A method to prevail false positive responses due to excess cations and viscous nature of Radiopharmaceuticals in Limulus Amebocyte Lysate Gel Clot test

Objective(s): Bacterial endotoxin test (BET) for detection and quantification of endotoxin in radiopharmaceuticals (RPs), used for therapy or diagnosis, is prerequisite to administration in patients. Out of the two established methods used for this purpose (Kinetic Chromogenic Assay: KCM and Gel Clot Bacterial Endotoxin Test: GC-BET), GC-BET is recommended by pharmacopeias to evaluate the interferences exhibited during the assay due to presence of various ingredients in samples. In the present study, the influence of excess of cations in [177Lu]Lu-DOTATATE, used for Peptide Receptor Radionuclide Therapy (PRRT), were studied and a protocol to negate the enhancement observed was developed. Additionally, a protocol for carrying out GC-BET for extremely viscous [131I]I-Lipiodol was standardized. Methods: GC-BET was performed for [177Lu]Lu-DOTATATE and [131I]I-Lipiodol at maximum valid dilution (MVD), using LRW as a diluent. To negate the false positivity observed in case of [177Lu]Lu-DOTATATE, various concentrations of calcium chloride (CaCl2) were added and evaluated for the reversal of the interference observed initially. To prevail the difficulty in performing GC-BET for [131I]I-Lipiodol various modification in the protocols like orbital vortexing at different rpm and time intervals were performed. KCM assays were also performed for studied RPs at MVD. Results: It was observed that at MVD, [177Lu]Lu-DOTATATE exhibited false positivity in GC-BET. However, all the individual reagents used in labeling of [177Lu]Lu-DOTATATE did not show any false positivity. Finally, performing the assay with an addition of 2mM CaCl2 (final concentration) nullified the false positivity. Further, intricacy in performing GC-BET for [131I]I-Lipiodol due to its viscosity was resolved by orbital vortexing at 3000 rpm for 5 minutes. Conclusions: Our study proved that false positivity was observed in GC-BET for [177Lu]Lu-DOTATATE due to the presence excess M3+ ions. Further, our study is the first of its kind which demonstrated methods for negating these false positive results by using modified protocol and hypothesizing the reason behind the enhancement. Additionally, ours is the first study which proved that a simple step of vortexing the viscous RPs like [131I]I-Lipiodol can resolved the problems encountered during performing GC-BET due to viscosity of RPs.


Introduction
The use of radio-labeled peptides like ([ 177 Lu]Lu-DOTATATE) for PRRT has enhanced effectiveness in cancer treatment because of selective delivery of radioisotope to tumor sites which further reduces toxic side effects to healthy tissues. Additionally, target specific radiolabeled fatty acid (RFA) like [ 131 I]I-Lipiodol 110 Asia Ocean J Nucl Med Biol. 2022; 10(2): [109][110][111][112][113][114][115][116] and [ 177 Lu]Lu-DOTATATE, a radio-lanthanide labeled RP (RL-RP), has been extensively used for treatment of liver metastases and have obtained an approval from European Medicines Agency (EMA) and United States Food and Drug Administration (USFDA) in recent years (1,2). The success of RPs lies on the selection of suitable peptides, fatty acids, chelating cores and their ability to form stable conjugate and further to achieve efficient radio-labeling with various radioisotopes like Lutetium-177, Yttrium-90, Rhenium-188 and Technetium-99m. Most of these target specific therapeutic RPs are approved by USFDA and the EMA for treatment of various cancers in humans and these therapeutic RPs are considered to be in generic form. Hence, it is mandatory to test these radiolabeled formulations for apyrogenicity prior to administration in patients, to ensure that endotoxin levels are within permissible limits.
For RL-RPs, Endotoxin Limit (EL) is 175 EU/V (3,4) whereas for intra-arterial infused RFA, EL is 17.8 EU/mL (5,6). The standard method for assessing the bacterial endotoxin levels in any of these studied RPs and their cold kits is by rabbit pyrogen test as described in United State Pharmacopoeia (USP) chapter 151. However, common limitations with this test are; its labor intensive nature, potential radioactive dose to analyst and problems during administering radioactivity to animals.
GC-BET is the most economical and technically uncomplicated method available for evaluation of bacterial endotoxin in radiopharmaceuticals. However, it is very sensitive towards concentration of divalent calcium ions and the change in concentration during assay, contributed by the samples, lead to interference either in the form inhibition or enhancement. Radiometals like 177 Lu, 90 Y, 188 Re, 225 Ac etc are used for preparation of rdaiopharmaceuticals for therapeutic purpose and these radiometals undergo radiometallation or radiochelation reaction with suitable cyclic / acyclic / bifunctional chelators to which target specific peptides / enzyme inhibitors / monoclonal antibodies are attached. These final radiopharmaceutical preparations contain various monovalent / divalent / trivalent non-radioactive cations which do not interfere with physiological uptake. However, these cations, crucially get involved during enzymatic reaction of GC-BET and compete with Ca 2+ ions, thereby increasing (enhancement) or decreasing (inhibition) the sensitivity of assay. The enhancement observed in the assay results in false positivity, while inhibition leads to false negativity.

