Background of the Invention In recent years, significant advances have been made in the use of diagnostic radio-imaging (radio-immunoscintigraphy) and of in vivo cytotoxic radiotherapy for the diagnosis and treatment of conditions such as cancer.

Radio-immunotherapy relies on the targeted delivery of conjugates of monoclonal antibodies with radioactive metal ions to specific tumour sites, the specificity being a function of the antibody chosen. In typical radiotherapy protocols, the active agent is prepared by incubating a metal chelate-antibody conjugate with an excess of the radio-isotopic metal ion for 0. 5-1. 0 hr. This is often followed by a series of time-consuming purification procedures to remove unbound or non-specifically bound metal ions. Typical purification protocols include dialysis and combinations of ion-exchange chromatography and gel filtration chromatography. Lengthy purification procedures lead to significant decreases in effective doses of radiation, particularly for conjugates comprising nuclideswith short half-lives. Additionally, multi-step purification procedures lead to undesirable dilution effects.

Representative examples of metal chelate, methods of preparation of metal chelate-protein conjugates, and the use of such conjugates in diagnostic and therapeutic applications, have been disclosed in several publications. See, for example, Krejcarek et a/., Biochem. Biophys. Res. Commun. , 1977, p. 581, vol. 77; Brechbiel et a/., Inorg. Chem. , 1986, p. 5783, vol. 25; Meares eta/., Jou.

Prot. Chem. , 1984, p. 215, vol. 3; Hnatowich et al., Science, 1983a, p. 613, vol.

220; Khaw et a/., Science. , 1980, p. 295, vol. 209; Scheinberg et al., Science, 1982, p. 1511, vol. 215; US-A-4479930; US-A-4472509; US-A-5130118; EP-A- 0484989 ; EP-A-0345723; and WO-A-96/15816.

Summary of the Invention According to the present invention, a metal ion-chelating adsorbent or another suitable material is used to capture radiactive metal ions in a liquid

sample. Such a method comprises using a syringe mounted at the outlet of a chamber to draw the sample through the chamber, wherein the chamber is housed within a radiation-absorbing shroud and contains an adsorbent for the component, and wherein the chamber also has an inlet and means for mounting a syringe thereon.

The component to be removed may be a radio-labelled antibody or other biological material. Passing the liquid sample through the chamber by use of negative pressure or vacuum, rather than using positive pressure, is an important safety feature of the invention, and reduces the risk of leakage/atomisation of radioactive materials.

This invention provides an expeditious procedure for the removal of contaminant metal ions from pharmaceutical and othertherapeuticformulations, e. g. utilising a terminally sterilized single-use disposable cartridge or element.

Brief Description of Drawings The accompanying drawings show devices suitable for use in the invention, and are described in greater detail below.

Description of Preferred Embodiments The invention utilises a device housing incorporating a syringe mounted at the outlet of a chamber to draw the sample through the chamber, wherein the chamber is housed within a radiation-absorbing shroud and contains an adsorbent for the said component, and wherein the chamber also has an inlet and means for mounting a syringe thereon. The device chamber is packed with a metal ion-chelating adsorbent capable of capturing radioactive metal ions from aqueous solutions with high efficiency. Preferably, the metal ion-chelating adsorbent comprises diethylenetriaminetetraacetic acid groups attached to a fluorocarbon matrix by means of a linking chemistry devoid of ester or amide bonds. Such materials facilitate dry packing and sterilisation by means of gamma irradiation. The device is used to remove contaminating metal ions from pharmaceutical preparations, including radio-labelled antibody conjugates, by drawing solutions of such pharmaceutical preparations through the packed chamber.

Preferably, the liquid sample is held in a vial contained within a radiation- adsorbing shroud which engages with the radiation-adsorbing shroud enclosing the chamber, to provide a continuous shield. It is also preferred that the syringe mounted at the chamber outlet is contained within a radiation-adsorbing shroud which engages with the radiation-adsorbing shroud enclosing the chamber, to provide a continuous shield. Another preference is that all wetted parts are rendered sterile before use.

Apparatus suitable for use in this embodiment is illustrated schematically in the accompanying drawings. Fig. 1 shows a chamber 1 having a syringe needle 2 mounted at its inlet, connected via a Luer fitting 3, and a seal 4. The combination is adapted to receive liquid sample 5 in a container 6. The chamber also has a syringe 7 mounted at its outlet and connected via a Luer fitting 8. The chamber includes an adsorbent 9. The combination of these components is surrounded by a radiation-absorbing shroud 10. In use, raising the syringe piston 11 draws liquid through the adsorbent 9. The syringe 7, containing less contaminated liquid, can then be removed.

