An improved method for the separation of 111Ag from irradiated natural palladium

Appl. Radiar. ISOI.Vol. 43, No. I, pp. 869-872, 1992 ht. J. Radial. Appl. Insrrum. Parr A Printed

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An Improved Method for the Separation of I1lAg from Irradiated Natural Palladium R.

ALBERTO,

P. BLAUENSTEIN, I. NOVAK-HOFER, and P. A. SCHUBIGER*

Radiopharmacy

Division,

Paul Scherrer

Institute,

5232, Villigen/PSI,

A. SMITH

Switzerland

(Received 12 November 1991)

A simple, high yield method for the separation of “‘Ag by liquid/liquid extraction from irradiated natural palladium for nuclear-medical purposes is described. In a first step ” 'Ag is extracted almost quantitatively, together with a small amount of palladium, from an aqueous solution into toluene by complexation with triphenylphosphine. Using the different chemical and physical properties of the two elements “‘Ag is subsequently re-extracted selectively into a convenient buffer solution. The overall yields of” ‘Ag are better than 70% and palladium is depleted by a factor of up to 27,000. The procedure takes about 2-3 h.

Introduction The use of antibodies labelled with various radionuclides to deliver therapeutic doses of radiation for human cancer treatment has now been shown to give clinically significant effects in a number of studies (Brady et al., 1990; Order et al., 1988). Such radioimmunotherapy (RAIT) is dependent on several contributing factors including the radiosensitivity of the target tumour, the characteristics of the chosen antibody and, of course, the nature of the nuclide employed. “‘Ag has been suggested to be more suitable for RAIT than the commonly used ‘“I, on the basis of its good beta emission characteristics, appropriate half-life and much more favourable gamma-ray component (342 keV, 6% for “‘Ag compared with 1989; Lederer 363 keV, 82% for 13’1) (Schubiger, et a/., 1967). Despite the suitability of “‘Ag for RAIT it has been largely ignored because of problems of availability and the complexity of the chemistry of linkage to the carrier antibody. We are now working towards the solution of these problems and have developed a protocol for the preparation of “‘Ag in good yield and with a greatly depleted level of palladium which would be expected to compete in subsequent ligand interactions. In natural palladium the isotopes “‘Pd and “‘Pd are of great significance for our purposes. “*Pd is transformed to ‘09Pd by neutron capture and decays with a 13 h half-life to the stable isotope lo9Ag. “‘Pd, on the other hand, forms “‘Pd which decays with a half-life of 23 min via “‘“Ag to “‘Ag. As a consequence, “‘Ag is always accompanied by larger

amounts of inactive ‘09Ag. In principle, “‘Ag could be produced from commercially available enriched “‘Pd and in this case the specific activity would be much greater. The maximum specific activity is limited to roughly 130 MBq/pg under our irradiation condition (see Experimental section) and the assumption that silver is completely separated from palladium. Silver and palladium are typical B-metal centers and prefer complexation with weak ligand atoms such as P, S, N etc. The approach of labelling biomolecules with “‘Ag consists of the complexation of the metal center to monodentate sulphydryl groups or to multidentate mixed NJ-ligands. However, palladium complexes are usually more stable than the corresponding silver complexes. It was therefore most important to develop a way to separate “‘Ag in high yield whilst minimizing the palladium content in the silver product so as to avoid competition during complexation. The methods described in the literature for this purpose are not very convenient for use in biological systems and do not have a very good yield (Lyle and Maghzian, 1968; Micheev et al., 1964; Miller and Toth, 1967; Okashita, 1965).

Experimental Glassware for re-extraction was siliconized with Si(CH,),Cl, prior to use as “‘Ag in trace amounts tends to adsorb on untreated vessels. Where convenient polyethylene or Teflon equipment should be used. (I) Pd/Ag

solution

A 100 mg sample of palladium black was packed in ‘Author for correspondence.

aluminium 869

foil and sealed in a quartz-tube

prior to

R. ALBERTO et al.

870

irradiation. Irradiation, in our research reactor, was for 26 h at a neutron flux of 3 x 10” n/cm2/s, giving an integrated neutron flux of 1.1 x 10” n. th. The sample was left to decay for 72 h to reduce the lo9Pd activity and thereby the dose to personnel. The average yield after this procedure was 1630 MBq of “‘Pd and 100 MBq of “‘Ag (1 pg of Ag). The sample was then refluxed in 10 mL of concentrated nitric acid to dissolve it completely. A 5 mL sample of the nitric acid was distilled off and the rest of the solution diluted with 35 mL of water. Part of this solution was used for the extraction procedure. (2) Extraction into tolueneltriphenylphosphine (TPP) Best results were obtained under the following conditions. A 25 mL sample of the above-mentioned solution was overlaid with 75 mL of toluene in a 150 mL round bottom flask equipped with a magnetic stirring bar. The stirring rate was 400 rpm so that no drops formed between the two layers. Subsequently 1200 PL of a 0.089 M TPP solution in toluene were added (final TPP concentration was 0.0014 M). The colour of the solution turned slowly to pale yellow and the increase of radioactivity in the organic phase was monitored by liquid scintillation counting or gamma-spectrometry. After 20-25 min it reached a maximum and stirring was stopped, the aqueous phase removed completely and the organic phase filtered through an inorganic filter (Anotop 0.2 pm) directly into a second round bottom flask containing 5 mL of 0.1 M acetate buffer, pH 6.0. The rate of stirring was increased and the silver re-extracted into acetate buffer, 0.1 M. pH 6.0 over a period of 60 min. Palladium remains in the organic phase as the TPP-complex or precipitates later, presumably as the insoluble Pd(TPP),(NO,), compound. The buffer was removed and filtered to give a clear colourless solution containing over 70% of the initial Ag activity and no detectable Pd activity (see Table 1). To check the characteristics of an individual protocol it is possible to run the first extraction without changing the phases to determine the exact time of maximum activity. Then the organic phase is replaced

