WO2002041989A1

WO2002041989A1 - Metal-chelating adsorbents and their use

Info

Publication number: WO2002041989A1
Application number: PCT/GB2001/005231
Authority: WO
Inventors: Mark Burton
Original assignee: Prometic Biosciences Ltd.
Priority date: 2000-11-27
Filing date: 2001-11-27
Publication date: 2002-05-30
Also published as: AU2002220830A1; GB0028879D0

Abstract

A metal-chelating ligand-support conjugate comprises coordination groups selected from COOH, PO3H and SO3H bound to a ligand backbone, 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 may be prepared by 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.

Description

METAL-CHELATING ADSORBENTS AND THEIR USE Field of the Invention

This invention relates to the preparation and use of novel affinity ligands for the removal of contaminant metal ions from diagnostic and therapeutic formulations. Background of the Invention

In recent years, significant advances have been made in the use of diagnostic radioimaging (radioimmunoscintigraphy) and of in vivo cytotoxic radiotherapy for the diagnosis and treatment of conditions such as cancer. Radioimmunotherapy 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 radioisotopic 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 nuclides with short half-lives. Additionally, multi-step purification procedures lead to undesirable dilution effects.

Representative examples of metal chelates, 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, rejcarek et al., Biochem. Biophys. Res. Commun., 1977, p.581, vol. 77; Brechbiel et al., Inorg. Chem., 1986, p.5783, vol. 25; Meares et al, Jou. Prot. Chem., 1984, p.215, vol. 3; Hnatowichetα/., Science, 1983a, p.613, vol.220;Khawetα/., Science., 1980, p.295, vol. 209; Scheinberg etβ/., Science, 1982, pJ511, vol. 215; US-A-4479930; US-A-4472509; US-A-5130118; EP-A-0484989; EP-A-0345723; and WO-A-96/15816.

A commonly used reagent for the covalent modification of proteins is the cyclic dianhydride of diethylenetriaminepentaacetic acid (DPTA), whereby an amide bond is formed between the protein and the chelator: The use of bifbnctional chelating reagents such as the dianhydride of DPTA results in cross-linking of the protein. Furthermore, the amide linkage between the protein and chelator is both acid- and base-labile. Thus, there remains the need for efficient and robust methods for the removal of contaminating metal ions from pharmaceutical preparations, including radiolabelled antibody conjugates, whilst overcoming the drawbacks of current procedures. Summary of the Invention

According to the present invention, in a metal-chelating ligand-support conjugate, the ligand comprises coordination groups selected from COOH, PO₃H and SO₃H, 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 embodying the present invention can be represented by formula (1)

CH X

(1)

wherein A represents a C,.₆ saturated hydrocarbon chain; each X is COOH, PO₃H or SO₃H; n is 2 or more;

M is an optional spacer arm; and Z is a support matrix. Description of the Invention

In compounds of formula 1, A maybe linear or branched alkylene such as divalent methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl ortert-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, soluble or insoluble, 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. Examples of soluble carriers include polymers of dextran, polyvinyl alcohol, polyethylene glycol and hydrolysed starch. The support matrix may be in the form of packed columns or cartridges, or of 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 (PNA). A preferred spacer M comprises a group of the formula -T- L- wherein T is O, S or ΝR, R is H or C,^ alkyl, and L is an optionally substituted C₂.₂₀ alkylene chain optionally including one or more ether or thioether linkages. A further aspect of this invention is a facile method for the preparation of conjugates represented by formula (1) with a stable C-Ν 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 examle, the method comprises the reaction of a polyamine of the formul H-[ΝH-A]_n-ΝH₂, 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.

In one embodiment of the invention, 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 embodying the invention, by the method shown in the following scheme

H₂N—(CH₂)₂— H—(CH₂)₂— H₂

Z-O CH,—CH- CH₂—NH—(CH,)₂— H—(CH₂)₂—NH

² I OH

Br—CH₂—COOH

CH,—COOH

I ² CH₂—COOH

/

Z-O—CH₂—CH—CH₂—N —(CH₂)₂—N —(CH₂)₂—N

OH I CH,—COOH

CH,—COOH ²

The method shown above can be adapted, as will be apparent to those skilled in the art, to produce any other embodiment of the invention.

