ISOTOPE PURIFICATION METHOD

Abstract

The invention provides a method for the purification of 227 Th from a mixture comprising 227 Th and 223 Ra, said method comprising: i) preparing a first solution comprising a mixture of 227 Th and 223 Ra ions dissolved in a first aqueous buffer; ii) loading said first solution onto a separation material such as a strong cation exchange resin; iii) eluting 227 Th from the separation material, whereby to generate a second solution comprising 227 Th; iv) Optionally rinsing said separation material using a first aqueous washing medium; The invention additionally provides a method for forming a radio pharmaceutical comprising complexing the purified 227 Th, the pharmaceutical product and its use in treatment of disease such as cancer and a kit for generation of such a product.

Claims

1) A method for the purification of .sup.227Th from a mixture comprising .sup.227Th and .sup.223Ra, said method comprising: i) preparing a first solution comprising a mixture of .sup.227Th and .sup.223Ra ions dissolved in a first aqueous buffer; ii) loading said first solution onto a separation material; iii) eluting .sup.227Th from said separation material whereby to generate a second solution comprising .sup.227Th; iv) Optionally rinsing said separation material using a first aqueous washing medium;

2) The method of claim 1 further comprising at least one of the following optional steps: v) assaying for the .sup.227Th content of said second solution; vi) evaporating the liquid from said second solution; vii) forming at least one radiopharmaceutical from at least a portion of the .sup.227Th contained in said second solution; viii) sterile filtering said radiopharmaceutical.

3) The method of claim 1 wherein the first aqueous buffer solution is at a pH of between 3 and 6.5.

4) The method of claim 1 wherein the first aqueous buffer solution comprises at least one organic acid buffer selected from citrate buffer, acetate buffer and mixtures thereof.

5) The method of claim 1 wherein the first aqueous buffer solution further comprises at least one radical scavenger and/or at least one chelating agent.

6) The method of claim 1 wherein the separation material is a strong cation exchange resin, preferably a silica based resin.

7) The method of claim 1 wherein the separation material is a cation exchange resin comprising at least one CH.sub.2SO.sub.3H moiety.

8) The method of claim 1 wherein the elution is by dry means, preferably under gravitational or centrifugal force or under gas pressure from above and/or vacuum from below.

9) The method of claim 8 wherein elution is by centrifugal force at a relative centrifugal force (RCF) of at least 5000 times the force of gravity.

10) The method of claim 8 wherein elution is by centrifugal force for a period of 10 seconds to 10 minutes.

11) The method of claim 1 not comprising any additional washing steps.

12) The method of claim 1 comprising washing said separation material with an aqueous washing medium.

13) The method of claim 1, additionally comprising assaying for the .sup.227Th content of said second solution by gamma detection or gamma spectroscopy, such as by use of a germanium semiconductor detector.

14) The method of claim 1, additionally comprising forming at least one radiopharmaceutical from at least a portion of the .sup.227Th contained in the second solution comprising .sup.227Th.

15) The method of claim 14 wherein the portion is between 0.1 MBq and 100 MBq .sup.227Th.

16) The method of claim 14 wherein the radiopharmaceutical is formed from said portion of .sup.227Th and at least one octadentate complexing agent.

17) The method of claim 16 wherein the octadentate complexing agent is conjugated to a targeting moiety selected from an antibody, antibody construct, antibody fragment or a construct of antibody fragments as well as nanoparticles and bisphosphonates.

18) The method of claim 14 wherein the radiopharmaceutical and/or targeting moiety has specificity for at least one target selected from cluster of differentiation (CD) cell surface markers.

19) The method of claim 14 wherein said forming comprises incubating the portion of the .sup.227Th contained in said second sample with a targeting conjugate comprising a complexing agent linked to a targeting moiety, wherein such incubation is carried out at a temperature below 50 C.

20) The method of claim 19 wherein said incubation is carried out for a period of less than 2 hours.

21) The method of claim 20 wherein said incubation is carried out in the first aqueous buffer.

22) .sup.227Th comprising less than 50KBq .sup.223Ra per 1MBq .sup.227Th.

23) .sup.227Th comprising less than 50KBq .sup.223Ra per 1MBq .sup.227Th formed or formable by a method of claim 1.

24) A pharmaceutical composition comprising the .sup.227Th as claimed in claim 22 and optionally at least one pharmaceutically acceptable diluent.

25) A kit comprising a mixture of .sup.227Th and .sup.223Ra, a first aqueous buffer solution, and a strong cation exchange resin.

26) A kit as claimed in claim 25 additionally comprising at least one of the following optional items: at least one sterile filter; at least one heat resistant vessel; at least one heating device; at least one .sup.227Th complexing agent;

Description

[0094] The invention will now be illustrated further by reference to the following non-limiting examples and the attached figures, in which:

[0095] FIG. 1 Shows the decay of .sup.227Th over time and the corresponding in-growth of .sup.223Ra and daughter isotopes over 90 days.

