Purification and Labeling Strategies for ⁶⁸Ga from ⁶⁸Ge/⁶⁸Ga Generator Eluate

Abstract

For successful labeling, ⁶⁸Ge/⁶⁸Ga generator eluate has to be concentrated (from 10 mL or more to less than 1 mL) and to be purified of metallic impurities, especially Fe(III), and ⁶⁸Ge breakthrough. Anionic, cationic and fractional elution methods are well known. We describe two new methods: (1) a combined cationic–anionic purification and (2) an easy-to-use and reliable cationic purification with NaCl solution. Using the first method, ⁶⁸Ga from 10 mL generator eluate was collected on a SCX cartridge, then eluted with 1.0 mL 5.5 M HCl directly on an anion exchanger (30 mg AG1X8). After drying with a stream of helium, ⁶⁸Ga was eluted with 0.4 mL water into the reaction vial. We provide as an example labeling of BPAMD. Using the second method, ⁶⁸Ga from 10 mL generator eluate was collected on a SCX cartridge, then eluted with a hydrochloric solution of sodium chloride (0.5 mL 5 M NaCl, 12.5 μL 5.5 M HCl) into the reaction vial, containing 40 μg DOTATOC and 0.5 mL 1 M ammonium acetate buffer pH 4.5. After heating for 7 min at 90°C, the reaction was finished. Radiochemical purity was higher than 95% without further purification. No ⁶⁸Ge breakthrough was found in the final product.

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1 Introduction

1.1 History

The first ⁶⁸Ga radiopharmaceuticals and ⁶⁸Ge/⁶⁸Ga radionuclide generators were developed at the beginning of the 1960s, in parallel with the creation of the first positron scintillation cameras, long before imaging with ¹⁸F (Rösch 2011). The first ⁶⁸Ge/⁶⁸Ga generators were self-made by radiochemical laboratories, mostly being based on EDTA elution (Hnatowich 1975). ⁶⁸Ge breakthrough was relatively high, as well as other metallic impurities. For a number of years now, a new type of ⁶⁸Ge/⁶⁸Ga generator has been commercially available (Obninsk, Russia), followed by diverse improvements from other manufacturers as well.

1.2 Current State

Molecular imaging of tumors by PET/CT using peptides radiolabeled for receptors is on the rise (Prasad and Baum 2010; Baum et al. 2010; Sainz-Esteban et al. 2010). The popularity of ⁶⁸Ga-labeled radiopharmaceuticals is rising due to their utility for molecular imaging, as well as the potential for on-demand production of ⁶⁸Ga for radiolabeling in the absence of cyclotron facilities. As generator technologies have matured to more consistently deliver high-purity ⁶⁸Ga over many months, the availability of highly adaptable postprocessing radiochemistry modules has also increased.

These developments suggest a rich future for generator-produced ⁶⁸Ga as an attractive candidate for radiolabeling of not only peptides but also other molecular targeting vectors such as carbohydrates, proteins, and oligonucleotides (Tolmachev et al. 2011; Fellner et al. 2010). Numerous methods for ⁶⁸Ga generator elution, purification, and radiolabeling of peptide radiopharmaceuticals have been advanced, being primarily based on three thematic ⁶⁸Ga eluate purification procedures found in the literature.

1.3 Basic Methods

1.3.1 Anionic Purification

In the first of these basic methods, Meyer et al. described ⁶⁸Ga labeling using an anionic purification step. In this procedure, ⁶⁸Ga chloride in the ⁶⁸Ge/⁶⁸Ga generator eluate is converted to the ⁶⁸Ga gallate anion by addition of concentrated HCl and then trapped on an anionic exchanger cartridge. After drying the cartridge with a stream of inert gas or air, the ⁶⁸Ga is eluted with water into the reaction vessel with chelator-modified peptide (Meyer et al. 2004). Although the obtained ⁶⁸Ga eluate has high chemical purity, an alternative to the intermediate conversion step using concentrated HCl may have advantages.

