Purpose: Monoclonal antibodies (mAbs), antibody fragments, and other biologics have increasingly been utilized for cancer treatment and other therapeutic interventions. Having alternative platforms to ligand binding assays such as liquid chromatography tandem mass spectrometry (LC-MS/MS) is necessary to obtain accurate and reliable methods to analyze these proteins in biological fluids. The overall purpose of this study was to optimize methods to evaluate bevacizumab (Avastin) and ranibizumab (Lucentis) in human plasma following intravitreal administration. Significantly lower quantification limits will be required for evaluation of safety following this administration, and to date, no studies have been published on bioanalytical method development of ranibizumab or bevacizumab in human plasma after intravitreal administration. Our goal is to validate individual selective methods for both ranibizumab and bevacizumab to reach clinically relevant levels by an optimal workflow of immunocapture, internal standardization, digestion, and signature peptide monitoring on two LC-MS/MS platforms, followed by comparison with a traditional LBA method.
Methods: In silico digestions were initially performed using Skyline and Blast to determine the signature peptides for Bevacizumab and Ranibizumab. Confirmation of the signature peptides and selection of parallel reaction monitoring (PRM) transitions were optimized using a Thermo Q-Exactive Plus hybrid quadrupole Orbitrap, and evaluated with samples prepared with either buffer solution or human plasma. Calibration curve and evaluation of the limit of detection were conducted throughout the optimization process. Optimization was evaluated by comparing component efficiency via analyte response. Parameters of the following general procedure were evaluated and optimized: immunocapture (using either VEGF-A(165) or HCA-182 (anti-bevacizumab fab), comparison in Figure 1), followed by denaturation, reduction, alkylation, and digestion using trypsin. Multiple volumes were evaluated regarding sample aliquot size. Optimized conditions are as follows: 250 μL sample aliquot is incubated with 25 μL of 10 μg/mL HCA-182 capture reagent (and 20 μL Strep-Tactin magnetic beads) for 2 hour or overnight, followed by denaturation / reduction at 90 °C for 15 minutes with 25 μL Rapigest and 10 μL 100mM dithiothreitol, followed by alkylation using 100mM iodoacetamide (in yellow room) for 30 minutes at room temperature; then samples are digested using 10 μL 0.250 mg/mL trypsin incubated for 1.5 hours at 37 °C. After digestion is terminated using 2N HCl, the SIL-peptide is added (20 μL 10 ng/mL VLIY-IS). The unique signature peptides that were used to represent bevacizumab, ranibizumab, and internal standards were then separated using 2D chromatography (to reduce background interference) on a UPLC reverse phase guard column (C4) and analytical column (C18), and analyzed using electrospray ionization on two instruments, 1) a triple quadrupole mass spectrometer (Sciex Triple Quad 6500) and 2) a hybrid quadrupole Orbitrap mass spectrometer (Thermo Q-Exactive Plus). Multiple reaction monitoring (MRM) (QqQ) or PRM (Q-Orbitrap) modes were employed. Instrumental conditions were optimized. In addition, VEGF-A(165) interference was assessed by evaluating the need for a dissociation step, and by evaluating control samples compared to samples in the presence of VEGF-A(165).
Results: Three unique signature peptides that are conserved between bevacizumab and ranibizumab were selected for quantitation (VLIYFTSSLHSGVPSR, FTFSLDTSK, STAYLQMNSLR). An additional tryptic peptide that is unique to ranibizumab only (LSCAASGYDFTHYGMNWVR) was also selected. MRM and PRM transitions were selected based on intensity. Students t-test was used to evaluate results. This method was validated with a linear range from 0.300 to 100 ng/mL in human plasma, with a correlation coefficient (R) greater than 0.9900. Results from intra- and inter-assay precision and accuracy were acceptable, between -15.7% to 16.8%. Selectivity was evaluated in both unfortified and fortified plasma lots (6 normal lots; 6 disease state lots); and was found to be acceptable. Stability testing (bench-top, frozen matrix, extract, freeze/thaw, whole blood) was all found to be acceptable, with accuracy and precision results between -6.95% to 6.20%. Matrix Factor and Recovery were evaluated, and all results were consistent at all levels and lots tested. All ancillary testing (hemolysis, lipemia, cross analyte interference, reinjection reproducibility, capture capacity, dilutional linearity) was found to be acceptable, with accuracy and precision results between -18.8% to 11.0%. As shown in Figure 2 below, VEGF-A(165) did not interfere with quantitative analysis of ranibizumab and bevacizumab, using the validated method. Samples were analyzed on both a Q-Exactive Plus and a Sciex Triple Quad 6500 (QqQ) (using a similar LC set up for both). Figure 3 below shows typical chromatograms.
Conclusion: Following optimization, this method was validated according to the May 2018 FDA Bioanalytical Method Validation guidance. Results were comparable on the Q-Exactive Plus and Sciex 6500, based on cross validation data (similar accuracy and precision results for all experiments tested). This method is now available to quantitatively access plasma drug concentrations in wet AMD patient samples. Comparison to a validated LBA method is currently in progress, using patient samples. Future research includes method development for a procedure quantitatively analyzing intact ranibizumab in vitreous fluid following intravitreal administration, on the Q-Exactive Plus platform.
Catherine Del Guidice– Ph.D. Student, Virginia Commonwealth University, Richmond, Virginia
Omnia Ismaiel– Senior Research Scientist, PPD Laboratories, Richmond, Virginia
Matthew Halquist– Assistant Professor, Virginia Commonwealth University, Richmond, Virginia
William Mylott– Associate Director, PPD Laboratories, Richmond, Virginia