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Time-efficient HPLC Validation Methodology for the Qualitative Analysis of 68Ga PSMA-11 in Routine Clinical Usage under Isocratic Method
Address for correspondence: Mr. Sachin Tayal, Mahamana Pandit Madan Mohan Malaviya Cancer Centre, A Unit of Tata Memorial Centre, BHU Campus, Sunadar Bagiya, Varanasi - 221 005, Uttar Pradesh, India. E-mail: sachintayal7@gmail.com
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Received: ,
Accepted: ,
This article was originally published by Wolters Kluwer - Medknow and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Background:
Prostate-specific membrane antigen (PSMA) has shown to be a promising agent for prostate cancer imaging under PET-CT. With the automation in radiolabeling with 68Ga, using iTG 68Ge/68Ga generator, it has helped introduce various new diagnostic agents and achieve good manufacturing practices (GMP) simultaneously. However, before any radiopharmaceutical is put into clinical usage, it should always be checked for its radiochemical purity and other quality parameters before injecting in the patient. Chromatography techniques such as Gas Chromatography (GC), High-Performance Liquid Chromatography (HPLC), and Thin-Layer Chromatography (TLC) are the most frequently utilized separation technique for purity analysis. A rapid quality control HPLC based methodology was required for radiopharmaceuticals.
Aim & Objective:
In our current setting, we conducted quality control analysis and standardized and validated HPLC method for the routine quality check of 68Ga-PSMA-11.
Materials and Methods:
The QC of 68Ga PSMA-11 was performed under ITLC and HPLC.
Results:
Linearity, accuracy, precision and specificity were assessed and quantified in accordance with International conference on harmonisation of technical requirements for registration of pharmaceuticals for human use (Q2 (R1) ICH) guidelines, which can be implemented in resource-limited settings to check the quality.
Conclusion:
The current HPLC based methodology is rapid, with a retention time of 2.24 min, rendering it a favorable analytical standard operating procedure for QC analysis of 68Ga-PSMA-11.
Keywords
Gallium-68 prostate-specific membrane antigen-11
high-performance liquid chromatography
isocratic
Introduction
Prostate cancer remains a significant health concern worldwide.[12] Although radiodiagnosis techniques such as magnetic resonance imaging and positron emission tomography (PET) scans have significantly improved detection and staging, challenges persist. However, prostate-specific membrane antigen (PSMA) has shown to be a promising agent for specific prostate cancer imaging[34] under PET-computed tomography. The radiolabeling of peptide (PSMA-11) with gallium-68 (68Ga) is well suited for rapid pharmacokinetics, resulting in low radiation doses for patients.[5] 68Ga-PSAM-11 proved a significant radiotracer in the management of prostate cancer patients.[6] Second, the automation in radiolabeling with 68Ga has helped introduce various new diagnostic agents and achieve good manufacturing practices (GMPs) simultaneously.[7] Due to their generator-based production method, 68Ga radiopharmaceuticals circumvent the substantial investment required for cyclotrons, which is necessary for 18F-labeled radiotracers.[8] However, before any radiopharmaceutical is put into clinical usage, it should always be checked for its high radiochemical purity (RCP) and other quality parameters before injecting in the patient/clinical usage. Hence, various quality control (QC) parameters contribute to substantiating the safety, quality, and efficacy of the prepared radiopharmaceutical.[9] Successful QC clearance confirms that the radiotracer is suitable for injection with its high radiochemical yield and RCP.[5101112131415] Nonetheless, a multitude of factors can result in chemical and radiochemical impurities, such as incomplete labeling and degradation of radiotracers over time. Chromatography techniques such as Gas Chromatography (GC), High-Performance Liquid Chromatography (HPLC), and Thin-Layer Chromatography (TLC) are the most frequently utilized separation technique for purity analysis.[161718] In our current setting, we conducted QC analysis and standardized and validated the HPLC method for the routine quality check of 68Ga-PSMA-11. A reverse-phase HPLC method was validated within our laboratory for the qualitative and quantitative analysis of 68Ga-PSMA-11. Linearity, accuracy, precision, and specificity were assessed and quantified in accordance with Q2 (R1) ICH guidelines, which can be implemented in resource-limited settings to oversee their quality.
