Bismuth generator method

FIELD OF THE INVENTION
The present invention relates generally to a method for purifying a solution of radionuclides or more specifically to a method for separation of .sup.213 Bi from a solution or mixture of radionuclides containing .sup.225 Ac, .sup.225 Ra, .sup.213 Bi and daughters therefrom.
BACKGROUND OF THE INVENTION
The widespread recognition of the use of radiation to kill or neutralize unwanted cell growth such as cancer has led to increasing interest in various species of radionuclides. Of particular interest are radionuclides, such as .sup.213 Bi, which emit alpha radiation, or alpha emitters, because the alpha radiation emitted by these radionuclides does not penetrate deeply into tissue. .sup.213 Bi is normally produced as a daughter product of .sup.229 Th (t.sub.1/2 =7,300 y). The radioactive decay chain in which .sup.213 Bi is found is well known: .sup.233 U (t.sub.1/2 =1.62.times.10.sup.5 yr).fwdarw..sup.229 Th (t.sub.1/2 =7,300 yr).fwdarw..sup.225 Ra (t.sub.1/2 =14.8 day).fwdarw..sup.225 Ac (t.sub.1/2 =10 day).fwdarw..sup.213 Bi (t.sub.1/2 =46 min). The daughters of interest for biological applications include .sup.225 Ra (t.sub.1/2 =14.8 d) which decays to .sup.225 Ac (t.sub.1/2 =10.0 d). .sup.225 Ac in turn decays through a series of steps to .sup.213 Bi (t.sub.1/2 =46 min). Thus, a Bi generator can have as the starting material either .sup.225 Ac, separated from the parents, or a mixture of .sup.225 Ra/.sup.225 Ac. There are advantages and disadvantages to the use of .sup.225 Ra as a starting material. If .sup.225 Ra is not separated from the .sup.225 Ac, the amount of Ac in terms of available radioactivity as a function of time is greatly extended. However, if the .sup.225 Ra also contains a fraction of .sup.224 Ra, because the original thorium "cow" contained both .sup.229 Th and a small percent of .sup.228 Th/.sup.224 Ra, separation to remove the radium may be desirable. The daughters of .sup.224 Ra include .sup.212 Bi and .sup.208 Tl which may be undesirable from a patient biological perspective.
Briefly, by placing alpha emitters adjacent to unwanted cell growth, such as a tumor, the tumor may be exposed to the alpha radiation without undue exposure of surrounding healthy tissue. In many such schemes, the alpha emitter is placed adjacent to the tumor site by binding the alpha emitter to a chelator which is in turn bound to a monoclonal antibody which will seek out the tumor site within the body. Unfortunately, in many instances, the chelator will also bind to metals (Fe, Cr, RE's) other than the desired alpha emitter. Since a tumor may have a limited number of sites available for binding with the monoclonal antibody, it is desirable that the number of monoclonal antibodies bonded to metals other than the desired alpha emitter be minimized. Thus, it is desirable that the alpha emitter be highly purified from other metal cations. In addition, alpha emitters such as .sup.213 Bi (t.sub.1/2 =46 min) have very short half-lives. Thus, to utilize these short lived radionuclides effectively in medical applications, they must be efficiently separated from other metals or contaminants in a short period of time to maximize the amount of the alpha emitter available. A more detailed description of the use of such radionuclides is found in the articles "Alpha-emitting bullet targets leukemia cells", Nuclear News, June 1996, pp 47-48, (Dr. David Scheinberg, Memorial Sloan Kettering Cancer Center, New York, N.Y.); and Pippin, C. Greg, Otto A. Gansow, Martin W. Brechbiel, Luther Koch, R. Molinet, Jaques van Geel, C. Apostolidis, Maurits W. Geerlings, and David A. Scheinberg, 1995; "Recovery of Bi-213 from an Ac-225 Cow: Application to the Radiolabeling of Antibodies with Bi-213", Chemists' Views of Imaging Centers, Edited by A. M. Emran, Pleaum Press, New York, N.Y.
