Keywords

1 Introduction

In cancer-associated fibroblasts, one of overexpressed proteins is called fibroblast activation protein (FAP), which was applied by researchers to develop new PET tracers for various cancers. Among different FAP inhibitors developed for PET imaging, 68Ga-FAPI-04 has demonstrated the most impressive properties with low nanomolar affinity to FAP, near-complete internalization of radioactivity bound to FAP, and rapid blood clearance.

Its first PET/CT demonstration in breast cancer patients was accomplished by Lindner et al. [1], whose work in the same group was prized the image of the year on 2019 Annual Meeting of the Society of Nuclear Medicine and Molecular Imaging (SNMMI).[2] The application of 68Ga-FAPI-04 has been extended to 28 different kinds of clinical cancer detection [3, 4]. All these exciting achievements quickly triggered a clinical research wave on FAPI-04 imaging and therapy globally [5,6,7].

The work on 68Ga-FAPI-04 clinical research has started since 2020 in Luzhou. There have been multiple works published on 68Ga-FAPI-04 PET imaging, which ranges from case studies to systematic research [8,9,10,11].

The manual synthesis of 68Ga-FAPI-04 is like other 68Ga-labeling peptides, such as PSMA-11 and DOTATATE.[12] However, because the radiochemical conversion (RCC) is about 90%, it is required to conduct a purification and isolation process to meet the required standard for clinical application.

To the best of our knowledge, there has been no previous study on quantitively characterizing the effect of eluting volumes to isolation efficiency (IE) in the manual synthesis of 68Ga-FAPI-04. The purpose of this work is to quantitively characterize the increase of IE by increasing the volume of formation liquid applied to elute the C18 column and the sterile filter.

We designed an experiment and measured the residual activity distribution on both C18 columns and sterile filters for different eluting volumes. From the measurements, we calculated IE with different eluting volumes. In addition, we characterized the change of activity residuals in the process of purification and isolation.

2 Materials and Methods

Ga-68 eluates were obtained from a 68Ge/68Ga generator with nominal activity 1850 MBq (ITG GmbH Germany) using 4 mL of 0.05 M HCl. FAPI-04 (60 μg, MedChemExpress LLC, China) was dissolved in 1 mL NaAc/HAc buffer (pH 4.0–5.0). The reaction mixture was incubated for 10 min at 95 ℃ (LAWSON DHS-100).

After the completion of radiolabeling, the reaction mixture passed over a C18 column (Sep-Pak Plus C18 Cartridge) and washed with 10 mL saline. The purified product was eluted with 1 mL 50-vol% ethanol followed by 5 mL saline and sterile filtered (Millex-GS, 0.22 μm) to get the final formulation. The activities of product, waste, and residuals on the C18 column and the sterile filter were measured with a dose calibrator (Capintec, CRC-55tR).

In order to study the effect of eluting volumes to IE, another 1 mL 50-vol% ethanol followed by 5 mL saline was applied to the C18 column and the sterile filter. For comparison, the activities of product, and residuals on the C18 column and the sterile filter were measured again by the dose calibrator.

The radiochemical purity (RCP) were determined by a radio-HPLC (LabAlliance). A C18 column (Agilent, ZORBAX Eclipse C18 Plus, 4.6 mm × 250 mm, 5 μm) was installed on the radio-HPLC. The gradient used mobile phase A and mobile phase B. Flow rate was 1 mL/min starting with 90% A to 10% A within 15 min.

This experiment was repeated by three different sophistic radio-labeling operators to suppress the effects caused by operators.

3 Results

Fig. 1.
figure 1

The radio-HPLC results of purified 68Ga-FAPI-04 product.

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The radio-HPLC results of the purified product are shown in Fig. 1 with an RCP of 99.4%. The free 68Ga peak appear near 2–4 min, which is not visible in the figure as a result of purification. The labeled 68Ga-FAPI04 peak appears near 5–7 min.

In the process of purification and separation, the labeled 68Ga-FAPI-04 normally could leave some percentages on C18 column and sterile filter. IE is calculated by,

$$ IE = \frac{{A_{product} }}{{A_{product} + A_{C18} + A_{filter} }} \times 100{\%}$$

where Aproduct is the activity of the product, AC18 is the residual activity on C18 column, and Afilter is the residual activity on sterile filter.

Similarly, the ratio of the C18 column residual over the labeled 68Ga-FAPI-04, RC18, and the ratio of sterile filter residual over the labeled 68Ga-FAPI-04, Rsf, are also calculated for each eluting volume case. The results are listed in Table 1.

Table 1. The calculated IE, RC18 and Rsf based on 3 different experiments, in which the average value and the standard deviation are shown.
Full size table

4 Discussion

From IE results, it was found that an obvious improvement was achieved by eluting the C18 column and the sterile filter twice. Although the volume of the product was increased as two times, the ratio of the radioactivity over the eluting volume is dropped only to 70% because of more product eluted off the C18 column and the sterile filter. In general, it is preferable to achieve more product activity in the production of 68Ga-FAPI-04, which means that more patients and better imaging results could be accomplished with one dose of production.

In addition, the standard deviation of IE representing the fluctuations was decreased by a factor of 2.5, from 11.3% to 4.5%. The fluctuations might come from the factor of different radiolabeling operators or the materials used in the experiment. In radiopharmaceutical production, the fluctuation should be reduced, which was also achieved by increasing the eluting volume.

In summary, the increase of eluting volume from 6 mL (1 mL 50-vol% ethanol followed by 5 mL saline) to 12 mL is more beneficial for clinical applications.

From the measurements, it was also found that more percentage of activity residual stays with the sterile filter than with the C18 column. There was about 3.0 times of 68Ga-FAPI-04 activity left on the sterile filter as much as on the C18 column after the first eluting. This factor was changed to 2.7 after the second eluting, which was not much with the consideration of the standard deviations.

Fig. 2.
figure 2

IE changes with eluting volume.

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Assuming a linear extrapolation (see Fig. 2), 100% of IE could be obtained at V = 15.0 mL, which might not be perfectly realized in practice, but would give a guidance on how much more one need to increase the eluting volume to gain more IE. For sure, the ratio of the radioactivity over the eluting volume will drop further down with the increase of the eluting volume.

5 Conclusion

In this work, we designed an experiment to quantitively characterize the change of manually labeled 68Ga-FAPI-04 IE by enlarging the eluting volume from 6 mL (1 mL 50-vol% ethanol followed by 5 mL saline) to 12 mL. It was found that the average IE was increase from 62.4% to 87.4%, with the standard deviation dropped from 11.3% to 4.5%, which are beneficial to clinical applications in general. In addition, it was found that there was 3.0 times activity left on the sterile filter as much as on the C18 column. Assuming a linear extrapolation, the highest IE could be expected with a third eluting on the C18 column and sterile filter.