2016 Volume 39 Issue 7 Pages 1187-1194
Presently, monoclonal antibodies (mAbs) therapeutics have big global sales and are starting to receive competition from biosimilars. We previously reported that the nano-surface and molecular-orientation limited (nSMOL) proteolysis which is optimal method for bioanalysis of antibody drugs in plasma. The nSMOL is a Fab-selective limited proteolysis, which utilize the difference of protease nanoparticle diameter (200 nm) and antibody resin pore diameter (100 nm). In this report, we have demonstrated that the full validation for chimeric antibody Rituximab bioanalysis in human plasma using nSMOL proteolysis. The immunoglobulin fraction was collected using Protein A resin from plasma, which was then followed by the nSMOL proteolysis using the FG nanoparticle-immobilized trypsin under a nondenaturing condition at 50°C for 6 h. After removal of resin and nanoparticles, Rituximab signature peptides (GLEWIGAIYPGNGDTSYNQK, ASGYTFTSYNMHWVK, and FSGSGSGTSYSLTISR) including complementarity-determining region (CDR) and internal standard P14R were simultaneously quantified by multiple reaction monitoring (MRM). This quantification of Rituximab using nSMOL proteolysis showed lower limit of quantification (LLOQ) of 0.586 µg/mL and linearity of 0.586 to 300 µg/mL. The intra- and inter-assay precision of LLOQ, low quality control (LQC), middle quality control (MQC), and high quality control (HQC) was 5.45–12.9% and 11.8, 5.77–8.84% and 9.22, 2.58–6.39 and 6.48%, and 2.69–7.29 and 4.77%, respectively. These results indicate that nSMOL can be applied to clinical pharmacokinetics study of Rituximab, based on the precise analysis.
In recent years, therapeutic monoclonal antibodies (mAbs) are featured for treatment of cancer, autoimmune diseases, and infectious diseases. Rituximab was the first therapeutic monoclonal antibody approved by the U.S. Food and Drug Administration (FDA) in November 1997 and by the European Medicines Agency (EMA) in June 1998. Rituximab is the top-selling cancer drug in the U.S. and had U.S. sales of about $3.59 billion in 2013. The patents on Rituximab will expire in the U.S. in September 2016 and expired in Europe in February 2013. This background is perhaps driving development in the many companies that are working on biosimilars of Rituximab. Some of the Rituximab biosimilars or in development have already proceeded, and the sum of developing biosimilars is number one worldwide in terms of biosimilar antibody drugs.
Rituximab, a chimeric murine/human monoclonal antibody against CD20, approved for the treatment of B-cell non-Hodgkin lymphoma and rheumatoid arthritis.1,2) Rituximab destroys CD20-overexpressed B cells and is therefore used to treat diseases that are characterized by excessive number of B cells. Moreover, Rituximab induces complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and direct cellular effects that lead to apoptosis. As observed with other antibodies, the many side effects of Rituximab are reported. Furthermore, treatment of Rituximab is effective in more than 50% of patients with relapsed or refractory CD20-positive follicular non-Hodgkin’s lymphoma, however this therapy is not curative treatment. Accordingly, binding of Rituximab to CD20 is not adequate to kill all of lymphoma cells, indicating that there are some unexplained mechanisms of resistance.3) To address these issues, treatment of Rituximab have to promote by individualized medicine. Hence, the credible methodologies for measurement of Rituximab in plasma are crucial for the assessment of exposure–response relationships in support of efficacy and safety evaluations, and significant dose selection by chemotherapeutic clinicians.
