Quality Assurance Investigations and Impurity Characterization during Upscaling of [177Lu]Lu-PSMAI&T

Abstract

[¹⁷⁷Lu]Lu-PSMA^I&T is widely used for the radioligand therapy of metastatic castration-resistant prostate cancer (mCRPC). Since this kind of therapy has gained a large momentum in recent years, an upscaled production process yielding multiple patient doses in one batch has been developed. During upscaling, the established production method as well as the HPLC quality control were challenged. A major finding was a correlation between the specific activity and the formation of a pre-peak, presumably caused by radiolysis. Hence, nonradioactive reference standards were irradiated with an X-ray source and the formed pre-peak was subsequently identified as a deiodination product by UPLC-MS. To confirm the occurrence of the same deiodinated side product in the routine batch, a customized deiodinated precursor was radiolabeled and analyzed with the same HPLC setup, revealing an identical retention time to the pre-peak in the formerly synthesized routine batches. Additionally, further cyclization products of [¹⁷⁷Lu]Lu-PSMA^I&T were identified as major contributors to radiochemical impurities. The comparison of two HPLC methods showed the likelihood of the overestimation of the radiochemical purity during the synthesis of [¹⁷⁷Lu]Lu-PSMA^I&T. Finally, a prospective cost reduction through an optimization of the production process was shown.

Keywords: HPLC; UPLC-MS; [177Lu]Lu-PSMA-I&T; quality assurance; radioligand therapy; radiolysis; upscaling.

Figure 1

Molecular structure of PSMA^I&T with Glu-urea-Lys-binding motif (red, left) and iodotyrosine moiety (center, blue).

Figure 2

Typical γ-HPLC chromatogram (method 1) of [¹⁷⁷Lu]Lu-PSMA^I&T with main product (R_t = 4.20 min) and radiolysis-induced side product (R_t = 3.88 min).

Figure 3

Graphical correlation between the integral of the suspected radiolysis-induced side product and the AC. Each blue dot refers to a single production batch. The orange line indicates the in-house specification benchmark of 5% impurities. All dots above the red line indicate batches with RCP < 95%.

Figure 4

Comparison between HPLC methods 1 and 2 through simultaneous measurements of [¹⁷⁷Lu]Lu-PSMA^I&T batches; three different batches with increasing activities (18–36 GBq, AC = 1–2 GBq/mL, N = 1) were synthesized according to the original synthetic procedure.

Figure 5

Total amount of radioactive impurities by using the new production and HPLC method in dependence of AC (blue dots: radiolysis pre-peak; orange circle: condensation side products; purple triangles: total impurities).

Figure 6

Representative chromatogram of a routine batch of [¹⁷⁷Lu]Lu-PSMA^I&T with HPLC method 2.

Figure 7

Molecular structure of the deiodinated PSMA^I&T by-product with the intact binding motif and missing iodine atom on the tyrosine side chain.

Figure 8

MS trace of an exemplary LC-MS measurement of a sample without buffer adjustments, irradiated with X-rays equivalent to 130 Gy. The blue arrows indicate the pre-peaks newly formed due to the irradiation.

Figure 9

MS spectra corresponding to the impurities detected (R_t = 7.095 (A); 7.442 (B) and 7.709 (C)); after irradiation of Gd-PSMA^I&T without adjusted buffer system, as described further above.

Figure 10

Integrals of the deiodinated compound determined via UPLC-MS in correlation with the applied dose in the two different reaction buffers. N = 1, performed in duplicate.

Figure 11

Overlay chromatogram of deiodinated [¹⁷⁷Lu]Lu-PSMA^I&T (blue) and a routine [¹⁷⁷Lu]Lu-PSMA^I&T batch (orange) from the same day.

Figure 12

UPLC-MS chromatogram of [^natLu]Lu-PSMA^I&T with the MS spectrum of the peak tail (Rt = 7.878 min); clearly visible are condensation products of ^natLu-PSMA (m/z = 835.0 + [M+2H]²⁺) with the mass of m/z = 827 [M-H₂O+2H]²⁺.

Figure 13

The PSMA binding motif and its possible heat-dependent condensation products, as proposed by Martin et al. [14].