PURIFICATION METHOD

Abstract

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

The invention additionally provides a purified .sup.227Th solution, a 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 complexed .sup.227Th from a mixture comprising complexed .sup.227Th and .sup.223Ra (complexed or in solution), the method comprising: i) preparing a first solution comprising a mixture of complexed .sup.227Th ions and .sup.223Ra ions in a first aqueous buffer; ii) loading the first solution onto a separation material; iii) eluting complexed .sup.227Th from the separation material whereby to generate a second solution comprising complexed .sup.227Th; and iv) optionally rinsing the separation material using a first aqueous washing medium.

2: The method of claim 1, further comprising step X): X) prior to step i), contacting .sup.227Th ions with at least one complexing agent in solution, whereby to form at least one aqueous solution of complexed .sup.227Th.

3: The method of claim 2, wherein the complexing agent is a chelating moiety conjugated to a targeting moiety.

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

5: The method of claim 1, wherein the first aqueous buffer is at a pH of between 3 and 6.5.

6: The method of claim 1, wherein the first aqueous buffer comprises at least one of citrate buffer, acetate buffer, and mixtures thereof.

7: The method of claim 1, wherein the first aqueous buffer solution further comprises at least one radical scavenger or at least one chelating agent, or both.

8: The method of claim 1, wherein the separation material is a silica based cation exchange resin.

9: The method of claim 8, wherein the cation exchange resin comprises at least one sulfonic acid moiety.

10: The method of claim 1, wherein the elution is under gravitational force, under centrifugal force, under gas pressure from above, under vacuum from below, or both under gas pressure from above and under vacuum from below.

11: The method of claim 10, wherein elution is under centrifugal force at a “relative centrifugal force” (RCF) of at least 5000 times the force of gravity.

12: The method of claim 10, wherein elution is under centrifugal force for a period of 10 seconds to 10 minutes.

13: The method of claim 1, wherein the method does not comprise any additional washing steps.

14: The method of claim 1, comprising washing the separation material with an aqueous washing medium.

15: The method of claim 1, additionally comprising assaying for the .sup.227Th content of the second solution by gamma detection or gamma spectroscopy.

16: 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 complexed .sup.227Th.

17: The method of claim 16, wherein the portion of the .sup.227Th contained in the second solution comprising .sup.227Th is between 0.1 MBq and 20 MBq .sup.227Th.

18: The method of any of claim 2, wherein the complexed .sup.227Th is formed from the .sup.227Th ions and at least one octadentate complexing agent.

19: The method of claim 18, wherein the at least one octadentate complexing agent is conjugated to a targeting moiety selected from an antibody, an antibody construct, an antibody fragment, or a construct of antibody fragments.

20: The method of claim 18, wherein the octadentate complexing agent is conjugated to a targeting moiety that has specificity for at least one target selected from “cluster of differentiation” (CD) cell surface markers.

21: The method of claim 2, wherein the contacting of step X) comprises incubating the .sup.227Th ions 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.

22: The method of claim 21, wherein the incubation is carried out for a period of less than 2 hours.

23: The method of claim 22, wherein the incubation is carried out in the first aqueous buffer.

24: A solution of complexed .sup.227Th comprising less than 50 KBq .sup.223Ra per 1 MBq .sup.227Th.

25: A solution of complexed .sup.227Th comprising less than 50 KBq .sup.223Ra per 1 MBq .sup.227Th, obtained by the method of claim 1.

26: A pharmaceutical composition comprising the solution of complexed .sup.227Th of claim 24 and at least one pharmaceutically acceptable diluent.

27: A kit comprising a mixture of .sup.227Th and .sup.223Ra, a complexing agent, a first aqueous buffer solution, and a strong cation exchange resin.

28: The kit of claim 27 additionally comprising at least one of the following: at least one sterile filter; at least one heat resistant vessel; at least one heating device; and at least one pharmaceutical excipient or diluent.

