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Title:
METHOD FOR THE QUANTIFICATION OF 227AC IN 223RA COMPOSITIONS
Document Type and Number:
WIPO Patent Application WO/2015/022074
Kind Code:
A1
Abstract:
A method for the quantification of 227Ac in a 223Ra composition comprising passing the composition through a first solid phase extraction column A, wherein said column comprises a thorium specific resin, passing the eluate of column A through a second solid phase extraction column B, wherein said column comprises an actinium specific resin and recovering the 227Ac absorbed onto the resin in column B and determining the amount thereof.

Inventors:
HJELLUM, Gro Elisabeth (Øvre Topenhaug 116, Bærums Verk, N-1353, NO)
Application Number:
EP2014/002222
Publication Date:
February 19, 2015
Filing Date:
August 13, 2014
Export Citation:
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Assignee:
ALGETA ASA (Kjelsåsveien 172A, Oslo, N-0884, NO)
International Classes:
B01D15/08; B01D15/18; B01D59/26; A61K51/00; C01F13/00; C22B60/00; G01N30/60; G21G4/08; G21G4/10
Foreign References:
US20030194364A12003-10-16
US5854968A1998-12-29
DE102006008023A12007-08-30
US5809394A1998-09-15
Other References:
MARTIN P ET AL: "Determination of <227>Ac by alpha-Particle Spectrometry", APPLIED RADIATION AND ISOTOPES, ELSEVIER, OXFORD, GB, vol. 46, no. 10, 1 October 1995 (1995-10-01), pages 1065 - 1070, XP004050685, ISSN: 0969-8043, DOI: 10.1016/0969-8043(95)00222-Y
R BOJANOWSKI ET AL: "DETERMINATION OF 227Ac IN ENVIRONMENTAL SAMPLES BY ION-EXCHANGE AND ALPHA SPECTROMETRY", JOURNAL OF RADIOANALYTICAL AND NUCLEAR CHEMISTRY, ARTICLES, 1 October 1987 (1987-10-01), pages 23 - 37, XP055145102, Retrieved from the Internet [retrieved on 20141008]
KOHLER M ET AL: "Simultaneous determination of Ra and Th nuclides, <238>U and <227>Ac in uranium mining waters by gamma-ray spectrometry", APPLIED RADIATION AND ISOTOPES, ELSEVIER, OXFORD, GB, vol. 52, no. 3, 1 March 2000 (2000-03-01), pages 717 - 723, XP027388517, ISSN: 0969-8043, [retrieved on 20000301]
Attorney, Agent or Firm:
GORDON, Jennifer (Dehns, St Bride's House10 Salisbury Square, London EC4Y 8JD, GB)
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Claims:
Claims

1. A method for the quantification of Ac in a Ra composition, said method comprising:

(i) passing said 223Ra composition through a first solid phase extraction column A, wherein said column comprises a thorium specific resin;

(ii) passing the eluate of column A through a second solid phase extraction column B, wherein said column comprises an actinium specific resin;

(iii) recovering the 227 Ac absorbed onto the resin in column B and determining the amount thereof.

2. A method as claimed in claim 1, wherein the thorium specific resin comprises a phosphonate extractant, preferably an alkyl phosphonate extractant.

3. A method as claimed in claim 1 or 2, wherein the thorium specific resin comprises a dialkyl alkyl phosphonate extractant of Formula I:

wherein each of R1-R3 is independently a C3-C8 straight or branched chain alkyl group, preferably a dipentyl pentylphosphonate extractant.

4. A method as claimed in any of claims 1 to 3, wherein the actinium specific resin comprises a diglycolamide extractant.

5. A method as claimed in any of claims 1 to 4, wherein the actinium specific resin comprises a tetra-alkyl diglycolamide extractant of Formula II: wherein R]-R4 are independently C3-Cj2 straight or branched chain alkyl groups, preferably a N,N,N',N'-tetra-n-octyldiglycolamide (DGA) extractant.

6. A method as claimed in any of claims 1 to 5, wherein column A and column B are arranged in series.

7. A method as claimed in any of claims 1 to 6, wherein the eluent used in both columns A and B comprises aqueous nitric acid.

227

8. A method as claimed in any of claims 1 to 7, wherein recovery of the "'Ac in step (iii) is achieved by washing column B with aqueous acid. 9. A method as claimed in claim 8 wherein the washing volume of aqueous acid is 16 to 400 times the volume of the column, preferably 40 to 200 times.

10. A method as claimed in any of claims 1 to 9, wherein the determination in step (iii) is achieved by γ-spectrometry via in-growth and detection of the daughter 227Th.

1 1. A method as claimed in any of claims 1 to 5, said method comprising:

(i) Placing a first solid phase extraction column A comprising a thorium specific resin (e.g. dipentyl pentylphosphonate UTEVA resin) and a second solid phase extraction column B comprising an actinium specific resin (e.g. N, N,

N', N' - tetra-n-octyldiglycolamide DGA resin) in series, preferably wherein the output of column A is connected to the input of column B; (ii) Adding a volume of a Ra composition corresponding to a known activity (e.g. 15 MBq) of 223Ra to an equal volume of nitric acid, preferably 8 mol/L nitric acid;

(iii) Transferring the sample from step (ii) to the input of the column A;

(iv) Passing said sample through both columns A and B

(v) Washing both columns with 20-100 times the combined volume of the two columns (e.g. 5-10 ml) nitric acid, preferably 4 mol/L nitric acid;

(vi) Disconnecting column A from column B;

(vii) Washing column B with 40-200 times its volume (e.g. 5-10 ml) nitric acid, preferably 4 mol/L nitric acid;

(viii) Washing column B with 40-200 times its volume (e.g. 5-10 ml) nitric acid at a concentration less than that used in step (vii), such as 0.05 mol/L nitric acid.

(ix) Determining the amount of 227Ac present in the eluate from column B

obtained in step (viii).

12. A method as claimed in claim 1 1, wherein the determination in step (ix) is achieved by γ-spectrometry via in-growth and detection of the daughter Th.

13. Use of a method as claimed in any of claims 1 to 12 in the quantification of 227 Ac in a 223Ra composition.

14. Apparatus for use in a method as claimed in any of claims 1 to 12, wherein said apparatus comprises a first solid phase extraction column A, wherein said column comprises a thorium specific resin (e.g. dipentyl pentylphosphonate UTEVA resin), and a second solid phase extraction column B, wherein said column comprises an actinium specific resin (a N, N, N', N'-tetra-n-octyldiglycolamide DGA resin).

15. Apparatus as claimed in claim 14, wherein column A and column B are arranged in series, preferably such that the output from column A is connected to the input of column B.

Description:
METHOD FOR THE QUANTIFICATION OF 227 C IN 223RA COMPOSITIONS

Field of the Invention The present invention relates to a novel method for quantifying levels of

Ac in Ra compositions, in particular a method which involves solid phase extraction followed by quantification via the in-growth of the Th daughter via γ- spectrometry. The invention further relates to the use of the method of the invention in determining the level of Ac in a Ra composition and to an apparatus for use in the method of the invention.

Background

A substantial percentage of cancer patients is effected by skeletal metastases. As many as 85% of patients with advanced lung, prostate and breast carcinoma develop bony metastates (Garret 1993, Nielsen et al, 1991). They are associated with a decline in health and quality of life, ultimately leading to death, often within a few years.

When tumors or metastases cannot be removed by surgery, the conventional approach is to apply external beam radiotherapy and chemotherapy. Both suffer from a lack of selectivity for tumor cells and tumor tissue. As a consequence, treatment most often cannot be applied at curative levels due to toxicity to healthy tissue.