Materials and Methods
Chemical and reagents
Pharmaceutical-grade chemicals and reagents were used for the synthesis of radiopharmaceutical products. Reference standard (500 μg) PSMA-11 peptides were purchased from ABX advanced biochemical compounds, Germany. We use an automated cassette-based synthesis module, a GMP-certified module to synthesize pure radiopharmaceutical products.[19] Specialized cassettes with consumables (HCl, saline, ethanol bottles, and C18 ion exchange cartridges) are required to synthesize 68Ga-based radiopharmaceuticals, which were also supplied by ABX advanced biochemical compounds, Germany. For RCP, QC experiments were performed under instant TLC medium (ITLC) and HPLC. HPLC grade solvents, acetonitrile (ACN), methanol, and trifluoroacetic acid (TFA) were procured from Merck, India. Ultra-pure water ≥18.2 MΩ was obtained from PURELAB® fle × 2 plus water purification system (ELGA LabWater, USA). ITLC paper strips were purchased from Sigma-Aldrich.
Instrument and equipment
iQS-TS Synthesis module, ITG, Germany, is an automated cassette-based synthesis module approved by a European agency as a GMP-certified module, and the itG 68Ge/68Ga generator has a nonmetallic column (silica gel [SG]-modified dodecyl gallate) which does not require prepurification steps unlike metallic column 68Ge/68Ga generator.
Synthesis and quality control procedures
The QC of 68Ga-PSMA-11 was performed under ITLC and HPLC.
Instant thin-layer chromatography
RCP assessment was performed on TLC SG 60 F254 aluminum sheets with an automatic peak finder with high sensitivity and accuracy (LabLogic Scan-RAM/Radio-TLC Scanner). An SG-ITLC strip measuring 10 cm × 1 cm and a 0.5 M sodium citrate buffer served as the solid and mobile phases, respectively. A drop of the final product was applied to the SG-ITLS strip at the point of spotting (PS), and the chromatogram was developed up to the solvent front (SF). 68Ga-labeled radiopharmaceuticals remained at PS, while the unlabeled fraction 68Ga migrated to SF [Figure 1]. Counting was performed in LabLogic Scan-RAM/Radio-TLC Scanner with the help of Laura™ radiochromatography data collection and analysis software version v6.1.1.20 and RCP was calculated [Figure 1], as shown in equation 1:[920]
Equation 1
where %RCP = Percentage RCP; Cnt (PS) = Count at PS; Cnt (SF) = Count at SF.
High-performance liquid chromatography
The equipment [Figure 2] consists of a solvent delivery system (S-1125 HPLC pump system-plus), an oven, a manual injection setup comprising a rotary valve, an Ultraviolet-Visible detector (S-3245 UV/Vis), and a gamma detector (S-3700). Data analysis was conducted using ChromStar workstation v. 7.0 software. Separation was achieved at 25°C utilizing an Acclaim 120, C18, 3 μm 120 Å (3.0 mm × 150 mm) column (Thermo Scientific, USA). The mobile phase comprised (a) water + 0.1% TFA, and (b) ACN + 0.1% TFA, delivered at a rate of 0.4 mL/min. Isocratic elution was performed to achieve separation, maintaining a mobile phase composition of 55% (a) water + 0.1% TFA and 45% ACN + 0.1% TFA throughout the runtime. Manual injection of 20 μL was performed, with detection wavelength set at 220 nm, and 68Ga radiotracer quantified by HPLC using a sodium iodide (NaI)-based detector.
Synthesis of radiopharmaceuticals
The routine production of 68Ga-labeled radiopharmaceuticals was conducted using the ITG 68Ge/68Ga generator, employing a metal-free modified silica matrix and having an automated cassette-based synthesis module. The synthesis process can be categorized into four main sections: mechanical testing, presynthesis procedures, radiopharmaceutical synthesis, and filter integrity assessment.[9] This generator allows elution with lower molarity HCl, ensuring consistently high labeling yields for various molecular imaging agents.[921] This module automates the elution, formulation, and purification processes of radiopharmaceuticals.