Description

[0103] The invention will now be illustrated further by reference to the following non-limiting examples and the attached figures, in which:

[0104] FIG. 1 Shows the decay of .sup.227Th over time and the corresponding in-growth of .sup.223Ra and daughter isotopes over 28 days.

[0105] FIG. 2 Shows the radioactive decay chain of .sup.227Th to stable .sup.207Ph via .sup.223Ra.

[0106] FIG. 3 Shows a schematic of the experimental steps for purification of a sample of complexed .sup.227Th on micro-spin columns where the .sup.227Th is partially decayed to .sup.223Ra.

[0107] FIG. 4 Shows the effect of pH (x-axis) on radiochemical purity of a .sup.227Th complex (y axis) in citrate buffered formulations. Plot a) contains NAP5 purity data. Plot b) is for iTLC purity data. Data series without pABA+EDTA (triangular data series) and with the presence of pABA+EDTA (square data series) are shown on each plot.

[0108] FIG. 5 SDS-PAGE chromatogram; samples 1 to 4 with application point with TTC (bound .sup.227Th) and front line (free .sup.227Th)

EXAMPLES

Materials

[0109] 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) and hydrochloric acid (fuming, 37%, for analysis) were purchased from Merck Millipore (Darmstadt, Germany). Citric acid monohydrate (analytical reagent) was purchased from VWR (West Chester, USA). Acetic acid (glacial, 100% anhydrous for analysis) was purchased from Merck (Darmstadt, Germany).

[0110] PSA (propylsulphonic acid) cation exchange resin based on silica was purchased from Macherey Nagel (Düren, Germany). NAP5 columns were purchased from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Pierce Micro-Spin Columns were purchased from Thermo Scientific Pierce (product number 89879 (Rockford, USA).

[0111] Trastuzumab from Herceptin® (150 mg powder for concentrate for solution for infusion) was used and is a trademark of Roche Registration Limited (Welwyn Garden City, Great Britain). To make the conjugate an in house chelator had been attached to the antibody. The resulting conjugate colloidal suspension was 5.0 mg/ml conjugate in sodium citrate buffer 0.10 M pH 5.1 and 0.90% (w/w) sodium chloride.

[0112] .sup.227Th (as thorium (IV)) in 0.05 M hydrochloric acid and metal free water (an in house product) was used as the radioactivity source. To build up to a near 1:1 ratio of .sup.227Th and .sup.223Ra (as radium (II)), .sup.227Th was stored in order to decay for approximately one half-life of 19 days.

[0113] iTLC-SG chromatography paper impregnated with silica gel from Agilent Technologies was used for instant thin-layer chromatography (iTLC) analyses (Santa Clara, Calif.).

[0114] The following materials were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); LDS (4×) sample buffer and NuPage 10% tris-bis gel from Novex (Carlsbad, Calif.). MES (20×) buffer from NuPage (Carlsbad, Calif.). Instant blue from Expedeon (Cambrideshire, UK), and Precision Plus Protein dual colour Standard from BioRad (Hercules, Calif.).

Example 1—Preparation of Buffered Formulations

[0115] Stock citrate buffers (0.10 M pH 4.0, 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 and 0.10 M pH 5.0) were prepared in and diluted with metal free water (if required) to the respective buffer concentrations used in the range of the DOE. pABA (2.0 mg/ml)+EDTA. (2.0 mM) and sodium chloride were subsequently added to the respective buffered formulations containing these excipients.

[0116] The pH of the stocks and final formulations were thoroughly controlled at ambient temperature with a calibrated sevenMulti pHmeter from Mettler Toledo (Oslo, Norway).

[0117] A calibrated sevenMulti pHmeter from. Mettler Toledo (Oslo, Noway) 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

[0118] A 100.0 mg/ml suspension of PSA resin was prepared in metal free water. To ensure homogeneity of the suspension, a vortex mixer was used and the required volume for 15.0, 30.0, and 22.5 mg resin was added to the micro-spin columns.