Bone-seeking β-emitters like Sr and Sm complexed with

ethylene-diaminetetramethylene-phosphonate (EDTMP) have been used as internal radiotherapy agents in the pain palliation of painful bone metastases especially in prostate cancer. The altered skeletal metabolic activity around many bone metastases results in a local increase in bone formation and uptake of calcium, which is used to construct the hydroxyapatite bone mineral. Bone-seeking radionuclides target this bone adjacent to the tumor deposits. Calcium mimetics, such as strontium 89 Sr, belong to the alkaline earth group of elements in the periodic table. They can be administered as an intravenous radioactive salt that will be incorporated into the newly formed hydroxyapatite in bone metastases. Other radionuclides, such as 153 Sm, require a carrier molecule to achieve selective uptake to the bone, for example, EDTMP. By selectively targeting areas of high metabolic activity in bone, a high therapeutic index is possible.

However, the β-particles are characterized by low-linear energy transfer

(LET) typically in the range of 0.2-1.0 keV/μηι and a modest relative biological effectiveness (RBE). The use of highly energetic β-particles is restricted by the radiation burden and cell damage to surrounding healthy tissue and especially by the suppression of blood cells in the red bone marrow. Hence, there is an unmet need for more effective bone-targeted treatments that improve quality of life and survival whilst maintaining a favorable safety profile.

The use of a-emitting radionuclides has a major advantage in radiotherapy of cancer. Compared to the low LET values of β-emitters, a-emitters have a mean LET value of 80-100 keV/μηι. 223 Ra has shown particular promise. For example, Alpharadin ® ( 223 RaCl 2 ) has completed a global phase-Ill clinical trial in patients with castration-resistant prostate cancer (CRPC) and bone metastases. Data shows that Alpharadin prolongs patient overall survival time while offering a well tolerated safety profile (Brady et al, Cancer J., 2013, 19, 71-78). 223 Ra, like 89 Sr, is a calcium mimic and also an alkaline earth element and can be administered as an intravenous radioactive salt. Due to the high LET-values of a-particles and, consequently, their short path-length in human tissue (< 100 μπι), a highly cytotoxic radiation-dose can be delivered to targeted cancer cells, while damage to the surrounding healthy tissue is limited.

Quality control is an essential part of pharmaceutical manufacture, to ensure the drugs sent to the market are safe and therapeutically active formulations have a performance which is consistent and predictable. The term quality control refers to the sum of all procedures undertaken on each batch to ensure e.g. the identity, activity and purity.

Radionuclidic purity is defined as the percentage of a contaminating radionuclide relative to the wanted radionuclide e.g. 227 Ac relative to 223 Ra with respect to activity in Bq. The primary reason for seeking radionuclidic purity in a radiopharmaceutical is to avoid unwanted administration of radiation to the patient. It is therefore extremely important to strictly control the levels of radionuclidic impurities in radiopharmaceuticals. Radionuclidic impurities may originate from several sources. For example, when a parent-daughter radionuclide generator system is used to produce the radionuclide of interest, the parent nuclides are defined as impurities in the product. Actions must be taken during production to ensure that the parent nuclides are separated from the nuclide of interest and, before release of the finished product for human use, it has to be confirmed that the radioactivity of the radionuclidic impurities are below the limit specified for the product.

Production of 223 Ra for pharmaceutical use is typically based on a radionuclide generator where the mother nuclide 227 Ac (t ½ = 21.77 years) is adsorbed on a column material. The daughter radionuclides are Th (t ½ = 18.68 days) and 223 Ra (t ½ = 1 1.43 days). 223 Ra is separated by column elution. 227 Ac and its daughter nuclide 27 Th must be strongly retained under conditions were 223 Ra can be

777 * ¾

eluted. Ac and Th do not have the same bone seeking properties as Ra and are regarded as impurities. Even very low amounts of these nuclides cannot be accepted in the pharmaceutical product. The acceptance criterion for Alpharadin has been set to not more than 0.004% for 227 Ac and not more than 0.5% for 227 Th relative to 223 Ra with respect to activity in Bq. Similar criteria would be expected for other 223 Ra products. Prior to formal release of the product to patients, each produced batch of radiopharmaceutical (e.g. Alpharadin) must be tested to show that it meets the acceptance criteria (adequately defined identity, strength, quality and purity). Due to the inherently short half-life of Ra, the radiopharmaceutical may be released before completion of all tests (e.g. sterility testing). This naturally has the disadvantage that patients could be exposed to a formulation which does not meet all the quality control criteria.

777 777

A quantitative determination of Ac is difficult as Ac decays almost entirely by emission of a low-energy β-particle (Ep >ma x = 0.0448 MeV), which is virtually undetectable in the presence of all the energetic a- and β-emitters of the

777 777

Ac chain (see Figure 1). Ac also decays by a-emission in 1.38% of its disintegrations. However, direct a-spectrometric determination of 227 Ac is complicated by interferences from the α-emissions of its rapidly growing decay products. Freshly purified 227 Ac emits no analytically useful γ-radiation. Consequently, many radiometric methods determine Ac indirectly by measurements of the a- and γ-radiations of its daughters, in particular by high- resolution γ-spectrometry of its daughter 227 Th. However, this cannot be determined until 10-12 months after release of the product as analysis must wait until there are

97 sufficiently measurable levels of Th. At this time, the potential amount of Ac

• 997 * contamination is in equilibrium with its daughter Th. Furthermore, the initial

077

amounts of Ra and any Th in the product would have decayed completely. These disadvantages not only lead to inaccuracy of results and increased costs but, more significantly, mean that the result comes too late for the 223 Ra pharmaceutical to be withdrawn from release to patients should it be shown to be contaminated with 227 Ac at levels which would be considered to jeopardise the efficacy of the treatment or the safety of the patient.

In view of the above, there remains a need to develop a new, reliable, accurate and cost-effective radiochemical method for early determination of the potential contamination of Ac in Ra pharmaceuticals, such as Alpharadin (RaCl 2 ). In particular, it would be an advantage to produce a method which is able to give a result in a matter of days rather than months. Ultimately, an analysis method which can be completed prior to release of the product and its administration to patients is attractive. The following criteria set out the desirable features of a new quantification method:

1. 227 Ac should selectively be separated from the precursors.

2. Recovery of 227 Ac > 70 % and precision > 30 %

3. Robustness i.e. the analytical result should remain unaffected by small variations in method parameters.

4. Easy to operate in routine production (in terms of time and cost).

5. Sample activity should be as low as possible due to cost and radiation exposure to the operators, and/or

6. Separation and quantification should be fulfilled before release of the product i.e. within 2 days after production of the 223 Ra pharmaceutical (e.g. 223-radium chloride). The present inventors have surprisingly found that an analytical method employing a tandem column arrangement comprising two different solid phase extraction resins can fulfil some or all of these requirements. In particular, the two columns enable facile separation and isolation of 227 Ac, which can be rapidly quantified.

Summary of the Invention

Thus, viewed from one aspect, the invention provides a method for the quantification of Ac in a Ra composition, said method comprising:

(i) passing said 223 Ra composition through a first solid phase extraction column A, wherein said column comprises a thorium specific resin (e.g. dipentyl pentylphosphonate UTEVA resin);

(ii) passing the eluate of column A through a second solid phase extraction column B, wherein said column comprises an actinium specific resin (e.g. N, N, N', N'-tetra-n-octyldiglycolamide DGA resin);

(iii) recovering the 227 Ac absorbed onto the resin in column B and determining the amount thereof.