with a new one and the protocol is executed as described above. It was found to be important to use very well siliconized glass vessels, especially for the re-extraction, as the silver sticks to the glass walls in neutral media resulting in a poor yield.

Results and Discussion The results of extractiomre-extraction experiments are summarized in Table 1. From the experiments l-3 it can be seen that increasing TPP concentration does not result in a better ‘l 'Ag yield but in a higher palladium concentration in the initial organic phase. Increasing the volume of the organic layer at different TPP concentrations results in an increased silver extraction (experiments 3-6). The combination of both (experiment 6) gives an almost quantitative separation of “‘Ag from the aqueous layer. Experiments 7-12 give typical examples of extraction and subsequent re-extraction into an aqueous buffer solution. Overall yields were usually higher than 70%. Palladium in the product solution could not be detected with sufficient accuracy either by liquid scintillation counting or by gamma-spectrometry and we therefore determined its content by the ICP-MS method. Typical amounts were of the same order of magnitude as the silver. The depleting factor for this simple and refined extractiomre-extraction protocol was therefore around 23,000 to 27,000. The protocol described for the separation of “‘Ag from irradiated natural palladium is uncommon in that the desired product changes phase twice in the same two-phase system. Palladium changes phase only once and then remains in the organic phase. In this way “‘Ag of high radiochemical purity, as required for radiopharmaceutical application, can be obtained. In liquid/liquid extractions differing thermodynamic behaviour (e.g. differing ligand affinity) normally suffices to give separation of the constituents. Various techniques allow the repeated performance of liquid/liquid extractions so that greater enrichment and purity can be achieved. In our method we take advantage of the markedly different

Table 1. Results from extraction/re-extraction experiments: (a) Starting solution (I M HNO,); (b) toluene/TPP (c) aqueous buffer (0.1 M Na acetate, pH 6.0) after re-extraction

Exp. NO.

Volume H,O/Tol

I

I:1 I:1 I:1 I:3 I:3 I:3 I:3 I:3 I:3 I:3 I:3 I:3

2 3 4 5 6 7 8 9 IO II I2

TPP m organic phase

WI 7.2.10 4 l.44.10-3 2.16.10-’ 4.7’ 10-4 9.4.10-4 1.41.10~’ I.41 .lO_’ 2.87. IO-’ I.41 IO_’ 1.41’10-’ l.41’10-3 1.41’10-3

Pd-109”

1MW 661 661 661 640 598 643 708 2262 874 524 513 770

Ag-lll”

1MW 54 54 54 65 68 71 81 254 II7 70 69 I08

Pd-109b

W&l 5 I3 17 6 7 19 I8 22 I8 I3 I2 I1

Ag-lllb

[M&I 32 40 36 50 51 66 80 221 II2 64 59 89

I I’ WW

Ag-I

Overall yield

phase after extraction;

Pd ICP-MS

WI

Irsl

Depleting factor

82 72 73 70 70 74

2.2 3.7 I.8 2.8 2.9 3.1

27,200 24,800 28,100 25,700 24,800 23,200

1 67 I81 82 49 48 80

and

Separation of “‘Ag

kinetic behaviour of Pd2+ and Ag+ as their thermodynamic behaviour tends to be similar. The stability constants with monosulphonated TPP in water were determined to be 8.15 and 5.40 for Ag+ (Ahrland et al., 1977) and 10.20 and 9.80 for Pd2+ (Chang and Bjerrum, 1972). Complex formation with phosphines is practically pH independent so that a similar affinity for TPP could also be expected in toluene. Most significant is that the K, and K2 values for Pd2+ are greater than those for Ag+. For thermodynamic reasons above all an extraction of Pd2+ into the organic phase would be expected, a situation that will in fact apply if the whole system is left standing. On the other hand, it is known that complex equilibria involving Ag+ are reached much more rapidly than with Pd2+. Consequently the TPP complex of Ag+ is extracted faster into toluene than the Pd2+ complex. Figure 1 shows the extraction kinetics of pure “‘Ag+ separated by our method from 1 M HNO, into toluene/TPP. For [TPP] > [Ag+ ] the extraction kinetics are always the same and appears to be first order (curve fitting). Depending on the speed of stirring, geometry etc., the Ag+ is completely extracted into the organic phase and remains there in the absence of Pd2 +. In the described method Pd2+ and TPP are in large excess over Ag+ but the amount of TPP is only about and, as a result, 10% of the Pd2+ concentration a combination of fast and slow kinetic processes occurs, and can be made use of. Figure 2 shows the movement of radioactive activity into the organic phase as a function of time. A very rapid increase in activity is observed, comparable to that depicted in Fig. 1. After 20-25 min more than 90% of the Ag+ is in the organic phase whereas the Pd2+ concentration is about the same as that of TPP: fast

Ag+ + TPP -

Ag(TPP)+

water

toluene

toluene

Pd2 + + TPP water

(1)

toluene

(2)

toluene

1.0

.. 0.6

.