[0119] For conditioning of the packed resin, 300 μl of the respective buffered formulations was added to the columns before spinning for 1 minute at 10000 ref on an Eppendorf thermomixer comfort (Hamburg, Germany (n=1, 2 or 3 for DOE samples and n=2 for center points) resulting in a dry resin bed before further use.

[0120] The columns were conditioned with 300 μl of the respective buffered formulations. The excess volume was removed by spinning for 1 minute at 10000 ref on the thermomixer resulting in dry columns (n=2 for test samples and center points).

Example 3—Complexation and Purification

[0121] The amount of radioactivity added to each sample was approximately 250 kBq .sup.227Th (as TTC) and 250 kBq 223Ra. Prior to use, the frozen trastuzumab-chelate conjugate colloidal suspension was allowed to equilibrate to ambient temperature. 50 μl of the conjugate was added to an Eppendorf tube with 500 kBq .sup.227Th and 500 kBq .sup.223Ra in 0.05 M hydrochloric acid (1-5 μl depending on the radioactive concentration) and mixed with 50 μl of the respective buffered formulations. The samples were then shaken 30 minutes (22° C., 750 rpm, 10 s cycles) on an Eppendorf thermomixer comfort in order to label the conjugate with decayed .sup.227Th and form the TTC. 250 μl buffered formulation was subsequently added and mixed with the labelled conjugate (TTC) before 170 μl of this sample was added to each micro-spin column (n=1, 2 or 3 for test samples and n=2 for center points). For samples with one or three parallels, the radioactivity and volumes were adjusted as required to maintain the same conditions as for two parallels described herein. The columns were spun for 1 minute at 10000 ref on the thermomixer to elute the columns and the purified material collected in eppendorf tubes.

Example 4—Radioassay

[0122] 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. Qrtec (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. The amount of .sup.227Th and .sup.223Ra on the columns and in the eluates after spinning was measured, and the distribution of the radionuclides between the column and the eluate was calculated by the aid of the HPGe-detector spectra. 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—Stability Studies: Radiochemical Purity of the TTC

[0123] The radiochemical purity (RCP) of a radiopharmaceutical is the relationship between .sup.227Th, in this case, present in a bound form (i.e. as TTC) to free .sup.227Th. Since only the radionuclides .sup.227Th and .sup.223Ra are measured on the High Purity Germanium. (HPGe)-detector GEM(15), the TTC data cannot be excluded from being free .sup.227Th. Some of the samples analysed for separation of TTC and .sup.223Ra on the columns and in the eluates were therefore also analysed for RCP (gel filtration, iTLC, SDS-PAGE) with the same detector at ambient temperature after measurement of the separation of the radionuclides (within the same day).

[0124] 5.1 Gel Filtration on NAP5 Columns

[0125] In order to analyse the radiochemical purity of the TTC, NAP5 columns were used (gel filtration with size exclusion). The standard procedure from the manufacturer was followed and a sample volume of 200 μl was added to the columns. HPGe-detector spectra were recorded in order to analyse the amount of TTC on the NAP5 column (n=2). See FIG. 4a)

[0126] 5.2 Instant Thin-Layer Chromatography

[0127] The iTLC-SG chromatography paper was cut and dried by heating for 20-30 min in an incubator at 110-120° C. in order to be activated. A beaker was filled with approximately 0.5 cm of 0.10M citrate buffer pH 5.5 with 0.90% (w/w) sodium chloride (mobile phase). 1-8 μl of the samples (TTC purified on micro-spin columns) was applied at the origin line of the paper strip (n=2). The strip was placed vertically into the beaker, carefully avoiding any damage to the surface. When the solvent had reached the solvent front line, the strip was removed from the beaker and allowed to dry. The strip was divided into upper and lower sections by cutting the strip in half, each section of the strip was then placed in counting tubes. The activity in each half was measured separately for 5 minutes and the percentage .sup.227Th at the front line and point of application was calculated by the aid of an Auto HPGe, Ortec gamma spectrometer with HPGe-detector (Oak Ridge, Tenn.). The results were corrected for the presence of .sup.223Ra from decayed .sup.227Th at the front line which had been building up during the time for analyses. See FIG. 4b)

[0128] 5.3 Gel Electrophoresis (SDS-PAGE)

[0129] Citrate buffered samples in Table 0.1 (below) were analyzed with SDS-PAGE (n=2).