Viewed from another aspect the invention provides a method as hereinbefore described, said method comprising

(i) Placing a first solid phase extraction column A comprising a thorium specific resin (e.g. dipentyl pentylphosphonate UTEVA resin) and a second solid phase extraction column B comprising an actinium specific resin (e.g. N, N, N', N' - tetra-n-octyldiglycolamide DGA resin) in series, preferably wherein the output of column A is connected to the input of column B;

(ii) Adding a volume of a 223 Ra composition corresponding to a known activity (e.g. 15 MBq) of Ra to an equal volume of nitric acid, preferably 8 mol/L nitric acid;

(iii) Transferring the sample from step (ii) to the input of the column A;

(iv) Passing said sample through both columns A and B (v) Washing both columns with 20-100 times the combined volume of the two columns (e.g. 5-10 ml) nitric acid, preferably 4 mol/L nitric acid;

(vi) Disconnecting column A from column B;

(vii) Washing column B with 40-200 times its volume (e.g. 5-10 ml) nitric acid, preferably 4 mol/L nitric acid;

(viii) Washing column B with 40-200 times its volume (e.g. 5-10 ml) nitric acid at a concentration less than that used in step (vii), such as 0.05 mol/L nitric acid.

(ix) Determining the amount of 227 Ac present in the eluate from column B

obtained in step (viii).

Viewed from another aspect the invention provides the use of a method as

997 99^

hereinbefore described in the quantification of Ac in a Ra composition.

Viewed from another aspect the invention provides apparatus for use in a method as hereinbefore described, wherein said apparatus comprises a first solid phase extraction column A, wherein said column comprises a thorium specific resin (e.g. a dipentyl pentylphosphonate UTEVA resin), and a second solid phase extraction column B, wherein said column comprises an actinium specific resin (e.g. a N, N, N', N'-tetra-n-octyldiglycolamide DGA resin).

Definitions

The 223 Ra composition of the invention will be understood to be any

9 * ¾ ·

composition which comprises the radionuclide Ra. The composition will typically be a pharmaceutical composition or a precursor to a pharmaceutical solution and will therefore usually contain the additional components often found in such compositions, e.g. pharmaceutically acceptable diluents, excipients and

9

carriers. Such components are well known in the art. The Ra may be in any form, however the most preferred form is as a salt such as a halide salt, preferably RaCl 2 (Alpharadin ® ), optionally in combination with other Ra salts. It will be appreciated that in order to be compatible with the method of the invention the 223 Ra composition must be in solution, typically an aqueous solution, such as an aqueous acid solution.

The method of the invention employs solid phase extraction. This technique is well known in the art, however a brief outline is provided here for completeness.

Solid-Phase Extraction (SPE) has become widely accepted as a substitute for traditional liquid-liquid extraction (LLE) in many types of separation procedures, and especially for those involving low to ultralow concentrations of analyte. SPE is based on the same principles as solvent extraction, which often involves

complexation to form a lipophilic compound of the analyte followed by transfer of this compound into an organic phase. In SPE the non-aqueous phase is solid instead of liquid as it is in LLE. SPE is generally faster, more efficient and generates less waste than LLE.

SPE comprises three major components; an inert support, a stationary phase and a mobile phase. The inert support usually consists of porous silica or particles of an organic polymer ranging in size from 50 to 150 μιη in diameter. The stationary phase, which is on the surface of the inert support, is selected appropriately depending on the analytes involved. The mobile phase is usually an aqueous acid solution, e.g. nitric or hydrochloric acid.

The method of the invention employs two different stationary phases (resins).

The first resin is a thorium specific resin, typically an UTEVA Resin (Uranium und TEtraValents Actinides), which is mainly used for the separation of uranium and tetravalent actinides. The extractant coated on the inert support is selected to specifically bind thorium in a solution mixture of radium, thorium and actinium. This specificity may be under all conditions, or the conditions used in the methods of the invention may be chosen to ensure specificity.

Extractants suitable for thorium specific resins include phosphonates, particularly alkyl phosphonates. Dialkyl alkyl phosphonates such as those of the following formula (Formula I) are preferred: wherein each of Ri-R 3 is independently a C 3 -C 8 straight or branched chain alkyl group. Preferably Ri-R 3 are straight chain alkyl groups. Preferably R] is a C 4 -C ( straight chain alkyl group, most preferably n-pentyl. R 2 and R 3 may be identical different. Preferably R 2 and R 3 are identical. Preferably each of R 2 and R 3 is a straight chain C 4 -C 6 alkyl group, most preferably n-pentyl. A high preferably extractant is dipentyl pentylphosphonate, which has the following structure:

The second resin is an actinium specific resin, typically selected to specifically bind actinium in a solution mixture of radium and actinium. This specificity may be under all conditions, or conditions used in the methods of the invention may be selected to ensure specificity.

In some embodiments, the conditions may be such that the actinium specific resin has some degree of affinity for radium as well as actinium and under those conditions both radium and actinium may bind to the second resin. It will be appreciated that, under such circumstances, the method of the invention may require a further step in which the conditions are altered such that any radium which has bound to the second resin may be specifically eluted whilst the actinium remains bound to the resin, before the actinium may be eluted from the second resin. Preferably, the conditions used in the methods of the invention are chosen such that the second resin does not have any affinity for radium and only actinium binds to the second resin. Thus, in a preferable embodiment, the conditions used in the method of the invention are such that the second resin is specific for actinium. A resin may be considered "specific" for one element over another if that resin will retain at least 90% of the first element under conditions that would elute at least 90% of the second element. This is preferably 95%, more preferably 99%.

Typically, the conditions chosen in the methods of the invention are certain concentrations of mineral acids (e.g. nitric acid) in water.

Extractants suitable for actinium specific resins include diglycolamides, particularly tetra-alkyl diglycolamides of the following formula (Formula II):

wherein Ri-Rj are independently C 3 -Ci 2 straight or branched chain alkyl groups, preferably C5-C10 straight or branched chain alkyl groups. R1 -R4 may be identical or different, preferably identical. R } may all be Cg alkyl groups. A preferred example is N,N,N',N'-tetra-n-octyldiglycolamide (DGA Resin, Normal), which has the following structure:

wherein the R-groups are straight chain Cg alkyl groups. The corresponding resin where the R-groups are branched Cg alkyl groups is also of value. In the context of the invention, the term "eluate" refers to the solution of solvent and dissolved matter resulting from elution, i.e. the mixture of components which elutes following separation using a solid phase extraction column.

The term "eluent" should be understood to be interchangeable with the term "mobile phase". Both terms are well known in the art and are used to refer to the solvent which is passed through a solid phase extraction column and is used to effect separation.

Detailed Description

The method of the invention comprises the following steps

""yyi 'y n 997

(i) passing a Ra composition (e.g. one containing Ac and Th contaminants) through a first solid phase extraction column A, wherein said column comprises a thorium specific resin (e.g. a dipentyl pentylphosphonate UTEVA resin);

(ii) passing the eluate of column A through a second solid phase extraction column B, wherein said column comprises an actinium specific resin (e.g. a N, N, N', N'-tetra-n-octyldiglycolamide DGA resin);

(ii) recovering the 227 Ac absorbed onto the resin in column B and determining the amount thereof.

In a preferable embodiment, column A and column B are arranged in series such that the eluate from column A passes directly into column B, i.e. wherein the output of column A is connected to the input of column B. The most preferable arrangement is for column A to be positioned above column B such that the eluate from column A drains directly into column B.

The method of the invention relies on the surprising finding that by choice of a resin and column configuration, contaminant 227 Ac can be purified from a mixture of Ra and Th to a sufficient degree to allow for accurate measurement of the

997 7 7

Ac via Th in-growth. For example, a UTEVA resin is capable of selectively retaining 227 Th out of a mixture of 223 Ra, 227 Ac and 227 Th and moreover that a DGA resin is capable of selectively retaining Ac from a mixture of Ac and Ra. This results in an efficient separation method. An outline of the process is provided in Figure 2.