Methods
In BET assay, it is mandatory to calculate the Maximum Valid Dilution (MVD) which is the maximum allowable dilution of a sample at which the endotoxin limit can be determined and interfering test conditions can be avoided. MVD = EL/λ (Where EL is Endotoxin Limit of RPs and λ is Sensitivity in EU/mL of LAL reagent used). Considering the EL of studied RPs, MVD is calculated and sensitivity at which assay is to be performed is described in Table 1. Detailed protocol for GC-BET is shown in Table 2, and the assays were performed for all the RPs at MVD. Positive Product Control 2λ † … 50 50 100 Test 3 and 4 were also performed initially for stock solution of CaCl2 solution, saline (0.9% sodium chloride) and also for ultrapure water to ensure absence of endotoxin in stock solutions. The tests, Negative Product Controls (NPC) and Positive Product Controls (PPC) were incubated at 37 o C for 60 minutes in a dry heating block. To demonstrate reproducibility of the results 3 batches of each of RPs were tested. To demonstrate that the usage of CaCl2 does not interfere in the gel-clot formation and also does not reduce or enhance the sensitivity of the test, we have carried out assays with positive water control at different standard endotoxin concentrations with and without calcium chloride. The assay has also been done with negative water control with and without calcium chloride. The assays were repeated 10 times to study reproducibility. For all the studied RPs and active product ingredients (APIs), KCM assays using PTS were performed at the same MVD at which GC-BET were performed. Detailed protocol for the procedure is shown in Table 3.  Table 4. Even at MVD as high as 400, it is unfeasible to perform successful GC-BET for [ 131 I]I-Lipiodol without any modification, due to high viscosity of lipiodol and thick precipitation were observed in NPC test sample. However, this precipitation in NPC can be overcome by carrying out orbital vortexing at 3000 rpm for 5-8 minutes, at first dilution of the test samples. This short orbital vortexing step allows the dispersion of the viscous layer formed during dilution of samples.    Table 7 shows the result obtained for the GC-BET carried out at different concentrations of standard endotoxin from 4λ to λ/4 (λ: sensitivity of lysate used i.e 0.125 EU/mL and 0.03 EU/mL) and it can be seen that the presence of CaCl2 does not interfere in the assay.

GC-BET results for [ 131 I]I-Lipiodol at 400 MVD are depicted in
Further, the experiments gave consistent results over 10 replications indicating the validity and reproducibility of the procedure of reversing the enhancement of gel clot formation after addition of excess CaCl2.  T2  T3  T1  T2  T3  T1  T2  T3  T1  T2  T3  T1  T2