For quantification of therapeutic mAbs in biological fluids, classical ligand binding assays such as enzyme-linked immunosorbent assay (ELISA) is the most widely used method. However, in some cases, an immunological-based assay is not the most versatile method for quantifying mAbs. For example, therapeutic mAbs can become analogs of endogenous immunoglobulin Gs (IgGs) in plasma with a minor change to their amino acid or nucleotide sequence by splicing valiant, the standard ELISA is unable to discriminate between endogenous and exogenous variants. Moreover, specificity, accuracy, and reproducibility of the ligand binding assays are inhibited by the presence of anti-mAbs.4,5) In these cases, mass spectrometry-based methodologies are available for quantifying the mAbs accurately, which offer superior selectivity over an immunoassay, and the method development is significantly short time and inexpensive. LC/MS is one of the most widely used method in pharmaceutical studies. Recently, tandem LC/MS (LC/MS/MS) has been applied to mAbs as a surrogate to ELISA for the bioanalysis of preclinical samples.6,7) Although LC/MS/MS provides high sensitivity and high specificity for quantifying target analytes in complicated biological matrices, the associated severe matrix effects result in disturbance of precise quantification. Furthermore, the selection of quantitative peptides for mAbs LC/MS/MS bioanalysis should be limited to the variable regions of the Fab fragments, to escape any interference from endogenous human immunoglobulins found in plasma. We developed a novel strategy for decreasing contaminant from various biological matrix using the nano-surface and molecular-orientation limited (nSMOL) proteolysis.8–10) The nSMOL method is designed as solid–solid proteolysis for Fab-selective limited proteolysis. This developed proteolysis has made it possible not only to minimize sample complexity, but also to collect only the mAbs signature peptides including complementarity-determining region (CDR), not existing endogenously in the biological matrix of interest. Hence, LC/MS/MS coupled with the nSMOL proteolysis is considered as an optimal method for mAbs bioanalysis by directly targeting CDR peptide quantitation. In this article, we have succeeded in the validation of Rituximab bioanalysis using nSMOL coupled with LC/MS/MS.
Trypsin-immoblized glycidyl methacrylate (GMA)-coated nano-ferrite particle FG beads with surface activation by N-hydroxysuccinimide (NHS) group was purchased from Tamagawa Seiki (Nagano, Japan). Toyopearl AF-rProtein A HC-650F resin was from Tosoh (Tokyo, Japan). Rituximab was obtained from Chugai Pharmaceutical (Tokyo, Japan). Individual male and female control human plasma ethylenediaminetetraacetic acid dipotassium salt (EDTA-2K) treated was from Kohjin Bio (Saitama, Japan). Trypsin gold was from Promega (Fitchburg, WI, U.S.A.). n-Octyl-β-D-thioglucopyranoside (OTG) was from Dojindo Laboratories (Kumamoto, Japan). P14R (proline repeats and C-terminal arginine) internal standard synthetic peptide was from Sigma-Aldrich (St. Louis, MO, U.S.A.). Ultrafree-MC GV centrifugal 0.22 µm filter was from Merck Millipore (Billerica, MA, U.S.A.). Other reagents, buffers, and solvents were from Sigma-Aldrich and Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Sequence Identification of Rituximab PeptidesFor general tryptic digestion, Rituximab (20 µg) was digested using trypsin gold (1 µg) in 150 µL of 25 mM Tris–HCl buffer (pH 8.0) at 37°C for 16 h. Trypsin reaction was quenched with additional 10% formic acid solution at a final concentration of 0.5%. For nSMOL reactions, 20 µg of Rituximab was collected with 25 µL of AF-rProtein A resin 50% slurry in 90 µL phosphate buffered saline (PBS) containing OTG with gentle vortexing at 25°C for 15 min. Protein A resin was harvested on an Ultrafree filter, then washed with 300 µL of PBS including OTG and 300 µL of PBS in twice by centrifugation (5000×g for 1 min), and finally substituted with 75 µL of 25 mM Tris–HCl (pH 8.0). nSMOL proteolysis was carried out using 1 µg of trypsin on FG-beads with gentle vortexing at 37°C for 16 h under saturated vapor atmosphere. Proteolysis reaction was stopped by adding 10% formic acid at a final concentration of 0.5%. The peptide solution was collected by centrifugation (5000×g for 1 min) to remove Protein A resin and trypsin FG-beads. Structure of tryptic peptides from Rituximab were analyzed by high-resolution liquid chromatography-linear ion trap time-of-flight MS (Nexera X2 ultra high performance liquid chromatograph and LC/MS-IT-TOF, Shimadzu, Kyoto, Japan), and fragment ions were assigned using an in-house Mascot Server and Distiller with Rituximab amino acid sequence information (Matrix Science, London, U.K.). The LC/MS conditions were as follows: solvent A, 0.1% aqueous formic acid; solvent B, acetonitrile with 0.1% formic acid; column, L-column2 ODS, 2.1×150 mm, 2 µm, 10 nm pore (Chemicals Evaluation and Research Institute, Tokyo, Japan); column temperature, 40°C; flow rate, 0.2 mL/min; gradient program, 0–5 min: %B=3, 5–35 min: %B=3–30 gradient, 35–46 min: %B=95, 46–55 min: %B=3. MS and MS/MS spectra were obtained using desolvation line and heat block at 250 and 400°C, respectively. Nebulizer nitrogen gas flows were set to 3 L/min. Drying gas pressure was 100 kPa. Ion accumulation time was 30 ms for MS, and 70 ms for MS/MS analysis. MS/MS analysis was performed using the automated data dependent mode. Ar pulse time into the ion trap cell was 125 µs. The electrode of collision-induced dissociation (CID) cell was set at −1.5 V.