223 223

The Ra composition used in the method of the invention comprises Ra. It will typically also comprise both Th and Ac contaminants. Thus, all three radionuclides are usually present in the starting mixture of analytes. As the mixture passes through the first column A, any Th present will absorb onto the thorium specific resin (e.g. UTEVA resin), leaving only Ra and Ac present in the eluate. As this eluate passes though the second column B, any 227 Ac will absorb onto the actinium specific resin (e.g. DGA resin). The 223 Ra will typically remain in the mobile phase. In some embodiments, the actinium specific resin may be washed with additional volumes of mobile phase so as to ensure all 223 Ra is eluted. Thus the total 227 Ac fraction may be obtained and isolated.

Importantly, the 227 Ac fraction, which is bound to the actinium specific resin (e.g. DGA resin), will be substantially free, preferably completely free, of Th and 223 Ra, thereby enabling more facile determination of its quantity via detection of the in-growth of its daughter nuclide, 227 Th at very low levels. In particular, results will

997 9 "¾ · ·

not be skewed by levels of Th initially present in the Ra composition or masked

997

by interferences due to other, more energetic, decay chains beginning at Th or 223 Ra. A first isotope may be considered "substantially free" of a second isotope if the second isotope is present at a concentration of less than 1%, preferably less than 0.01%, relative to the concentration of the first isotope. Correspondingly,

"completely free" may be considered to correspond to a concentration of less than 0.001% of the second isotope relative to the first isotope.

The mobile phase (eluent) is typically a solution comprising an acid, such as hydrochloric acid or nitric acid. The most preferable acid is nitric acid. Typically, the concentration of any acid used in the method of the invention will be in the range 0.01 to 10 mol/L, preferably 0.02 to 8 mol/L, such as 0.05 to 4 mol/L.

Column A comprises a thorium specific resin, such as a UTEVA resin. The inventors have found that the affinity of an UTEVA resin for "Th increases with increasing nitric acid concentration. This is thought to arise because as the concentration of the nitric acid increases so too does the propensity with which the 227 Th will form nitrate complexes. It is believed to be these complexes for which the resin has affinity. Column B comprises an actinium specific resin, such as a DGA

227

resin. DGA resin has been found to have particular affinity for Ac.

The 223 Ra composition used in the methods of the invention typically comprises 223 Ra at a concentration in the range 2 to 30 MBq/ml (e.g. 2.4 to 30 MBq/ml), such as 5 to 20 MBq/ml. The composition will usually be used in the form of an aqueous acid solution, such as nitric acid. The acid will typically have a concentration in the range 4-10 mol/L, for example, 8 mol/L.

In step (iii) the 227 Ac absorbed onto the resin of column B is removed. This may be carried out by a variety of methods but is typically achieved by washing the column with an aqueous acid solution of lower concentration than that which was used as eluent in step (ii), such as 0.05 mol/L nitric acid. The volume of aqueous acid solution used to wash the column may be in the range 16 to 400 times the volume of the column (e.g. 2-20 ml), preferably 40-200 times (e.g. 5-10 ml). The

227

eluate obtained from column B after step (iii) contains Ac. Preferably this eluate is substantially free of 227 Th. For example, the eluate may contain 227 Th at a molar concentration of less than 5%, preferably less than 1% or less than 0.1% and more preferably less than 0.01% relative to the concentration of 227 Ac.

In a highly preferred embodiment, the method of the invention comprises the following steps:

(i) Place a first solid phase extraction column A comprising a thorium specific resin (e.g. a dipentyl pentylphosphonate UTEVA resin) and a second solid phase extraction column B comprising an actinium specific resin (e.g. a N, N, Ν', N' - tetra-n-octyldiglycolamide DGA resin) in series, preferably wherein the output of column A is connected to the input of column B;

(ii) Add a volume of a Ra composition corresponding to a known activity (e.g.

15 MBq) of Ra to an equal volume of nitric acid, preferably 8 mol/L nitric acid;

(iii) Transfer the sample from step (ii) to the input of the column A;

(iv) Pass said sample through both columns A and B

(v) Wash both columns with 20-100 times the combined volume of the two

columns (e.g. 5-10 ml) nitric acid, preferably 4 mol/L nitric acid;

(vi) Disconnect column A from column B; (vii) Wash column B with 40-200 times its volume (e.g. 5-10 ml) nitric acid, preferably 4 mol/L nitric acid;

(viii) Wash column B with 40-200 times its volume (e.g. 5-10 ml) nitric acid at a concentration less than that used in step (vii), such as 0.05 mol/L nitric acid.

• 997

(ix) Determining the amount of Ac present in the eluate from column B

obtained in step (viii).

Following isolation of the 227 Ac from the actinium specific resin (e.g. DGA resin), its amount may be quantified by any known method in the art. Typical percentage recoveries of 227 Ac using the method of the invention are in the range 70 100%, such as 72-98%, preferably 74-97% (e.g. 80 to 97% or 80 to 90%).

Evidently, for an analytical method reproducibility in recovery of Ac is as important as the absolute recovery. The distribution of such recoveries will thus typically have a standard deviation of no more than 20%, preferably no more than 10%.

997

Typical methods used to determine the quantity of Ac may involve γ- spectrometry, a-spectrometry and liquid scintillation counting (LSC) with pulse- shape discrimination. The preferred technique is γ-spectrometry, which enables

997 997

quantification of Ac via in-growth and detection of the daughter Th. Methods for performing γ-spectrometry are well known in the art.

The activity of 227 Ac, which is not directly determinable by γ-ray

997

spectrometry, can be calculated from measurement of the daughter Th. As the specification limit for a 223 Ra pharmaceutical is 0.004% 227 Ac, relative to 223 Ra, an

99 * 5 997 activity of 15 MBq Ra should give an activity of 600 Bq of Ac. The in-growth

997 997

of Th from Ac is calculated by Equation 1.

^Ingrowth Th)

(1)

Due to regulatory requirements for radiopharmaceuticals, the result from radionuclidic purity of a 223 Ra pharmaceutical should be available before release of the product. In order to meet these requirements the maximum in-growth period of Th from Ac should preferably not be more than two days to avoid a

prohibitively high loss of 223 Ra by decay before administration. The calculated activity after 24 and 48 hours in-growth of 227 Th from 600 Bq 227 Ac is shown in Table 1.

Table 1. Ingrowth of z h from 600 Bq Ac

After separation of Ac from Th and Ra, potential traces of Th can be left in the sample (minimum detectable value < 1.6 Bq). By counting only one

997

spectrum, the activity of Ac can be overestimated. In the present invention, this

997 issue has been addressed by utilizing two consecutive measurements of the Th daughter: one counted after 24 hours and one after 48 hours from separation. 227 Th activity obtained from analyses after 24 hours is subtracted from Th activity obtained after 48 hours assuming that the in-growth of 227 Th is almost linear in the period. Correction of decay of potential traces of 227 Th (ti^ = 18.68 days) between 24 and 48 hours after separation, has not been taken into account. It is considered to be sufficiently accurate and within the uncertainty of the measurement.