Preparation of reagents
The initial peptide stock, consisting of 500 μg of PSMA-11, was dissolved in 3 mL of ultrapure water. Subsequently, 30 μL aliquots were dispensed into 1 mL polypropylene tubes (Eppendorf tube) under aseptic conditions and stored at − 20°C. On the day of synthesis, one tube containing PSMA-11 peptide was retrieved and allowed to equilibrate to room temperature before use.[9]
Radiopharmaceutical synthesis
The synthesis is initiated on the software platform, with the entire process lasting approximately 17 min. The synthesis encompasses various steps as outlined in a previous paper.[9] A schematic flowchart illustrating these steps can be found in a previous paper.[15]
Quality control of radiopharmaceuticals under high-performance liquid chromatography
In the present study, our primary emphasis focuses on validating the HPLC method for QC purposes. The The analytical method for assessing the chemical and radiochemical purity of 68Ga-PSMA-11 was validated according to the Q2 (R1) ICH guideline. The validation process was assessed through the evaluation of specificity, linearity, accuracy, and precision.[2223] In addition to this, ITLC assessment, pH determination (utilizing pH strips), and verification of radionuclide purity (via examination of principal γ photons) using γ-ray spectrometry[24] was conducted as mentioned in work done by Tayal et al.[9]
Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present. Typically, these might include impurities, degradants, and matrix. The assay’s specificity was evaluated by repeated measurement of the content or potency of the analyte obtained from the final product, 68Ga-PSMA-11 solution with different concentrations of radioactivity.
Linearity
Linearity is the ability of an analytical procedure to produce test results that are directly proportional to the concentration of the analyte within the standard curve range. To cover a range of 68Ga-PSMA-11 concentration values, three sets of calibration standards of radioactivity concentration in (MBq) ranging from 100% to 12.5% were injected into the HPLC systems under the above-specified chromatographic conditions (injection volume was 20 μl) and analyzed for a linearity analysis. Using the peak area of the (68Ga-PSMA-11) analyte, (y) versus the amount (x), the calibration curve was created for evaluating the slope, intercept, and regression coefficient (R2) with the help of linear regression.
Accuracy
The accuracy of the obtained concentrations compared to the known concentration was assessed in this method. The accuracy was expressed by the percentage of recovery (R%), calculated as the ratio of found concentration to actual concentration. The quantitative recovery values that are higher than 95% are an acceptance criterion for expressing high accuracy. For determination of accuracy, we performed three sets of runs with radioactivity concentration in (MBq) ranging from 100% to 12.5% being injected into the HPLC systems under the above specified chromatographic conditions. 68Ga-PSMA-11 being a radionuclide showed the decay inevitably leading to a decrease over time of the radioactivity and recalculating the obtained peak area values with the below-mentioned decay equation:
Equation 2
A0 = corrected peak area; A = measured peak area; and t1/2 = half-life (68Ga = 67.63 min).
Precision
Precision or repeatability was calculated based on the content of 68Ga-PSMA-11, and the statistical parameter of concern evaluated was coefficient of variation (CV%), which is determined using the equation: CV% = s/m × 100, where m is the average of the concentrations and s is the standard deviation. Three different radioactivity concentrations in (MBq) of 68Ga-PSMA-11 ranging from 75% to 25%, (HQC – 75%, MQC – 50%, and LQC – 25%) were prepared and injected into the HPLC system to measure repeatability or intermediate precision using the regression equation with 6 replicates and 12 replicates for each concentration on intraday and interday, respectively. Normalized peak area values after decay correction were compared to ensure consistent statistical analysis.
Results
Quality control results
The QC tests for 68Ga-PSMA-11 are illustrated in Table 1. The QC experiment methodology is described in Table 1.[9]
| QC results 68Ga-PSMA-11 radio-labeled product | |||
|---|---|---|---|
| QC data | Run - 1 | Run - 2 | Run - 3 |
| Appearance | Colorless | Colorless | Colorless |
| Particulate matter | Not observed | Not observed | Not observed |
| Radionuclidic identity (min) | 67.71 | 67.71 | 67.71 |
| Radionuclidic purity (%) | 100.00 | 100.00 | 100.00 |
| Radiochemical identity (min) | 2.10 | 2.11 | 2.10 |
| Radiochemical purity (%) | 99.45 | 99.78 | 99.75 |
| 68Ge impurity (%) | 4.9×10−5 | 4.7×10−5 | 4.8×10−5 |
| pH | 5.5 | 5.5 | 5.5 |
| Yield (%) | 63.34 | 63.71 | 64.01 |
| Activity (MBq) | 463 | 459 | 464 |
| Final volume (mL) | 10 | 10 | 10 |
PSMA 11: Prostate-specific membrane antigen-11, QC: Quality control, 68Ga: Gallium-68
Specificity
The concentration changes did not affect the retention time and show evidence of any interference from another compound (single peak) as depicted in Figure 3a. The average recovery obtained from the accuracy experiment range was 99.99%–100%, while the range of CV came to be 0.766%–2.239%. Therefore, the method was found to have acceptable specificity for the assay of 68Ga-PSMA-11 with high accuracy.