[0096] FIG. 2 Shows the radioactive decay chain of .sup.227Th to stable .sup.207Pb via .sup.223Ra.

[0097] FIG. 3 Shows the purification and labelling steps carried out shortly before administration in order to separate .sup.227Th from in-grown .sup.223Ra and complex the purified .sup.227Th to an antibody/ligand conjugate.

[0098] FIG. 4 Shows the effect of buffer concentration (M) (y-axis) and pH (x-axis) on uptake of .sup.223Ra from citrate buffered formulations at 15.0 mg resin (a) and 30.0 mg resin (b).

[0099] FIG. 5 Shows the effect of pH (x-axis) and resin amount (mg) (y-axis) on uptake of .sup.227Th from acetate buffered formulations without additives (a) and with pABA and EDTA (b).

[0100] FIG. 6 Shows the radioactive decay chain of .sup.227Ac to .sup.223Ra.

[0101] The following legends apply to the corresponding Figures of this application:

[0102] FIG. 3Purification of decayed .sup.227Th and preparation of Targeted Thorium Conjugate (TTC); sequestering of .sup.223Ra from buffered formulation by purification on micro-spin column, followed by labelling of purified 227Th on conjugate (antibody with chelator)

[0103] FIG. 4Effect of varying citrate buffer concentration and pH on uptake of .sup.223Ra (in percentage) onto (a) 15.0 mg and (b) 30.0 mg of PSA resin.

[0104] FIG. 5Effect of varying PSA resin mass and buffer pH on uptake of .sup.227Th (in percentage) onto PSA from acetate buffer a) without additives pABA/EDTA and b) with pABA/EDTA additives.

EXAMPLES

Materials

[0105] Sodium acetate trihydrate (99.0%), Sodium citrate tribasic dihydrate, (99.0%), 4-aminobenzoic acid sodium salt (pABA, 99%), Edetate disodium (EDTA, meets USP testing specifications), and sodium hydroxide (98.0-100.5%) were purchased from Sigma-Aldrich (Oslo, Norway). Metal free water (TraceSELECT) was purchased from FLUKA (Buchs, Switzerland). Sodium chloride (for analysis), hydrochloric acid (fuming, 37%, for analysis) and acetic acid (glacial, 100% anhydrous for analysis) was purchased from Merck Millipore (Darmstadt, Germany). Citric acid monohydrate (analytical reagent) was purchased from VWR (West Chester, USA). PSA (propylsulphonic acid) cation exchange resin based on silica was purchased from Macherey Nagel (Dren, Germany). NAPS columns were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Micro-Spin Columns were purchased from Thermo Scientific Pierce (product number 89879 (Rockford, USA).

[0106] The conjugate was an in house product and consisted of 5 mg/ml trastuzumab in sodium citrate buffer 0.10 M pH 5.5 and 0.90% (w/w) sodium chloride. The conjugate was made from an in house chelator attached to the trastuzumab antibody. Trastuzumab from Herceptin (150.0 mg powder for concentrate for solution for infusion) is a trademark of Roche Registration Limited (Welwyn Garden City, Great Britain).

[0107] The available radioactivity source was decayed .sup.227Th (as thorium(IV)) in 0.05 M hydrochloric acid and metal free water (an in house product). .sup.227Th was left to decay for approximately one half-life of 19 days until which the quantity of .sup.223Ra (as radium(II)) builds up to a near 1:1 ratio of .sup.227Th and .sup.223Ra.

EXAMPLE 1

Preparation of Buffered Formulations

[0108] Stock citrate buffers (0.10 M pH 4.0, 0.10 M pH 5.5, 0.05 M pH 5.0, and 0.07 M pH 4.8) and stock acetate buffers (0.10 M pH 4.0, 0.10 M pH 6.0, 0.10 M pH 5.5, 0.07 M pH 4.8, and 0.10 M pH 5.0) were prepared in metal free water and further diluted if required. pABA (2.0 mg/ml) and EDTA (2.0 mM) was subsequently added to selected formulations. In addition, selected citrate buffered formulations were added sodium chloride (0.45 or 0.90% (w/w) to adjust ionic strength.

[0109] All excipients used are included in the inactive ingredient list from FDA for approved drug products suitable for i.v. injection.

[0110] A calibrated sevenMulti pHmeter from Mettler Toledo (Oslo, Norway) was used to measure pH of stocks and final formulations at ambient temperature.

EXAMPLE 2

Preparation of Micro-Spin Columns with PSA Cation Exchange Resin

[0111] 1500.00 mg PSA silica resin was suspended in 15.00 ml metal free water. The suspension was shaken on a vortex mixer to ensure homogeneity before the appropriate volume was transferred to the micro-spin columns to give a resin amount of 15.0, 22.5, and 30.0 mg, respectively. The columns were subsequently spun for 1 minute at 10000 rcf to remove the water by an Eppendorf thermomixer comfort (Hamburg, Germany).