Various methods to separate bismuth from other radionuclides have been developed over the last few years. However, with the need for increasing amounts of alpha emitting radionuclides, it has been recognized that better generator designs are required (see Scheinberg). Recent work designed to develop Bi generators has focused on the use of an actinium-loaded organic cation exchange resin and is described in Pippin, C. Greg, Otto A. Gansow, Martin W. Brechbiel, Luther Koch, R. Molinet, Jaques van Geel, C. Apostolidis, Maurits W. Geerlings, and David A. Scheinberg, 1995, "Recovery of Bi-213 from an Ac-225 Cow: Application to the Radiolabeling of Antibodies with Bi-213", Chemists' Views of Imaging Centers, Edited by A. M. Emran, Pleaum Press, New York, N.Y.; Wu, C., M. W. Brechbiel, and O. A. Gansow, 1996, An Improved Generator for the Production of Bi-213 from Ac-225, American Chemical Society Meeting, Orlando, Fla., August, 1996; and Mirzadeh, Saed and Stephen J. Kennel, 1996, Optimization of Radiolabeling of Immunoproteins with Bi-213, American Chemical Society Meeting, Orlando, Fla., August, 1996.
The major problem with these organic cation exchange methods is that, with the need for larger amounts of ".sup.225 Ac cow" (1-20 mCi), the generator is limited by the early destruction of the actinium-loaded organic cation exchange resin, which is exposed to alpha radiation damage (usually for 20 days during its use). Attempts to minimize this destruction have been employed by Dr. Wu at the National Institute of Health (see Wu). Under this type of approach, the actinium is absorbed onto a small portion of the organic resin in a batch mode. The loaded ion exchange beads are then mixed with non-loaded beads to "dilute" the destructive effect, when placed in an ion exchange column used for Bi separation. Still, under this approach, the separation of the Bi from the organic resin requires at least two loading and elution steps resulting in a total separation time exceeding one hour.
Another inorganic ion exchange "generator" concept, has been developed by Gary Strathearn, Isotope Products Laboratories, Burbank, Calif. and is described in Ramirez Ana.R. and Gary E. Strathearn, 1996, Generator System Development of Ra-223, Bi-212, and Bi-214 Therapeutic Alpha-Emitting Radionuclides, American Chemical Society Meeting, Orlando, Fla., August, 1996. In this approach, inorganic polyfunctional cation exchangers are used to avoid damage from the intense alpha bombardment. A column of Alphasept-1.TM. is pretreated with nitric acid (HNO.sub.3), the .sup.225 Ac in 1M HNO.sub.3 feed is then loaded on to the column and the .sup.213 Bi product is eluted with 1M HNO.sub.3. The product HNO.sub.3 must then be evaporated to dryness to remove the nitric acid. It is then brought back into solution with a suitable buffered solution to prepare the final binding of the alpha emitter to a chelator and monoclonal antibody. The evaporation step extends the time required to prepare the final product and thus limits the usefulness of this approach. Thus, a need still exists for a method for rapid separation of high purity .sup.213 Bi from a solution or mixture of radionuclides containing .sup.225 Ac, .sup.225 Ra, .sup.213 Bi and daughters therefrom.
SUMMARY OF THE INVENTION
The present invention is thus a new .sup.213 Bi generator system. Several goals for this new generator system include resistance to the intense alpha bombardment from the .sup.225 Ac "cow", a high recovery of .sup.213 Bi with minimal ion exchange column breakthrough of .sup.225 Ac, minimization of the steps and time required to obtain the radiopharmaceutical, high purity of the .sup.213 Bi which can be easily and reproducibly linked to monoclonal antibody systems ("smart bullets"), and a system which can be easily handled by radiopharmacy technicians.
