Research Article Special Focus Issue: Bioanalysis of Large Molecules by LC–MS
For reprint orders, please contact [email protected]
Application of high-resolution MS in the quantification of a therapeutic monoclonal antibody in human plasma
Background: Monoclonal antibodies are the fastest growing class of protein therapeutics. Ligand-binding assays have been the technique of choice for the quantification of these large proteins; however, LC–MS and more recently LC–HRMS have been gaining momentum as robust alternatives for the bioanalysis of antibodies in biological matrices. Results: Optimization of sample preparation and LC–HRMS analysis in MRMHR mode has allowed us to develop a highly specific dual-peptide targeted assay for the quantification of Rituximab, in human plasma. The linearity of the assay was established from 1.0 to 200 μg/ml for both light and heavy chain surrogate peptides, with accuracy and precision within 15%. Conclusion: LC–HRMS can be an effective tool for the quantification of monoclonal antibodies in regulated bioanalysis.
Background In the last few years, therapeutic monoclonal antibodies (mAbs) have emerged as one of the most important drug classes in the biopharmaceutical industry. At present, more than 30 mAbs are marketed for a wide range of therapeutic areas such as oncology, autoimmunity, infectious diseases and metabolic disorders [1,2] . The increasing importance of protein therapeutics necessitates performance improvements in the bioanalytical methods used in the pharmacokinetic assessments at the preclinical and clinical stages of drug development. Conventionally, ligandbinding assays (LBAs) have been the analytical technique of choice for quantifying proteins in biological fluids due to their high sensitivity and throughput [3,4] . However, the development of highly specific reagents used in these immunoassays is both costly and time-consuming. Moreover, the assay specificity, accuracy and reproducibility can be compromised by the crossreactivity with endogenous Abs and by the possible presence of anti-mAb antibodies [5,6] . Recently, liquid chromatography coupled to MS (LC–MS) has been gaining momentum and interest as an alternative technology
10.4155/BIO.14.111 © 2014 Future Science Ltd
Kevork Mekhssian1, Jean-Nicholas Mess1 & Fabio Garofolo*,1 Algorithme Pharma Inc., 575 Armand-Frappier, Laval, Québec, Canada, H7V 4B3 *Author for correspondence: Tel.: +1 450 973 6077 ext. 2301 Fax: +1 450 973 7866 [email protected]
1
for the quantitative bioanalysis of biotherapeutics in complex matrices [7–9] . In comparison with LBAs, LC–MS-based methods have several advantages such as high specificity, fast method development, wide linear dynamic range and the ability to use internal standards that correct for different sources of analytical variability. There are two main analytical approaches for the quantification of proteins in biological fluids by LC–MS [10,11] . For peptides and small proteins (typically 5), is at 25 μg/ml using the TOF-MS mode and 1.0 μg/ml using the MRMHR mode (Table 3) . Additionally, closer examination of the heavy chain peptide (25 μg/ml) chromatograms shows that a higher peak intensity is achieved by TOF-MS, however due to the presence of a high background, this approach is less selective (Figure 3) . To further enhance the sensitivity of the assay, the addition of trifluoroethanol (TFE) in the high-performance LC (HPLC) mobile phases was explored. Due to its protein solubilizing properties, TFE is often used to reduce carryover in LC systems by its addition to the needle and column wash steps or directly in mobile phases [33,34] . During chromatography optimization, the intensity and peak areas for both heavy and light chain peptides were monitored
Table 4. Inter-assay precision and accuracy results for Rituximab heavy and light chain surrogate peptides quantification by LC–HRMS. LLOQ QC
Low QC
Mid QC
High QC
1.00 μg/ml
3.00 μg/ml
25.00 μg/ml
150.00 μg/ml
Mean (μg/ml); n = 18
1.01
2.97
23.87
155.54
SD (μg/ml)
0.10
0.21
0.78
14.3
100.8
99.1
95.5
103.7
10.1
7.2
3.3
9.2
HC peptide
Accuracy (% nominal) Precision (%CV) LC peptide Mean (μg/ml); n = 18
1.00
3.01
24.55
147.16
SD (μg/ml)
0.13
0.21
1.14
15.65
Accuracy (% nominal)
99.8
100.4
98.2
98.1
Precision (%CV)
12.8
6.9
4.6
10.6
% Mean difference
1.00
−1.30
−2.78
5.55
*The % mean difference was calculated as follows: (%nominal HC peptide - %nominal LC peptide)/average %nominal of LC and HC peptides × 100% HC: Heavy chain; LC: Light chain.