1.3.2 Cationic Purification

In a second approach, a labeling procedure based on a purification step of the ⁶⁸Ga eluate using a cation exchange resin was described by Zhernosekov et al. (2007). In this method, ⁶⁸Ga eluted from the generator is trapped by a cation exchange resin and then washed and eluted with two different hydrochloric acid/acetone solutions. The majority of the acetone is removed during the radiolabeling step, which is carried out at 100°C. This method has formed the basis for numerous effective strategies for purification of ⁶⁸Ga generator eluates and radiolabeling of peptides. To ensure that the final product is of sufficient radiochemical purity (>95%), a variation on this basic approach to ⁶⁸Ga purification and radiolabeling applies the same principles (Meyer et al. 2004) but includes also a final product purification using a C-18 cartridge, from which the final product is extracted with ethanol. The use of acetone/HCl may lead to detectable presence of mesityl oxide (4-methyl-3-penten-2-on) in the final product, although this can be avoided by using fresh preparations of the acetone/HCl eluent.

1.3.3 Fractional Elution

The third fundamental approach to ⁶⁸Ga labeling procedures uses the generator eluate directly based on fractional elution of the ⁶⁸Ge/⁶⁸Ga generator. In this method, early and late fractions of the 0.1 M HCl eluent containing ⁶⁸Ga are discarded, and an intermediate volume of eluted ⁶⁸Ga is collected and buffered to an appropriate pH for radiolabeling (e.g., with HEPES). Theoretically, a significant fraction of impurities are removed in early fractions of eluent (Breeman et al. 2005), and the fraction of eluent containing the highest concentration of pure ⁶⁸Ga is used for preparation of the radiopharmaceutical. Following the radiolabeling step, impurities such as free ⁶⁸Ga are removed by use of a C-18 cartridge and extraction with ethanol (as described above). The potential disadvantages of this method lie in that the procedure uses only a fraction of the elutable ⁶⁸Ga activity, which may reduce the achievable specific activity of the final radiopharmaceutical product, as well as the need for gas chromatographic analysis to establish the concentration of ethanol as an excipient in the final product.

2 Combined Cationic–Anionic Purification of ⁶⁸Ge/⁶⁸Ga Generator Eluate for Labeling of Fragile Peptides and Proteins

2.1 Aim

The combination of cationic and anionic purification of ⁶⁸Ga eluate should reduce the amount of concentrated HCl as well as the eluate volume. On the other hand, in the case of larger and fragile peptides or proteins such as DOTA-conjugated Affibody, the cationic concentration with the help of acetonic HCl followed by labeling with ⁶⁸Ga (Zhernosekov et al. 2007) requires high temperature to evaporate the acetone. The basic idea for this procedure is trapping of ⁶⁸Ga using a cation exchanger in a first step followed by elution of the precleaned ⁶⁸Ga with a minimal amount of 5.5 M HCl. The resulting ⁶⁸Ga tetrachlorogallate can then be collected using an anion exchanger in a second purification step (Mueller et al. 2009). This purification procedure for the ⁶⁸Ge/⁶⁸Ga eluate also eliminates acetone or other organic solvents.

2.2 Description

The ⁶⁸Ge/⁶⁸Ga generator was eluted with a total of 10 mL 0.1 M HCl, and the ⁶⁸Ga was collected on a SCX cartridge (Varian, Bond Elut-SCX, 100 mg, 1 mL). The activity was then eluted with 1.0 mL 5.5 M HCl and directly collected again on an anion exchanger cartridge (AG1X8, 30 mg, preconditioned with 1 mL 5.5 M HCl). After drying of the cartridge with a stream of helium (or air) for 1 min, 60% of the ⁶⁸Ga was eluted with 0.4–1.0 mL water into the reaction vial with 0.4 mL 1.5 M HEPES buffer and 100 μg DOTA-Affibody (DOTA-Z_{Her2:342-pep2}). The reaction mixture was heated to 80°C for 5 min.

For the anionic purification step, the authors developed a combined cartridge. For this purpose a 100 mg SAX cartridge (Alltech SAX Extract Clean SAX, 100 mg, 1.5 mL) was covered with 70 mg AG1X8 (BIO-RAD, 200–400 Mesh). If this cartridge is used, up to 80% of ⁶⁸Ga is elutable.