The present invention relies on the use of an organic anion exchange resin to adsorb .sup.213 Bi from an ".sup.225 Ac cow." Instead of storing the .sup.225 Ac on a cation resin for .about.20 days as is common in prior art separation schemes during use and milking the .sup.213 Bi off of the cation resin, the ".sup.225 Ac cow" is stored in an acid solution and passed quickly through an anion exchange resin, preferably an anion exchange resin bond in a thin film or "web", to collect the .sup.213 Bi when needed. This greatly accelerates the separation and purification process and has been demonstrated to allow separation and purification of .sup.213 Bi in times of under 6 minutes. The ability to extract bismuth as an anion as a function of HCl concentration is well known and is described in Kraus, K. A. and F. Nelson, 1955, Proceedings of the International Conference on the Peaceful Uses of Atomic Energy, Nuclear Chemistry and the Effect of Irradiation, Vol. VII, P/837, "Adsorption of the elements from hydrochloric acid", held in Geneva, Aug. 8-20, 1955. Briefly, the distribution for the bismuth chloride complex anion in HCl increases with decreasing acid concentration, while the distribution for Fe increases with acid concentration. Other chelator interfering ions of interest, i.e., rare earths, radium (Ra), francium (Fr), and actinium (Ac) do not extract as chloride anions using anion exchange resin. Therefore, the use of the anion exchange resin allows .sup.213 Bi to be effectively removed from these and other interfering ions which do not extract as chloride anions using anion exchange resin.
Bismuth(III) is a cation in most solutions. However, when placed in a solution containing chloride ions (Cl.sup.-), Bi may, under the correct conditions, be absorbed from solution as an anionic complex. The efficiency of the absorption is dependent on the hydrogen ion concentration. Thus, according to the present invention, an .sup.225 Ac cow is first contained in a hydrochloric acid solution wherein the concentration of the chloride ions and the hydrogen ions are adjusted to levels which allow the formation of the chloride complex. Preferably, the HCl concentration is at a level which maximizes the absorption of the chloride complex, or between about 0.25M and about 1M, however, much higher concentrations do allow absorption, but at distribution levels below optimum. The cow solution is then brought into contact with an anion exchange resin, whereupon the daughter product of the .sup.225 Ac, .sup.213 Bi, is absorbed from the cow solution and adhered onto the anion exchange resin in the chloride complex. By using organic anion exchange resin, the bismuth extraction distribution ratio increases with decreasing acid concentration. As will be appreciated by those skilled in the art, a wide variety of anion exchange resins, including but not limited to Anex, manufactured by Sarasep Corp., Santa Clara, Calif., and Dowex-1, manufactured by Dow Chemical Company, are suitable for the practice of the present invention, the selection of the particular of the resin being dependant upon the needs or convenience of the particular user. As practiced by the present invention, the anion exchange resin is configured to allow rapid adherence and stripping of the .sup.213 Bi, thereby minimizing the time necessary for the separation and purification of the .sup.213 Bi. In a preferred embodiment of the present invention, the anion exchange resin is provided as a thin membrane, which allows the rapid adherence and stripping of the .sup.213 Bi.
Once the chloride complex containing the .sup.213 Bi is absorbed on the anion exchange resin, the resin may then be washed to remove traces of the ".sup.225 Ac cow" and acid from the anion exchange resin, leaving the anion exchange resin with purified .sup.213 Bi adhered as a chloride complex. Finally, the .sup.213 Bi is removed from the anion exchange resin by washing the anion exchange resin with a stripping solution. The stripping solution, free of chloride ions, and with a reduced hydrogen ion concentration in solution, breaks the chloride anionic complex and releases the .sup.213 Bi as a cation. Again, as will be recognized by those skilled in the art, a wide variety of stripping solutions are suitable for breaking the chloride complex and releasing the .sup.213 Bi. The selection of the stripping solution is thus made based upon considerations for further use of the .sup.213 Bi. In general, elution solutions having a pH in the neutral �pH 4-10! range, and more preferably a pH of greater than about 2, and which are substantially free of chloride ions are effective. Suitable stripping agents thus include water, complexants such as DTPA, and sodium acetate (NaOAc). By way of example, a preferred eluant for purification of .sup.213 Bi for use in medical applications may be a solution that will provide for the addition and linking of the .sup.213 Bi to a chelator and monoclonal antibody. The stripping solution may thus also contain the proteins conjugated with bifunctional chelating agents in vivo to receive the .sup.213 Bi as it is being released from the anion exchange resin, thereby shortening the total time for linking preparation before being received by the patient.