Prediction of Rituximab Signature PeptidesAmino acid sequences of mAb drugs were obtained from Kyoto Encyclopedia of Genes and Genomes (KEGG). Multiple alignment analysis was performed using the amino acid sequence of Rituximab (KEGG DRUG entry D02994), Cetuximab (D03455), and Infliximab (D02598) by ClustalW algorithm on the GENETYX software (GENETYX, Tokyo, Japan). In this analysis, theoretical tryptic peptides containing the CDR sequence, amino acid substitution, positions of conserved cysteine residue, and insertion or deletion sequences were aligned.
Quantitative LC/MS/MS for Rituximab PeptidesThe peptide quantification was performed using an LC-electrospray ionization-MS (LC-ESI-MS) with triple quadrupole (Nexera X2 and LC/MS-8050, Shimadzu). Chromatographic separation was achieved on a Shim-pack GISS C18, 2.1×50 mm, 1.9 µm, 20 nm pore (Shimadzu). The column temperature was set at 50°C. Mobile phase A consisted of 0.1% aqueous formic acid and mobile phase B consisted of acetonitrile with 0.1% formic acid. The gradient cycle was as follows: flow rate, 0.4 mL/min; gradient program, 0–1.5 min: %B=1, 1.5–5 min: %B=1–21 gradient, 5–5.8 min: %B=95 with flow rate 1 mL/min, 5.8–6.2 min: %B=1 with flow rate 1 mL/min, and 6.2–7 min: %B=1. MS was operated in electrospray positive mode with the ESI probe temperature, desolvation line, and heat block at 350, 200, and 400°C, respectively. Nebulizer, heating, and drying nitrogen gas flows were set to 3, 15, and 5 L/min, respectively. Interface voltage was set at 1.5 kV. The dwell time was set to 10 ms for each transition. Multiple reaction monitoring (MRM) monitor ions of peptide fragments were imported from the measured values of structure-assigned fragments by high-resolution LC/MS analysis. CID Ar partial pressure in the Q2 cell was set to 270 kPa. The electrode voltage of Q1 pre bias, collision cell Q2, Q3 pre bias, and parent and fragment ion m/z were examined optimization support software (LabSolutions LCMS software, Shimadzu). For MRM transition, one fragment ion of b- or y-series selected for quantitation, and two ions selected for structural confirmation, according to the optimized MRM ion yield are described in Table 1. We have checked the peak and retention time using the standard antibody peptides by nSMOL preparation. And peptide ion area is detected by microchannel plate. We used the peak area ratio by internal standard (P14R). For optimization of MRM condition, the electrode voltage of Q1 pre bias, collision cell Q2, Q3 pre bias, and parent and fragment ion m/z were examined optimization support software by a maximal peptide ion yield at individual electrode parameters and m/z values.