1776
Bioanalysis (2014) 6(13)
future science group
Application of high-resolution MS in the quantification of a therapeutic monoclonal antibody in human plasma Research Article
in the presence of different levels of TFE (1, 2.5 and 5%) in the mobile phase. At 2.5% TFE, the highest increase in signal intensity was achieved with 87% for heavy chain peptide and 66% for light chain peptide (Figure 4) . Similarly, peak area increased 92% for heavy chain peptide and 57% for light chain peptide, with no apparent increase in background noise levels (data not shown). The reduction in carryover and sample loss can explain this increase in sensitivity, however the shift in retention time in the presence of TFE can also lead to less ion suppression at the new elution time. Precision & accuracy
The intra- and inter-assay precision and accuracy were assessed using six replicates at four QC concentrations (LLOQ, low, medium and high QCs) in three different batches. Representative chromatograms for extracted blank, LLOQ and ULOQ are shown in Figure 5. For both surrogate peptides, accuracy (% nominal) and precision (%CV) levels below 15% (20% for LLOQ) were achieved for all levels of QCs ranging from 1.00 to 200.0 μg/ml. These results indicated that pellet digestion in the presence of SIL-IS flanked peptides provided a reproducible and efficient digestion in different lots of human plasma. Furthermore, since two peptides are used to quantify one protein using the same calibration range, we applied the acceptance criteria put forward by Furlong et al., which stipulates that the percentage difference between the two surrogate peptide-derived mAb concentrations must be less than 20% for all individual calibrants and QC samples [13] . Inter-assay precision, accuracy and % mean differences are shown in Table 4. Additionally, the % difference of individual calibrants and QC values of Rituximab heavy and light chain peptides were all within 20% (data not shown). Furthermore, specificity was evaluated on ten different lots of blank human plasma (K 2EDTA) including one lipemic and one hemolyzed. All samples were found free of significant interference at the retention times of both light and heavy chain peptides (less than 20% of LLOQ) and IS peptides (less than 5% of IS response), demonstrating the selectivity of the assay.
Conclusion A highly specific method using HRMS was developed to quantify a human mAb in human plasma. Using the TOF-MS mode for the selection of surrogate peptides followed by the MRMHR mode for protein quantitation has allowed us to conduct both qualitative and quantitative facets of method development using one instrument. The dual peptide approach increases the reliability on the structural integrity of the antibody and enhances the confidence on the concentration data obtained. Moreover, the optimization of sample processing steps of protein precipitation, reduction, alkylation and tryptic digestion improved the digestion efficiency with high reproducibility. In addition, by selecting narrow mass selection windows over multiple fragments has allowed us to develop a specific and sensitive assay on a par with similar assays developed with triple quadrupoles. Finally, we believe that using HRMS in a targeted approach such as MRMHR, can be an effective tool in the bioanalysis of large molecules in a regulated environment, as observed by the linearity, precision and accuracy results obtained in the Rituximab assay. Future perspective With the increasing popularity of biotherapeutic agents and the numerous drug-development programs ongoing in the industry on compounds such as fusion proteins, pegylated proteins, mAbs or antibody–drug conjugates, there will be a growing demand for rapid and reliable quantification assays to support pre-clinical and clinical studies of these drugs. Due to the inherent characteristic of LBAs, which rely on the availability of high-selectivity reagents, LC–HRMS instruments could definitively meet this demand due to their versatility and their ability to effectively perform qualitative analysis, such as peptide mapping and characterization, and quantitative analysis to fit the strictest acceptance criteria of the industry. Although standard triple quadrupole mass spectrometers are still considered more sensitive, robust and adapted to quantitative analysis, these instruments will never provide the resolution and mass accuracy of HRMS instruments. Alternatively, the upcoming generation of HRMS instruments can
Executive summary • A dual-peptide targeted approach was used to quantify Rituximab by high-resolution MS in human plasma. • Pellet digestion with pre-treatment resulted in a significant increase in digestion yield compared with direct method with no pre-treatment. • The use of multiple reaction monitoring at high resolution mode for quantification showed increased sensitivity and specificity compared with the time-of-flight MS mode. • Addition of trifluoroethanol in mobile phase considerably increased peak area for both surrogate peptides. • Good linearity, precision, accuracy and specificity were achieved in the assay. • High-resolution MS in MRMHR mode has proven to be an effective tool in the bioanalysis of large molecules.
future science group
www.future-science.com
1777
Research Article Mekhssian, Mess & Garofolo only increase in sensitivity, reliability and speed while keeping this high selectivity level.