A desirable key feature of metal-chelating ligand-matrix conjugates prepared 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.

Another 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), e.g. using an inert support matrix such as polytetrafluoroethylene (PTFE). 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.

This invention may be applied to the removal of any suitable metal ion including radioactive and non-radioactive metal ions. The use of 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.

Another aspect of the present invention is the provision of an expeditious procedure for the removal of contaminant metal ions from pharmaceutical and other therapeutic formulations and comprises a terminally sterilized single-use disposable cartridge or element containing a metal-chelating adsorbent of formula (1). In one embodiment of the invention, the conjugate is used as the adsorbent for a radiactive component 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.

Apparatus suitable for use in this embodiment is illustrated schematically in the accompanying drawing. The drawing 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 sryinge 7, containing less contaminated liquid, can then be removed.

The novel conjugate may also be reused, if necessary or desired; its robustness means that NaOH can be used for sterilisation.

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 (128ml) and 10M sodium hydroxide (16ml) 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 200ml 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. IM 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 (50ml) and IM sodium bicarbonate (50ml). 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), IM sodium hydroxide (10 x 5ml portions) and RO water (10 x 5ml portions). The gel was then mixed with 5 ml RO water and the slurry gravity packed into a 10ml column.

A solution of non-radioactive ⁸⁹yttιium chloride (2.191mg/ml in RO water; 25ml) 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

Thus, Total Load 24.625mg Flow through 21.540mg Amount bound 3.085mg Capacity 0.617mg ⁸⁹Y³Jg gel

6.9μmol ⁸⁹Y³Jg gel

Eluted 0.9675mg

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 (2ml) and 1 OOmg sodium borohydride was reacted with 1,4-butanediol diglycidyl ether (30ml) 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 for 24 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 IM sodium hydrogen carbonate (25ml) 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 1ml glass column connected up to a Gilson peristaltic pump and washed sequentially with IM sodium hydroxide (10ml) and RO water (10ml) at a flow rate of 0.5ml/min. A stock solution of copper sulphate (16mg/ml in RO water; 11.35ml) was loaded onto the column at a flow rate of 0.5ml/min and the flow through collected (10ml). The column was rinsed withRO water ( 10ml) to remove any unbound copper sulphate. Bound copper sulphate was eluted from the column using nitric acid (OJM; 3.5ml). All fractions were assayed for copper sulphate spectrophotometrically. The results are shown in Table 2.

Table 2

Thus, Total Load 176.13mg

Flow through + RO rinse = 174.43mg

Eluted 0.401mg

Capacity = 2.512μmol Cu²Jg gel

Yttrium-binding capacity

A20%,(v/v) ethano 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), IM sodium hydroxide (10ml) and RO water (10ml) at a flow rate of 0.5ml/min. A stock solution of non-radioactive⁸⁹yttrium chloride (1. Img/ml in RO water; 10ml) was loaded onto the column, washed with RO water (20ml) and the bound yttrium was eluted with nitric acid (0. IM; lOrnl). All fractions were assayed for yttrium by ICP- MS. The results are shown in Table 3.

Table 3

Thus, Total Load = 4923 μg

Flow through = 3014μg

RO rinse = 1681μg

Eluted = 221μg Capacity = 58.2μg ⁸⁹Y³⁺/g gel

0.654μmol ⁸⁹Y³7g gel Radioactive Yttrium-Binding Efficiency

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.) containing a leur fitting at the inlet port and a 0.45mm filter connected to a leur 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, OJM pH 5.5) was pulled through the cartridge. A solution of radioactive ⁹⁰yttrium chloride (0.5ml) made up in sodium acetate buffer (0. IM, pH 5.5) containing 37MBq of radioactivity units was loaded onto the cartridge followed by acetate buffer (5 x 1ml, 0. IM 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 (OJM,