2.3 Advantages

This procedure leads to a final product with radiochemical purity greater than 95% without further purification steps. The described purification delivers ⁶⁸Ga in high chemical and radiochemical purity. Use of acetone or ethanol during the labeling procedure is not necessary, so that GC investigation is not essential for release. This method allows labeling of fragile peptides with molecular mass higher than DOTA-D-Phe1-Tyr3-octreotide (DOTATOC) such as DOTA-Affibody (DOTA-Z_{Her2:342-pep2}) or proteins (Mueller et al. 2006, 2009) (Figs. 1, 2, 3).

Fig. 1

Purification procedure, schematic drawing

Fig. 2

HPLC of the final product. Column: RP-18, LiChroCART 250-4, LiChrospher 100, RP-18e (5 μm); solvent A: acetonitrile solution in water (5%), 0.1% TFA; solvent B: 95% acetonitrile solution in water, 0.1% TFA; flow rate: 1.2 mL/min; gradient: from 0–2 min 100% A, 3–15 min to 100% B. ⁶⁸GaCl₃: (R _t = 2.5 min) 0.04%, ⁶⁸Ga-Affibody: (R _t = 9.4 min) 99.96%

Fig. 3

HPLC of ⁶⁸GaCl₃ samples at different HCl concentrations. Column RP-18, LiChroCART 250-4, LiChrospher 100, RP-18e (5 μm); solvent A: acetonitrile solution in water (5%), 0.1% TFA; solvent B: 95% acetonitrile solution in water, 0.1% TFA; flow rate: 1.2 mL/min; gradient: from 0–2 min 100% A, 3–5 min to 30% B

2.4 Example: Synthesis of ⁶⁸Ga-BPAMD

2.4.1 Introduction

For the ⁶⁸Ga-labeled bisphosphonate monoamide derivative of DOTA (BPAMD) for patient studies, the combined cationic/anionic ⁶⁸Ga eluent purification was successfully used in our department. The widely used cationic labeling procedure (Zhernosekov et al. 2007) leads to a nonphysiologic final product with pH lower than 2. Neutralization of the final product with sodium hydrogencarbonate (8.4%) is not possible, because of the decomposition of ⁶⁸Ga-BPAMD. Furthermore, the final product contains nonreacted and free ⁶⁸Ga³⁺ in different concentrations, so that a purification step is necessary. The combined purification method avoids these problems.

2.4.2 Labeling Procedure

The ⁶⁸Ga generator was eluted with a total of 10 mL 0.1 M HCl, and the ⁶⁸Ga was collected on a SCX cartridge (Varian, Bond Elut-SCX, 100 mg, 1 mL). This cartridge was then eluted with 5.5 M HCl directly through an anion exchanger cartridge.

For this anionic purification step we used a combined SAX cartridge (Alltech SAX Extract Clean SAX, 100 mg/1.5 mL), covered with 70 mg AG1X8 (BIO-RAD, 200–400 Mesh, hydroxide form), preconditioned with 1 mL 5.5 M HCl. The cartridge was dried with a stream of inert gas or air for 1 min, and ⁶⁸Ga was then eluted with 1 mL water followed by elution with 0.5 mL aqueous 1.5 M ammonium acetate solution into the reaction vial with 1 mL water, 0.5 mL aqueous 1.5 M ammonium acetate solution, and 20 μg BPAMD. Then, the reaction mixture was heated to 100°C for 12 min.

2.4.3 Results

The ⁶⁸Ga³⁺ was completely bonded, and after sterile filtration the radiochemical yield was about 55% (n.d.c.). This procedure leads to a final product with radiochemical purity greater than 95% without further purification steps. The pH of the final solution is about 4. Determination of other impurities by gas chromatography is not necessary. No organic solvents were added. The use of our developed combined SAX/AG1X8 cartridge increased the yield significantly. The instant thin layer chromatography (ITLC) quality control (Fig. 4) determines free ⁶⁸Ga³⁺ as well as potentially formed ⁶⁸Ga hydroxide.

