Preliminary Communication Special Focus Issue: Bioanalysis of Large Molecules by LC–MS
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An exploratory universal LC–MS/MS assay for bioanalysis of hinge region-stabilized human IgG4 mAbs in clinical studies Background: Due to the increasing number of monoclonal antibody (mAb) drug candidates entering clinical development, bioanalytical laboratories can benefit from generic liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays capable of quantifying a variety of human mAb-based therapeutic drug candidates in plasma/serum samples from clinical studies. Results: We have developed and evaluated an exploratory LC–MS/MS assay capable of quantifying hinge region-stabilized IgG4 therapeutic mAb drugs and drug candidates in clinical samples. The exploratory assay is based upon a single ‘universal IgG4’ surrogate peptide. Conclusion: The novel exploratory LC–MS/MS assay reported herein, upon further refinement and full validation, is predicted to enable bioanalytical scientists to quantify all hinge region-stabilized human IgG4 therapeutic mAbs in human studies without having to develop a new assay for every new stabilized IgG4 mAb entering clinical development.
Monoclonal antibody (mAb)-based therapeutic agents occupy an increasingly important role in the treatment of a variety of human diseases [1–4] . The growing number of mAbs entering drug development represents a significant challenge to bioanalytical laboratories engaged in the quantification of these candidates in biological matrices. Bioanalysis of human therapeutic mAbs by LC–MS/MS is typically based upon quantification of ‘signature’ surrogate peptides whose amino acid sequences are unique to the protein analyte of interest [5,6] . Signature peptides are found in the antibody variable regions of the human therapeutic mAb light and heavy chains. The signature surrogate peptide approach is labor-intensive, particularly in the realm of assay development and validation, wherein a new signature surrogate peptide must be identified and a new assay must be developed for each human therapeutic mAb candidate entering development. To address this challenge for nonclinical studies, we have previously developed and successfully deployed a LC–MS/MS assay that relies upon ‘universal’ surrogate peptides rather than ‘signature’ surrogate peptides [7–9] . These universal peptides possess four key attributes necessary for a single universal peptide LC–MS/MS assay capable of
10.4155/BIO.14.64 © 2014 Future Science Ltd
Michael T Furlong*‡1,2, Craig Titsch‡1, Weifeng Xu1, Hao Jiang1, Mohammed Jemal3 & Jianing Zeng1 1 Bristol-Myers Squibb, R&D, Route 206 & Province Line Road, Princeton, NJ 08543, USA 2 Current: Molecular & Bioanalytical Pharmacology Amicus Therapeutics 3 931 Edinburg Road, Hamilton Square, NJ 08690, USA *Author for correspondence: Tel.: +1 609 662 2071 [email protected] ‡ These authors contributed equally
quantifying a diversity of human therapeutic mAbs in animal PK/TK studies: • Universality: the sequences are present in the constant regions of human therapeutic mAb candidates, thus enabling their general applicability across multiple mAb drug candidates and development programs (Figure 1A) ; • Selectivity: the sequences are not found in the antibody constant region or any other plasma/serum protein sequences present in any animal species commonly used in nonclinical drug development studies, thus ensuring quantification that is free of plasma protein-derived interferences (Figure 1A) ; • The sequences are reliably produced from trypsin digestion of human mAbs; • The sequences possess favorable LC–MS/MS characteristics, such as good chromatographic peak shape, adequate chromatographic retention and efficient ionization. The amino acid sequences of the universal surrogate peptides identified and used in
Bioanalysis (2014) 6(13), 1747–1758
part of
ISSN 1757-6180
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Preliminary Communication Furlong, Titsch, Xu, Jiang, Jemal & Zeng
Key Terms Surrogate peptides: Peptide derived from proteolytic digestion of a protein analyte. The peptide is quantified by LC–MS/MS as a ‘surrogate’ for the protein analyte. Antibody variable regions: These regions are found on the amino terminal portion of antibody light and heavy chains. The amino acid sequences of the variable regions vary greatly from antibody to antibody. Signature surrogate peptide: Surrogate peptide that is unique to the protein analyte of interest. In monoclonal antibodies, signature surrogate peptides can be found in the variable regions of either the light or heavy chains. Universal peptide LC–MS/MS assay: Single LC–MS/ MS assay capable of bioanalytical support of a diversity of proteins that contain a shared universal surrogate peptide. Antibody constant region: These regions are found on the carboxy terminal portion of antibody light and heavy chains. The amino acid sequences of the constant regions are identical across all antibodies within a species and subclass. Universal surrogate peptides: Single surrogate peptide that is found in many variants of a particular therapeutic protein class. In monoclonal antibodies, universal surrogate peptides can be found in the constant regions of either the light or heavy chains.
our laboratories are as follows: light chain (k class) – TVAAPSVFIFPPSDEQLK – and heavy chain (IgG1 and IgG4 subclasses) – VVSVLTVLHQDWLNGK [7,9] . Preliminary experiments indicate that the closely related IgG2 variant of the heavy chain universal peptide (VVSVLTVVHQDWLNGK) could be incorporated into a universal LC–MS/MS assay to support bioanalysis of human IgG2-based therapeutic mAbs [7] . Currently, the universal peptide LC–MS/MS assay is applicable only to animal studies. Herein, we report the extension of the universal assay concept to potentially enable bioanalytical support of clinical studies. The new exploratory LC–MS/MS assay is based upon a single tryptic peptide that is located in the constant region of hinge region-stabilized IgG4-based human therapeutic mAb drugs and drug candidates, and is not present in the constant regions of endogenous antibodies present in the human plasma/serum samples. This single LC– MS/MS assay, upon further refinement and full validation, is predicted to enable reliable quantification of all hinge region-stabilized human IgG4 therapeutic mAb drug candidates in clinical PK studies. Experimental In silico analyses
Antibody sequences were downloaded from the Immunogenetics information system website [10] . In silico trypsin digests were carried out using the ExPASy Bioinfomatics Resource Portal [11] .