5ml), RO water (5ml), sodium hydroxide (IM, 5ml) and finally with RO water (10ml). A preparation of antibody immunoconjugate labelled with ⁹⁰yttrium was prepared by incubating ⁹⁰yttrium chloride (37Mbq, 200μl in 0. IM acetate buffer pH 5.5) with antibody immunoconjugate (5μl, 30μg) for 30 minutes. The radiolabelled reaction mixture was passed through the cartridge and immunoconjugate eluted with sodium acetate (3 x lml, 0. IM pH 5.5). The radiochemical purity of the conjugate prior to purification was 96% and increased to 98% after purification using the cartridge. Human Serum Albumin Binding Capacity

An aqueous slurry of DETATA-PTFE was packed into a glass chromatography column (10mm i.d.) to a bed height of 49mm (3.85ml column volume) and washed sequentially with sodium hydroxide (IM, 10ml) and RO water (10ml) at a flow rate of lml/min. Copper was loaded onto the adsorbent by passing an aqueous solution of copper sulphate (10ml, 16mg/ml) through the column. The column was then washed with RO water (10ml) to remove excess copper sulphate and equilibrated with loading buffer (0. IM sodium acetate, 0.5M sodium chloride pH 7.7, 10ml). A sample of human serum albumin (2ml, lmg/ml) prepared in loading buffer was loaded onto the column at a flow rate of lml/min and the column was washed with loading buffer (10ml) to remove all unbound protein. Bound serum albumin was eluted from the column by applying 50mM tris/HCl, 0.15M ammonium chloride pH 8.0 (4.0ml). All fractions were assayed for albumin using the Coomassie protein assay. The results are summarized below.

Total serum albumin loaded = 2000 μg

Total serum albumin in flow through = 1870 μg

Total serum albumin in elution = 84.9 μg

Thus albumin binding capacity = 22.1 μg/ml of adsorbent.

Claims

1. A metal-chelating ligand-support conjugate in which the ligand comprises coordination groups selected from COOH, PO₃H and SO₃H bound to a ligand backbone, 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.

2. A conjugate according to claim 1, of the formula

(1)

wherein A represents a C,^ saturated hydrocarbon chain; each X is COOH, PO₃H or SO₃H; n is 2 or more;

M is an optional spacer arm; and Z is a support matrix.

3. A conjugate according to claim 2, wherein M is present and comprises a group of the formula -T-L- wherein T is O, S or NR, R is H or C_x_₆ alkyl, and L is an optionally substituted C₂.₂₀ alkylene chain optionally including one or more ether or thioether linkages.

4. A conjugate according to claim 2, wherein M is absent.

5. A conjugate according to any of claims 2 to 4, wherein n is 2.

6. A conjugate according to any preceding claim, wherein the coordination group/X is COOH.

7. A conjugate according to any preceding claim, wherein the support matrix is a perfluorocarbon polymer or a perfluorocarbon copolymer, either of which is optionally coated with a hydrophilic polymer.

8. A conjugate according to any preceding claim, capable of chelating yttrium ions.

9. A method for the preparation of a conjugate according to any preceding claim, which 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.

10. A conjugate according to any of claims 1 to 8, obtainable by a method according to claim 9.

11. A complex of a conjugate according to any of claims 1 to 8 and 10, and chelated metal ions.

12. Use of a conjugate according to any of claims 1 to 8 and 10, for the removal of metal ions from a sample.

13. Use according to claim 12, wherein the metal ions are radioactive.

14. Use of a complex according to claim 11 , for the separation, isolation, purification, characterisation, identification or quantification of proteins or nucleic acids.

15. A method for the removal of a radioactive component from a liquid sample, which 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 said component, and wherein the chamber also has an inlet and means for mounting a syringe thereon.

16. A method according to claim 15, wherein the adsorbent.is a conjugate according to any of claims 1 to 8.