Objects
It is therefore an object of the present invention in one of its embodiments to provide a method for separating and purifying .sup.213 Bi from a solution of radionuclides.
It is a further object of the present invention in one of its embodiments to combine a solution containing radiochemical .sup.225 Ac and hydrochloric acid, with an anion exchange resin, whereupon the daughter product of the .sup.225 Ac, .sup.213 Bi, is absorbed the from the "cow" solution and adheres onto the anion exchange resin as a chloride complex.
It is a further object of the present invention in one of its embodiments to remove traces of the .sup.225 Ac cow and the acid from the anion exchange resin, leaving a chloride complex which adheres the .sup.213 Bi to the anion exchange resin.
It is a further object of the present invention in one of its embodiments to provide a stripping solution which weakens or destroys the chloride complex, thereby releasing the .sup.213 Bi from the anion exchange resin.
It is a further object of the present invention in one of its embodiments to provide the anion exchange resin in a configuration which minimizes the time necessary to absorb, wash, and strip the .sup.213 Bi from the anion exchange resin.
It is a further object of the present invention in one of its embodiments to provide the anion exchange resin as a thin membrane to minimize the time necessary to adhere and strip the .sup.213 Bi from the anion exchange resin.
It is a further object of the present invention in one of its embodiments to provide the stripping solution as a solution which will allow final preparation and attachment of a monoclonal antibody to the separated and purified .sup.213 Bi for patient treatment for leukemia or other forms of cancer.
It is a further object of the present invention in one of its embodiments to provide the stripping solution containing proteins conjugated with bifunctional chelating agents to receive the .sup.213 Bi as it is being released from the anion exchange resin to allow in vivo initiation of the linking process.
The subject matter of the present invention is particularly pointed out and distinctly claimed in the concluding portion of this specification. However, both the organization and method of operation, together with further advantages and objects thereof, may best be understood by reference to the following description taken in connection with accompanying drawings wherein like reference characters refer to like elements.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1a illustrates the configuration of a thin film of an anionic exchange resin contained within a cartridge placed in-line with a syringe at the start of extraction of a solution containing .sup.213 Bi.
FIG. 1b illustrates the extraction of a solution containing .sup.213 Bi.
FIG. 1c illustrates the adherence of the .sup.213 Bi on the anionic exchange resin after extraction.
FIG. 1d illustrates the recovery of the .sup.225 Ac "cow" after extraction of the .sup.213 Bi.
FIG. 2a illustrates the wash solution utilized in the preferred embodiment.
FIG. 2b illustrates the wash step of the preferred embodiment.
FIG. 2c illustrates the recovery of the wash solution as practiced in the preferred embodiment.
FIG. 3a illustrates the elution solution utilized in the strip step of the preferred embodiment.
FIG. 3b illustrates the loading of the stripping solution in the strip step of the preferred embodiment.
FIG. 3c illustrates the recovery of the .sup.213 Bi into the stripping solution in the strip step of the preferred embodiment.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)
The process of the present invention is preferentially accomplished in a three step process; an extraction step, a wash step and a strip step.