[0130] The standard procedure from the manufacturer of the NuPAGE® Bis-Tris Mini Gels was followed. MES Running Buffer was prepared by mixing 950 ml Milli Q water and 50 ml MES buffer. The samples were prepared by dilution to achieve a conjugate concentration of 1.0 mg/ml (in 0.03 M citrate buffer pH 5.5 and 0.90% w/w sodium chloride) with LDS sample buffer, Milli Q water and MES buffer. The samples were then mixed and stored on ice until, use. 5 μg of the conjugate was loaded in each well (n=2). The gel electrophoresis was run manually at a constant voltage of 200 V on the XCell SureLock Mini-Cell (Invitrogen, Carlsbad, Calif.) with Power Pac Adaptor, 4 mm and Power Pac Basic (BioRad, Hercules, Calif.). The gel was stained with Instant Blue and incubated for 60 minutes at ambient temperature. The staining reaction was stopped by washing the gel with water. The gel was then moved to a transparency film and pictures were taken. See FIG. 5

TABLE-US-00002 TABLE 1 Sample pABA + EDTA Spin denomination pH (w/wo) (w/wo) 1 4.0 w w 2 5.5 wo w 3 4.0 wo wo 4 4.0 wo w

Example 6—Separation Optimisation

[0131] 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-00003 TABLE 2 DoE variable Denomination Range pH citrate/acetate buffer A 4.0-6.0 pABA + EDTA B w-wo Buffer concentration (M) C 0.05-0.10 Resin mass (mg) D 15.0-30.0 Sodium chloride concentration E 0.45-0.90 (% w/w)

[0132] Each of the DoE variables was investigated using the separation and analysis methodology indicated in Examples 1 to 5. The results are shown in Table 3, which illustrate the effect of various parameters on radioisotope uptake onto PSA resin.

TABLE-US-00004 TABLE 3 Variables and correlation to uptake (positive↑ Model Pooled or negative↓) Model 95% Model Pooled 95% Response (p < 0.05) Uptake, % SD, % CI R SD, % CI Citrate TTC ↓A, ↑C.sup.1, ↑D↓E.sup.1  3.9-24.2 1.9  ±3.8.sup.  0.93 1.3 ±2.6 Citrate .sup.223Ra ↓A, ↓C, ↑D, ↓E, AC.sup.1 15.3-98.6 7.5 ±15.0.sup.  0.96 2.7 ±5.4 Acetate TTC ↓A, ↑D 12.4-37.2 2.9  ±5.8.sup.  0.91 2.2 ±4.4 Acetare .sup.223Ra ↓A, ↓B.sup.1, ↑D 82.3-99.3 3.3.sup.2  ±6.6.sup.2 0.52 1.7 ±3.4 .sup.1bordeline significant, .sup.2run with pABA/EDTA, high pH and low resin mass contribute to increased uncertainty in predicted .sup.223Ra

[0133] Some examples of highly effective separation conditions were found to be:

TABLE-US-00005 TABLE 4 pABA/EDTA Buffer Resin NaCl (w/w Predicted Predicted Buffer pH (w-w/o) conc. (M) mass (mg) %) TTC, % .sup.223Ra, % Citrate 4.0 w 0.05 15.0 0.45 13 82 Citrate 4.0 w 0.05 30.0 0.45 22 100 Acetate 4.0 w 0.05-0.10 15.0 0.45-0.90 21 96 Acetate 6.0 w 0.05-0.10 30.0 0.45-0.90 28 96

[0134] Where predicted TTC % is the predicted uptake of the TTC onto the resin and predicted .sup.223Ra % is the predicted uptake of .sup.223Ra onto the resin. High separation efficiency should combine low TTC uptake and high .sup.223Ra uptake.