997 997

With the Th activity at measurement time one (24 h) and "'Th activity at measurement time two (48 h), the unknown, but time independent, activity of the long-lived mother 227 Ac can be calculated by:

A.( 227 Th) = A spec ^J 227 Th)-A spectmm ( 227 Th) (2)

The activity of 227 Ac in the sample at time 0, is based on in-growth of 227 Th. The equations used are given below.

where:

A 0 ( 227 Ac) = the activity of 227 Ac in the 223 Ra pharmaceutical (Bq) A & ( 227 Th) = the activity of 227 Th produced between measurement time one and measurement time two, e.g. 24 and 48 hours after separation (Bq) λπι-227 = In 2 / 18.68 days As seen from Table 1 , the activity 24 and 48 hours after in-growth of 227 Th from 600 Bq 227 Ac is 21.9 and 42.9 Bq, respectively. Based on the theoretical calculation of the in-growth of 227 Th and the fact that the separation gives highly purified 227 Ac it was assumed that a counting time of 10000 s in the closest calibrated position (position 5 cm) from the detector surface was sufficient, and satisfactory counting uncertainties were achievable.

997

Consequently, in the methods of the invention, the quantity of Ac is preferably determined by γ-spectrometry via the in-growth of the daughter nuclide 227 Th, wherein two measurements of the activity of the 227 Th daughter are made. Preferably these measurements are taken at n and 2n hours, wherein n is 12 to 36, preferably at 24 hours and 48 hours, after performing the separation method of the invention. Activity is typically measured over a period of 10000s at each time point.

In one variant, the method of the invention could be performed in the presence of 225 Ac as a tracer for the chemical yield of 227 Ac to check the accuracy of the results. However, 225 Ac is not commercially available hence it cannot be used in routine analysis at present. Instead, initial verification of the method may be carried

99 * } 99S

out by spiking the Ra composition with Ac, providing quality control data of critical process steps such as correct preparation of acid samples, weighting of correct resins and elution with the correct acid and acid concentration. 225 Ac can be

997

presumed to have the same resin absorption properties as Ac but is easier to detect. 225 Ac may be quantified by the in-growth of the daughter 213 Bi via γ- spectrometry. The decay chain for 225 Ac is shown in Figure 3.

The method of the invention is suitable for routine analysis of Ac in Ra compositions and can be performed quickly on the same day as preparation of the 223 Ra composition. Preferably separation steps (i) to (iii) can be completed in no more than 2 hours, preferably no more than 1 hour (e.g. 5 minutes to 1 hour).

Advantageously, the results from the method of the invention are typically available two days after production i.e. before the product is released and administrated to the patient.

The invention further relates to the use of the methods as hereinbefore described in quantifying levels of Ac in Ra compositions. It should be appreciated that all previous discussion relating to preferable aspects of the invention relate equally to this embodiment.

The invention further relates to apparatus for use in a method as hereinbefore described. The apparatus comprises a first solid phase extraction column A, wherein said column comprises a thorium specific resin (e.g. a dipentyl pentylphosphonate UTEVA resin), and a second solid phase extraction column B, wherein said column comprises an actinium specific resin (e.g. a N, N, N', N'-tetra-n-octyldiglycolamide DGA resin). Preferably, column A and column B are arranged in series, preferably such that the output of column A is connection to the input of column B. Most preferably, column A is positioned above column B such that the eluate from column A drains directly into column B. It should be appreciated that all previous discussion relating to preferable aspects of the invention relate equally to this embodiment.

Figures

77 "7 Π7

Figure 1 - Decay scheme of Ac to stable Pb. Branches with less than 2% probability are omitted. Figure 2 - Process flow-chart for actinium, thorium and radium separation and purification using the method of the invention - extraction is shown using aqueous HN0 3 of particular concentrations by way of example only. Figure 3 - The decay scheme of Ac and daughter radionuclides to stable Bi.

Figure 4 - HPGe γ-spectrum of 225 Ac daughters 221 Fr and 213 Bi

000 000

Figure 5 - HPGe γ-spectrum of in-growth of Th from Ac 24 hours after

00"\

separation from Ra-chloride drug substance

001 ΟΟ'

Figure 6 - Linearity of measured versus theoretical Th amount in Ra chloride drug substance Examples

The 223 Ra composition utilised in the Examples is RaCl 2 (Alpharadin), hereinafter referred to as "Ra-chloride drug substance".

Gamma-spectra were measured with a High-Purity Germanium detector (HPGe) of 50% efficiency (relative to a 3 inch x 3 inch Nal detector for a 60 Co source at a distance of 25 cm from the detector surface) coupled to a 8192-channel Multi Channel Analyser (MCA). Spectra were analysed using GammaVision Software (GammaVision-3.2 software, v 6.01, Ortec, Oak Ridge, USA). Calibration of the energy dependent efficiency of the HPGe detector at two fixed positions was performed with a reference source (γ-mixed standard from Eckert & Ziegler) with an overall uncertainty below 4%. The fixed calibration positions were 5 and 20 cm from the detector. The HPGe detector was energy calibrated in an energy range from 59-1400 keV.

00 " ·

In order to evaluate Ra with an activity in the MBq range, an ionization chamber ("dose calibrator", Capintec-CRC15-R) was used. Accurate activity measurements of radionuclides using commercial ionization chambers require that the correct calibration setting ("dial setting") must be applied. For many nuclides, the manufactures of dose calibrators, recommend those calibration settings.

223 Ra is a relatively novel radionuclide in nuclear medicine and a calibration setting for the radionuclide is therefore not available from commercial manufactures of ionization chambers. A primary standardization of 223 Ra to establish dial settings was performed by the National Institute of Standards and Technology (NIST). The

* * 223 reason is to assure quality-controlled measurements of the radioactivity of Ra during production, quality control and preparation of patient doses.

Measurements with 223 Ra Standard Reference Material (SRM) from NIST were performed in Capintec dose calibrators. The determined calibration setting (dial setting) for the dose calibrator used in the present invention is presented in Table 2.

Table 2. Ra dose calibration setting

The instruments were qualified, which means verification that the instrument is installed correctly and is capable of operating as intended according to the specifications. Control of the HPGe instrument was performed daily before use by

7A 1 "Ϊ7

measuring the long-lived radionuclide Ra. The radionuclides Co and Cs are used for daily control of the dose calibrator. The purpose of the quality control of the instruments is to ensure that the instrument provides reliable and consistent results.

777

Detection of Ac is difficult due to the low energy of its β-radiation and no useful γ-rays. Therefore, in order to easily obtain rapid information concerning the

777 777 77"ϊ

efficiency of the separation of Ac from Th and Ra using the method of the invention the process was carried out using Ac as a radioactive tracer in place of

77 77^ 777

Ac. Ac can be presumed to have the same resin absorption properties as Ac but is easier to detect. Examples 1 -3 were carried out with Ac and Examples 4

777 77 ¾ "11 "2 and 5 with "'Ac. " J Ac may be quantified by the in-growth of the daughter Bi via γ-spectrometry. Uncertainties were calculated as follows:

Uncertainty in the recovery (Examples 1 , 2. 3 and 5)

There is an uncertainty σΑ in the activity of the "known" (spiked) samples, A. There is an uncertainty OB in the activity of the "found" sample (eluate), B.

R = Recovery (%)

R = - 100

A

Uncertainty in the deviation (Example 4)

There is an uncertainty σΑ in the calculated activity, A.

There is an uncertainty σΒ in measured activity, B.

Calculation of the combined uncertainty (Example 5) There is an uncertainty OA in the activity of the ingrowth of 227 Th after 24 hours, A. There is an uncertainty OB in the activity of the ingrowth of 227 Th after 48 hours, B.

Calculation of the combined uncertainty: Example 1 - Separation of 25 Ac from Z27 Th and " J Ra using Solid-Phase Extraction Columns

Sample preparation, 225 Ac

The 225 Ac used was supplied from the Institute for Transuranium Elements,

225

Karlsruhe, Germany. The solution had a nominal total activity of 6 MBq Ac at the day of receipt and the activity was diluted in 4 mol/L HN0 3 to an activity of 3.1 Bq/μΤ.