In a preferred embodiment of the present invention, the Extraction step, as illustrated in FIG. 1a, 1b, 1c and 1d, is accomplished utilizing a cartridge holder 1 containing an organic anion exchange resin 2. As practiced in a preferred embodiment of the present invention, the resin 2 is configured as a thin "web" film, and is manufactured by 3M, St. Paul, Minn. The anionic exchange resin 2 is preferably selected as Anex.TM., which may be acquired from Sarasep Corp., Santa Clara, Calif. The resin 2 is first ground to a fine powder and is then secured in a PTFE (polytrifluoroethylene) "web" film in accordance with the method described in U.S. Pat. No. 5,071,610, incorporated herein by reference.
The resin 2 in web form and cartridge 1 is placed in-line with a syringe 3. 0.5M HCl is first drawn through the cartridge 1 to wet the resin 2 and to assure that the resin 2 is in the chloride form. As will be apparent to those skilled in the art, both the cartridge size, the type of anion exchange resin used, and the chemistry of the "web" structure, can be varied depending on the amount of .sup.213 Bi to be produced. A sample of .sup.225 Ac is then provided in a solution of 0.5M HCl which forms a chloride complexed solution 4. Preferably, the .sup.225 Ac is first prepared by the method described in co-pending U.S. Patent Application entitled METHODS OF SEPARATING AND PURIFYING SHORT HALF-LIFE RADIONUCLIDES FROM A MIXTURE OF RADIONUCLIDES, Ser. No. 08/766,684, filed Dec. 13, 1996. As illustrated in FIG. 1b, and 1c, the .sup.225 Ac/.sup.213 Bi chloride complexed solution 4 is then drawn through the resin 2 whereupon the anionic .sup.213 Bi complex in the solution absorbs on the resin 2. As illustrated in FIG. 1c, the remaining solution 7 and .sup.225 Ac passes through the resin 2 and into the syringe. By removing the cartridge 1, the .sup.225 Ac may then be returned to the original "cow" container 8 as illustrated in FIG. 1d, for storage and future "in-growth of .sup.213 Bi.
As illustrated in FIG. 2a, a wash solution 9 is then provided to accomplish the washing step of the preferred embodiment. The cartridge 1 containing the absorbed .sup.213 Bi on the resin 2 is again placed in line with the syringe 3. In the preferred embodiment of the present invention, as illustrated in FIG. 2b, a wash solution of 0.005M HCl 9 is drawn through the resin 2 to remove traces of .sup.225 Ac and HCl from the chloride complexed solution 4 which may be adhered to the interstitial surfaces of the resin 2. After the wash step, purified .sup.213 Bi is left attached to the resin 2. The cartridge 1 is again removed to allow the wash solution 9 to be ejected from the syringe 3 as illustrated in FIG. 2c. As will be apparent to those skilled in the art, it is not necessary that the chloride complexed solution 4 and the wash solution 9 be drawn through the resin 2 in the same direction, however, it is preferred that they be drawn in the same direction to maximize the .sup.213 Bi recovery.
As illustrated in FIG. 3a, a stripping solution 10 is then provided to accomplish the stripping step of the preferred embodiment. As illustrated in FIG. 3b, a stripping solution 10 selected as having a pH above about 2 and substantially free of Cl.sup.- ions is drawn through into the syringe 3 with the cartridge 2 removed. Once the syringe 3 is loaded with the stripping solution 10, the cartridge 1 containing the absorbed .sup.213 Bi on the resin 2 is again placed in line with the syringe 3. The stripping solution 10 is then pushed out of the syringe 3 through the resin 2 and into a recovery vessel 11. In this manner, the now purified .sup.213 Bi is removed from the resin 2. As illustrated by FIG. 3c, the purified .sup.213 Bi in the stripping solution 10 is thus available for further use. As will be apparent to those skilled in the art, it is not necessary that the stripping solution 10 be pushed through the resin 2 in a direction opposite to the direction which the chloride complexed solution 4 and the wash solution 9 were drawn, however, it is preferred that the stripping solution 10 be pushed in the opposite direction to maximize the .sup.213 Bi recovery. As will also be apparent to one skilled in the art, the relative direction of flow may also be reversed, however, the feed and wash should be sent through the web in one direction and the product eluted in the opposite direction. Preferable stripping solutions 10 include, but are not limited to, between about 0.01M and about 0.2M NaOAc. A preferable stripping solution is 0.05M NaOAc, pH 5.5; or NaOAc, pH 5.5, containing a suitable protein conjugated with bifunctional chelating agent (such as DTPA).