No writing assistance was utilized in the production of this manuscript.
Financial & competing interests disclosure
Ethical conduct of research
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies. Biomed. Chrom. 26(8), 1024–1032 (2012).
References Papers of special note have been highlighted as: • of interest. 1
Beck A, Wurch T, Bailey C, Corvaia N. Strategies and challenges for the next generation of therapeutic antibodies. Nat. Rev. Immunol. 10(5), 345–352 (2010).
2
Nelson AL, Dhimolea E, Reichert JM. Development trends for human monoclonal antibody therapeutics. Nat. Rev. Drug Discov. 9(10), 767–774 (2010).
3
DeSilva B, Smith W, Weiner R, Kelley M et al. Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm. Res. 20(11), 1885–1900 (2003).
4
5
Hoofnagle AN, Wener MH. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. Immunol. Methods 347(1–2), 3–11 (2009).
6
Ezan E, Bitsch F. Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodies. Bioanalysis 1(8), 1375–1388 (2009).
7
1778
Damen CW, de Groot ER, Heij M et al. Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma. Anal. Biochem. 391, 114–120 (2009).
Li F, Fast D, Michael S. Absolute quantitation of protein therapeutics in biological matrices by enzymatic digestion and LC–MS. Bioanalysis 3(21), 2459–2480 (2011).
8
Mesmin C, Fenaille F, Ezan E, Becher F. MS-based approaches for studying the pharmacokinetics of protein drugs. Bioanalysis 3(5), 477–480 (2011).
9
Ezan E, Dubois M, Becher F. Bioanalysis of recombinant proteins and antibodies by mass spectrometry. Analyst 134(5), 825–834 (2009).
10
Ewles M, Goodwin L. Bioanalytical approaches to analyzing peptides and proteins by LC–MS/MS. Bioanalysis 3(12), 1379–1397 (2011).
11
van den Broek I, Niessen WMA, van Dongen WD. Bioanalytical LC–MS/MS of protein-based biopharmaceuticals. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 15 (929), 161–179 (2013).
•
Good review article highlighting several critical factors affecting bioanalytical LC–MS of protein-based biotherapeutics.
12
Furlong MT, Ouyang Z, Wu S et al. A universal surrogate peptide to enable LC–MS/MS bioanalysis of a diversity of
Bioanalysis (2014) 6(13)
13
Furlong MT, Zhao S, Mylott W, Jenkins R, Gao M et al. Dual universal peptide approach to bioanalysis of human monoclonal antibody protein drug candidates in animal studies. Bioanalysis 5(11), 1363–1376 (2013).
14
Morin LP, Mess JN, Garofolo F. Large-molecule quantification: sensitivity and selectivity head-to-head comparison of triple quadrupole with Q-TOF. Bioanalysis 5(10), 1181–1193 (2013).
•
Detailed comparison between high-resolution MS (HRMS) and triple quadrupoles instruments for the quantification of six different peptides. The study lists the advantages of HRMS along with basic guidelines on how to use HRMS for large-molecule quantification in regulated environment.
15
Dillen Lieve, Cools W, Vereyken L, Lorreyne W et al. Comparison of triple quadrupole and high-resolution TOFMS for quantification of peptides. Bioanalysis 4(5), 565–579 (2012).
16
Huang MQ, Lin Z, Weng N. Applications of high-resolution MS in bioanalysis. Bioanalysis 5(10), 1269–1276 (2013).
17
Ramagiri S, Garofolo F. Large molecule bioanalysis using Q-TOF without predigestion and its data processing challenges. Bioanalysis 4(5), 529–540 (2012).
18
Liu H, Manuilov AV, Chumsae C, Babineau ML, Tarcsa E. Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography-mass spectrometry. Anal. Biochem. 414, 147–153 (2011).
19
Reff ME, Carner K, Chambers KS, Chinn PC et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody. Blood 83, 2861–2868 (1994).