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mAbs & peptides
mAbs 1, 2 and 3 were produced at Bristol-Myers Squibb. mAb 1 is comprised of a human IgG4 subclass heavy chain and a human k-light chain. The heavy chain of mAb 1 contains a serine-to-proline amino acid substitution at position 241 (Kabat numbering [12]). mAb 2 is a stable isotope-labeled (SIL) version of mAb 1, at all arginine (13C615N4) and lysine (13C615N2) positions. mAb 2 served as the analytical IS for quantification of mAb 1. mAb 3 is a mouse mAb that binds to the variable region of mAb 1 and interferes with the ability of mAb 1 to bind to its pharmacological target. Peptides 1 and 2 used for method development were custom-synthesized at Bristol-Myers Squibb. The amino acid sequences of the peptides are shown in Table 1. Chemicals & reagents
Trypsin from bovine pancreas, iodoacetamide, dithiothreitol, HPLC grade acetonitrile, HPLC grade methanol and PBS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid was purchased from EMD Chemicals (Billerica, MA, USA). Ammonium bicarbonate was purchased from MP Biomedicals (Solon, OH, USA). Calibration standards & QC samples
The mAb 1 calibration curve and QC samples were prepared by dilution of the mAb 1 stock solution (10 mg/ml) into blank human serum followed by further serial dilution in blank serum to obtain the desired final concentrations. Calibration standard concentrations were 5.00, 10.0, 25.0, 50.0, 100, 200, 450 and 500 µg/ml. QC concentrations were 5.00, 15.0, 25.0, 60.0, 125, 250 and 400 µg/ml. Sample preparation
Samples were processed using our previously reported ‘pellet digestion’ methodology in 96-well plates as follows: serum samples (25 µl) were fortified with 25 µl of IS working solution (mAb 2 at 200 µg/ml in PBS) [8,13] . Matrix blank samples were fortified with PBS instead of IS working solution. The mixtures were briefly vortexed and then centrifuged for 1 min at 200 g. Methanol (200 µl) was added to each well followed by vigorous vortexing for 2 min and centrifugation at 200 g for 2 min. Following removal of the supernatant, the pellets were suspended in 100 mM aqueous ammonium bicarbonate (50 µl) by vigorous vortexing for 2 min. The resulting suspensions were further fortified with 100 mM dithiothreitol (10 µl), followed by centrifugation at 200 g for 1 min, vigorous vortexing for 2 min and incubation for 1 h at 60°C in an Eppendorf Thermomixer® (NY, USA) at 1000 rpm.
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An exploratory universal LC–MS/MS assay
Human therapeutic mAb analyte
A
Variable region
Constant region
Constant region
Endogenous animal plasma/serum antibodies
{
{
Non-identical constant regions
Human therapeutic mAb analyte
B
Variable region
Preliminary Communication
Endogenous human plasma/serum antibodies
{
{
Identical constant regions
Figure 1. Surrogate peptide options for bioanalysis of human therapeutic monoclonal antibodies are based in part on the species of the matrix sample. (A) For LC–MS/MS quantification of human therapeutic mAb analytes in animal samples, universal surrogate peptides, located in the constant regions of the human mAb light and heavy chains, may be used as alternatives to signature surrogate peptides from the variable regions. The locations of universal surrogate peptides in human mAb analytes are depicted as green bars. (B) For LC–MS/MS quantification of human mAb analytes in human samples, signature surrogate peptides, located in the variable regions of the human mAb light and heavy chains, must be used. The locations of the signature surrogate peptides in human mAb analytes are depicted as yellow bars. mAb: Monoclonal antibody. Please see colour figure at www.future-science.com/doi/full/10.4155/BIO.14.64
Cysteine thiol alkylation was carried out by addition of 100 mM iodoacetamide (25 µl), centrifugation at 200 g for 1 min, vigorous vortexing for 2 min and incubation in the dark for 30 min at 30°C in a thermomixer (1000 rpm). Trypsin solution (25 µl of an 8.0 mg/ml solution in 0.1% aqueous formic acid) was added, followed by centrifugation at 200 g for 1 min, vigorous vortexing for 1 min and incubation in the dark for 30 min at 50°C in a thermomixer (1000 rpm). The digested samples were treated with 10% aqueous formic acid (25 µl), followed by centrifugation at 200 g for 1 min and vigorous vortexing for 2 min. A final
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high speed centrifugation (5000 g; 5 min) was carried out to pellet residual particulates. The supernatants (120 µl) were transferred to 96-well collection plates for LC–MS/MS analysis. Chromatographic & mass spectrometric conditions
Experiments were conducted using a Shimadzu Nexera HPLC system and SIL-30AC autosampler (Columbia, MD, USA) coupled to an AB Sciex API 5500 triple quadrupole mass spectrometer equipped with a TurboIonspray™ source (Concord, Ontario, Canada).
