Optimization of automated radiosynthesis of gallium-68-labeled PSMA11 with two 68Ge/68Ga generators: fractional elution or prepurification?

doi:10.21203/rs.3.rs-2727479/v1

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Optimization of automated radiosynthesis of gallium-68-labeled PSMA11 with two 68Ge/68Ga generators: fractional elution or prepurification?

https://doi.org/10.21203/rs.3.rs-2727479/v1

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Background

Prostate cancer is one of the most common forms of cancer in men. An imaging technique for its diagnosis is [68Ga]-prostate specific membrane antigen (⁶⁸Ga-PSMA-11) positron emission tomography (PET). Gallium 68 (⁶⁸Ga) is typically obtained using a germanium 68/gallium 68 (⁶⁸Ge/⁶⁸Ga) generator, allowing for the diagnostic drug to be made readily available in the nuclear medicine department through elution. To meet the increasing demand for ⁶⁸Ga labeled peptides and to reduce the cost of radiosynthesis, it is therefore necessary to optimize the elution process of ⁶⁸Ge/⁶⁸Ga generators. This study aims to identify the most effective approach for optimizing radiosynthesis using double elution in parallel of two ⁶⁸Ge/⁶⁸Ga generators. Two methods have been tested: one using prepurification, and the other using fractionated elution.

Results

Five synthesis sequences were conducted using each method. The mean labeling yields for double elution with prepurification were 45.8 ± 29.4 (mean ± standard deviation) and none met the required criteria. The mean labeling yields for the fractionated double elution were 97.5 ± 1.9 (mean ± standard deviation) meeting the criteria, significantly superior to the prepurification method (p=0.012), and similar to those of simple elution. There was no significant difference in the elution yields of both methods.

Conclusions

This study showed that fractionated double elution from ⁶⁸Ge/⁶⁸Ga generators produced a significantly higher labeling yield than double elution with prepurification, resulting in a larger activity recovered by radiosynthesis, and thereby allowing for more diagnostic tests to be performed. Additionally, this method does not add complexity or synthesis time compared to simple elution labeling, and could also be applied to other ⁶⁸Ga labeled peptides.

Radiopharmacy

gallium 68

double elution

Prostate cancer is the 4th most common form of cancer in Europe and the most common in men (1). According to the European Society for Medical Oncology (ESMO) guidelines, the diagnosis of high-risk forms relies on imaging techniques including magnetic resonance imaging (MRI), [¹⁸F]-fluorocholine positron emission tomography (PET), and [68Ga]-prostate specific membrane antigen (⁶⁸Ga-PSMA-11) PET (2). In France, ⁶⁸Ga-PSMA-11 is a radiopharmaceutical drug with compassionate access for patients with biologically recurrent prostate cancer, defined as a re-increase in serum prostate specific antigen (PSA) concentration (3). This radiopharmaceutical drug has high sensitivity and excellent specificity for the diagnosis of prostate cancer and is increasingly used in routine practice (4).

Gallium 68 can be obtained via cyclotron production with a ⁶⁸Zn target solution or from elution of a germanium 68/gallium 68 (⁶⁸Ge/⁶⁸Ga) generator. These generators can be eluted on-site, making radiolabeled PSMA available free from industrial production constraints, several times a day, and up to one year due to the ⁶⁸Ge period of 271 days. However, they come with limitations which are their cost and a limited activity (only 1850 MBq available in France). Because of the short half-life of ⁶⁸Ga (68 min), the dosage of ⁶⁸Ga-PSMA-11 (2 MBq/kg), and the time between injections (25 minutes), not many patients can be injected per elution. To address the growing demand for ⁶⁸Ga-PSMA-11 and reduce the cost of the radiopharmaceutical / patient, optimization of radiosynthesis has been implemented. Two possible options are to elute a generator calibrated with high activity or to elute two generators in parallel (5).

In the present study, we investigate the radiosynthesis optimization of ⁶⁸Ga-PSMA-11 based on the double elution in parallel of two ⁶⁸Ge/⁶⁸Ga generators with calibration dates six months apart. There is limited literature on the subject, and this double elution is expected to increase the activity of radiosynthesis and ensure a more consistent supply of ⁶⁸Ga-PSMA, which would improve the flow of PET exams over time.

Our objective was to compare two methods in order to determine the most suitable, i.e. the one that guarantees the best synthesis yield, following the Good Manufacturing Practices for Preparations:

Double elution with prepurification to concentrate the gallium 68
Double fractional elution to extract the most concentrated fraction in activity of the two eluates

The equipment used for each radiosynthesis method is detailed in Table 1 and the preparation steps are summarized below.