20
Du J, Wang H, Zhong C, Peng B, Zhang M et al. Crystal structure of chimeric antibody C2H7 Fab in complex with a CD20 peptide. Mol. Immunol. 45, 2861–2868 (2008).
21
Mok CC. Rituximab for the treatment of rheumatoid arthritis: an update. Drug Des. Devel. Ther. 8, 87–100 (2014).
22
Samonig M, Huber C, Scheffler K. LC/MS analysis of the monoclonal antibody Rituximab using Q Exactive benchtop Orbitrap mass spectrometer. Thermo Scientific, Application note 591: www.planetorbitrap.com
23
Bronsema KJ, Bischoff R, van de Merbel NC. Internal standards in the quantitative determination of protein
future science group
Application of high-resolution MS in the quantification of a therapeutic monoclonal antibody in human plasma Research Article
digestion: comparison with traditional digestion methods in LC–MS/MS bioanalysis. Bioanalysis 4(24), 2887–2896 (2012).
biopharmaceuticals using liquid chromatography coupled to mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 15, 893–894 (2012). 24
Heudi O, Barteau S, Zimmer D, Schmidt J et al. Towards absolute quantification of therapeutic monoclonal antibody in serum by LC–MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry. Anal. Chem. 80(11), 4200–4207 (2008).
25
Li H, Ortiz R, Tran L, Hall M, Spahr C, Walker K et al. General LC–MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies. Anal. Chem. 84, 1267–1273 (2012). Ocaña MF, Neubert H. An immunoaffinity liquid chromatography-tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum. Anal. Biochem. 399, 202–210 (2010).
26
27
Neubert H, Muirhead D, Kabir M, Grace C et al. Sequential protein and peptide immunoaffinity capture for mass spectrometry-based quantification of total human β-nerve growth factor. Anal. Chem. 85(3), 1719–1726 (2013).
28
Ouyang Z, Furlong MT, Wu S et al. Pellet digestion: a simple and efficient sample preparation technique for LC–MS/MS quantification of large therapeutic proteins in plasma. Bioanalysis 4(1), 17–28 (2012).
29
Yuan L, Arnold ME, Aubry A-F, Ji QC. Simple and efficient digestion of a monoclonal antibody in serum using pellet
future science group
30
Yu YQ, Gilar M, Lee PJ, Bouvier ES, Gebler JC. Enzymefriendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins. Anal. Chem. 75, 6023–6028 (2003).
31
Chen EI, Cociorva D, Norris JL, Yates JR III. Optimization of mass spectrometry compatible surfactants for shotgun proteomics. J. Proteome Res. 6(7), 2529–2538 (2007).
32
Proc JL, Kuzyk MA, Hardie DB, Yang J et al. A qualitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin. J. Proteome Res. 9(10), 5422–5437 (2010).
33
Mitulovic G, Stingl C, Steinmacher I, Hudecz O et al. Preventing carryover of peptides and proteins in nano LC– MS separations. Anal. Chem. 14, 5955–5960 (2009).
34
Chambers EE, Lame ME, Bardsley J, Hannam S et al. High sensitivity LC–MS/MS method for direct quantification of human parathyroid 1–34 (teriparatide) in human plasma. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 938, 96–104 (2013).
•
Good overview of assay optimization for large hydrophobic peptides with poor solubility to reach detection limits close to levels observed in the immunoassay.
www.future-science.com
1779
For reprint orders, please contact [email protected]
Application of high-resolution MS in the quantification of a therapeutic monoclonal antibody in human plasma
Background: Monoclonal antibodies are the fastest growing class of protein therapeutics. Ligand-binding assays have been the technique of choice for the quantification of these large proteins; however, LC–MS and more recently LC–HRMS have been gaining momentum as robust alternatives for the bioanalysis of antibodies in biological matrices. Results: Optimization of sample preparation and LC–HRMS analysis in MRMHR mode has allowed us to develop a highly specific dual-peptide targeted assay for the quantification of Rituximab, in human plasma. The linearity of the assay was established from 1.0 to 200 μg/ml for both light and heavy chain surrogate peptides, with accuracy and precision within 15%. Conclusion: LC–HRMS can be an effective tool for the quantification of monoclonal antibodies in regulated bioanalysis.