0.8

0

. . . .. . . . .. ,~~~_*_.~~_~-~_~-._._.-

. . .

/’ -7

20

40

60

80

100

120

140

160

180

200

Time (min) Fig. 2. ” 'Ag-activity (arbitrary units) in the toluene/TPP

layer as a function of time (a) without and (b) with re-extraction. If the system is left standing long enough, it is to be expected that the more slowly accumulated palladium complex will eventually, in accordance with equation (3), displace the Ag+ by virtue of its higher stability, so that Ag+ will be re-extracted into the aqueous phase. Figure 2(a) shows the continual reduction of activity indicating the displacement of Ag+. Ag(TPP)+ + Pd2+ + Ag+ + Pd(TPP)2+ toluene

water

water

(3)

toluene

After 2-3 h the Ag+ is again completely transferred to the aqueous phase while the Pd2+ in the organic phase remains constant. On the other hand, if the original aqueous phase is replaced by a fresh buffer solution, equation (3) can not occur as no uncoordinated Pd2+ is left. In this case competition must take place with Pd(TPP)2+ Ag(TPP)+ + Pd(TPP)2+ + Ag+ + Pd(TPP);+ toluene

Slow Pd(TPP)2 +.

871

toluene

water

(4)

toluene

Figure 2(b) shows again the continual reduction of reactivity in the organic phase as a result of the re-extraction of Ag+ into the aqueous buffer phase. The endpoint (dependent upon physical factors) shows only “‘Pd remaining in the organic phase. When this extraction/re-extraction process and particularly the changing of the aqueous buffer phase are carefully executed the Ag+ can be obtained in a highly pure form.

Conclusion

I

I

10

20

III1

30

J

III

40

50

60

70

60

90

100

Time (min) Fig. 1. Extraction kinetics of pure “‘Ag into toluene/TPP.

The protocol described stands out from others in the literature by virtue of its simplicity, greater efficiency and higher enrichment of the “‘Ag. The method also offers the added advantage that the silver can be re-extracted into the desired buffer system and is ready for further application to radiopharmaceutical purposes.

872

R. ALBERTO et al.

Acknowledgements-This project is supported in part by Grant No. 31-30127.90 from the Schweizerischer Nationalfonds zur Fijrderung der Wissenschaftlichen Forschung. We thank Maria Iftimia and Judith Miiller for their technical assistance. We also thank Drs Kopajtic and Wernli for the ICP-MS investigations.

References Ahrland S., Berg T. and Trinderup P. (1977) Thermodynamics of complex formation in dimethylsulfoxide with ligands coordinating via N, P, As, Sb, or Bi. 1. silver complexes. Acta Chem. Stand. A31, 775. Brady L. W., Woo D. V., Markoe A. M., Dadparvar S. and Karlsson U. (1990) Treatment of malignant gliomas with ‘251-labeled antibody against epidermal growth factor receptor. Antibody Immunoconj. Radiopharm. 3, 169. Chang J. C. and Bjerrum J. (1972) The complex formation of Diphenylphosphino benzene m-sulphonate with Pd(II) and Pt(I1). Acta Chem. Stand. 26, 815. Lederer C. M., Hollander J. M. and Perlman I. (1967) Table of Isotopes. Wiley, New York.

Lyle S. J. and Maghzian R. (1968) Separation of carrier free Ag-I 11 from irradiated palladium. Talanta 15, 712. Micheev N. B.. Abdel-Rassoul A. A. and Fouad H. (1964) Application of co-crystallization technique for the production of carrier free Ag-I 11 from pile irradiated palladium. Z. Anorg. Allg. Chem. 332, 209. Miller J. and Toth G. (1967) Removal of silver traces from palladium by means of selective adsorption. Isoropenpraxis 3, 19. Okashita H. (1965) Rapid separation of radio-nuclides by spontaneous deposition on metallic mercury. Radiochimica Acta I, 85. Order S. E., Vriesendorp H. M., Klein J. L. and Leichner P. K. (1988) A phase 1 study of “yttrium Antiferritin: Dose escalation and tumor dose. Antibody Immunoconj. Radiopharm. 2, 163. Schubigkr P. A. (1989) Neue Radionuklide in der Diagnostik and Therapie-Trends zur Reduzierung der Strahlenbelastung. In: Die Wirkung niedriger Strahlendosen (Eds, Koehnlein W., Trout H. and Fischer M.). Springer Verlag, Berlin.