| Selected peptide | Region | Optimal MRM condition | Role | |||
|---|---|---|---|---|---|---|
| Transition mass filter [m/z] | Q1 [V] | Collision [V] | Q3 [V] | |||
| GLEWIGAIYPGNGDTSYNQK | CDR2 of H-chain | 1092.1→1180.6 (y11+) | −32 | −35 | −46 | Quantitation |
| 1092.1→1343.6 (y12+) | −32 | −33 | −30 | Structure | ||
| 1092.1→840.4 (b8+) | −32 | −33 | −24 | Structure | ||
The parameters are defined as follows: Selected peptide; peptide sequence for Rituximab quantitation, Region; region of selected peptide, Transition mass filter; fragment ion m/z for quantitation from the parent ion m/z, Q1 [V]; voltage condition of the quadrupole cell Q1, Collision; electrode voltage of collision cell Q2, Q3 [V]; voltage condition of the quadrupole cell Q3, Role; purpose of each ion m/z.
In the current study, we performed a bioanalytical validation of Rituximab in plasma using the nSMOL method as described in our previous report with a minor improvement.8–10) The nSMOL proteolysis coupled with LC/MS/MS method was validated in accordance with the Guideline on Bioanalytical Method Validation in Pharmaceutical Development from Notification 0711-1 of the Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, the Ministry of Health, Labour and Welfare, dated July 11, 2013. Briefly, all validation sample sets were prepared and stored at −20 or −80°C for 24 h or longer before each validation assay. Ten microliter of Rituximab-spiked human plasma samples were diluted 10-fold in PBS (pH 7.4) containing 0.1% OTG. The immunoglobulin (Ig) fraction was collected with 25 µL of PBS-substituted AF-rProtein A resin (50% slurry) in 90 µL of PBS containing OTG with gentle vortexing at 25°C for 15 min. Protein A resin was harvested onto an Ultrafree filter and washed twice with 300 µL of PBS containing OTG and then with 300 µL of PBS, centrifuged (5000×g for 1 min), and substituted with 75 µL of 25 mM Tris–HCl (pH 8.0) containing 10 fmol/µL P14R. nSMOL proteolysis was carried out using 10 µg trypsin gold on FG-beads with gentle vortexing at 50°C for 6 h under saturated vapor atmosphere. nSMOL proteolysis reaction was stopped by adding 10% formic acid at a final concentration of 0.5%. The peptide solution was collected by centrifugation (5000×g for 1 min) with to remove Protein A resin and trypsin FG-beads. These analytes were transferred into low protein binding polypropylene vials, and then performed LC/MS analysis with 15 µL injection of each samples. Calibration standard concentrations of Rituximab in plasma samples were set from 0.586 to 300 µg/mL with two-fold serially dilution for 10 calibration samples. QC concentrations were set 0.586, 1.76, 14.1, and 240 µg/mL, as lower limit of quantification (LLOQ), low quality control (LQC), middle quality control (MQC), and high quality control (HQC), respectively.
The advantage of Rituximab quantification using nSMOL proteolysis was compared with other LC/MS bioanalysis called pellet digestion in Fig. S1. Calibration curve in human plasma bioanalysis using nSMOL showed significant improvement of LLOQ and ion intensity. This observation could be achieved in the minimizing sample complexity by the successful Fab-selective proteolysis by nSMOL proteolysis. Using pellet digestion method, dynamic range was decreased because of the excess peptides caused ionization suppression in LC/MS ion path compared with nSMOL chemistry.
Selection of Rituximab Signature Peptides by LC/MS-IT-TOF MS and ClustalW AnalysisFor bioanalysis of mAbs in human plasma, the selection of signature peptides should be selected to the variable regions containing CDRs, to avoid any interference from endogenous human plasma protein. The identification of tryptic Rituximab peptides by LC/MS-IT-TOF MS and Mascot analysis showed that five tryptic peptides were identified using the nSMOL proteolysis, and three peptides were from CDR containing peptides in heavy- or lignt-chain (Figs. 1a, b). It was revealed that the nSMOL proteolytic sites were mainly limited the flexible and unfolding region on CDRs. Furthermore, Rituximab specific peptides were selected with some criteria for accurate quantification like our prvious report of Trastuzumab,9) Bevacizumab,10) and Cetuximab (in submission) bioanalysis. Finally, we have selected four candidate signature peptides, GLEWIGAIYPGNGDTSYNQK, ASGYTFTSYNMHWVK, and FSGSGSGTSYSLTISR of the CDR containing region and QVQLQQPGAELVKPGASVK of heavy-chain N-terminal for Rituximab quantification.