Discussion
The use of peptides, enzyme inhibitor and other molecules, radiolabeled with different radioisotopes of different half-lives and energies, is ever increasing in treatment as well as diagnosis. In view of the spot preparations of RPs, in house endotoxin evaluation before administration to patients, has become mandatory in hospital radiopharmacy unit of the Nuclear Medicine Departments of hospitals. As mentioned earlier the endotoxin in samples can be estimated using GC-BET or KCM. Coagulation cascade reaction in GC-BET is based on presence of natural substrates i.e. proline rich proteins and 8.6 kDa proteins of limulus transglutaminase present in non-granular hemocytes (L granules) of horse shoe crab8 and also on optimum proteolytic activity of transglutaminase enzyme. Optimum proteolytic activity of transglutaminase is dependent on the balanced free divalent cations concentration9, 10,11,12 and overall this concentration in ionic form (Ca 2+ ) has to be maintained for optimized transglutaminase activity (13).
Transglutaminase binds to three Ca 2+ ions at three different sites, which means optimum concentration should be 1.0 Ca 2+ /mol in absence of any other metals. The Ca 2+ ion in site one is constitutively bound with binding affinity of Kd~0.3 µM (ΔH ~ -6.70 kcal/mol) and is required for stabilization of the enzyme (14). Upon proteolysis transglutaminase exothermally acquires two more Ca 2+ ions at site 2 and 3, which activates the transglutaminase by opening a channel which exposes tryptophan-236 (Trp-236) and tryptophan-327 (Trp-327) residues that controls substrate access to the active site (15).
[ 177 Lu]Lu-DOTATATE is used for PRRT for patients with neuroendocrine tumors (NET) and in present study, it was prepared at Centralized Radiopharmacy Centre (CPC) with RCP of ≥95 to 99%, using carrier added [ 177 Lu]LuCl3 as per the standard procedures (7). At CPC when GC-BET was performed at 30 MVD (total production volume is 7-9 ml) along with other QC analysis, the preparation did not show any false positive results .
However  (12), we reported that some of the 99m Tc-labeled radiopharmaceuticals ( 99m Tc-RPs) and their cold kits (TCKs) exhibited inhibition during GC-BET. The PPC test samples ( 99m Tc-DMSA-III, 99m Tc-DMSA-V, 99m Tc-EC, 99m Tc-DTPA and their respect-tive cold kits) did not form any firm clots and false negative results were observed. These false negative results were reversed by adding excess 300 µM Ca 2+ ions (CaCl2 form) in test samples at MVD. We reported that, the free carboxyl groups (-COOH) present in some of the 99m Tc-RPs and their respective TCKs inhibit GC-BET by depleting Ca 2+ ions concentration, thereby inhibiting transglutaminase activity. However, though DOTATATE acetate, was with presence of free -COOH groups, no false negative results in PPC test samples were observed. This may be because the molar concentration for DOTATATE acetate was 0.565 µmoles at final GC-BET, which is very low as compared to TCKs. Thus the molar concentrations of DMSA-III and DMSA-V were 2.74 µmoles at final GC-BET assay which was almost 5 times the molar concentration of DOATATATE acetate. While molar concentrations for EC and DTPA were as high as 3.73 and 29.51 µmoles at final GC-BET .
Highly viscous [ 131 I]I-Lipiodol (EL <17 EU/mL) (6,18) forms two different phases (viscous lipophilic and aqueous hydrophilic phase) on diluting with LRW (MVD -400), thereby making this product unfeasible to perform GC-BET. We used orbital vortexing at 3000 rpm for 5-8 minutes for [ 131 I]I-Lipiodol at first dilution (200) which resulted in uniform dispersion of [ 131 I]I-Lipiodol in LRW allowing successful GC-BET of this product. Use of heating prior to performing GC-BET for RFA is not recommended as heating of diluted [ 131 I]I-Lipiodol at 125 o C though may solubilize highly viscous lipiodol but will volatilize highly radioactive Iodine-131 which is very hazardous. Centrifugation of 131 I-Lipiodol is strictly not recommended for dispersion of RFA as it will lead to separation of hydrophilic and lipophilic phase. BET by KCM using PTS cannot be performed for [ 131 I]I-Lipiodol at 400 MVD, since these highly viscous RFA will not mix thoroughly with lyophilized LAL, synthetic CS and CSE present in channel of PTS test cartridges (PTS-TC) and lead to blockage of channel which has diameter of 2 mm.

Conclusion
The present study is the first one to report that simple 3000 rpm orbital vortexing can solve the problem of performing GC-BET for [ 131 I]I-Lipiodol. Further, this is also the first study to report addition of 2 mM CaCl2 solution for [ 177 Lu]Lu-DOTATATE to overcome the enhanced false positive result due to increased transglutaminase enzymatic activity and formation of 4 to 6 fold stiffer gel. The adaptation of these modified LAL test methods provides a reliable BET quality control testing option for both the RPs at Hospital Radiopharmacy Settings. Thus an expensive assay like KCM-BET can be replaced by these modified GC-BET for the RPs mentioned in our study.