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Table 1. Amino acid sequences, locations and SRM transitions/charge states for surrogate peptide analytes and IS. Peptide
Sequence
Universal IgG4 peptide from heavy YGPPCPPCPAPEFLGGPSVFLFPPKPK chain constant region (Peptide 1) YGPPCPPCPAPEFLGGPSVFLFPPK‡PK‡ (unlabeled and labeled)† Confirmatory peptide from heavy chain variable region (Peptide 2) (unlabeled and labeled)
ASGITFSNSGMHWVR ASGITFSNSGMHWVR
§
Precursor ion mass (m/z units) and charge state
Product ion mass (m/z units) and charge state
985.3 [M+3H] 3+
912.1 [M+3H] 3+
990.6 [M+3H] 3+
917.4 [M+3H] 3+
825.5 [M+2H] 2+
1073.5 [MH] +
830.5 [M+2H]
1083.5 [MH] +
2+
The thiol groups on all cysteine residues in the Universal IgG4 peptide and IS were carboxamidated with iodoacetamide during sample preparation (see ‘Experimental’ section). ‡ Denotes lysine labeled with 13C615N2; used as IS. § Denotes arginine labeled with 13C615N4; used as IS. †
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Chromatographic separation was performed at 60°C on a 1.7 µm, 2.1 × 100 mm, Acquity UPLC™ BEH C18 column (Waters Corporation, Milford, MA, USA). Mobile phase A consisted of 0.1% formic acid in water; mobile phase B consisted of 0.1% formic acid in acetonitrile. Samples were separated by gradient elution using the following program: 0.0–0.2 min, 5% B; 0.2–9.5 min, 5–35% B; 9.5–9.6 min, 35–95% B; 9.6–10.6 min, 95% B; 10.6–10.7 min, 95–5% B; 12.0 min, stop. The flow rate was 600 µl/min. The sample injection volume was 2 µl. Mass spectrometer parameters employed for detection of all analytes and IS detection were as follows: ionspray voltage (IS) +4000 V, temperature 600°C, nebulizer gas (GS1) 40, TurboIonSpray gas (GS2) 50, collision-activated dissociation gas 8, curtain gas 30, dwell time 50 ms and entrance potential 10 V. Analyte-specific mass spectrometer parameters were as follows: Peptide 1 analyte and SIL IS: declustering potential +50 V, collision energy +38 V and collision exit potential +5 V; Peptide 2 analyte and SIL IS: declustering potential +100 V, collision energy +40 V and collision exit potential +20 V. The SRM transitions used to monitor Peptides 1, 2 and the corresponding SIL IS peptides are shown in Table 1.
and precision statistics for QC samples were calculated using Watson LIMS.
LC–MS/MS data acquisition, processing & sample quantification
QCs
Analyst software (Version 1.5.1) was used for raw LC–MS/MS data acquisition and chromatogram processing. Data regression using peak area ratios of the analyte to the IS was carried out using Watson LIMS (version 7.3; Thermo Fisher Scientific Inc., MA, USA). Calibration curves were constructed using peak area ratios of the calibration standards by applying a linear, 1/x² weighted, least-squares regression algorithm. All calibration curves, QCs and study sample concentrations were then calculated from their peak area ratios against the calibration line. Mean accuracy
The predicted concentrations of at least two-thirds of all analytical QC samples (i.e., all QCs except for the LLOQ QC, 5 µg/ml) had to be within ±15.0% of their nominal concentrations. At least 50% of the analytical QC samples and their mean had to be within ±15.0% of their nominal concentrations at each level. The predicted concentrations of at least 50% of the LLOQ QC samples and their mean had to be within ±20.0% of the nominal concentration. Additionally, %CV values for all analytical QC levels could not exceed 15.0% (20.0% for the LLOQ QC level).
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Conduct of & acceptance criteria for precision & accuracy runs
The exploratory universal IgG4 surrogate peptide assay was subjected to three precision and accuracy runs. Calibration curves were extracted and injected at the beginning and end of each run. Seven QC levels were evaluated (n = 6 replicates at each level). Acceptance criteria were as follows: Calibration standards
The predicted concentrations of at least three-quarters of all calibration standards had to be within ±15.0% of nominal values with the exception of the LLOQ, which had to be within ±20.0%. At least 50% of the calibration standards at all concentration levels had to meet the above criteria. Standards not meeting these criteria were excluded from the regression. At least one replicate of the lowest concentration in the standard curve had to meet the above criteria and be included within the final regression for that level to qualify as the LLOQ. If this criterion was not met, the next standard level was subjected to the same test and the LLOQ was raised accordingly.