Experiment
A series of experiments were undertaken to demonstrate the efficacy of a preferred embodiment of the present invention. According to those experiments, the following procedure was followed. All acids, salts or bases used to "milk" .sup.213 Bi from the "Bi-Generator" were made up using "ULTREXTM.TM." purity or equivalent to eliminate cation impurities in the final .sup.213 Bi product.
Extraction Steps
1. Obtain the "Bi-generator" stored in a bottle or appropriate container. The radiochemical contents consist of .sup.225 Ac and daughters, taken to dryness in HCl to form a chloride salt for interim storage or off-site shipment.
2. Obtain a 3M anion exchange disc. Pre-treat the disk with 0.5M HCl by placing the acid in a syringe, locking or attaching the disc to the syringe, and by pushing down on the syringe plunger, forcing the acid through the membrane. Discard the pre-wash acid. This step insures that the anion resin is in the chloride form.
3. Take a known volume of 0.5M HCl into a pipettor.
4. Remove the screw lid from the "Bi-generator". Eject the HCl into the "cow" storage bottle allowing the .sup.221 Ac to dissolve in the solution.
5. Attach a pre-treated 3M filter to the syringe outlet and an appropriate plastic needle or tip to the outlet side of the 3M filter. With the same plastic tip or needle, pull the dissolved "cow" containing the .sup.225 Ac and its daughters (including .sup.213 Bi) up through the 3M anion exchange filter and up into the syringe barrel.
6. Remove the plastic tip or needle and remove the Bi-loaded 3M anion exchange disc and eject the .sup.225 Ac--0.5M HCl solution.
Wash Step
7. The .sup.213 Bi "product has now absorbed on to the anion exchange disc, along with minor traces of .sup.225 Ac and HCl (which adhere to the interstitial surfaces of the resin).
8. The used syringe and plastic needle or tip are replaced to eliminate any traces of .sup.225 Ac and acid. A new syringe is attached to the .sup.213 Bi 'loaded" anion exchange disc and a 0.005M HCl wash solution is pulled up through the disc. The disc is then removed and the acid wash, containing traces of interstitial "cow" solution, is expelled into a waste bottle. The "waste" HCl is discarded. Other acid concentrations (0.001-0.5M HCl) can be used. 0.005M HCl was selected because it provides an efficient wash without major loss of Bi product, while removing the 0.5M HCl which will interfere with the target pH value of 5.5 in the 0.05M NaOAc Product.
.sup.213 Bi Product Elution
9. The .sup.213 Bi "loaded" 3M disc, free of interstitial .sup.225 Ac and feed HCl, is eluted down-flow with one of several solutions of choice. This elution solution is placed in a clean syringe and forced through the .sup.213 Bi loaded disc and into a product bottle. (Optional) The above step can be repeated to obtain maximum product.
Summary of Selected Test Results
.sup.213 Bi loading, washing and elution experiments were performed to determine, 1) maximum loading of .sup.213 Bi from a 0.5M HCl feed solution; 2) loss of Bi during anion exchange disc washing, to remove .sup.225 Ac contamination and to reduce the acid concentration; and 3) to develop the most efficient method to remove the purified Bi product with an elution solution that can then be used directly to allow final preparation and attachment of the monoclonal antibody.