A known amount of 227 Th and 225 Ac was added to 15 MBq 223 Ra-chloride drug substance. The activity corresponded to approximately the specification limits of 227 Th and 227 Ac in a Ra-chloride drug substance. The specification states that the activity of 227 Th should be less than 0.5% of 223 Ra activity and the activity of 227 Ac should be less than 0.004% of 223 Ra. Table 3 gives the activities of 225 Ac and 227 Th used in the experiments.

Table 3. Activities (Bq) of 227 Th and 225 Ac in experiment I

and II.

Uncertainty in the activity (2 σ)

Activities were determined using the following methods: Ra-chloride drug substance was transferred to two 20 mL vials (each with an activity of 15 MBq) and measured in a dose calibrator in dial setting 262. The γ- rays of 225 Ac and 227 Th solutions were measured with an HPGe detector in the

calibrated position 5 cm and 20 cm, respectively.

For determining the areas of the γ-peaks in the energy spectrum, ORTEC

GammaVision software was used. The energy and photon yield data are taken from Evaluated Nuclear Data File (ENSDF - available from

http://www.nndc.bnl.gov/nudat2/ - 21 June 2013). The most abundant γ-lines, given in Table 4, are used for the activity calculation.

Table 4. γ-ray energies and emission probabilities (percent) used for the determination of radionuclide activities. Data was taken from ENSDF.

Production of Ac is based on a Th generator from which Ac is eluted.

The γ-lines from 229 Th used for activity calculation are seen in Table 4. No traces of 229 Th were found in the sample. Preparation and conditioning procedure of the UTEVA and PGA columns The extraction-chromatographic resins as well as the prefilter material were packed in 2 ml disposable polystyrene plastic gravity-feed columns (obtained from Fisher Scientific). The following steps were carried out:

• Weigh in approximately 100 mg of UTEVA resin and 50 mg of DGA resin.

• Transfer the resin to two 20 ml plastic vials and add approximately 3 ml of 4 mol/L HNO3 to each. Swirl to mix.

• Prior to packing of the two columns, transfer a filter to the columns. Push the filter down to the base of the columns.

• Transfer the solution into the reservoir. Place a filter on the top of the

UTEVA resin and DGA resin.

• Push the filter and the resin down.

• Discard the acid above the top filter. Add 2-3 ml of 4 mol/L HN0 3 . Discard the acid again.

• Remove the bottom plug from the columns.

• Add 2-3 ml 4 mol/L HN0 3 . Allow to drain.

Two-Column separation procedure

• Place one UTEVA resin column and one DGA resin column in the column rack in series, i.e., solutions from the UTEVA resin column (on top) will drain into the DGA resin column (on bottom) (see Figure 1).

• Based on 223 Ra-chloride drug substance radioactivity concentration,

MBq/ml, pipette accurately a volume corresponding to 15 MBq into a 20 ml vial. Add an equal amount of 8 mol/L HN0 3 .

• Known activities of 227 Th and 225 Ac were added to the 223 Ra-chloride

solution, according to Table 3.

• Use a plastic pipette to transfer the sample to the top of the UTEVA column.

2 25 Ac and 223 Ra will elute from the UTEVA column into the DGA column while 227 Th will absorb to the UTEVA column.

• Wash the columns with 5 ml of 4 mol/L HN0 3 .

• Disconnect the UTEVA column from the DGA column. • Transfer 5 ml of 4 mol/L HN0 3 onto the top of the DGA column. Ra will elute from the column while 227 Ac will stay trapped on the DGA column.

• Elute the DGA column with 5 ml 0.05 mol/L HN0 3 . This will remove 227 Ac from the column.

111 111 11"

It will be appreciated that in routine analysis (separation of Ac, Th and Ra) m 11$ ιΐ"\

no addition of Th and Ac to the Ra -chloride solution was carried out, the solution was transferred directly to the UTEVA column.

Two separation experiments with Ac were conducted. After complete separation, the columns and eluates were counted the day after separation with an HPGe detector. 225 Ac has no suitable γ-rays and therefore it is quantified through its

'yy c 11$ daughter " J Bi. "Ac is in secular equilibrium with is daughters after 24 hours. Ac

221 213

was identified through the measurement of the daughters Fr and Bi. A spectrum of the highly purified 225 Ac eluate is shown in Figure 4. The γ-ray energies and

11 $ 111

intensities, used for the identification and quantification of Ac and Th, are presented in Table 4.

Percentage recovery for 225 Ac obtained from experiments I and II was 97% and 86% respectively. Results are reported in Table 5. These results show that the extraction resins UTEVA and DGA give an effective, reproducible, robust and rapid

'yyc 111 11"X

separation of " J Ac from "'Th and "Ra. The DGA resin shows a strong retention of Ac in nitric acid and efficient stripping of Ac in dilute nitric acid. As seen from Table 5, the breakthrough ("recovery" in Table 5) of 227 Th and 223 Ra in the final eluate was less than 2- 10 "3 and 8-10 "4 , respectively. Moreover, only traces of the

11"\ 111

initial amounts of Ra and Th were detected in the eluates. This shows that the

11 $ 111 11"X

separation procedure of Ac from Th and Ra with UTEVA and DGA column is highly effective. Thus, the method demonstrates that separation of 227 Ac, 227 Th and 223 Ra will also be effective. Table 5. Activity (Bq) of 25 Ac, 2 h and J Ra on the UTEVA and DGA column after separation and in the eluate obtained by γ-ray spectrometry. Uncertainty in the activity (2 σ). Activities added at the start of the experiment are seen in Table 3.

minimum detectable amount (MDA)

In Experiment II, three fractions of 1.7 ml of the eluate were collected and counted

Example 2 - Testing of the Robustness of the Method

Robustness of the method was tested by using new batches (new lot number) of UTEVA and DGA resin. The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small variations in method parameters and provides an indication of its reliability during normal usage.

A total of three experiments (I, II and III) were conducted with 15 MBq 2 3 Ra-chloride drug substance spiked with known activity of 225 Ac. The activity of 2 25 Ac is shown in Table 6. In addition, approximately 75 kBq of 227 Th was added. The separation was performed according to the procedure given in Example 1. Percentage recovery of Ac obtained after eluting with 5 ml 0.05 mol/L HNO3 was 68, 81 and 77%. The recoveries obtained were lower than for the UTEVA and DGA batches used in Example 1. It was therefore decided to increase the eluting volume (in addition to the 5 ml already added) by adding 2 times a volume of 2.5 ml of 0.05 mol/L HNO3. The results are presented in Table 6. The percent recovery of 225 Ac increased to 81, 85 and 84% and this is comparable to the previously obtained result. This shows that the method is robust with regards to new batches (new lot) of resins. However, the eluting volume may require adjustment to obtain sufficient recoveries (> 70%).

Table 6. Measured Ac activity (Bq). Testing of robustness of the method by new batch of UTEVA and DGA resins. Uncertaint in the activit (2 σ .

Example 3 - Testing of the range of the method

Analytical methods developed by a pharmaceutical company must be validated. The method should be validated in the range from reporting limit to at least 120% of the specification limit. In the previous experiments, the amount of Ac has been added related to the specification limit. As the specification limit is 0.004% relative to 2 23 Ra, approximately 600 Bq of 225 Ac has been added to 15 MBq 223 Ra. The purpose of Example 3 was to add lower and higher activities of Ac. The reason was to cover the range of amounts of Ac which would be used during validation. Three experiments were conducted with various amounts of Ac.

Results

Percent recovery of 225 Ac obtained after elution of 10 ml 0.05 mol/L HN0 3 were 91, 74 and 92%. The results are presented in Table 7. This shows that the recovery is acceptable (> 70%) from 46 to 172 % of the specification limit.