Fig. 2 is a cross-sectional view of a device attached to a single vial holder, whilst Fig. 3 shows a multi-vial holder. The significance of this is that there are different designs of radio-labelling vial which are of different heights and widths.

Consequently, it may be advantageous if the device is used in conjunction with a holder capable of holding multiple vials of different types, as well as single vials.

Fig. 2 shows many of the same components, and uses the same reference numerals for them, as Fig. 1. The syringe needle 2 is optionally vented to allow pressure equilibration in vial. The vial 6 is preferably of glass.

The adsorbent 9 may be any adsorbent packing material capable of binding and separating free radio-label from a (non-bound) radio-labelled biological molecule or compound. In addition, Fig. 2 shows a filter 12 and, for guidance only, dimensions (each including a decimal point).

In preferred metal-chelating ligand-matrix conjugates for use in the invention, the ligand comprises coordination groups selected from COOH, P03H and SO3H1 the ligand backbone is linked, optionally by means of a spacer, to a

support matrix, and the ligand backbone/spacer is free of ester and amide bonds. Such a conjugate can be used for the capture of metal ions from water, aqueous solutions, blood, plasma, and pharmaceutical and therapeutic products and proteins.

Metal-chelating ligand-matrix conjugates suitable for use in the present invention can be represented by formula (1) wherein A represents a C16 saturated hydrocarbon chain; each X is COOH, P03H or S03H ; n is 2 or more; M is an optional spacer arm; and Z is a support matrix.

In compounds of formula 1, A may be linear or branched alkylen such as divalent methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert- butyl. X is preferably COOH. n is preferably 2, so that the conjugate has 4 coordination groups, and usually no more than 3,4 or 5.

The support matrix may be any compound or material, particulate or non- particulate, porous or non-porous, which may be used for the immobilization of metal-chelating ligands, to form a metal-chelating ligand-matrix conjugate, thereby providing a convenient means of binding metal ions from solution.

Examples of particulate support matrices include natural polymers such as agarose, dextran, cellulose or starch, synthetic polymers and co-polymers such as perfluorocarbons, polystyrene, polyacrylamide, polyvinyl alcohol and polymethyl methacrylate, and inorganic compounds such as silica, glass, alumina and metal oxides. The use of fluorocarbon materials such as PTFE as

a chromatographic support in this invention has specific advantages over other commercially available support matrices, including chemical and biological inertness, compatibility with gamma-irradiation and minimal swelling upon wetting which minimizes any sample dilution effects during use. The support matrix may be in the form of particles, membranes or sheets comprising such polymers.

Covalent attachment of ligand structures as represented by formula (1) to the support matrix Z may be achieved by use of a variety of activation agents including, but not limited to, cyanogen bromide, epichlorohydrin, 1, 4-butanediol diglycidyl ether, tosyl chloride, tresyl chloride, and cyanuric chloride. The spacer group M may be absent, although it will be understood by those skilled in the art that the non-functional part of any functional groups used to conjugate Z and the coordination groups may be present in the product. If present, M may be any group which is conveniently capable of holding the ligand at a distance from the support matrix. Such spacer groups include diaminoalkanes and polyvinyl alcohol (PVA). A preferred spacer M comprises a group of the formula-T-L- wherein T is O, S or NR, R is H or C_g alkyl, and L is an optionally substituted 2-20 alkylen chain optionally including one or more ether or thioether linkages.

Conjugates represented by formula (1) can readily be prepared with a stable C-N bond formed between the ligand and the support matrix Z. A variety of methods is available, which avoid introducing an ester or amide bond. For example, the method comprises the reaction of a polyamine of the formula H- [NH-A] n-NH2, with an activated support matrix Z-Y, wherein Y is a reactive group, optionally in the presence of or after reaction of either component with a compound introducing M; and reaction of the product with a compound of the formula Q-CH2-X, wherein Q is a group reactive with NH.