The results (Table 1) show that, the .sup.213 Bi extraction and recovery generator system will recover 85 to 93% of the .sup.213 Bi in 0.05M NaOAc (pH 5.5), free of all other radionuclides. Although not shown, the entire extraction, wash and strip operation requires approximately six minutes. Three elutions of the bismuth from the "web" disc were performed per test to show the efficiency of the first "Product" elution. The second and third elutions recover an additional 4-7 % of the final product. Solutions suitable for removal of the .sup.213 Bi from the disc include: 1) 0.05M NaOAc; or, (1) NaOAc containing a suitable protein conjugated with bifunctional chelating agent (such as DTPA). The concentration of NaOAc may also be varied, i.e. 0.01-0.2M. The percent of .sup.213 Bi loaded and eluted with any of the above solutions is improved by loading only in one direction, washing in the same direction as the loading step, and eluting in the opposite direction from loading. The feed, wash and elution solutions were maintained at 4 milliliters for the initial investigation (based on 5 mCi per mL for a 20 mCi .sup.225 Ac "cow"). No attempt was made to optimize these volumes.
Extraction Step
Even though the anion exchange "system" is a paper thin sheet of organic ion exchange media, the initial "web" loading surface acts like the top of a standard ion exchange column and the back surface acts as the bottom of a top loading column. In addition, it should be recognized that the feed "cow" solution does not contain grams of bismuth to be loaded on to the exchanger, but only about 5.3.times.10.sup.-8 mg Bi per millicurie (.sup.221 Ac "cows" from 1 to 20 mCi are proposed). Therefore, the loading experiments show that if the loading solution is pulled out of the generator storage container, up through the exchanger (up-flow) and then sent back to the bottle in the reverse direction that the loss of .sup.213 Bi to the effluent is 2-5% (13A and B). When the loading feed is passed only in one direction through the anion exchange media, the loss to the effluent is <0.1% (13C and D, 16A). The original two directional flow was conceived to permit the return of the .sup.225 Ac "cow" to the shielded container by the most expeditious route. However, when the feed flows through the "web", the Bi is absorbed on the front surface, but reversing the direction allows the "clean" 0.5M HCl to "kick" a small portion of the Bi off of the surface back into the original "generator" bottle.
Wash Step
The objective of the wash solution is to remove traces of residual feed solution that had not contacted the ion exchange media (to eliminate the interstitial .sup.225 Ac/Ra), and to reduce the acid concentration in the ion exchange media. The goal is to elute the .sup.213 Bi product at a pH (.about.5.5) required to link the .sup.213 Bi with a suitable protein conjugated with bifunctional chelating agent (such as DTPA). The wash solution combination of up-flow and down-flow gave .sup.213 Bi losses of .about.3.6G (13A-D). When the wash solution was passed only in the opposite direction as the feed, the loss was 13-17 % (12C). When the wash solution was passed in the same direction as the feed flow, the wash loss was reduced to <1%. (16A).
Product Elution Step
Several types of eluants were tested including 0.05M NaOAc and 0.05M NaOAc+0.001M DTPA. They were selected based on known buffer solutions to be used with a suitable protein conjugated with bifunctional chelating agent (such as DTPA). No major differences were noted (13A, D and 13B, C).