[0112] The columns were conditioned with 300 l of the respective buffered formulations. The excess volume was removed by spinning for 1 minute at 10000 rcf on the thermomixer resulting in dry columns (n=2 for DOE samples and center points).

EXAMPLE 3

Purification

[0113] 600 l of the respective buffered formulations was mixed with approximately 400 kBq .sup.227Th and 400 kBq .sup.223Ra in 0.05 M hydrochloric acid (i.e. 1-5 l decayed .sup.227Th in hydrochloric acid, dependent on the radioactive concentration). Half the volume was subsequently added to each column (n=2). The columns were then spun for 1 minute at 10000 rcf on the thermomixer, leaving the columns dry. The eluate was collected in an Eppendorf tube below the column.

EXAMPLE 4

Radioassay

[0114] The amount of .sup.223Ra and.sup.227Th on the cation exchange columns and in the eluates after the separation method of Example 3 was measured before calculating the distribution of the radionuclides between the column and the eluate. HPGe spectra from a High Purity Germanium (HPGe)-detector (GEM(15) from Ortec (Oak Ridge, Tenn.) was used. This detector identifies and quantifies radionuclides with gamma energies ranging from approximately 30 to 1400 keV. All samples analyzed by the HPGe-detector were placed in the same position and counted for 1 min. This method could be used to assay the radioisotope concentration in the eluate prior to preparing the radiopharmaceutical both to ensure a standard activity and to validate radiochemical (radioisotope) purity.

EXAMPLE 5

Labelling of Trastuzumab Conjugate with Purified .SUP.227.Th

[0115] 350 l of the respective buffered formulations was mixed with approximately 1000 kBq .sup.227Th and 1000 kBq .sup.223Ra in 0.05 M hydrochloric acid (i.e. 1-5 l decayed .sup.227Th in hydrochloric acid, dependent on the radioactive concentration). Half the volume was added to each micro-spin column (n=2). The columns were then for spun 1 minute at 10000 rcf on the thermomixer, leaving the columns dry with the eluate in an Eppendorf tube below the column. The amount of .sup.227Th and .sup.223Ra on the cation exchange columns and in the eluates was measured with the High Purity Germanium (HPGe)-detector GEM(15) before calculating the amount of .sup.227Th in the eluate for further use to label the conjugate.

[0116] A frozen sample of the trastuzumab-chelator conjugate (5.00 mg/ml) was allowed to equilibrate to ambient temperature. 160 l of the conjugate was then transferred to an Eppendorf tube and mixed with 160 l eluate in selected citrate buffered formulations from the micro-spin columns (approximately 500 kBq .sup.227Th). The formulations tested were 0.10 M citrate buffer pH 5.5 and the equivalent buffered formulation containing pABA+EDTA (n=2). The samples were then shaken for 30 minutes (22 C., 750 rpm, 10 s cycles) on the thermomixer.

EXAMPLE 6

Validation of the Labelling Reaction

[0117] The radiochemical purity (RCP) of a radiopharmaceutical is the relationship between .sup.227Th (in this case) present in a bound form (the TTC) to .sup.227Th in its unbound form (free radionuclide). The RCP was calculated by adding 200 l of the respective labelled conjugated sample (TTC) to a NAP5 column and, following the standard procedure for the column given by the manufacturer, the amount of 227Th uptake on the NAP5 column (size exclusion chromatography) and in the eluate was analysed by the aid of the HPGe-detector spectra (n=2).

[0118] According to Bayer AS standards, a successful labelling reaction will give a radiochemical purity above 90% for the TTC (Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer, Wiley, 2010.). The labelling of trastuzumab conjugate with purified decayed 227Th was within requirements for both the formulation containing 0.10 M citrate buffer pH 5.5 and the equivalent formulation also containing pABA+EDTA (n=2).

EXAMPLE 7

Separation Optimisation

[0119] A Design of Experiment (DOE) was devised to investigate and optimise the conditions for separation of .sup.223Ra from .sup.227Th on a silca/PSA micro spin column. For each buffer (citrate and acetate), the following variables were investigated:

TABLE-US-00002 TABLE 1 DoE variable Denomination Span pH A 4.0-5.5 pABA (2 mg/ml) + EDTA (2 B w/wo (with or without) mM) Buffer concentration (M) C 0.03 0.10 Resin mass (mg) D 15.0 30.0

[0120] Each of the DoE variables was investigated using the separation methodology indicated in Examples 1 to 6. The results are shown in FIGS. 4 and 5, which illustrate the effect of various parameters on radioisotope uptake onto PSA resin.

Optimal separation conditions were found to be:

TABLE-US-00003 TABLE 2 Buffer Resin pABA/EDTA conc., mass, Predicted Predicted Formulation PH w or w/o M mg .sup.227Th, % .sup.223Ra, % Citrate 4.0 w or w/o 0.03 15.0 2.4 97.5 Acetate 4.0 w* 0.03- 15.0 1.6 96.2 0.10 *According to Pooled SD and response surface the uncertainty is expected to be lower with than without pABA/EDTA