Amino acid sequence of three mAbs amino acid sequences of Rituximab, Cetuximab, and Infiximab were aligned in (a) heavy chain and (b) light chain. Black area shows the matched sequence, as common frameworks, and gray area is to highlight similar amino acids. The red underlines showed the observed signature peptides in Rituximab. The green one showed the observed common peptide in IgGs.
The analytical interference of four selected Rituximab peptides in plasma was examined by the nSMOL proteolysis coupled with LC/MS/MS. To evaluate potential interference of Rituximab peptides from human plasma, each candidate peptide was analyzed using 200, 250, and 300 µg/mL of each sample with two-fold serial dilution in plasma. The heavy-chain peptide GLEWIGAIYPGNGDTSYNQK was position of CDR2 position and determined as the specific Rituximab peptide. The signature peptide showed no interference from human plasma and a good correlation with Rituximab concentrations. The optimized MRM transition of GLEWIGAIYPGNGDTSYNQK for quantitation and structure confirmation were shown in Table 1. Following assay validation, we used molecule’s unique peptide GLEWIGAIYPGNGDTSYNQK.
Results of Full ValidationSelectivityAgainst six individual human plasma from three males and three females, any interference peaks in the MRM chromatogram were not detected for the internal standard. On the other hand, minimal response was observed from interfering substance at the retention time of 3.49 min corresponding to GLEWIGAIYPGNGDTSYNQK signature Rituximab peptide. The response attributable to interfering components was not higher than 20% of the response in the LLOQ for the Rituximab peptide. Representative MRM chromatograms of the signature Rituximab peptide are shown in Fig. 2.
The significant Rituximab peptide peak GLEWIGAIYPGNGDTSYNQK (fragment m/z 1180.6 from parent 1092.1) was observed at the retention time 3.49 min in the LLOQ sample. The horizontal axis shows the retention time (min) and vertical axis shows the ion count (cps). Representative chromatograms from (a) none-spiked plasma, (b) Rituximab-spiked LLOQ plasma sample of a male person, and (c) Rituximab-spiked HQC plasma sample of a male person were shown. Arrows showed target peptide.
The LLOQ was defined as the lowest concentration of the calibration curve with acceptable precision and accuracy, which were determined to be 0.586 µg/mL (Table 2). The mean accuracy and precision at the LLOQ was within ±20% derivation of the theoretical concentration and not more than 20%, respectively. Accuracy is defined as the error from correct value.
| Nominal concentration (µg/mL) | Back-calculated concentration (µg/mL) | Accuracy (%) | ||||
|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 1 | 2 | 3 | |
| 0.586 | 0.569 | 0.579 | 0.579 | 97.2 | 98.9 | 98.9 |
| 1.17 | 1.32 | 1.20 | 1.14 | 113 | 103 | 97.7 |
| 2.34 | 2.18 | 2.03 | 2.55 | 93.0 | 86.8 | 109 |
| 4.69 | 4.49 | 4.65 | 4.64 | 95.8 | 99.1 | 99.0 |
| 9.38 | 9.74 | 9.40 | 9.48 | 104 | 100 | 101 |
| 18.8 | 18.7 | 19.8 | 18.0 | 99.7 | 106 | 96.1 |
| 37.5 | 36.6 | 34.6 | 35.4 | 97.6 | 92.3 | 94.4 |
| 75.0 | 83.5 | 79.1 | 74.3 | 111 | 105 | 99.0 |
| 150 | 152 | 153 | 155 | 102 | 102 | 103 |
| 300 | 283 | 319 | 309 | 94.4 | 106 | 103 |
The linearity of the nSMOL method was evaluated by analyzing ten calibration standards (zero sample, 0.586, 1.17, 2.34, 4.69, 9.38, 18.8, 37.5, 75.0, 150, 300 µg/mL) using the linear regression model. The calibration plot of weighting was performed using the 1/area2 method. The typical standard curve of the Rituximab peptide in plasma was shown in Fig. 3. The calibration fit formulas of the triplicate runs were Y=0.00583X+0.000254 (r=0.998), Y=0.00587X−0.00165 (r=0.998), Y=0.00643X−0.00115 (r=0.997) (r: correlation coefficient). The accuracy at LLOQ was 97.2–98.9%, and other concentrations were 86.8–113% (Table 2).