By way of example, a diamine such as diethylenetriamine is coupled to an activated support matrix followed by reaction with a halogenoacetic acid such as bromoacetic acid or chloroacetic acid to give a conjugate by the method shown in the following scheme:

A desirable feature of preferred metal-chelating ligand-matrix conjugates for use according to this invention is the ultrastable C-N linkage between the support matrix and the metal-chelating ligand, thereby enabling the use of such conjugates for sequestering metal ions at extremes of pH, ionic strength and temperature, without the risk of potentially toxic metal ion leachates. Such chemistries allow sterilisation of the adsorbent by sterilisation techniques such as gamma irradiation. The conjugate may be reused, if necessary or desired, following cleaning and sanitisation with, for example, solutions of nitric acid and sodium hydroxide.

An aspect of this invention is a one-step chromatographic method for the removal of contaminant metal ions from diagnostic and therapeutic products. In a preferred embodiment of the invention, unbound and/or loosely bound metal ions are scavenged from a pharmaceutical product by passing the preparation

through a terminally sterilized column containing a conjugate represented by formula (1). Such adsorbents have a high affinity for heavy metals, and the method yields a therapeutic product that is substantially free of unbound metal ions and is suitable for administration to humans in vivo.

The following Examples illustrate the invention. DETATA is diethylenetriaminetetraacetic acid.

Example 1 DETATA-agarose Stage 1-Preparation of epichlorohydrin-activated agarose A slurry of Sepharose CL-6B (200g settled weight), RO water (128mi) and 1 OM sodium hydroxide (16mut) was reacted with epichlorohydrin (14. 4ml) at 40°C for 3.5 hours. The epoxy activated Sepharose CL-6B was washed exhaustively with RO water (10 x 200mi portions) to remove excess reactants and used immediately in stage 2.

Stage 2-Preparation of diethylenetriamine-agarose Epoxy-activated Sepharose CL-6B (94g settled weight) from stage 1 was mixed with 0.2M sodium bicarbonate solution (225ml) and diethylenetriamine (150ml) and the reaction mixture stirred for 23 hours at 50°C. The resulting amino gel was washed with RO water (10 x 100ml portions), 0. 1 M acetic acid (10 x 100ml portions) and finally with RO water (10 x 100ml portions). This gel was used in stage 3 of the reaction.

Stage 3-Preparation of DETATA-Agarose The diethylenetriamine-agarose (stage 2) was mixed with bromoacetic acid (16g), 2M sodium hydroxide (50moi) and 1 M sodium bicarbonate (50moi). The resulting reaction mixture was adjusted to pH 7.0 with sodium hydroxide and left to react at room temperature for 16 hours. The gel was then washed with RO water (10 x 100ml portions) to remove excess reactants.

Yttrium-binding capacity DETATA-agarose (5g) was washed sequentially with RO water (10 x 5ml portions), 1M sodium hydroxide (10 x 5ml portions) and RO water (10 x 5ml portions). The gel was then mixed with 5ml RO water and the slurry gravity packed into a 1 Oml column.

A solution of non-radioactive 89yttrium chloride (2.191 mg/mi in RO water; 25mi) was loaded onto the column under gravity followed by RO water (5ml) to wash out any unbound metal ion. Elution of bound yttrium was achieved by passing 1 OmM hydrochloric acid (25ml) through the column. The flow-through, eluted fractions and the stock yttrium solutions were analysed for elemental yttrium by Inductively Coupled Plasma Atomic Emission Spectrometry. Results are shown in Table 1.

Table 1 Solution [89y3+] (mg/L) Stock solution 985 Flow-through fraction 718 Eluted fraction 38. 7 Thus, Total Load = 24. 625 mg Flow through 21. 540 mg Amount bound-3. 085 mg Capacity = 0. 617 mg 89Y3+/g gel = 6.9 µmol 89Y3+/g gel Eluted = 0. 9675 mg Example 2 DETATA-PTFE Stage 1-Preparation of 1, 4-butanediol diglycidyl ether-activated PTFE A suspension of polyvinyl alcohol-coated PTFE particles (20g wet PTFE), RO water (47ml), 10M sodium hydroxide solution (2mi) and 100mg sodium borohydride was reacted with 1, 4-butanediol diglycidyl ether (30mil) for 7 hours at room temperature. The epoxy-activated PTFE material was washed with excess RO water to remove reactants.

Stage 2-Preparation of diethylenetriamine-PTFE The activated, settled product was mixed with an equal volume of diethylenetriamine and allowed to react for24 hours at 50° C. Diethylenetriamine was removed at the end of the reaction by washing the gel with excess RO water.

