Table 6 Acceptance limits and parameters for the validation of analytical methods (to assure that the analytical methods can properly determine specifications set in Table 2)
From: Optimized, automated and cGMP-compliant synthesis of the HER2 targeting [68Ga]Ga-ABY-025 tracer
HPLC | Specificity | Sample matrix spiked with ABY-025: Area of ABY-025 peak in spiked matrix differ maximum ± 20% and that no peaks ≥ 10% of the area of 50 µg/mL ABY-025 in the range 6–10 min in UV Sample matrix spiked with 68Ga: 68Ga should elute only in the void peak in the RD Difference in Rt between [68Ga]Ga-ABY-025 (radio chromatogram) and Rt ABY-025 (UV-chromatogram of SST) ≤ 60 s |
Linearity of chemical purity for ABY-025 | Peak area in at least 5 levels in the range 30–100 µg/mL. Linear regression should give a r2 ≥ 0.99 Carry over: The ABY-025 peak in blank after the 100 µg/mL sample should not exceed 20% of peak in previous injection | |
Linearity of radiochemical purity for [68Ga]Ga-ABY-025 | Linearity is tested for samples from 10 MBq/mL to 150% of normal product activity (75 MBq/mL) Linear range for the method are defined as the activity range where %ROI [68Ga]Ga-ABY-025 differs ≤ 0.5%ROI for the average (of samples within range) | |
Repeatability | The repeatability is tested in the range 30–100 µg/mL ABY-025. The relative response, as compared with the SST should show an RSD% ≤ 10% on each concentration level (3 injections) and for all injections (9 injections) within the range 30–100 µg/mL (3 concentrations / 3 replicates). Relative response = (Area/Conc.)Sample / (Area/Conc.)SST/Ref | |
Intermediate precision | On 3 occasions 3 triplicates on 3 concentrations are performed. Different days or analysts, at least two columns from different lots. The relative response, as compared with the SST should show an RSD% ≤ 10% on each concentration level (9 injections) and for each analysist (9 injections) | |
Accuracy | Accuracy can be inferred once precision, linearity and specificity has been established. Accuracy should be shown in the range 30–100 µg/mL | |
GC | Linearity | R2 ≥ 0.99; Slope, y-intercept and residual sum of square must be given. A minimum of 5 concentrations must be used LOD and LOD: Calculation based the standard deviation of the response and the slope (based on calibration curve) |
Repeatability | RSD% ≤ 10% for a minimum of 9 determinations over 3 concentrations within the range (e.g. 3 concentrations /3 replicates from Accuracy) | |
Intermediate precision | RSD% ≤ 10%; for a minimum of 9 determinations over 3 concentrations within the range (e.g. 3 concentrations/3 replicates from Accuracy) performed a different day or by a different analyst | |
Accuracy | Diff% ≤ 10% between calculated and actual concentrations for 3 concentrations within the range using a minimum of 3 replicate determinations from each concentration | |
TLC | Specificity | i.e. its ability to determine free 68Ga and/or unidentified radioactive impurities. The following criteria must be met: • The peak corresponding to free 68Ga and/or unidentified radioactive impurities must be completely separated from the peak corresponding to [68Ga]Ga-ABY-025 • The radioactivity of free 68Ga and/or unidentified radioactive impurities must be ≤ 3% of the total radioactivity to be released • Retardation factors (Rf) ~ 0–0.1 for free 68Ga and/or unidentified radioactive impurities Rf ~ 0.2–0.6 and Rf > 0.6 for [68Ga]Ga-ABY-025 |
Endotoxins | Optimal dilution factor | Interference screen on worst case simulation of [68Ga]Ga-ABY-025 formulation using maximum allowed ethanol concentration The optimal dilution factor is the one that yields a spike recovery close 100% (related to the known endotoxin level in the cartridge) |
Sterility | MST | MST at the external contractor according to their certified protocol |