Standard range was tested 0.586–300 µg/mL of Rituximab in human plasma.
The intra- and inter-assay precision and accuracy were investigated by analysis of human plasma validation sample at LLOQ, LQC, MQC, and HQC of Rituximab concentrations. The intra-day and inter-day precision and accuracy were determined by analyzing five replicates of QC samples at four concentration levels on three different days. Precision and accuracy data were: run 1, 10.3 and 99.3% at LLOQ, 2.81–6.58 and 94.4–110% at other concentrations; run 2, 5.45 and 112% at LLOQ, 4.99–8.84, 101–104% at other concentrations; run 3, 12.9 and 93.3% at LLOQ, 2.58–5.77, 99.5–110% at other concentrations; intra-assay (N=15), 11.8 and 101% at LLOQ, 4.77–9.22, 103–104% at other concentrations, respectively (Table 3).
| Run | Nominal concentration | Concentration (µg/mL) | |||
|---|---|---|---|---|---|
| 0.586 | 1.76 | 14.1 | 240 | ||
| 1 | Observed | 0.578 | 1.60 | 14.4 | 247 |
| 0.564 | 1.56 | 15.9 | 249 | ||
| 0.610 | 1.70 | 16.9 | 264 | ||
| 0.660 | 1.83 | 15.8 | 260 | ||
| 0.498 | 1.61 | 14.7 | 253 | ||
| Mean | 0.582 | 1.66 | 15.5 | 255 | |
| S.D. | 0.06 | 0.11 | 0.99 | 7.16 | |
| CV (%) | 10.3 | 6.58 | 6.39 | 2.81 | |
| Accuracy (%) | 99.3 | 94.4 | 110 | 106 | |
| 2 | Observed | 0.651 | 1.70 | 15.1 | 248 |
| 0.648 | 2.00 | 14.8 | 268 | ||
| 0.706 | 1.94 | 14.6 | 247 | ||
| 0.606 | 1.62 | 13.3 | 225 | ||
| 0.660 | 1.87 | 14.9 | 228 | ||
| Mean | 0.654 | 1.83 | 14.54 | 243 | |
| S.D. | 0.04 | 0.16 | 0.72 | 17.72 | |
| CV (%) | 5.45 | 8.84 | 4.99 | 7.29 | |
| Accuracy (%) | 112 | 104 | 103 | 101 | |
| 3 | Observed | 0.590 | 2.05 | 13.6 | 256 |
| 0.430 | 1.79 | 14.2 | 259 | ||
| 0.611 | 2.01 | 14.4 | 245 | ||
| 0.539 | 1.98 | 13.9 | 250 | ||
| 0.563 | 1.84 | 13.7 | 244 | ||
| Mean | 0.547 | 1.93 | 14.0 | 251 | |
| S.D. | 0.07 | 0.11 | 0.36 | 6.74 | |
| CV (%) | 12.9 | 5.77 | 2.58 | 2.69 | |
| Accuracy (%) | 93.3 | 110 | 99.5 | 104 | |
| Mean (N=15) | 0.59 | 1.81 | 14.7 | 250 | |
| S.D. (N=15) | 0.08 | 0.17 | 0.95 | 11.90 | |
| CV (%) | 11.8 | 9.22 | 6.48 | 4.77 | |
| Accuracy (%) | 101 | 103 | 104 | 104 | |
Matrix factor is defined as the effect of ionization suppression in the biological matrix. Six individual plasma samples from three males and three females were analyzed for matrix effect test at the LQC and HQC concentrations. The response ratio of the Rituximab peptide in the presence of plasma prior to each sample preparation step was compared to that in PBS. The average of matrix factors (MF) at LQC and HQC were 3.06 and 0.85, respectively, which were within the accepted performance criteria of precision (CV) levels below 15% (Table 4).