Background In the last few years, therapeutic monoclonal antibodies (mAbs) have emerged as one of the most important drug classes in the biopharmaceutical industry. At present, more than 30 mAbs are marketed for a wide range of therapeutic areas such as oncology, autoimmunity, infectious diseases and metabolic disorders [1,2] . The increasing importance of protein therapeutics necessitates performance improvements in the bioanalytical methods used in the pharmacokinetic assessments at the preclinical and clinical stages of drug development. Conventionally, ligandbinding assays (LBAs) have been the analytical technique of choice for quantifying proteins in biological fluids due to their high sensitivity and throughput [3,4] . However, the development of highly specific reagents used in these immunoassays is both costly and time-consuming. Moreover, the assay specificity, accuracy and reproducibility can be compromised by the crossreactivity with endogenous Abs and by the possible presence of anti-mAb antibodies [5,6] . Recently, liquid chromatography coupled to MS (LC–MS) has been gaining momentum and interest as an alternative technology
10.4155/BIO.14.111 © 2014 Future Science Ltd
Kevork Mekhssian1, Jean-Nicholas Mess1 & Fabio Garofolo*,1 Algorithme Pharma Inc., 575 Armand-Frappier, Laval, Québec, Canada, H7V 4B3 *Author for correspondence: Tel.: +1 450 973 6077 ext. 2301 Fax: +1 450 973 7866 [email protected]
1
for the quantitative bioanalysis of biotherapeutics in complex matrices [7–9] . In comparison with LBAs, LC–MS-based methods have several advantages such as high specificity, fast method development, wide linear dynamic range and the ability to use internal standards that correct for different sources of analytical variability. There are two main analytical approaches for the quantification of proteins in biological fluids by LC–MS [10,11] . For peptides and small proteins (typically 5), is at 25 μg/ml using the TOF-MS mode and 1.0 μg/ml using the MRMHR mode (Table 3) . Additionally, closer examination of the heavy chain peptide (25 μg/ml) chromatograms shows that a higher peak intensity is achieved by TOF-MS, however due to the presence of a high background, this approach is less selective (Figure 3) . To further enhance the sensitivity of the assay, the addition of trifluoroethanol (TFE) in the high-performance LC (HPLC) mobile phases was explored. Due to its protein solubilizing properties, TFE is often used to reduce carryover in LC systems by its addition to the needle and column wash steps or directly in mobile phases [33,34] . During chromatography optimization, the intensity and peak areas for both heavy and light chain peptides were monitored
Table 4. Inter-assay precision and accuracy results for Rituximab heavy and light chain surrogate peptides quantification by LC–HRMS. LLOQ QC
Low QC
Mid QC
High QC
1.00 μg/ml
3.00 μg/ml
25.00 μg/ml
150.00 μg/ml
Mean (μg/ml); n = 18
1.01
2.97
23.87
155.54
SD (μg/ml)
0.10
0.21
0.78
14.3
100.8
99.1
95.5
103.7
10.1
7.2
3.3
9.2
HC peptide
Accuracy (% nominal) Precision (%CV) LC peptide Mean (μg/ml); n = 18
1.00
3.01
24.55
147.16
SD (μg/ml)
0.13
0.21
1.14
15.65
Accuracy (% nominal)
99.8
100.4
98.2
98.1
Precision (%CV)
12.8
6.9
4.6
10.6
% Mean difference
1.00
−1.30
−2.78
5.55
*The % mean difference was calculated as follows: (%nominal HC peptide - %nominal LC peptide)/average %nominal of LC and HC peptides × 100% HC: Heavy chain; LC: Light chain.