Intra-run
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Evaluation of an exploratory universal LC–MS/MS assay
Inter-run
The mean predicted analytical QC concentrations across all three precision and accuracy runs had to be within ±15.0% of their nominal concentrations (±20.0% for the LLOQ QC) and the %CV of the mean predicted analytical QC concentrations could not exceed 15.0% (20.0% for the LLOQ QC). Conduct of & acceptance criteria for matrix sample stability experiments
Independent mAb 1 matrix QC samples sets (n = 3 replicates; 15.0, 60.0 and 400 µg/ml concentrations) were prepared and subjected to the following stresses: three freeze–thaw cycles, 26 h at room temperature and 29 days at -70°C. Stressed QCs were extracted and quantified against freshly prepared and extracted calibration curves. Stability for a given stress condition was established if the mean observed concentration of the stressed samples was within ±15.0% of the nominal concentration. Evaluation of assay performance in the presence of anti-mAb 1 antibodies
A set of blank human serum samples were spiked with mAb 1 (20 µg/ml), as well as 0–500 µg/ml of cynomolgus monkey affinity-purified polyclonal antiserum (anti-mAb1 antibodies). A second set of human serum samples was spiked with mAb 1 (40 µg/ml), as well as 0–800 µg/ml of mAb 3 (mouse monoclonal anti-mAb1 antibodies). Details of polyclonal and mAb preparation, purification and characterization will be reported separately. Samples were extracted in triplicate and quantified against a mAb 1 calibration curve prepared in human serum. Results & discussion Limitations of the existing universal LC–MS/MS assay approach to mAb quantification
The heretofore described universal LC–MS/MS assay approach to mAb quantification is limited to animal studies (Figure 1A) and is not applicable to human studies [7,9] . This limitation is related to assay specificity – the universal surrogate peptide sequences are present in both human therapeutic mAbs and endogenous human antibodies present in human matrix samples, thus disqualifying these universal peptides from use in quantification of human therapeutic mAbs in human samples. Based upon these assay selectivity considerations, signature surrogate peptides from the variable regions of the human mAb light and/or heavy chains must be used for LC–MS/MS quantification of human mAb analytes in human samples (Figure 1B) .
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Preliminary Communication
Key Terms Affinity-purified polyclonal antiserum: Highly purified preparation of antidrug antibodies harvested by affinity purification from the serum of immunized animals.
Potential expansion of the universal LC–MS/MS assay concept into human studies
Human IgG4 subclass antibodies are distinct from other subclasses in that they exhibit the inherit capacity to exchange half-molecules (Figure 2) [14] . This phenomenon, commonly called ‘Fab-arm exchange’, occurs both in vitro [14,15] and in vivo [16,17] . The exchange can occur between: two human IgG4-based therapeutic mAb molecules; two endogenous human IgG4 antibody molecules; or between human IgG4-based therapeutic mAb molecules and endogenous human IgG4 antibody molecules upon administration of the therapeutic mAb to human subjects. Fab-arm interchange is potentially undesirable from a drug-stability and an efficacy perspective. Angal et al. [18] and others [16,17] have shown that incorporation of a single amino substitution – serine to proline at position 241 (Kabat numbering [12]) – into the hinge region of human IgG4 mAbs substantially reduces Fab-arm exchange. This structure-stabilizing substitution has subsequently been incorporated into many human IgG4-based therapeutic mAb drugs and drug candidates [10,16] . The S241P amino acid substitution creates a predicted tryptic peptide with an amino acid sequence and mass that is distinct from the corresponding sequence in the endogenous human IgG4 antibodies present in clinical plasma/serum samples (Figure 3) . We reasoned that a single LC–MS/MS assay based upon a tryptic peptide that contains the S241P substitution as the surrogate peptide (Peptide 1; Figure 3 and Table 1) should in theory enable quantification of all hinge region-stabilized (S241P-containing) human IgG4 mAbs, free from interference from the large excess of endogenous IgG4 antibodies that would be present in human matrix samples, thus rendering the assay ‘universal’ for this class of human therapeutic mAb drugs and drug candidates. Development & evaluation of an exploratory universal IgG4 clinical LC–MS/MS assay
An exploratory universal IgG4 LC–MS/MS assay based upon Peptide 1 was developed in human serum using mAb 1 as a model hinge region-stabilized human IgG4 therapeutic mAb. The assay was subjected to three method performance evaluation runs. As shown in Table 2, acceptance criteria were met for precision and accuracy. Representative chromatograms for Peptide 1 are shown in Figure 4. Calibration curve
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Human IgG4 therapeutic mAb
Fab-arm exchange
Hybrid IgG4 antibodies Endogenous human IgG4 antibodies Figure 2. IgG4-based human therapeutic monoclonal antibodies and endogenous IgG4 human antibodies undergo ‘Fab-arm exchange’ in vitro and in vivo. Shown is an exchange between an IgG4-based therapeutic mAb and an endogenous IgG4 human antibody; this exchange occurs in the bloodstream after administration of human IgG4-based mAbs to human subjects. mAb: Monoclonal antibody.
