New sensitive method for HEPES quantification in 68Ga-radiopharmaceuticals

Antunes, Inês Farinha; Franssen, Gerben M.; Zijlma, Rolf; Laverman, Peter; Boersma, Hendrikus H.; Elsinga, Philip H.

doi:10.1186/s41181-020-00093-x

Cited by 8 publications

(8 citation statements)

References 6 publications

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“…However, it has been proven that the physical separation of the components by TLC, as well as their coloring through the exposure under iodine beads, is neither reliable nor suitable to detect the presence of residual HEPES [ 31 , 32 , 33 , 34 ], indicating the need to improve this method or develop a new one.…”

Section: Discussionmentioning

confidence: 99%

A Specific HPLC Method to Determine Residual HEPES in [68Ga]Ga-Radiopharmaceuticals: Development and Validation

et al. 2022

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Background: Nowadays, in Nuclear Medicine, clinically applied radiopharmaceuticals must meet quality release criteria such as high radiochemical purity and radiochemical yield. Many radiopharmaceuticals do not have marketing authorization and have no dedicated monograph within European Pharmacopeia (Ph. Eur.); therefore, general monographs on quality controls (QCs) have to be applied for clinical application. These criteria require standardization and validation in labeling and preparation, including quality controls measurements, according to well defined standard operation procedures. However, QC measurements are often based on detection techniques that are specific to a certain chromatographic system. Several radiosyntheses of [68Ga]Ga-radiopharmaceuticals are more efficient and robust when they are performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity to be assessed in the QC procedure, prior to clinical use. Thus, Ph. Eur. has introduced a thin-layer chromatography (TLC) method to quantify the HEPES amount that is present in [68Ga]Ga-radiopharmaceuticals. However, this is only qualitative and has proven to be unreliable. Here we develop and validate a new high-performance liquid chromatography (UV-Radio-HPLC) method to quantify the residual amount of HEPES in 68Ga-based radiopharmaceuticals. Method: To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document that was adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. A range of concentrations of HEPES (100, 80, 60, 40, 20, 10, 5, 3 μg/mL) were analyzed. Moreover, to test the validity and pertinence of our new HPLC method, we analyzed samples of [68Ga]Ga-DOTATOC; [68Ga]Ga-PSMA; [68Ga]Ga-DOTATATE; [68Ga]Ga-Pentixafor; and [68Ga]Ga-NODAGA-Exendin-4 from different batches that were prepared for clinical use. Results: In the assessed samples, HEPES could not be detected by the TLC method that was described in Ph. Eur. within 4 min incubation in an iodine-saturated chamber. Our developed HPLC method showed excellent linearity between 3 and 100 μg/mL for HEPES, with a correlation coefficient (R2) for calibration curves that was equal to 0.999, coefficients of variation (CV%) < 2%, and percent deviation value of bias from 100% to 5%, in accordance with acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed and the limit-of-quantification (LOQ) was 3 μg/mL, confirming the high sensitivity of the method. The amount of HEPES that was detected with our developed HPLC method in the tested [68Ga]Ga-radiopharmaceuticals resulted well below the Ph. Eur. limit, especially for [68Ga]Ga-NODAGA-Exendin-4. Conclusions: The TLC method that is described in Ph. Eur. to assess residual HEPES in [68Ga]-based radiopharmaceuticals may not be sufficiently sensitive and thus unsuitable for QC release. Our new HPLC method was sensitive, quantitative, reproducible, and rapid for QCs, allowing us to exactly determine the residual HEPES amount in [68Ga]Ga-radiopharmaceuticals for safe patient administration.