Table 7. Measured Ac activity (Bq). Three experiments with an Ac

997

activity ranging from 46-172% of the specification limit of Ac. The

uncertainties are given as 2 σ.

Example 4 - Determination of counting conditions

Separation of 227 Ac, 227 Th and 223 Ra was performed by UTEVA and DGA columns as described in Example 1, with the difference that a sample of 534 ± 21 Bq (2σ)

997 9 S 997 Ac was added to the column rather than the Ac spike and supplemental Th.

Prior to separation, the sample was counted with a HPGe detector and quantified via

997 997

its daughter Th as Ac was in equilibrium with its daughters for this sample. Results

227 Ac was eluted from the DGA column with 0.05 mol/L HNO3 and counted in the calibrated position 5 cm from the detector surface for 10000 s after approximately 1 and 2 days. Results are given in Table 8.

Table 8. Results from γ-ray spectrometry ingrowth of Th from

uncertainties are given as 2 σ.

As seen from Tabl, satisfactory counting uncertainties (< 7%, 2σ) were achievable for a sample counted for 10000 s in position 5 cm from the detector. There was no difference between calculated and measured activity.

Example 5 - Validation of the method

Analytical methods developed by a pharmaceutical company must be validated. Analytical method validation is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use, i.e. to ensure reliability, consistency and accuracy of the analytical data. In order to demonstrate the applicability of the methods of the invention to commercial applications it was validated according to ICH Harmonized Guideline. Prior to a formal method validation it is mandatory to set up a protocol with test parameters to be evaluated and appropriate acceptance criteria.

The method was validated in terms of selectivity, accuracy, precision

(repeatability/ intermediate precision), linearity, range, limit of detection (LOD) and limit of quantification (LOQ). Robustness of the method was performed in Example 2 in terms of different lots of resins and was hence was not repeated in this Example.

The ICH guideline says nothing about acceptance criteria for the different parameters. However, accuracy in terms of recovery between 80-120% and a precision of ± 20% is normally regarded as acceptable. This is applicable for impurities > 0.1% of the active ingredient. As the specification for the impurity 227 Ac in 223 Ra-chloride drug substance is set as low as 0.004% relative to 223 Ra, a broader acceptance was required.

Samples of 223 Ra-chloride drug substance were spiked with known amounts of 227 Ac and 227 Th. Validation parameters and corresponding acceptance criteria for the method validation are given in Table 9.

Table 9. Validation arameters and acce tance criteria

NA = Not applicable

Experimental parameters

According to ICH, accuracy and repeatability should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e.g. 3 concentrations / 3 replicates). The recommended range for validation of an impurity method is from reporting limit to at least 120% of the specification. Samples from 60-140% of the specification limit were prepared according to Table 10. Table 10. Overview of samples used in method validation. Ra is spiked with 227 Ac and 2 7 Th. The 227 Ac activity is from 60-140% of the specification limit.

The method stipulates a sample size of 15 MBq. According to specifications, the amount of 227 Ac and the amount of 227 Th should be less than 0.004% and less than 0.5% relative to 223 Ra activity, respectively. As the decided validation range was from 60 to 140% of the specification, spikes of 227 Ac from 360 to 840 Bq were prepared. The content of 227 Th was held constant i.e. 75 kBq (0.5% of the specification). The Ac and Th stock solutions were both made in 4 mol/L HN0 3 and the activities were approximately 5 Bq^L and 0.5 kBq^L, respectively.

997

To determine the exact activity of Ac spike solutions, counting with a HPGe detector in position 5 cm for 1000 s was performed. The counting time was selected to give a counting uncertainty (1 σ) of less than approximately 3%, which was regarded as adequate. Counting of 227 Th stock solutions was performed in position 20 cm for 300 seconds.

Ra from three Ra-chloride drug substance batches were pooled.

Contamination of 227 Ac in the pooled batch was determined. An aliquot of 15 MBq was taken from the pooled sample and analyzed according to the method described in Example 1. This was done to ascertain if any correction needed to be made to the above results on account of background contamination of the samples with 227 Ac. No traces of Ac in the pooled Ra-chloride drug substance were found. Hence, no correction was performed.

Results - Selectivity Selectivity is the ability of the measurement to assess the analyte without any interference from other components in the matrix. At the time of analysis, radionuclides present in 223 Ra-chloride drug substance have been separated from

777 " 7

Ac according to the method presented in Example 1. β-decay of Ac does not produce emissions of γ-rays that are appropriate for γ-detection. Traces of 223 Ra and its daughters can remain in the sample after purification and the selectivity of the method is demonstrated by comparing the energies of the γ-rays of 227 Th with the energies of 223 Ra and its γ-emitting daughters, 219 Rn, 21 1 Pb and 21 'Bi, and by showing that they are clearly separated and identifiable. The γ-ray used for quantification of 227 Th is 236.0 keV, which is the most abundant γ-line of 227 Th (12.9%).

The γ-ray energies characteristic of Th, Ra and daughters are shown in

227 223

Table 1 1. A spectrum acquired 24 hours after separation of Ac from Ra- chloride drug substance is shown in Figure 5.

Table 11. γ-ray energies of 223 Ra and its daughters and 227 Th (Data taken from Evaluated Nuclear Structure Data File (ENSDF) database )

219 Rn 227 Th

223 Ra 211 Pb 2n Bi

144.2

154.2

210.6

236.0

256.2

269.5

271.2

286.1

300.0

304.5

323.9

329.9

338.3

351.1

401.8

404.9

427.1

704.6

832.0

As seen from Figure 5, the γ-ray used for quantification of Th is distinctly and visibly separated from the energies of other nuclides. There are no interferences from the matrix. The method is considered specific and the acceptance requirement was fulfilled.

Results - Accuracy

The accuracy of the method was determined by performing recovery experiments on 223 Ra-chloride drug substance spiked with five levels of 227 Ac at 60%, 80%, 100%, 120%, and 140% of the specification limit of 227 Ac. The solutions were additionally spiked with 227 Th amounts corresponding to the 227 Th specification limit. For the 60%, 100% and 140% levels the solutions were prepared in three-fold. For the 80% and 120% level the solutions were prepared once. Solutions were analyzed as described in Example 1. Each solution was measured twice. First measurement was performed 24 ± 1 hour after sample preparation, the second measurement was performed 48 ± 1 hour after sample preparation.

Accuracy as the percent recovery was determined using the measured content calculated as described in Equation 4.

Measured content

Recovery - x lOO

Nominal content

(4)

Results are presented in Table 12.

Table 12. Results for the accuracy (as recovery) of the method. Samples in the range of 60- 140% of the specification limit for 227 Ac relative to 223 Ra.

Nominal h after "Th after Calculated Diff between Recovery activity 24 hours 48 hours activity measurement [%] 2

227 1 and 2

Ac [Bq] 1 [Bq] 1 227 Ac

2

[Bq] 1 [Bq] [Days]

60 323 ± 22.0 20.4 ± 2.7 31.6 ± 3.0 303 ±4.0 1.0 94.0 ± 6.5

322 ± 22.5 10.9 ±2.6 25.7 ±2.9 397 ±3.9 1.0 123.2 ± 8.7

359 ±23.0 16.9 ±2.0 26.5 ±2.3 263 ± 3.0 1.0 73.3 ± 4.8

80 460 ±27.6 16.9 ±2.5 30.5 ±2.9 369 ±3.8 1.01 80.3 ± 4.9

100 540 ± 30.2 22.1 ±2.0 42.7 ±2.8 578 ±3.4 0.98 107.0 ± 6.0

591 ±31.9 24.0 ±2.1 43.8 ±2.9 556 ±3.6 0.97 94.1 ± 5.1

527 ±29.5 21.1 ±3.2 40.1 ±4.1 535 ±5.2 0.97 101.5 ± 5.8

120 715 ±35.8 26.7 ±2.3 53.8 ±2.9 655 ±3.7 1.14 91.6 ± 4.6

140 868 ± 39.9 36.0 ±4.9 62.8 ±5.1 735 ±7.1 1.00 84.7 ± 4.0

861 ±41.3 31.0±3.2 56.7 ±3.6 706 ± 4.8 1.00 82.0 ± 4.0

803 ±38.5 36.8 ±4.6 61.0 ±4.5 662 ± 6.4 1.00 82.4 ± 4.0

Mean recovery [%] (n= 11 ) 92.2

Relative standard deviation of the recovery [%] (n=l 1) 15.5

Confidence interval (95%) of recovery [%] 82.2-102.1

Uncertainty in the activity (2 σ).