1776
Bioanalysis (2014) 6(13)
future science group
Application of high-resolution MS in the quantification of a therapeutic monoclonal antibody in human plasma Research Article
in the presence of different levels of TFE (1, 2.5 and 5%) in the mobile phase. At 2.5% TFE, the highest increase in signal intensity was achieved with 87% for heavy chain peptide and 66% for light chain peptide (Figure 4) . Similarly, peak area increased 92% for heavy chain peptide and 57% for light chain peptide, with no apparent increase in background noise levels (data not shown). The reduction in carryover and sample loss can explain this increase in sensitivity, however the shift in retention time in the presence of TFE can also lead to less ion suppression at the new elution time. Precision & accuracy
The intra- and inter-assay precision and accuracy were assessed using six replicates at four QC concentrations (LLOQ, low, medium and high QCs) in three different batches. Representative chromatograms for extracted blank, LLOQ and ULOQ are shown in Figure 5. For both surrogate peptides, accuracy (% nominal) and precision (%CV) levels below 15% (20% for LLOQ) were achieved for all levels of QCs ranging from 1.00 to 200.0 μg/ml. These results indicated that pellet digestion in the presence of SIL-IS flanked peptides provided a reproducible and efficient digestion in different lots of human plasma. Furthermore, since two peptides are used to quantify one protein using the same calibration range, we applied the acceptance criteria put forward by Furlong et al., which stipulates that the percentage difference between the two surrogate peptide-derived mAb concentrations must be less than 20% for all individual calibrants and QC samples [13] . Inter-assay precision, accuracy and % mean differences are shown in Table 4. Additionally, the % difference of individual calibrants and QC values of Rituximab heavy and light chain peptides were all within 20% (data not shown). Furthermore, specificity was evaluated on ten different lots of blank human plasma (K 2EDTA) including one lipemic and one hemolyzed. All samples were found free of significant interference at the retention times of both light and heavy chain peptides (less than 20% of LLOQ) and IS peptides (less than 5% of IS response), demonstrating the selectivity of the assay.
Conclusion A highly specific method using HRMS was developed to quantify a human mAb in human plasma. Using the TOF-MS mode for the selection of surrogate peptides followed by the MRMHR mode for protein quantitation has allowed us to conduct both qualitative and quantitative facets of method development using one instrument. The dual peptide approach increases the reliability on the structural integrity of the antibody and enhances the confidence on the concentration data obtained. Moreover, the optimization of sample processing steps of protein precipitation, reduction, alkylation and tryptic digestion improved the digestion efficiency with high reproducibility. In addition, by selecting narrow mass selection windows over multiple fragments has allowed us to develop a specific and sensitive assay on a par with similar assays developed with triple quadrupoles. Finally, we believe that using HRMS in a targeted approach such as MRMHR, can be an effective tool in the bioanalysis of large molecules in a regulated environment, as observed by the linearity, precision and accuracy results obtained in the Rituximab assay. Future perspective With the increasing popularity of biotherapeutic agents and the numerous drug-development programs ongoing in the industry on compounds such as fusion proteins, pegylated proteins, mAbs or antibody–drug conjugates, there will be a growing demand for rapid and reliable quantification assays to support pre-clinical and clinical studies of these drugs. Due to the inherent characteristic of LBAs, which rely on the availability of high-selectivity reagents, LC–HRMS instruments could definitively meet this demand due to their versatility and their ability to effectively perform qualitative analysis, such as peptide mapping and characterization, and quantitative analysis to fit the strictest acceptance criteria of the industry. Although standard triple quadrupole mass spectrometers are still considered more sensitive, robust and adapted to quantitative analysis, these instruments will never provide the resolution and mass accuracy of HRMS instruments. Alternatively, the upcoming generation of HRMS instruments can
Executive summary • A dual-peptide targeted approach was used to quantify Rituximab by high-resolution MS in human plasma. • Pellet digestion with pre-treatment resulted in a significant increase in digestion yield compared with direct method with no pre-treatment. • The use of multiple reaction monitoring at high resolution mode for quantification showed increased sensitivity and specificity compared with the time-of-flight MS mode. • Addition of trifluoroethanol in mobile phase considerably increased peak area for both surrogate peptides. • Good linearity, precision, accuracy and specificity were achieved in the assay. • High-resolution MS in MRMHR mode has proven to be an effective tool in the bioanalysis of large molecules.
future science group
www.future-science.com
1777
Research Article Mekhssian, Mess & Garofolo only increase in sensitivity, reliability and speed while keeping this high selectivity level.
No writing assistance was utilized in the production of this manuscript.
Financial & competing interests disclosure
Ethical conduct of research
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies. Biomed. Chrom. 26(8), 1024–1032 (2012).
References Papers of special note have been highlighted as: • of interest. 1
Beck A, Wurch T, Bailey C, Corvaia N. Strategies and challenges for the next generation of therapeutic antibodies. Nat. Rev. Immunol. 10(5), 345–352 (2010).
2
Nelson AL, Dhimolea E, Reichert JM. Development trends for human monoclonal antibody therapeutics. Nat. Rev. Drug Discov. 9(10), 767–774 (2010).