Fig. 4

TLC of sterile filtered reaction solution synthesized by the combined cationic–anionic purification method (ITLC-SG; solvent: 0.1 M citric acid)

We thereby developed a reproducible and applicable labeling procedure for synthesis of ⁶⁸Ga-BPAMD. The reaction delivers the product with radiochemical purity higher than 95%. Subsequent purification is not necessary, and the pH of the reaction solution is about 4.

3 A New Highly Efficient NaCl-Based Cationic ⁶⁸Ge/⁶⁸Ga Generator Eluate Purification: The Basis for Effective ⁶⁸Ga Labeling

3.1 Aim

The aim is to develop efficient ⁶⁸Ga labeling procedures for routine application in clinical practice. The purification procedure for the ⁶⁸Ge/⁶⁸Ga eluate should reduce handling with concentrated HCl, and should form the labeled final product in high yield with high purity. Use of acetone or other organic solvents or compounds such as HEPES should be avoided. Handling should be reduced to a minimum of simple steps and should allow transfer to an automated system.

3.2 Labeling Strategy

Our strategy was influenced in part by the methods mentioned above, with the finer points of the new approach inspired by a reported procedure in which a rinse step for an OASIS WAX cartridge with 5 M NaCl solution was used to remove the 5.5 M HCl (de Blois et al. 2011). The use of only one ion exchange cartridge, but without use of organic solvents and high purification ability, were part of this method, combined with a search for a suitable buffer system. For DOTA-conjugated peptides, we exemplarily used DOTATOC.

3.3 Description of the Method

All reagents were purchased from commercial sources and used as received. The mentioned cartridges are commercially available. For all experiments a ⁶⁸Ga generator from Obninsk (Eckert and Ziegler Europe) and IGG100 ⁶⁸Ga generator (Eckert and Ziegler Europe) were used. The generator was eluted with 10 mL 0.1 M HCl (Merck, Germany). The ⁶⁸Ga of the generator eluate was collected on a SCX cartridge (VARIAN, Bond Elut-SCX, 100 mg, 1 mL; preconditioned with 1 mL 5.5 M HCl, 10 mL water) and then eluted with hydrochloric solution of sodium chloride (0.5 mL 5.0 M NaCl, 12.5 μL 5.5 M HCl) into the reaction vial with 40 μg DOTATOC, 3 mL water, and 250 μL 1 M ammonium acetate buffer (3.9 g NH₄Ac, 50 mL water, 1 mL HCl conc., adjusted with acetic acid to pH 4.5). After heating the solution for 7 min at 90°C, the reaction was finished.

This method works very reliably on an automated synthesis module. The concentration of unbound ⁶⁸Ga is lower than 5%. The radiochemical purity of the labeled DOTATOC is higher than 95%. The reaction mixture contains no toxic substances or substances of concern, so subsequent purification is not required. After sterile filtration, the radiochemical yield is about 82% (n.d.c.).

3.4 Results

We developed a new, easy-to-handle, highly effective NaCl-based cationic method for ⁶⁸Ge/⁶⁸Ga generator eluate purification. With this procedure, the radiochemical yield for labeling of DOTATOC with ⁶⁸Ga is about 82%. A subsequent purification step is not necessary. The final solution contains no organic solvents, and a GC investigation is not essential for release (Figs. 5, 6, 7).

Fig. 5

ITLC-SG of the final reaction mixture; eluent: acetonitrile/water 1:1; ⁶⁸Ga-hydroxide/-chloride (R _f = 0): 2.4% ⁶⁸Ga-DOTATOC (R _f = 0.5): 97.6%

Fig. 6

HPLC of the final product. Column: RP-18, LiChroCART 250-4, LiChrospher 100, RP-18e (5 μm); solvent A: 5% acetonitrile in water, 0.1% TFA; solvent B: 95% acetonitrile in water, 0.1% TFA; flow rate: 1.2 mL/min; gradient: from 0–2 min 100% A, 3–15 min to 100% B. ⁶⁸Ga³⁺ (R _t = 2.3 min) 0.6%, ⁶⁸Ga-DOTATOC (R _t = 8.4 min): 99.4%

Fig. 7

ITLC-SG of the final product after sterile filtration; eluent: acetonitrile/water 1:1