data and representative calibration curves are provided in Supplementary Figure 1 & Supplementary Table 1. mAb 1 matrix stability experiments were also performed using Peptide 1 as the surrogate peptide. Matrix stability was demonstrated for three freeze–thaw cycles, 26 h at room temperature and 29 days at -70°C. Reliability evaluation of the exploratory universal IgG4 LC–MS/MS assay: quantitative comparison with a confirmatory surrogate peptide
To further evaluate the reliability of the universal IgG4 assay, a second confirmatory ‘signature’ surrogate peptide (Peptide 2; Table 1), located in the variable Endogenous human plasma/serum IgG4
Human IgG4 therapeutic mAb analyte
Serine-Proline substitution
YGPPCPSCPAPEFLGGPSVFLFPPKPK (average mass: 2830.4) YGPPCPPCPAPEFLGGPSVFLFPPKPK (average mass: 2840.4) Figure 3. A single amino substitution (S241P) is deployed in the hinge region of many human IgG4-based therapeutic monoclonal antibodies. Distinct predicted peptide sequences arising from trypsin digestion of S241P-stabilized human IgG4 therapeutic mAbs and endogenous human IgG4 antibodies are also shown. mAb: Monoclonal antibody. Please see colour figure at www.future-science.com/doi/full/10.4155/ BIO.14.64
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region of the heavy chain of mAb 1, was incorporated into the universal IgG4 LC–MS/MS assay. Representative chromatograms for Peptide 2 are shown in Figure 5. Precision and accuracy acceptance criteria were met for Peptide 2 (Table 2) . Peptide 1 and Peptide 2 QC datasets were compared using an incurred samples reanalysis-based calculation [9,19] . Very good quantitative agreement between the two peptides was observed in all three precision and accuracy runs, Table 3 and Supplementary Tables 2A–C . These data indicate that quantitative data obtained with the universal IgG4 LC–MS/MS assay would be reliable, even though the universal assay relies upon a single surrogate peptide (Peptide 1). The confirmatory peptide contains a methionine residue, which could potentially undergo oxidation during calibration curve and QC samples preparation, extraction and analysis. This possibility was not directly assessed. However, a SIL mAb protein was deployed as the analytical IS in these studies. Any confirmatory peptide methionine oxidation that may occur in each sample would be compensated for by oxidation to an equivalent extent in the corresponding labeled methionine of the mAb protein IS. Therefore, quantitative data from the confirmatory peptide can be considered reliable in the context of these studies. Evaluation of the exploratory universal IgG4 LC–MS/MS assay with simulated human serum study samples: anti-mAb 1 antibodies do not interfere with the universal IgG4 LC–MS/MS assay
The administration of protein drugs to humans or animals can elicit the production of antidrug
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Evaluation of an exploratory universal LC–MS/MS assay
Preliminary Communication
Table 2. Intra- and inter-day QC accuracy and precision summary for surrogate peptides. Surrogate peptide
Nominal mAb concentration (µg/ml)
Intra-day (n = 6)
Inter-day (n = 18: 3 days × 6 replicates)
Mean (µg/ml) %DIFF
CV (%)
Mean (µg/ml) %DIFF
CV (%)
Universal IgG4 peptide (Peptide 1)
5.00
5.31
6.2
4.7
5.29
5.8
5.3
15.0
15.3
2.0
2.3
15.7
4.7
3.8
25.0
26.5
6.2
2.1
26.8
7.2
3.3
60.0
63.9
6.5
2.1
64.2
6.9
3.2
125
125
0.0
2.4
129
3.0
4.3
250
246
-1.6
3.0
248
-0.7
5.1
400
363
-9.3
4.3
369
-7.8
7.3
Confirmatory peptide 5.00 (Peptide 2) 15.0
5.04
0.8
3.4
5.11
2.2
6.8
14.4
-4.1
6.7
15.2
1.0
7.6
25.0
25.3
1.0
4.2
25.5
2.1
4.2
60.0
63.8
6.3
4.7
62.7
4.6
4.8
125
125
-0.3
1.7
126
0.8
3.4
250
252
0.9
3.6
257
2.7
3.3
400
410
2.6
4.6
405
1.3
3.5
DIFF: Percentage difference from the nominal concentration.
antibodies (ADA). ADA are typically polyclonal (heterogeneous) in nature, that is, they can bind to multiple unique regions of the protein drug (Figure 6) . The binding of ADA to protein drugs in study samples can potentially interfere with accurate bioana lysis of the protein drug, particularly if ligand binding assays (LBA) are deployed [6,20,21] . The susceptibility of LBA formats to ADA interference is due to their reliance upon binding interactions between the protein analyte and LBA reagents. ADA present in study samples can potentially prevent LBA reagents from binding to the protein analyte, thus impacting LBA reliability (Figure 6) . In contrast, typical LC–MS ana lysis-enabling sample-processing procedures can disrupt ADA–protein interactions, rendering LC–MS assays potentially less susceptible to ADA-mediated assay interference (Figure 6) [22] . In order to evaluate potential ADA impacts on quantification of clinical samples using the universal IgG4 LC–MS/MS assay, blank human serum samples were co-spiked with mAb 1 and either affinity-purified polyclonal antisera raised in monkeys against mAb 1 or mAb 3, a mAb that binds to the target binding region of mAb 1 and interferes with the ability of mAb 1 to bind to its drug target. As shown in Figure 7A & B, the presence of up to 25-fold molar excess of ADA did not adversely impact the quantification of mAb 1 in the samples. These results suggest that a fully validated version of this universal IgG4 LC–MS/MS assay should be able to support reliable
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quantification of hinge region-stabilized human IgG4 therapeutic mAbs in human samples, even if ADA are present. The LLOQ for the exploratory universal IgG4 assay described herein was 5 µg/ml, which may be of insufficient sensitivity to support clinical studies in many cases. Preliminary experiments indicate the LLOQ of this assay could be lowered by approximately tenfold if the serum tryptic digests quantified in these preliminary studies were subjected to solid-phase extraction prior to LC–MS/MS analysis. Conclusion We have developed and evaluated a single exploratory LC–MS/MS assay that is potentially capable of quantifying all hinge region-stabilized human IgG4 therapeutic mAb drugs and drug candidates in human plasma/serum samples. The assay uses a single ‘universal IgG4’ surrogate peptide that was chosen based upon the incorporation of a single amino acid substitution into the hinge region of many human IgG4-based therapeutic mAbs. The assay was developed and evaluated using a model hinge region-staKey Terms Antidrug antibodies: Antibodies often produced by the immune systems of humans or animals in response to dosing of drugs or drug candidates. Antidrug antibodies are typically polyclonal (heterogeneous) in nature; they can bind to multiple unique regions of the drug.