Table 1

Material used for radiosynthesis with prepurification and fractional double elution.
Double elution with prepurification	Double fractional elution
High energy shielded enclose: H700hotcell, Trasis® (Ans, Belgium)
MiniAIO® synthesis module, Trasis®
Peptide PSMA-11, Iason® (Graz-Seiersberg, Austria)
Generators ⁶⁸Ge/⁶⁸Ga GalliaPharm® Eckert & Ziegler (Berlin, Germany)
Elution solvent: 1 M hydrochloric acid Eckert & Ziegler
Acetate buffer 1 M, Trasis®	Acetate buffer 0.7 M, prepared extemporaneously
Cassette with prepurification ® ref: S10886 Trasis® C18 prepurification cartridge, Oasis® Suprapur® NaCl/HCl 30%	Cassette without prepurification, Trasis® (Ref: S7577)
HLB cartridge
NaCl 0.9%
Absolute ethanol

The double elution with prepurification method

Cassette with prepurification is placed on the MiniAIO® synthesizer
10 µg of PSMA-11 are reconstituted with 3 mL of sodium acetate buffer from Trasis^® and then transferred into the reaction vial (step 1 in Fig. 1)

Elution of the two ⁶⁸Ge/⁶⁸Ga generators is performed in parallel, by 5 mL of HCl 1M (2 mL/min) each. A female Luer lug style tee and a ventilated filter are positioned at the outlet of the two generators (step 2)
⁶⁸Ga solution is transferred to the prepurification cartridge which is eluted with 5 mL of a Suprapur® NaCl/HCl 30% mixture (step 3)
Labeling reaction is performed at 95 ± 5°C during 6.5 min (step 4)
The solution is loaded onto an HLB cartridge to retain the ⁶⁸Ga-PSMA-11. This cartridge is rinsed with 0.9% NaCl. The free ⁶⁸Ga is evacuated in waste (steps 5 and 6)
Elution of the cartridge with 0.7 mL of absolute ethanol, recovering the ⁶⁸Ga-PSMA-11 (step 7)
Transfer of the solution to the final vial
Formulation with 9.3 mL of 0.9% NaCl. Volume of the final product is 10 mL (step 8)

The double fractional elution method

Cassette without prepurification is placed on the MiniAIO® synthesizer.
10 µg of PSMA-11 is reconstituted with 1 mL of 0.7 M sodium acetate solution, prepared and filtered in radiopharmacy under laminar flow (ISO 5), and then transferred into the reaction vial (step 1 in Fig. 3)

Elution of the two ⁶⁸Ge/⁶⁸Ga generators is performed in parallel, by 5 mL of HCl 1M (2 mL/min) each. A T device and a ventilated filter are positioned at the outlet of the two generators (step 2)
The first 3 mL, i.e. 1,5 mL for each generator and the last 1 mL, i.e. 0,5 mL for each generator, of the elution are eliminated in waste (steps 3a and 3b). These volumes were established according to a study (5) showing that fractionation of the elution from the ⁶⁸Ge/⁶⁸Ga generators resulted in a higher activity volume. The following diagram from the study shows this result (Fig. 2).

6 mL of the elution, the most active fraction, are transferred to the reaction vial containing the peptide in solution in acetate buffer (step 4)
Labeling reaction is performed at 95 ± 5°C for 6.5 min (step 5)
The solution is loaded onto a C18 cartridge (SEP-Pak) which retains the ⁶⁸Ga-PSMA-11. The cartridge is rinsed with 0.9% NaCl (step 6)
Elution of the cartridge with 0.7 ml of absolute ethanol removing the ⁶⁸Ga-PSMA-11 transferred to the final vial. The final product is then diluted with 9.3 mL of 0.9% NaCl (steps 7 and 8)

The acceptance criteria

For each synthesis procedure, quality controls were carried out:

Visual examination of organoleptic character: the product must be clear and colorless
pH measured with a pH strip must be between 4 and 8
Radiochemical purity (RCP), measured by two methods:

High performance liquid chromatography (HPLC) with C18 column Waters^® 250 x 4.6 mm, flow: 1 ml/min, with solvent gradient Acetonitril/TFA 0,1% and water/TFA 0,1%. The programming is 00:00–3:00 min: 97% A -3% B; 06:00–9:00 min: 0% A -100% B et de 12:00–15:00 97% A -3% B. The retention time is 3.7 min for free ⁶⁸Ga and 8.3 min for ⁶⁸Ga-PSMA-11. The addition of the stereoisomers is at least 95% of the total radioactivity due to ⁶⁸Ga.