| Analyte | Corresponding concentration (µg/mL) | Blank matrix No. | P14R-Normalized MF | Mean | S.D. | CV (%) |
|---|---|---|---|---|---|---|
| Rituximab | 1.76 | M1 | 2.92 | 3.06 | 0.30 | 9.87 |
| M2 | 2.95 | |||||
| M3 | 3.16 | |||||
| F1 | 2.91 | |||||
| F2 | 2.79 | |||||
| F3 | 3.62 | |||||
| 240 | M1 | 0.81 | 0.85 | 0.06 | 7.34 | |
| M2 | 0.95 | |||||
| M3 | 0.78 | |||||
| F1 | 0.83 | |||||
| F2 | 0.90 | |||||
| F3 | 0.84 |
Carryover is a remaining substances in autosampler and columns. The carryover was examined by analyzing three replicates for Rituximab signature peptide and one run for internal standard immediately after the highest concentration of calibration standard (300 µg/mL) analysis. Each carryover was calculated as percent response in the blank plasma compared with the LLOQ sample. Rituximab peptide carryover was observed at 15.8–18.1% of the analyte response of Rituximab peptide at the LLOQ and 2.3% of the analyte response of P14R was observed, confirming minimal levels of carryover influencing in nSMOL analysis (Table 5).
| Compound | Run | Peak area | Peak area rate (%) | |
|---|---|---|---|---|
| LLOQ | Carry over sample | |||
| Rituximab | 1 | 2441 | 442 | 18.1 |
| 2 | 1857 | 293 | 15.8 | |
| 3 | 2137 | 372 | 17.4 | |
| P14R | 1 | 289435 | 6543 | 2.3 |
The effect of dilution on the nSMOL analysis of Rituximab concentration was assessed by preparing spiked validation human plasma at the concentration of 500 µg/mL. The 10- and 25-fold dilution of at least five validation samples were analyzed within the calibration range. The precision and accuracy of the diluted samples were 2.97 and 111%, 3.94 and 104%, for 10- and 25-fold dilutions, respectively. These values achieved the performance criteria of decision and suggested absence of interference from dilution (Table 6).
| Defined concentration (µg/mL) | Dilution factor | Observed (µg/mL) | Mean | S.D. | CV (%) | Accuracy (%) |
|---|---|---|---|---|---|---|
| 500 | 10 | 55.1 | 556 | 1.65 | 2.97 | 111 |
| 55.7 | ||||||
| 56.7 | ||||||
| 57.3 | ||||||
| 53.1 | ||||||
| 500 | 25 | 21.6 | 521 | 0.82 | 3.94 | 104 |
| 20.1 | ||||||
| 21.5 | ||||||
| 19.8 | ||||||
| 21.1 |
The stability following five freeze-thaw cycles at −20 and −80°C with at least 24 h of frozen time, short-term stability at room temperature for 4 h, long-term stability at −20 and −80°C for 20 d prior to sample treatment, and processed sample stability at 5°C for 24 and 48 h were demonstrated at the LQC and HQC concentrations. The mean accuracy in the measurements for five freeze and thaw cycles stability at LQC and HQC in −20°C was 108 and 97.8%, respectively, and in −80°C was 105 and 109%, respectively. That for short-term stability (at room temperature for 4 h) at LQC and HQC was 95.3 and 104%, respectively. For long-term stability, the mean accuracy at LQC and HQC in −20°C was 101 and 101%, respectively, and in −80°C was 109 and 99.3%, respectively. Stability of the processed samples at LQC and HQC at 5°C for 24 h was 104 and 107%, respectively, and for 48 h was 101 and 99.4%, respectively (Table 7).