Stage 3-Preparation of DETATA-PTFE The settled gel from stage 2 was suspended in 1M sodium hydrogen carbonate (25moi) and 2M sodium hydroxide solution (25ml). Bromoacetic acid (8g) was added and the pH of the suspension adjusted to pH 7.0 by the drop- wise addition of 2M sodium hydroxide. The reaction mixture was stirred for 16 hours at room temperature. The final chelate adsorbent was washed with an excess of RO water at the end of the reaction.

Copper-binding capacity An aqueous slurry of DETATA-PTFE (1. 02g) was loaded into a 1 mi glass column connected up to a Gilson peristaltic pump and washed sequentially with 1M sodium hydroxide (10ml) and RO water (10mut) at a flow rate of 0. 5ml/min.

A stock solution of copper sulphate (16mg/ml in RO water; 11-35moi) was loaded onto the column at a flow rate of 0. 5ml/min and the flow through collected (1 Oml).

The column was rinsed with RO water (10ml) to remove any unbound copper sulphate. Bound copper sulphate was eluted from the column using nitric acid (0. 1 M ; 3. 5ml). All fractions were assayed for copper sulphate spectrophotometrically. The results are shown in Table 2.

Table 2 Solution [Copper sulphate] (mg/ml) Stock solution 15.52 Flow-through + RO rinse fraction 8.72 Eluted fraction 0. 114 Thus, Total Load 176. 13mg Flow through + RO rinse = 174. 43mg Eluted = 0.401mg Capacity = 2.512µmol Cu2+/g gel Yttrium-binding capacity A 20% (v/v) ethanol : water slurry of gamma-irradiated DETATA-PTFE (3. 975g) was loaded into a 10ml glass column connected up to a peristaltic pump and washed with RO water (10ml), 1M sodium hydroxide (10ml) and RO water (10ml) at a flow rate of 0. 5ml/min. A stock solution of non-radioactive89yttrium

chloride (1. 1 mg/ml in RO water; I Oml) was loaded onto the column, washed with RO water (20ml) and the bound yttrium was eluted with nitric acid (0. 1 M; 1 owl).

All fractions were assayed for yttrium by ICP-MS. The results are shown in Table 3.

Table 3 Solution [89Y3+] (Ug/L Stock solution 495000 Flow through fraction 303000 RO rinse fraction 84000 Eluted fraction 22100 Thus, Total Load = 4923 µg Flow through 3014 pg RO rinse = 1681 pg Eluted = 221 µg Capacity = 58. 2 ug 89y3+/g gel 0. 654 µmol 89Y3+/g gel Example 3 Removal of Radioactive Yttrium DETATA-PTFE adsorbent (1.2g dry weight) was dry packed to a bed height of 20mm into a cartridge (40mm height x 8mm i. d. ) as shown in Fig. 2, containing a luer fitting at the inlet port and a 0.45mm filter connected to a luer fitting at the outlet port. The packed cartridge was housed within a radiation shield to minimize exposure to radiation during use.

As a control, in the absence of immunoconjugate, a syringe was connected to the outlet port of the cartridge and sodium acetate buffer (5ml, 0.1 M pH 5.5) was pulled through the cartridge. A solution of radioactive 90yttrium chloride (0. 5ml) made up in sodium acetate buffer (0. 1M, pH 5.5) containing 37MBq of radioactivity units was loaded onto the cartridge followed by acetate buffer (5 x 1 ml, 0.1 M pH 5.5). Measurement of radioactivity in the wash fractions showed that 99. 1% of the radioactivity was retained by the cartridge.

The cartridge was regenerated by sequentially washing with nitric acid (0. 1 M, 5ml), RO water (5ml), sodium hydroxide (1M, 5ml) and finally with RO

water (1 Oml). A preparation of antibody immunoconjugate labelled with 90yttrium was prepared by incubating 90yttrium chloride (37Mbq, 200pi in 0. 1M acetate buffer pH 5.5) with antibody immunoconjugate (5µl, 30µg) for 30 minutes. The radio-labelled reaction mixture was passed through the cartridge and immunoconjugate eluted with sodium acetate (3 x 1 ml, 0. 1 M pH 5.5). The radio- chemical purity of the conjugate prior to purification was 96% and increased to 98% after purification using the cartridge.