have different end points for the different electrolyte solutions. It is currently believed that this may be attributed to the differences in conductance of the electrolytes.

Example 9 In this example, narrow pH gradients were established that were stable over time. The narrow gradients were formed from a 5% ampholyte solution which yielded the same pH gradient as that produced in Example 7. After focusing, individual channels were collected, diluted to a 1%

solution, distributed in all 8 channels of the isoelectric focusing device, and refocused. Figure 19 illustrates the pH established by the subsequent focusing of the contents of channels 1 and 2. Data showing the results from focusing the other channels of the 5% run are not included, but they show similar narrow gradients. The gradients formed by the 1% runs have an average pH of the corresponding 5% channel fractionation.

It is currently believed that spiking is caused by ions entering the focusing cell due to electrolysis at the electrodes. During focusing, the high voltage at the electrodes surface causes electrolysis of water. At the cathode, the electrolysis reaction is:

2H ₂ 0 + 2e- => H ₂ + 20H ^"

Hydroxy1 ions (OH ^" ) are generated at the cathode during the electrolysis of water. Because conventional isoelectric focusing devices only use an anion selective membrane to separate the electrolyte from the focusing channels, it is suggested that H ^* ions pass through the anion selective membrane into the focusing chamber and move under the influence of the electric field into the channel bounding the anode, thereby eventually lower the pH of that channel. At the anode, the electrolysis reaction is:

2H ₂ 0 => 0 ₂ + 4H ⁺ + 4e ^'

Hydrogen ions (H ⁺ ) are generated at the cathode during the electrolysis of water. Because conventional isoelectric focusing devices only use a cation selective membrane to separate the electrolyte from the focusing channels, it is suggested that OH ^" ions pass through the cation selective membrane into the focusing chamber and move under the

influence of the electric field into the channel bounding the cathode, thereby eventually raising the pH of that channel.

The use of a single bipolar membrane or dual anionic and cationic selective membranes to separate the electrolyte chambers and the focusing chambers dramatically reduces the movement of positively and negatively charged ions from the electrode compartments into the focusing cell compartments. As a result, almost no spiking or acid notching is observed over time so that very shallow pH gradients can be produced enabling high resolution separations.

In practice, the sample fluid from a first reservoir of each reservoir pair is pumped through the isoelectric focusing cell and is returned to the second reservoir of the reservoir pair which is initially empty. Reservoir inlet and outlet valves at the bottom and top of the reservoir housing, preferably in the form of solenoid driven pistons will automatically direct the fluid flow in an alternating fashion between the two reservoirs with the solenoids activated by fluid level sensing devices at the base of each reservoir. As one reservoir empties initially, the other reservoir slowly fills. When the reservoir empties, its fluid level sensor actuates the corresponding reservoir outlet valve for the reservoir pair and closes the reservoir exit for that reservoir and near instantaneously opens the reservoir exit for the other, recently filled, reservoir of the reservoir pair.

After appropriate time delay, the reservoir inlet valve at the top of the reservoir housing switches the returning fluid flow from the isoelectric focusing cell to start filling the empty reservoir. Thus, as fluid sensors inside each reservoir detect fluid levels dropping, they send signals to a controller which activates the solenoids

connected to the reservoir inlet and outlet valves connected to each reservoir pair. These reservoir switching actions take place independently for all separation channels. A reservoir pair is preferably coupled to each separation channel.

The number of focusing cell passageways and accompanying reservoir pairs could vary from two (2) to fifty (50) , but will normally be in the range from about eight (8) to about twelve (12) . Thus, in addition to the reservoirs for anolyte and catholyte solutions, the reservoir housing would include two reservoirs for each separation channel in the isoelectric focusing cell. For the isoelectric focusing cell illustrated in Figure 2 having eight (8) separation channels, a total of 18 reservoirs would be needed in the reservoir cooling chamber, two (2) reservoirs for each separation channel, and one (1) anolyte reservoir, and one (1) catholyte reservoir.

The basic isoelectric focusing system within the scope of the present invention can include both pH and temperature sensors at the inflow or outflow side of each reservoir, which could be interfaced with a microcomputer for real time monitoring of both pH and temperature of the fluid coming from the focusing cell. The computer may also control the power supply to the focusing cell and also control the flow rate of the multichannel pump which directs the fluid movement in all separation channels.

The computer can also control other valves (not shown) at the inlet and outlet ports to the isoelectric focusing cell which would empty the contents of each reservoir at the end of a separation run into a separate collection vessel. The computer would then activate valves and pumps to refill the reservoirs with starting material from a central holding reservoir and start a new run from the

beginning, thereby permitting continuous processing of fluid samples.

It has been observed that periodic disruption of the fluid flow and of the electric current flow may improve the separation efficiency of the isoelectric focusing apparatus. Although not fully understood, it is currently believed that "dead spots" form within the focusing cell during operation. By periodically turning the apparatus "off," amphoteric substances within the dead spots is removed and circulated with the remainder of the fluid through the isoelectric focusing apparatus.

Although the optimum disruption protocol has not yet been established, it has been observed that more efficient separation is achieved by turning the power off for about 30 seconds approximately every 15 minutes and by briefly reversing the pump.

From the foregoing, it will be appreciated that the present invention provides apparatus and methods for isoelectric focusing of amphoteric substances which combine high resolution separation with large quantitative sample capacity.

Additionally, it will be appreciated that the present invention provides apparatus and methods for recycling isoelectric focusing of amphoteric substances which do not mix semipurified sample with the crude sample.

Likewise, it will be appreciated that the present invention provides apparatus and methods for isoelectric focusing of amphoteric substances which efficiently remove heat generated during the process, thereby permitting increased power input.

It will also be appreciated that the present invention provides apparatus and methods for isoelectric focusing of amphoteric substances which rapidly separate the desired substance from accompanying impurities.

In addition, it will be appreciated that the present invention provides apparatus and methods for isoelectric focusing of amphoteric substances which rapidly prefocus to establish a stable pH gradient.

It will be also be appreciated that the present invention provides apparatus and methods for isoelectric focusing of amphoteric substances which inhibit spiking or acid notching.

Finally, it will be appreciated that the present invention provides apparatus and methods for isoelectric focusing of amphoteric substances which enable narrow pH gradients to be obtained and maintained without significant spiking in the separation channels adjacent the electrodes.

It will be appreciated that the apparatus and methods of the present invention are capable of being incorporated in the form of a variety of embodiments, only a few of which have been illustrated and described above. The invention may be embodied in other forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive, and the scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.