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Section: Discussionmentioning

confidence: 99%

A Specific HPLC Method to Determine Residual HEPES in [68Ga]Ga-Radiopharmaceuticals: Development and Validation

et al. 2022

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“…A limit test for the HEPES content was performed using a validated HPLC method based on a 150 × 4.6 mm XBridge C18 5 µm column (186003116, Waters, Milford, Massachusets) and 20 mM solution of ammonium formate (pH 8) as mobile phase together with a flow rate of 0.7 mL/min modified from Antunes et al [ 36 ]. The HEPES reference solution (40 µg/mL) and the sample were measured at UV λ = 195 nm.…”

Section: Methodsmentioning

confidence: 99%

Automated Synthesis of 68Ga-Labeled DOTA-MGS8 and Preclinical Characterization of Cholecystokinin-2 Receptor Targeting

et al. 2022

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The new minigastrin analog DOTA-MGS8 targeting the cholecystokinin-2 receptor (CCK2R) used in this study displays the combination of two site-specific modifications within the C-terminal receptor binding sequence together with an additional N-terminal amino acid substitution preventing fast metabolic degradation. Within this study, the preparation of 68Ga-labeled DOTA-MGS8 was validated using an automated synthesis module, describing the specifications and analytical methods for quality control for possible clinical use. In addition, preclinical studies were carried out to characterize the targeting potential. [68Ga]Ga-DOTA-MGS8 showed a high receptor-specific cell internalization into AR42J rat pancreatic cells (~40%) with physiological expression of rat CCK2R as well as A431-CCK2R cells transfected to stably express human CCK2R (~47%). A favorable biodistribution profile was observed in BALB/c nude mice xenografted with A431-CCK2R cells and mock-transfected A431 cells as control. The high tumor uptake of ~27% IA/g together with low background activity and limited uptake in non-target tissue confirms the potential for high-sensitivity positron emission tomography of stabilized MG analogs in patients with MTC and other CCK2R-related malignancies.

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“…This very strict limit translates to a maximum HEPES dose of 200 μg per dose. Labelling of specific radiopharmaceuticals may require different volumes and concentrations of HEPES as reported by Antunes et al (Antunes et al 2020 ).Using these examples, in a worst-case scenario where no HEPES is removed during the labelling process, a total single dose between 283 and 714 mg HEPES would be intravenously administered to a patient. This translates to a HEPES dose of 4.0–10.2 mg/kg for a 70 kg patient.…”

Section: Use Of Hepes In Pharmaceutical Productsmentioning

confidence: 97%

“…The European Pharmacopoeia prescribes a strict limit of 200 μg per dose for the HEPES content in radiopharmaceuticals intended for intravenous administration (European Pharmacopeia 2017 ). However, the method described in the European Pharmacopoeia to test for HEPES in radiopharmaceutical preparations has proven to be unreliable (Antunes et al 2020 ; Pfaff et al 2018 ; Sasson et al 2010 ). The low limit has hampered the use of HEPES in gallium-68 radiopharmaceuticals, despite its superior buffering properties.…”

Section: Use Of Hepes In Pharmaceutical Productsmentioning

confidence: 99%

The use of HEPES-buffer in the production of gallium-68 radiopharmaceuticals – time to reconsider strict pharmacopoeial limits?

Kleynhans

Rubow

2021

EJNMMI radiopharm. chem.

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HEPES (4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid) is a buffer that is used in the radiolabelling of gallium-68 compounds. The beneficial effects of HEPES on molar activity in bioconjugates have been well described. Current strict regulations on the HEPES content in radiopharmaceuticals limit its use when intended for parenteral administration.This short communication summarizes data from the literature on the toxicity of HEPES in dogs after intravenous infusion and the subsequent use in humans. We also highlight the use of HEPES in an FDA labelled intravenous drug formulation. Regulatory institutions may consider this data to review current strict limits.

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New sensitive method for HEPES quantification in 68Ga-radiopharmaceuticals

Cited by 8 publications

References 6 publications

A Specific HPLC Method to Determine Residual HEPES in [68Ga]Ga-Radiopharmaceuticals: Development and Validation

A Specific HPLC Method to Determine Residual HEPES in [68Ga]Ga-Radiopharmaceuticals: Development and Validation

Automated Synthesis of 68Ga-Labeled DOTA-MGS8 and Preclinical Characterization of Cholecystokinin-2 Receptor Targeting

The use of HEPES-buffer in the production of gallium-68 radiopharmaceuticals – time to reconsider strict pharmacopoeial limits?

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