2Combined and recovery uncertainty

As seen from Table 12, the single percent recovery and the mean (n=l 1) is all within the criteria of acceptance (70 to 130%, see Table 9). The method is

considered sufficiently accurate for the determination of 227 Ac content in the range from 60% to 140% of the specification limit which corresponds to 0.002% - 0.006%

227 223

of Ac in Ra-chloride drug substance at release. The requirement is thereby fulfilled. The uncertainties in recovery were in the range from 4-8.7%, comparable to 2σ in the counting statistics, and it was the lowest activity which gave rise to the largest uncertainties. A contribution to the uncertainties is the uncertainty in the spiked value. This is not relevant for analyses of "normal" 223 Ra-chloride drug substance samples and hence the real uncertainties are lower.

Results - Precision

The repeatability of the method was determined by calculating the relative standard deviation (RSD) for three replicates of Ac in Ra-chloride drug substance at three different levels at 60% (corresponding to 0.002% of 227 Ac), 100% (0.004% of 227 Ac), and 140% (0.006% of 227 Ac) of the specification limit. For each level, the solutions were prepared in triplicate and analyzed as described in Example 1. Results are presented in Table 13.

As seen from Table 13, the mean relative standard deviations were < 30% for all three levels. The method is considered sufficiently precise and the acceptance requirement was fulfilled (see Table 9).

Table 13. Results for the precision of the method

Level Recovery Mean RSD

[%] 227 Ac (n=3) (n=3)

[%] [%] [%]

60 94.0 96.8 25.9

123.2

73.3

100 107.0 100.9 6.4

94.1

101.5

140 84.7 83.0 1.7

82.0

82.4 Results - Intermediate precision

Intermediate precision expresses within-laboratory variations in terms of e.g. different days, different analysts and different equipment. The intermediate precision was in this case determined in terms of different days. Separation was performed on four different days and the results are given in Table 14.

Table 14. Intermediate precision.

Level Measured activity

[Days] Ac-227

[Bq]

1 94.0

1 123.2

2 73.3

3 107.0

3 94.1

3 101.5

4 84.7

4 82.0

4 82.4

Mean recovery [%] (n=9) 93.6

RSD [%] (n=9) 16.3

As seen from Table 14, the mean relative standard deviation was < 30% for all four days. Data show that the results from different days are comparable and that the acceptance requirement are thereby fulfilled. Results - Linearity

Linearity is the ability to generate a response which is directly proportional to the concentration of an analyte in a sample. To demonstrate the linearity of the method, a sample with an activity of 359 ± 23 Bq 227 Ac was used. The sample was separated according to the procedure described in Example 1. The in-growth of 227 Th from 227 Ac was measured 6 times in a period from 1 to 6 days after separation. The corresponding theoretical activity was calculated using Equation 1. Results are presented in Table 15 and the plot of signals is displayed in Figure 6.

Table 15. Results for the linearity of the method

Theoretical Measured activity

227 Th 227 Th

Time from

separation [Bq] [Bq]

[Days]

1.0 13.2 ± 0.8 16.9 ± 2.0

2.0 25.8 ± 1.6 26.5 ± 2.3

2.4 30.2 ± 2.0 30.5 ± 2.4

3.1 39.1 ± 2.5 43.7 ± 1.5

4.3 53.0 ± 3.4 48.0 ± 3.0

5.9 70.6 ± 4.5 67.0 ± 3.5

Regression line

Slope 0.8629

Intercept [Bq] 5.4606

Correlation coefficient (r) 0.9797

The linearity curve was measured with Th activities ranging from 17-67 Bq. This range covers the Th activities which are measured from decay of Ac in specification levels of 78-156% (100% gives an activity of 21.9 Bq after 24 hours and 42.9 Bq after 48 hours, see Table 1). The measured 227 Th activity is plotted as a function of the theoretical 227 Th activity. The correlation coefficient was determined to be r = 0.98 and is well above the criteria of acceptance (> 0.95). The method gives a linear response and the requirement is fulfilled (see Table 9).

Results - Range

The method is validated in the specific range of 227 Ac content from 0.002% to 0.006% relative to Ra with respect to activity (Bq). Linearity, accuracy, and precision of the method were demonstrated over a range of 227 Ac amounts as listed in Table 16.

Table 16. Tested range for linearity, accuracy, and precision of the method

Validation characteristics 22 Ac

[%]

Linearity 0.003% to 0.006%

Accuracy 0.002% to 0.006%

Precision 0.002%, 0.004%, and 0.006 %

Results - Limit of Quantification and Limit of Detection

A part of a formal validation of the method is to determine limit of detection (LOD) and limit of quantification (LOQ). Limit of blank (LOB) is the highest apparent analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested. LOD is the lowest analyte concentration likely to be reliably distinguished from the LOB and at which detection is feasible. LOQ is the lowest amount one can quantify with sufficiently good (and preselected) accuracy and precision. The γ-peak, 236 keV, which is the most abundant Th

007 007

peak, is used for quantification of the ingrowth of Th from Ac. The LOD and LOQ were determined as described using the equations:

LOD = 2.71 + 3.29(5(1 +— ) (5 )

2m

LOQ - 50(1 + (1 + ~- ) (6)

25m

Where:

n = Number of channels in the peak region

m = Number of channels used for the background estimation

B = Background correction

Calculated LOD and LOQ from equations 5 and 6 are given in counts and the corresponding activity (Bq) was calculated using the following equation:

A E = — E (7)

ε Ε · ί · γ

AE : The activity in Bq of a nuclide based on a γ-peak with energy E

NE: the net peak area for a γ-peak at energy E (counts)

£E: the detector efficiency at energy E

γ : emission probability

t: counting time

The LOD was calculated to be 1.8 Bq. This corresponds to 8% of the specification limit as the ingrowth after 24 hours from 100% of specification (600 Bq) corresponds to 22 Bq. The method is suitable to detect an 227 Ac content of 0.0003% (LOD). The LOQ was calculated to 7 Bq of 227 Th this corresponds to 32% of the specification limit. The method is suitable to quantify an Ac content of 0.0013% (LOQ).

A summary of the validation results is presented in Table 17. Accuracy and

227 precision were assessed on drug substance sample solutions spiked with Ac activity ranging from 60 to 140% of the specification limit. 100% of the

77 * 7 77 * ¾

specification limits corresponds to 0.004% Ac relative to Ra.

Table 17. Summary of validation results

As seen from Table 17, LOD is 2 Bq and LOQ is 7 Bq. This corresponds to approximately 8% and 32% of the specification limit, respectively. Investigation of the specificity shows that the γ-ray energy for quantification of Th from Ac is clearly resolved from interfering γ-ray energies. There are no interferences from the matrix.

All validation parameters met the pre-specified acceptance criteria. The method is considered suitable for its intended use.