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A 2.5e2
Intensity (cps)
Intensity (cps)
6.9e2
8.2 B
8.4 Time (min)
8.6
2.9e2
8.4 Time (min)
8.6
8.2
8.4 Time (min)
8.6
8.2
8.4 Time (min)
8.6
5.4e4 Intensity (cps)
Intensity (cps) C
8.2
8.2
8.4 Time (min)
8.6
3.0e3
Intensity (cps)
Intensity (cps)
5.6e4
8.2
8.4 Time (min)
8.6
Figure 4. Representative Peptide 1 (Universal IgG4 peptide) LC–MS/MS chromatograms. (A) Blank human serum; (B) blank human serum spiked with monoclonal antibody 2-IS; and (C) serum spiked with monoclonal antibody 1 at the LLOQ (5 µg/ml) and monoclonal antibody 2-IS. Left panels: analytes; right panels: IS. The SRM transitions monitored were 985.3→912.1 and 990.6→917.4 for Peptide 1 analyte and IS, respectively.
bilized human IgG4 therapeutic mAb. Precision and accuracy of the assay was demonstrated using three performance evaluation runs. Reliability of the assay was further demonstrated by quantitative comparison between the universal IgG4 surrogate peptide and an independent surrogate peptide in the same mAb analyte. The assay was successfully applied to the quantification of the model human IgG4 therapeutic mAb in simulated human serum study samples that contained ADA. Based on the results of these exploratory studies, the assay is expected to be capable, after refinement as needed and full validation, of providing reliable hinge region-stabilized human IgG4 mAb concentrations, with little or no anticipated interferences from ADA that may be present in clinical study samples.
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Future perspective LC–MS/MS is a useful platform for the bioanalysis of therapeutic mAbs. LC–MS/MS assays used for bioanalysis of human therapeutic mAbs frequently rely upon quantification of a ‘signature’ surrogate peptide whose amino acid sequence is unique to the protein analyte of interest. The signature peptide approach can be time-, labor- and cost-intensive due to the fact that a new LC–MS/MS assay must be developed for each new mAb that requires bioanalytical support during drug development. These challenges can, in principle, be substantially overcome by the deployment of universal assays capable of quantifying a variety of human therapeutic mAb candidates. We have recently developed and reported a single universal assay that is capable of
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Evaluation of an exploratory universal LC–MS/MS assay
Preliminary Communication
A 2.6e2
Intensity (cps)
Intensity (cps)
6.9e2
5.2 B
5.4 Time (min)
5.6
5.2
5.4 Time (min)
5.6
5.2
5.4 Time (min)
5.6
5.2
5.4 Time (min)
5.6
3.8e4
Intensity (cps)
Intensity (cps)
2.1e2
5.2 C
5.4 Time (min)
5.6
1.3e3
Intensity (cps)
Intensity (cps)
4.4e4
5.2
5.4 Time (min)
5.6
Figure 5. Representative Peptide 2 (confirmatory peptide) LC–MS/MS chromatograms. (A) Blank human serum; (B) blank human serum spiked with monoclonal antibody 2-IS; and (C) serum spiked with monoclonal antibody 1 at the LLOQ (5 µg/ml) and monoclonal antibody 2-IS. Left panels: analytes; right panels: IS. The SRM transitions monitored were 825.5→1073.5 and 830.5→1083.5 for Peptide 2 analyte and IS, respectively.
quantifying a variety of human therapeutic mAbs in animal studies [7,9] . In this report, we have carried out exploratory studies based upon a novel universal IgG4based peptide, which supports the possible extension
of the universal assay concept into the clinical realm. As a consequence of their applicability to variety of mAbs, these two universal assays will be very useful to bioanalytical scientists supporting both nonclinical
Table 3. Quantitative comparison of universal IgG4 peptide and confirmatory peptide. Analytical run
Percentage of individual QC replicates with percentage difference ≤20%†‡
Mean percentage difference §
1
95.2
-0.337
2
97.6
-1.19
3
97.6
-0.0294
See Supplementary Table 2A–C for individual QC replicate percentage difference values. N = 42 QC samples from each run (6 replicates × 7 QC concentration levels) were compared. ‡ Percentage difference = (confirmatory peptide observed concentration - universal IgG4 peptide observed concentration)/(mean of the two values) × 100. § Mean percentage difference computed as the overall mean of the individual percentage difference values from each run.
†
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Preliminary Communication Furlong, Titsch, Xu, Jiang, Jemal & Zeng
mAb
1. Protein(s) denaturation 2. Disulfide bond reduction/alkylation 3. Trypsin digestion
Tryptic peptides
ADA
LC–MS/MS quantification of surrogate peptide
X LBA assay reagent
Figure 6. Antidrug antibody binding to protein drugs (e.g., monoclonal antibodies) in vivo – potential impact on bioanalysis. ADA can potentially prevent ligand binding assay reagents from binding to the protein drug analyte, thus potentially impacting ligand binding assay performance. In contrast, typical LC–MS analysis-enabling sample processing procedures can disrupt ADA–protein drug interactions, rendering the LC–MS assay less susceptible to ADA-mediated assay interference. ADA: Antidrug antibody; LBA: Ligand binding assays; mAb: Monoclonal antibody.
and clinical studies involving a diversity of therapeutic mAb drug candidates.