TABLE 1__________________________________________________________________________Experimental ResultsFeed: 4 mL, 0.5M HCl, .sup.225 Ra/Ac and .sup.213 BiTD = Solution fed two directions (Up-flow/Down-flow)UP = Up-flow onlyDF = Down-flow only% = .sup.213 BiTest Effluent Scrub Product #1 Product #2 Product #3 Final Filter Matl. Bal.__________________________________________________________________________Elution: 0.05M NaOAc, pH 5.62 (initial)13A 4 mL 4 mL 4 mL 4 mL 4 mL 3.2% 90.5% 2.6% 3.2% 76.0% 4.0% 1.5% (81.5%) TD TD DF DF DF FpH 1.63 FpH 5.28 FpH 5.54 FpH 5.53Elution: 0.05M NaOAc, pH 5.62 (initial)13D 4 mL 4 mL 4 mL 4 mL 4 mL 2.6% 104.5% .about.0% 3.6% 92.3% 4.5% 1.5% (98.3%) UF TD DF DF DF FpH 1.58 FpH 5.27 FPH 5.54 FpH 5.49Elution: 0.05M NaOAc, pH 5.62 (initial)16A 4 mL 4 mL 4 mL 4 mL 4 mL 7.7% 100.0% .about.0% 0.9% 84.0% 5.4% 2.0% (91.4%) UF UF DF DF DF FpH 1.57 FpH 5.27 FpH 5.50 FpH 5.41Elution: 0.05M NaOAc + 0.001M DTPA, pH 5.62 (initial)13B 4 mL 4 mL 4 mL 4 mL 4 mL 2.9% 98.1% 2.9% 3.4% 83.5% 4.2% 1.2 (88.9%) TD TD DF DF DF FpH 1.57 FpH 5.23 FpH 5.40 FpH 5.54Elution: 0.05M NaOAc + 0.001M DTPA, pH 5.62 (initiaL)13C 4 mL 4 mL 4 mL 4 mL 4 mL 4.4% 103.0% .about.0% 4.3% 89.0% 4.0% 1.3% (94.3%) UF TD DF DF DF FpH 1.38 FPH 5.27 FpH 5.48 FpH 5.48Elution: 0.05M NaOAc, pH 5.62 (initial)12C 4 mL 4 mL 4 mL 4 mL 4 mL .about.3.5% 97.6% 4.8% 13.5% 69.2% 4.6% 2.0% (71.8%) TD DF DF DF DF FpH 1.83 FpH 5.49 FpH 5.67 FpH 5.58Elution: 0.05M NaOAc, pH 5.62 (initial)12D 4 mL 4 mL 1 mL 1 mL 1 mL 21.3% 99.35% 1.85% 17.3% 38.2% 11.8% 5.6% (58.9%) TD DF DF DF DF F pH Not Det.Elution: 0.05M NaOAc, pH 5.62 (initial)11A 4 mL 4 mL 4 mL 4 mL 4 mL Not Det. -- 3.8% 3.5% 64.7% 11.6% 3.6% (79.9%) TD TD TD TD TD FpH 1.67 FpH 5.50 FpH 5.61 FpH 5.61Elution: 0.05M NaOAc, pH 5.62 (initial)11B 4 mL 4 mL 4 mL 4 mL 4 mL Not Det. -- 4.8% 3.0% 56.0% 9.1% 4.3% (69.4%) TD TD TD(3-times) TD TD FpH Not Det.Elution: 0.05M NaOAc + 0.1M NaCl, pH 5.58 (initial)12A 4 mL 4 mL 4 mL 4 mL 4 mL 17.8% .about.100% 1.79% 3.7% 57.9% 13.4% 5.5% (76.8%) TD TD TD TD TD FpH 1.72 FpH 5.50 FpH 5.51 FpH 5.50Elution: H.sub.2 O12B 4 mL 4 mL 4 mL 4 mL 4 mL .about.16.5% .about.100% 4.7% 4.6% 36.2% 32.1% 5.8% (74.1%) TD TD TD TD TD FpH 1.77 FpH 3.04 FpH 4.87 FpH 6.66Elution: 0.1M NaOAc, pH 5.68 (initial)11C 4 mL 4 mL 4 mL 4 mL 4 mL Not Det. -- 3.3% 3.3% 67.0% 8.9% 3.3% (80.6%) TD TD TD TD TD FpH Not Det.__________________________________________________________________________
While a preferred embodiment of the present invention has been shown and described, it will be apparent to those skilled in the art that many changes and modifications may be made without departing from the invention in its broader aspects. The appended claims are therefore intended to cover all such changes and modifications as fall within the true spirit and scope of the invention.

Number	Name	Date
3758663	Kirby	Sep 1973
4018561	Siemens et al.	Apr 1977
4663129	Atcher et al.	May 1987
5355394	van Geel et al.	Oct 1994

Bismuth generator method

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