Thin layer chromatography (TLC) with ITLC-SG plates, where the retention factor of ⁶⁸Ga-PSMA-11 is 0.8-1, that of ⁶⁸Ga 3 + is 0-0.1 and that of the ⁶⁸Ga colloid is 0.7. No less than 95% of the total radioactivity is due to ⁶⁸Ga-PSMA-11.

Bacterial endotoxins, measured by a kinetic and chromogenic LAL method (Endosafe®, Charles River), the preparation is diluted to the 10th and deposited on PTS-10 cassettes with a sensitivity of 0.01EU/ML - PTS2001. The expected result is a bacterial endotoxin content lower than 15.1 IU/mL
Radiolabeling yield, measured with an ISOMED2010^® dose calibrator, which is expected to be higher than 80%
Elution yields were calculated. The manufacturer specification of elution yield is > 62%, but the prepurification/fractioning steps tend to decrease this yield (about 10%) and therefore we set the specification to elution yield > 55%.

Statistical tests

A Mann-Whitney test was performed to compare elution and labeling yields as well as the overall radiosynthesis yields (multiplication of the elution and labeling yields) of the two methods.

A total of 10 radiosynthesis sequences were performed: 5 with prepurification and 5 with fractionated elution. All yields (elution and labeling) were measured at the time of elution.

The labeling yields of the elution with prepurification were between 6 and 74% and therefore did not comply with the expected yields. Furthermore, the radiolabeling success was inconsistent (mean of labeling yield: 45,8 ± 29,4%). For the third radiosynthesis, the quality controls were not compliant with a TLC-assessed RCP = 71,8%. For this radiosynthesis, HPLC was not performed because the TLC was already not compliant. The elution yields were compliant to specifications (63,8 ± 4,8%). Radiosynthesis results are detailed in Table 2.

Table 2: Results of double elution radiosynthesis with prepurification

As for double fractional elution, yield and quality control requirements were met for all sequences. The labeling yields were between 94% and 99%, as shown in Table 3, and the elution yields were compliant to specifications (59,4 ± 2,2%)

There was no significant difference between the elution yields of both methods (p = 0.151), but there was a significant difference in labeling yields between the prepurification and the fractionated elution methods (p = 0.012), in favor of the latter. Regarding overall radiosynthesis yields, the difference was also significant (p = 0.008).

Table 3: Results of fractionated double elution radiosynthesis

In this study, we show that double elution with prepurification led to insufficient and greatly varying radiolabeling yield of ⁶⁸Ga-PSMA-11, whereas fractionated double elution resulted in consistent and higher labeling/overall yield.

There are several potential explanations for the results of the synthesis with elution prepurification. One possibility is that there were issues with pH. pH is a critical factor in the radiosynthesis of ⁶⁸Ga-PSMA-11, and a too acidic pH can result in the protonation of the chelator. On the other hand, if the pH is too basic, it can lead to the formation of insoluble \(Ga({OH)}_{3}\). In this method, 5 mL of an HCl/NaCl mixture were added to 3 mL of acetate buffer. In comparison, in the double fractionation elution method, 6 mL of ⁶⁸Ga in HCl 0,1M are added to 1 mL of acetate buffer. As the reaction medium is different, the pH may vary. Unfortunately, the pH at this step is not an “in-process” checkpoint leaving doubt as to the role of pH in the synthesis failure (6)(7).

Another hypothesis might have been the presence of impurities in the reaction vial. Metallic impurities can be caused by degradation of the generator column or the purification process. Ferric impurities may compete with ⁶⁸Ga, preventing complexation with the chelator and thereby decreasing the yield. However, this hypothesis was effectively ruled out since single elution was performed and quality checks/yields met requirements (see supplementary information, Table 1) (8)(9).

As a result of this, we started to question the overall preparation process. Interestingly, Reverchon et al (10) showed that the best conditions for the preparation of ⁶⁸Ga-PSMA-11 may involve no purification and no heating, which significantly reduced the preparation time. Under these conditions, quality controls showed a RCP of over 99% and a radiolabeled yield of over 99%. This approach, however, creates other issues. Removing the purification step leads to a larger elution volume (10 mL), and requires a complete modification of the radiosynthesis process including changes in acetate buffer volume, heating profile, and volume activity for labeling.

Finally, all fractional elution tests produced results within specifications. Fractional elution appears to be the method of choice for ⁶⁸Ga-PSMA-11 radiosynthesis for several reasons. Firstly, as previously mentioned, fractional elution allows for the removal of the eluate portion containing metallic impurities, while retaining the part with the highest activity for radiolabeling with an almost identical volume to that of simple elution. Although the elution yields may be slightly lower, the labeling yields are significantly higher. Secondly, the preparation process is simplified as there is no longer a need for prepurification, making it similar to simple elution which shows high labeling yields. Lastly, the simplified preparation process saves time and therefore results in higher activity.