Observed mAb 1 concentration (µg/ml)
A 20
10 Spiked polyclonal ADA (µg/ml)
Observed mAb 1 concentration (µg/ml)
0
500
40
The authors acknowledge Martin Corbett, Mian Gao, Lin Cheng and Yongmi An (Bristol-Myers Squibb, NJ, USA) for production of the stable-isotope labeled antibody mAb 2. Kimberly Voronin (Bristol-Myers Squibb) is acknowledged for production of peptide used for method development. Roger Demers (Tandem Labs, a Labcorp Company, NJ, USA) is acknowledged for preliminary method development experiments. Qin Ji and Mark E Arnold (Bristol-Myers Squibb) are acknowledged for careful review of and comments on this manuscript.
Financial & competing interests disclosure
20 Spiked monoclonal ADA (µg/ml) 0
0
800
Figure 7. Impact assessment of antidrug antibodies on the performance of the exploratory universal IgG4 LC–MS/MS assay. (A) Blank human serum was spiked with mAb 1 (20 µg/ml) along with a series of concentrations (0–500 µg/ml) of affinity purified cynomolgus monkey polyclonal anti-mAb 1 antiserum. (B) Blank human serum was spiked with mAb 1 (40 µg/ml) along with a series of concentrations (0–800 µg/ml) mAb 3, a mouse monoclonal antibody that binds to the target binding region of mAb 1. Samples were extracted and quantified in triplicate by LC–MS/MS. Data plotted are mean concentrations. CV values for all replicate determinations were under 3%. ADA: Antidrug antibody; mAb: Monoclonal antibody.
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To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/full/10.4155/BIO.14.64
Acknowledgements
0 B
Supplementary data
Bioanalysis (2014) 6(13)
All of the authors are current or former employees of Bristol-Myers Squibb. The authors have no other 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 apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations.
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Evaluation of an exploratory universal LC–MS/MS assay
Preliminary Communication
Executive summary Development & evaluation of an exploratory universal IgG4 clinical LC–MS/MS assay • An exploratory universal LC–MS/MS assay has been developed to potentially enable reliable quantification of many human IgG4-based therapeutic monoclonal antibodies (mAbs) in clinical studies. • The exploratory universal IgG4 assay is based upon quantification of a mAb-specific universal surrogate tryptic peptide found in the constant region of IgG4-based therapeutic mAbs that contain a structurally stabilizing serine to proline substitution in the heavy chain constant region.
Reliability evaluation of the exploratory universal IgG4 LC–MS/MS assay: quantitative comparison with a confirmatory surrogate peptide • Three method performance evaluation runs in human serum matrix demonstrated the accuracy and precision of the exploratory assay. • Reliability of the exploratory assay was confirmed by quantitative comparison with data from an independent confirmatory peptide in the same mAb analyte.
Evaluation of the exploratory universal IgG4 LC–MS/MS assay with simulated human serum study samples: anti-mAb 1 antibodies do not interfere with the assay • The exploratory assay was shown to perform well even in the presence of antidrug antibodies, which can often be present in clinical study samples.
References
8
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).
•
Describes the first application of the universal surrogate peptide to support bioanalysis of a human monoclonal antibody-based analyte in an animal PK study. The paper also details the first application of pellet digestion as a simple and efficient sample preparation technique.
9
Furlong MT, Zhao S, Mylott W et al. Dual universal peptide approach to bioanalysis of human monoclonal antibody protein drug candidates in animal studies. Bioanalysis 5(11), 1363–1376 (2013).
•
Desribes the second generation ‘dual’ universal peptide approach, wherin a light chain constant region-based universal peptide has been incorporated into the original universal LC–MS/MS assay.
Papers of special note have been highlighted as: • of interest •• of considerable interest 1
Beck A, Wagner-Rousset E, Bussat M-C et al. Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. Curr. Pharm. Biotechnol. 9(6), 482–501 (2008).
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Strohl WR. Therapeutic monoclonal antibodies: past, present, and future. In: Therapeutic Monoclonal Antibodies: From Bench to Clinic. An Z (Ed.). John Wiley & Sons, Inc., Hoboken, NJ, USA, 3–50 (2009).
3
Beck A, Wurch T, Bailly C, Corvaia N. Strategies and challenges for the next generation of therapeutic antibodies. Nat. Rev. Immunol. 10(5), 345–352 (2010).
4
Nelson AL, Dhimolea E, Reichert JM. Development trends for human monoclonal antibody therapeutics. Nat. Rev. Drug Discov. 9(10), 767–774 (2010).
5
Halquist MS, Thomas Karnes H. Quantitative liquid chromatography–tandem mass spectrometry analysis of macromolecules using signature peptides in biological fluids. Biomed. Chromatogr. 25(1–2), 47–58 (2011).
10
Immunogenetics Information System www.imgt.org
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ExPASy. Bioinformatics Resource Portal. www.expasy.org
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Ezan E, Bitsch F. Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodies. Bioanalysis 1(8), 1375–1388 (2009).
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•
Reviews the relative strengths and weaknesses of ligand binding assays and LC–MS-based bioanalysis of therapeutic monoclonal antibodies.
Kabat E, Wu TT, Reid-Miller M, Perry HM, Gottesman KS. Sequences of Proteins of Immunological Interest. United States Department of Health and Human Services, Washington DC, USA (1987).
