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Abstract Introduction:
Monoclonal antibodies (mAbs) are increasingly being used in combination with other mAbs for cancer and COVID-19 treatments. In breast cancer treatment, trastuzumab and pertuzumab are commonly co-administered to target unique human epidermal growth factor receptor 2 (HER2) epitopes. With the increasing application of mAb co-administration, simple and robust quantification methods are necessary to meet the needs of pharmacokinetic (PK) studies.
Conventional analytical methods involving ligand-binding assays, such as enzyme-linked immunosorbent assays (ELISAs), are employed during PK evaluations due to high sensitivity and sample throughput. However, multiplexing can be a significant challenge for such assays with limited selectivity. In addition to selectivity, sensitivity is also a critical factor because PK studies are often challenged by low sample volumes. Therefore, LC-MS/MS based techniques are being increasingly adopted for PK measurements of combination mAb therapeutics given their enhanced specificity, selectivity and sensitivity. In this study, an immunoaffinity purification workflow with on-bead digestion was employed to simultaneously quantify trastuzumab and pertuzumab in human serum on the ZenoTOF 7600 system.
Methods:
A 3 mg/mL stock solution of trastuzumab was prepared by dissolving 1 mg of lyophilized trastuzumab in 333 µL of phosphate buffered saline (PBS) with a pH of 7.4. A 3 mg/mL stock solution of pertuzumab was prepared by adding 300 µL of PBS to a 30 mg/mL stock solution. Subsequent dilutions were performed in normal human serum to prepare spiked serum samples ranging from 0.03 µg/mL to 300 µg/mL. A 500 µg/mL stock solution of SILuMAB was prepared by dissolving 0.1 mg of lyophilized SILuMAB in 0.2 mL of PBS. The stock was diluted 250:1 (v/v) in PBS to prepare a final solution at a concentration of 2 µg/mL. Protein A beads were diluted 8:1 with PBS and washed three times with PBS before use.
Each sample contained a 10 µL sample of normal human serum, 20 µL of 2 µg/mL SILuMAB, 200 µL of diluted protein A beads and 200 µL of PBS. After samples were shaken for 30 minutes at room temperature, the beads were washed twice with PBS. The beads were resuspended in 150 µL of digestion buffer containing 150 mM ammonium carbonate and 1 mM calcium chloride, and they were denatured at 95°C for 5 minutes. After allowing it to cool to room temperature, 2 µg of trypsin/lys-c was added to each sample and on-bead digestion was performed for 2 hours at 50°C. Digestion was stopped by adding 3 µL of formic acid. Samples were separated from the beads and placed in vials or plates for LC-MS/MS analysis.
Chromatography was performed on a Shimadzu LC-40 X3 system using a Phenomenex Biozen XBC18 (2.1 × 100 mm, 2.6 µm, 100 Å) column. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The operating flow rate was 0.5 mL/minute. Column temperature was set at 40°C. A 20 µL sample was injected for analysis.
Results:
Trastuzumab and pertuzumab were selected as model mAb therapeutics to evaluate the quantification of combination mAbs in human serum on the ZenoTOF 7600 system. The calibration curve included concentrations ranging from 0.15 µg/mL to 300 µg/mL for trastuzumab and pertuzumab. The concentration of the internal standard (IS), SILuMAB, was 0.25 µg/mL. Each calibration point was measured in three replicate injections.
At the level of the LLOQ, the %CV was required to be <20% with accuracy within ±20% of the nominal concentration. For the remaining concentrations, the %CV was required to be <15%, and the accuracy was required to be within ±15% of the nominal concentration.
An LLOQ of 0.15 µg/mL was achieved for trastuzumab and pertuzumab. Matrix interferences in the blank were more than 5x lower than the peak areas in the LLOQ. The overall %CV was <10.1% for trastuzumab and <8.12% for pertuzumab. The overall accuracy was within ±8% of the nominal concentration for trastuzumab. While for pertuzumab, the overall accuracy was within ±12% of the nominal concentration. Strong linearity was achieved with correlation coefficients (r) of >0.996 for both trastuzumab and pertuzumab.
Conclusions:
A highly sensitive method for the quantification of trastuzumab and pertuzumab in human serum using the ZenoTOF 7600 system and on-bead digestion was demonstrated. Using this approach LLOQs of 0.15 µg/mL were reached for both trastuzumab and pertuzumab in human serum due to the improved MS/MS sampling efficiency offered by the Zeno trap.
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= Discovery stage. (24.37%, 2023)
= Translation stage. (39.50%, 2023)
= Clinically available. (36.13%, 2023)
