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Abstract Background: Risankizumab (RISA) is a humanized IgG1 kappa therapeutic monoclonal antibody (tmAb) that targets interleukin 23 (IL23). RISA is used to treat patients with moderate to severe plaque psoriasis or those with Crohn’s disease. Therapeutic drug monitoring is used to identify loss of response to therapy. A novel lab developed test (LDT) was developed and validated utilizing IL23 coupled to magnetic beads to enrich RISA from patients’ serum. Samples were analyzed using an Agilent Infinity II LC in front of a Sciex 7600 ZenoTOF mass spectrometer.
Methods:
IL23 bead coupling: Approximately 25 mL of 10 mg/mL DynabeadsTM M-280 Streptavidin (ThermoFisher Scientific; Waltham MA) were added to a 50 mL conical tube. Beads were washed with 12.5 mL 1x PBS + 5 mg/mL BSA and mixed on an Eppendorf Thermomixer for 1 minute at room temperature (RT) at 1000 RPM. A DynaMagTM-50 Magnet was used to collect the magnetic beads against the tube wall and allow for the removal of the supernatant. The coupling mixture consisting of 25 mL 1x PBS + 5 mg/mL BSA and 500 µg IL23 protein (Sino Biological; Wayne, PA) was added to the beads and mixed for 30 minutes at RT at 1000 RPM. The beads were washed three times with 25 mL 1x PBS. The beads were then resuspended in 25 mL 1x PBS to give a final concentration of 20 µg/mL coupled protein.
Sample enrichment: Samples were prepared by adding 100 µL IL23 coupled bead slurry, 150 µL 1x PBS, 20 µL unknown/standard/QC, and 30 µL of 2.5 µg/mL stable isotopically labeled (SIL)-RISA (Promise Proteomics; Grenoble, France) to a 96-well filter plate (Sigma-Aldrich; St. Louis, MO). Samples were mixed at RT for two hours at 1250 RPM. The sample wells were then washed twice with 150 µL 1x PBS and twice with 150 µL HPLC water, filtering via positive pressure after each wash. Elution was performed by adding 50 µL 5% acetic acid and mixing at RT for 15 minutes at 1250 RPM. The eluent was then pushed to a 96-well PCR plate after attaching the PCR plate underneath the filter plate and centrifuging for 3 minutes at 3000 RPM. The eluent was reduced with 25 µL 100 mM DTT in 1 M ammonium bicarbonate. The plate was mixed at 55°C for 45 minutes at 800 RPM.
LC-MS: The enriched and reduced samples were analyzed using an Agilent 1290 Infinity II LC connected to an AB Sciex 7600 ZenoTOF mass spectrometer. A volume of 20 µL was injected onto an Agilent Poroshell 300SB C3, 2.1x75mm column with 5 µm particles heated to 60°C, running a 7-minute gradient from 27 to 40% B at a flow rate of 400 µL/min. Mobile phases consisted of (A) 1% formic acid in water and (B) 90% acetonitrile, 10% isopropyl alcohol, and 0.1% formic acid. Sciex OS was used for data analysis. The +11, +12, and +13 charge states for the RISA light chain were combined to give the XIC used for quantitation. A calibration curve from 1 to 100 µg/mL using the Hill Equation and 1/x2 weighting was obtained from standards prepared by spiking RISA in normal human serum.
Development: During development, optimization experiments were performed to minimize the amount of IL23 coupled beads that would be needed per sample well. This was done to try and make the assay cost effective while also ensuring there was sufficient IL23 coupled beads to avoid saturation when enriching the RISA in patient serum samples and the spiked internal standard. The final coupled bead slurry of 100 µL consists of 1 mg beads coupled to 2 µg IL23. Using 100 µL was proven to be able to enrich the quantity of RISA in the highest standard (2 µg) and the internal standard (0.075 µg) while having the RISA area counts be linear across the analytical measuring range.
Results: Intra-assay and inter-assay precision was analyzed at four levels; 1, 5, 30, and 70 µg/mL. Intra-assay precision, N = 20 replicates, had coefficients of variation of 5.7%, 4.9%, 4.7%, and 4.7% respectively. Inter-assay precision was measured over twenty analytical runs and had coefficients of variation of 8.5%, 7.8%, 9.6%, and 5.2% respectively. The assay was proven to be linear from 1 to 100 µg/mL by re-extracting standards of 1, 5, 10, 25, 50, 75, and 100 µg/mL as unknowns. Carryover was assessed over twenty analytical runs by dividing the area counts of a matrix blank extracted after the highest standard (100 µg/mL) and the area counts of the low standard (1 µg/mL) to get the %LLOQ (lower limit of quantitation) area. The average %LLOQ of the carry over blank across twenty analytical runs was 9% with a range of 1-30%, which was below our acceptance criteria of <50% of the LLOQ. The LOD (limit of detection) was determined to be approximately 0.17 µg/mL, which is 17% of the LLOQ of 1 µg/mL. Residual samples, N=37, for patients being treated with RISA were analyzed during validation. Using patient chart review it was determined that N=4 patients that had a RISA quantitation below the LLOQ (limit of quantitation) had not received a RISA dose before their blood was drawn. Patients being treated for Crohn’s Disease, N=21, had an average RISA concentration of 25.1 µg/mL, while patients treated for Psoriasis, N=12, had an average RISA concentration of 6.4 µg/mL. These values coincided with the RISA drug package insert.
Conclusion: We have successfully developed and validated a new LDT for RISA therapeutic drug monitoring which can be used to monitor potential loss of response to therapy. This quantitative assay will soon be paired with an ADA (anti-drug antibody) assay and will be offered as a panel to add more value to patients receiving RISA therapy.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