| Parameters for stability studies | Concentrations of Rituximab in human plasma (µg/mL) | |||
|---|---|---|---|---|
| 1.76 | 240 | |||
| Mean (µg/mL) | Accuracy (%) | Mean (µg/mL) | Accuracy (%) | |
| Stability in plasma during freeze (−20°C) and thaw cycles | ||||
| Cycle 5 | 1.91 | 108 | 235 | 97.8 |
| Stability in plasma during freeze (−80°C) and thaw cycles | ||||
| Cycle 5 | 1.84 | 105 | 262 | 109 |
| Short-term stability in plasma for 4 h at room temperature | ||||
| 1.68 | 95.3 | 251 | 104 | |
| Long-term stability in plasma for 20 d at −20°C | ||||
| 1.78 | 101 | 241 | 101 | |
| Long-term stability in plasma for 20 d at −80°C | ||||
| 1.93 | 109 | 238 | 99.3 | |
| Processed sample stability in HPLC set at 5°C | ||||
| For 24 h | 1.83 | 104 | 257 | 107 |
| For 48 h | 1.77 | 101 | 239 | 99.4 |
In this study, we described Rituximab fully validated reports using nSMOL proteolysis. Three nSMOL proteolytic peptides including CDR in Rituximab were significantly validated according to requirements of Guideline on Bioanalysis Method Validation for small molecule in Pharmaceutical Development. The parameters of Rituximab full validation bioanalysis using nSMOL proteolysis were achieved. We have already demonstrated that nSMOL was developed as one of the useful methods for mAbs bioanalysis with LC/MS/MS. Several antibody drugs fulfilled the validation guideline criteria in the measurement using the nSMOL proteolysis.
Many types of mAbs are being explored for therapeutic application, resulted in requirement of bioanalytical methods for such protein drugs in the pharmacokinetic assessments at the preclinical and clinical studies of drug development and therapy. Recently, bioanalysis of antibody drugs using LC/MS/MS has been attracting attention as an alternative method.11–14) In comparison with reported LC/MS/MS based approach, nSMOL proteolysis has some advantages such as high selectivity of analyte, suppress the matrix effect to a minimum, and convenient internal standard. nSMOL is limited proteolysis and is possible to solve the matrix effect problems which must be overcome in LC/MS/MS bioanalysis.
For Rituximab pharmacokinetics, most used methods are ELISA, based on the affinity of Rituximab by polyclonal or monoclonal antibodies.15–18) ELISA assay of Rituximab has linearity of 0.5 to 6.6 µg/mL. The mean values of AUC and Cmax in the four consecutive weekly administrations of 375 mg/m2 Rituximab were 118237±53412 µg/mL·h and 194.3±58.3 µg/mL, respectively. In this our validation of Rituximab, we have effectively developed the full bioanalytical validation using multiple signature CDR peptides from one peptide in heavy and two in light chains without interfere of biological matrix. The linearity of the quantification by nSMOL is from 0.586 to 300 µg/mL. Precision and accuracy values below 15% (20% for LLOQ) were achieved for all levels of QCs. From these findings indicate that using nSMOL proteolysis in quantification of Rituximab is enough to measure the serum concentration under clinical therapy and is sufficient available as a substitute for ELISA.
Assessment of the comparability of mAbs biosimilars to the original drug should follow the guidelines laid down by the FDA and EMA. Clinical pharmacokinetics (PK) of the biosimilars is required for evidence of the biosimilar similarity to the innovator drug. Bioanalytical methods applied to mAbs must contribute to not only the quantification of therapeutic mAbs but also the development of biosimilars. The reasonable method for mAbs bioanalysis is considered ELISA, but for some criteria reason, LC/MS/MS analysis becomes more valuable approach to support biosimilars studies. Because of development biosimilars, effective sample preparation for using LC/MS/MS bioanalysis of mAbs is an essential issue. We expect that our established bioanalysis method for mAbs quantification may contribute to accelerate of biosimilars development.
This work was partly supported by Grant-in-Aid from the Ministry of Health and Labor Sciences Research of Japan (AH).
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