3
DeSilva B, Smith W, Weiner R, Kelley M et al. Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm. Res. 20(11), 1885–1900 (2003).
4
5
Hoofnagle AN, Wener MH. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. Immunol. Methods 347(1–2), 3–11 (2009).
6
Ezan E, Bitsch F. Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodies. Bioanalysis 1(8), 1375–1388 (2009).
7
1778
Damen CW, de Groot ER, Heij M et al. Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma. Anal. Biochem. 391, 114–120 (2009).
Li F, Fast D, Michael S. Absolute quantitation of protein therapeutics in biological matrices by enzymatic digestion and LC–MS. Bioanalysis 3(21), 2459–2480 (2011).
8
Mesmin C, Fenaille F, Ezan E, Becher F. MS-based approaches for studying the pharmacokinetics of protein drugs. Bioanalysis 3(5), 477–480 (2011).
9
Ezan E, Dubois M, Becher F. Bioanalysis of recombinant proteins and antibodies by mass spectrometry. Analyst 134(5), 825–834 (2009).
10
Ewles M, Goodwin L. Bioanalytical approaches to analyzing peptides and proteins by LC–MS/MS. Bioanalysis 3(12), 1379–1397 (2011).
11
van den Broek I, Niessen WMA, van Dongen WD. Bioanalytical LC–MS/MS of protein-based biopharmaceuticals. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 15 (929), 161–179 (2013).
•
Good review article highlighting several critical factors affecting bioanalytical LC–MS of protein-based biotherapeutics.
12
Furlong MT, Ouyang Z, Wu S et al. A universal surrogate peptide to enable LC–MS/MS bioanalysis of a diversity of
Bioanalysis (2014) 6(13)
13
Furlong MT, Zhao S, Mylott W, Jenkins R, Gao M et al. Dual universal peptide approach to bioanalysis of human monoclonal antibody protein drug candidates in animal studies. Bioanalysis 5(11), 1363–1376 (2013).
14
Morin LP, Mess JN, Garofolo F. Large-molecule quantification: sensitivity and selectivity head-to-head comparison of triple quadrupole with Q-TOF. Bioanalysis 5(10), 1181–1193 (2013).
•
Detailed comparison between high-resolution MS (HRMS) and triple quadrupoles instruments for the quantification of six different peptides. The study lists the advantages of HRMS along with basic guidelines on how to use HRMS for large-molecule quantification in regulated environment.
15
Dillen Lieve, Cools W, Vereyken L, Lorreyne W et al. Comparison of triple quadrupole and high-resolution TOFMS for quantification of peptides. Bioanalysis 4(5), 565–579 (2012).
16
Huang MQ, Lin Z, Weng N. Applications of high-resolution MS in bioanalysis. Bioanalysis 5(10), 1269–1276 (2013).
17
Ramagiri S, Garofolo F. Large molecule bioanalysis using Q-TOF without predigestion and its data processing challenges. Bioanalysis 4(5), 529–540 (2012).
18
Liu H, Manuilov AV, Chumsae C, Babineau ML, Tarcsa E. Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography-mass spectrometry. Anal. Biochem. 414, 147–153 (2011).
19
Reff ME, Carner K, Chambers KS, Chinn PC et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody. Blood 83, 2861–2868 (1994).
20
Du J, Wang H, Zhong C, Peng B, Zhang M et al. Crystal structure of chimeric antibody C2H7 Fab in complex with a CD20 peptide. Mol. Immunol. 45, 2861–2868 (2008).
21
Mok CC. Rituximab for the treatment of rheumatoid arthritis: an update. Drug Des. Devel. Ther. 8, 87–100 (2014).
22
Samonig M, Huber C, Scheffler K. LC/MS analysis of the monoclonal antibody Rituximab using Q Exactive benchtop Orbitrap mass spectrometer. Thermo Scientific, Application note 591: www.planetorbitrap.com
23
Bronsema KJ, Bischoff R, van de Merbel NC. Internal standards in the quantitative determination of protein
future science group
Application of high-resolution MS in the quantification of a therapeutic monoclonal antibody in human plasma Research Article
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Good overview of assay optimization for large hydrophobic peptides with poor solubility to reach detection limits close to levels observed in the immunoassay.
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