The literature on fractional elution for PSMA-11 radiolabeling remains limited, and our study demonstrates that this method applied on a ⁶⁸Ge/⁶⁸Ga generator can optimize radiosynthesis. Fractional elution allows for a higher activity product enabling more patients to benefit from ⁶⁸Ga-PSMA-11 PET for diagnosis, while decreasing the frequency of radiosynthesis per week. This optimization can help to plan for the anticipated huge increase in ⁶⁸Ga-PSMA-11 PET-scans. Additionally, double fractional elution could also be applied to the labeling of other peptides such as edotreotide.

⁶⁸Ga-PSMA-11

[68Ga]-prostate specific membrane antigen

⁶⁸Ge

Germanium 68

⁶⁸Ga

Gallium 68

HPLC

High performance liquid chromatography

PET

Positron emission tomography

PSA

Prostate specific antigen

RCP

Radiochemical purity

TLC

Thin layer chromatography

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

All data generated or analysed during this study are included in this published article

Competing interests

The authors declare that they have no competing interests.

Funding

Nuclear medicine department, CHU Lille, F-59000 Lille, France

Authors' contributions

FD, BD and JFL performed the radiosynthesis and worked on preparation method. BD, JFL, AR, EM, FS, PO reviewed the manuscript. All authors approved the final manuscript.

Acknowledgements

Not applicable.

Dyba T, Randi G, Bray F, Martos C, Giusti F, Nicholson N, et al. The European cancer burden in 2020: Incidence and mortality estimates for 40 countries and 25 major cancers. Eur J Cancer Oxf Engl 1990 nov. 2021;157:308–47.
Parker C, Castro E, Fizazi K, Heidenreich A, Ost P, Procopio G, et al. Prostate cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol sept. 2020;31(9):1119–34.
PROTOCOLE, D’UTILISATION THERAPEUTIQUE ET DE RECUEIL, D’INFORMATIONS «. PSMA-11 10 µg, poudre pour solution injectable » Janvier 2022.
Perera M, Papa N, Roberts M, Williams M, Udovicich C, Vela I, et al. Gallium-68 Prostate-specific Membrane Antigen Positron Emission Tomography in Advanced Prostate Cancer—Updated Diagnostic Utility, Sensitivity, Specificity, and Distribution of Prostate-specific Membrane Antigen-avid Lesions: A Systematic Review and Meta-analysis. Eur Urol avr. 2020;77(4):403–17.
Velikyan I. 68Ga-Based Radiopharmaceuticals: Production and Application Relationship. Molecules. 16 juill 2015;20(7):12913–43.
Boukhlef S, Buziau B, Zajac C, Dekyndt B, Legrand JF. Automatisation du fractionnement d’élution d’un générateur de Gallium-68 en utilisant un automate MiniAIO®. French society of Nuclear Medicine congress, short presentation sept 2021
Kulprathipanja S. D.J.Hnatowich. A method for determining the pH stability range of gallium radiopharmaceuticals.The International journal of applied radiation and isotopes1977
Meyer GJ, Mäcke H, Schuhmacher J, Knapp WH, Hofmann M. 68Ga-labelled DOTA-derivatised peptide ligands. Eur J Nucl Med Mol Imaging [Internet] août. 2004. 10.1007/s00259-004-1486-0. [cité 16 févr 2023];31(8). Disponible sur:. http://link.springer.com/.
Morgat C, Hindié E, Mishra AK, Allard M, Fernandez P. Gallium-68: Chemistry and Radiolabeled Peptides Exploring Different Oncogenic Pathways. Cancer Biother Radiopharm mars. 2013;28(2):85–97.
Chomet M, Cross T, Géraudie B, Chipan AS, Talbot JN, Nataf V. Gallium-68: considérations pratiques pour le succès des applications cliniques au sein d’un service de médecine nucléaire. Médecine Nucl sept. 2014;38(4):229–34.
Reverchon J, Khayi F, Roger M, Moreau A, Kryza D. Optimization of the radiosynthesis of [68Ga]Ga-PSMA-11 using a Trasis MiniAiO synthesizer: do we need to heat and purify? Nucl Med Commun sept. 2020;41(9):977–85.

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Optimization of automated radiosynthesis of gallium-68-labeled PSMA11 with two 68Ge/68Ga generators: fractional elution or prepurification?

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Version 1

Abstract

Figures

Background

Methods

The acceptance criteria

Statistical tests

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

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