Linearity
The regression analysis of three independent runs of 68Ga-PSMA-11 indicated no deviation with an R2 value to be 0.9983, as shown in Figure 3b. Linearity was ≥0.99% and met acceptance criteria.
Accuracy
The accuracy of the obtained concentrations in comparison to the known concentration was assessed for the procedure. The accuracy range came out to be between 99.99% and 100%, while the range of CV is 0.766%–2.239%, as shown in Figure 3c. The outcome shows good accuracy for varying radioactivity concentration in MBq (100%–12.5%) of 68Ga-PSMA-11.
Precision
The method was considered precise, obtaining coefficients of variation ranging between 0.6306% and 1.316% for intraday and 1.562%–2.472% for the interday precision, as shown in Figure 4a and b. The intraday and interday accuracy range was 99.98%–100% and 99.99%–100%, respectively.
Discussion
Radiotracers directed at the PSMA are employed in clinical settings for PET scans to visualize the disease burden of prostate cancer. FDA approval for 68GA-PSMA came a decade after its discovery in 2010.[24] Despite the ongoing advancements in imaging technology, the progress in developing, standardizing, and validating QC parameters for in-house prepared radiopharmaceuticals has not kept pace.[25] The absence of consensus guidelines in India regarding the validation of radiopharmaceutical QC methods prompted us to develop and utilize an isocratic radio-HPLC method to validate the cassette-based production of 68Ga-PSMA-11 at our institute. The process is straightforward, employing the isocratic method for QC testing of synthesized 68GaPSMA-11. Moreover, the mobile phase used in our study has been previously employed for various PSMA variants by other researchers.[20262728] The optimal amount of starting peptide per production of 68Ga-PSMA-11 was 16 μg at our facility. The peak intensity and retention time observed for both PSMA-11 and 68Ga-PSMA-11 under ultraviolet and radionuclide detectors were relatively similar. Our method not only demonstrated favorable analytical characteristics but was also validated for linearity, accuracy, and precision, yielding results indicative of excellent reproducibility and method robustness. Therefore, it is advisable to adhere to GMPs and implement a validation method using analytical techniques for radiopharmaceutical analysis. This practice instills confidence in a clinical setting when conducting studies with in-house labeled radiotracers, ensuring patient safety and optimal image quality while accurately quantifying radiotracer accumulation for planning therapeutic studies. Furthermore, the integration of automation has significantly enhanced reproducibility while concurrently reducing operator variability.[729] The iTG 68Ge/68Ga generator had a stable production, and it is highly encouraged to have regular elution and at least one elution before preparing the radiopharmaceutical after a gap of few days. Our study concurs with Assadi and Dadgar[30] that 68Ga obtained from a generator system serves as a cost-effective tracer compared to cyclotron-produced alternatives. The radiopharmaceutical production interval of every 3 h is particularly advantageous. Our obtained RCP consistently exceeded 95%, aligning with earlier findings.[831] Previously established validation methods by Migliari et al.[26] and according to Pharmeuropa 2017 involved retention times of 7 min and 16 min, respectively. In our study, we have developed a novel method to expedite this process, achieving a retention time of 2.245 min for rapid validation and release for clinical application[26] under isocratic conditions. There is also HPLC-based method validated assay for 68Ga-PSMA-11 analysis, providing estimation in 7-min retention time and a relatively narrower linearity range.[26] A recently published EP monograph utilized a TFA/water/ACN mixture as a mobile phase with a 16-min runtime (Pharmeuropa, 2017). Hence, our suggested assay demonstrates superiority in assay time, a critical factor during the quality assessment of radiopharmaceuticals.
Conclusion
The current HPLC-based methodology is rapid, with a retention time of 2.24 min, rendering it a favorable analytical standard operating procedure for QC analysis of 68Ga-PSMA-11. In conclusion, our study indicates that this method is easily reproducible and dependable for assessing RCP during cassette-based 68Ga-PSMA-11 production, ensuring the reliability of its results based on isocratic flow parameters of mobile phase.
Limitation
We evaluated the graph only under radio detector of HPLC for the isocratic method. We wanted to have a faster method of isocratic protocol to minimize radioactive decay.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
The authors are thankful to Ms. Ankita Pal, Biostatistician, for her assistance in the manuscript.
Nil.
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