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Furlong MT, Ouyang Z, Wu S et al. A universal surrogate peptide to enable LC–MS/MS bioanalysis of a diversity of human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies. Biomed. Chromatogr. 26(8), 1024–1032 (2012).
Jiang H, Zeng J, Titsch C et al. Fully validated LC–MS/MS assay for the simultaneous quantitation of coadministered therapeutic antibodies in cynomolgus monkey serum. Anal. Chem. 85(20), 9859–9867 (2013).
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King DJ, Adair JR, Angal S et al. Expression, purification and characterization of a mouse-human chimeric antibody and chimeric Fab’ fragment. Biochem. J. 281(2), 317–323 (1992).
•
Describes the in vitro Fab-arm exchange between human IgG4 antibodies.
15
Deng L, Wylie D, Tsao Yung S et al. Detection and quantification of the human IgG4 half-molecule, HL,
7
•• Describes the universal peptide concept along with the identification/characterization of the original universal peptide located in the heavy chain Fc region of many human monoclonal antibody drugs/drug candidates.
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Preliminary Communication Furlong, Titsch, Xu, Jiang, Jemal & Zeng GLP bioanalysis: assay reproducibility for incurred samples – implications of Crystal City recommendations. AAPS J. 11(2), 238–241 (2009).
from unpurified cell-culture supernatants. Biotechnol. Appl. Biochem. 40(Pt 3), 261–269 (2004). 16
•
Describes the in vivo Fab-arm exchange between human IgG4 therepeutic mAbs and endogenous human IgG4 antibodies.
17
Stubenrauch K, Wessels U, Regula JT, Kettenberger H, Schleypen J, Kohnert U. Impact of molecular processing in the hinge region of therapeutic IgG4 antibodies on disposition profiles in cynomolgus monkeys. Drug Metab. Dispos. 38(1), 84–91 (2010).
18
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Labrijn AF, Buijsse AO, van den Bremer ETJ et al. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo. Nat. Biotechnol. 27(8), 767–771 (2009).
Angal S, King DJ, Bodmer MW et al. A single amino acid substitution abolishes the heterogeneity of chimeric mouse/ human (IgG4) antibody. Mol. Immunol. 30(1), 105–108 (1993).
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Demonstrates that a single engineered serine to proline substitution in the hinge region of human IgG4 antibodies significantly reduces the Fab-arm exchange reaction.
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Fast DM, Kelley M, Viswanathan CT et al. Workshop report and follow-up – AAPS workshop on current topics in
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White JT, Golob M, Sailstad J. Understanding and mitigating impact of immunogenicity on pharmacokinetic assays. Bioanalysis 3(16), 1799–1803 (2011).
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Describes the problem of of antidrug–antibody-mediated interference with ligand binding assay-based bioanalysis. Approaches to the identification and mitigation of antidrug–antibody interferences are also presented.
21
Kelley M, Ahene AB, Gorovits B et al. Theoretical considerations and practical approaches to address the effect of anti-drug antibody (ada) on quantification of biotherapeutics in circulation. AAPS J. 15(3), 646–658 (2013).
22
Wang SJ, Wu ST, Gokemeijer J et al. Attribution of the discrepancy between ELISA and LC–MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies. Anal. Bioanal. Chem. 402(3), 1229–1239 (2012).
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An exploratory universal LC–MS/MS assay for bioanalysis of hinge region-stabilized human IgG4 mAbs in clinical studies Background: Due to the increasing number of monoclonal antibody (mAb) drug candidates entering clinical development, bioanalytical laboratories can benefit from generic liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays capable of quantifying a variety of human mAb-based therapeutic drug candidates in plasma/serum samples from clinical studies. Results: We have developed and evaluated an exploratory LC–MS/MS assay capable of quantifying hinge region-stabilized IgG4 therapeutic mAb drugs and drug candidates in clinical samples. The exploratory assay is based upon a single ‘universal IgG4’ surrogate peptide. Conclusion: The novel exploratory LC–MS/MS assay reported herein, upon further refinement and full validation, is predicted to enable bioanalytical scientists to quantify all hinge region-stabilized human IgG4 therapeutic mAbs in human studies without having to develop a new assay for every new stabilized IgG4 mAb entering clinical development.
Monoclonal antibody (mAb)-based therapeutic agents occupy an increasingly important role in the treatment of a variety of human diseases [1–4] . The growing number of mAbs entering drug development represents a significant challenge to bioanalytical laboratories engaged in the quantification of these candidates in biological matrices. Bioanalysis of human therapeutic mAbs by LC–MS/MS is typically based upon quantification of ‘signature’ surrogate peptides whose amino acid sequences are unique to the protein analyte of interest [5,6] . Signature peptides are found in the antibody variable regions of the human therapeutic mAb light and heavy chains. The signature surrogate peptide approach is labor-intensive, particularly in the realm of assay development and validation, wherein a new signature surrogate peptide must be identified and a new assay must be developed for each human therapeutic mAb candidate entering development. To address this challenge for nonclinical studies, we have previously developed and successfully deployed a LC–MS/MS assay that relies upon ‘universal’ surrogate peptides rather than ‘signature’ surrogate peptides [7–9] . These universal peptides possess four key attributes necessary for a single universal peptide LC–MS/MS assay capable of
10.4155/BIO.14.64 © 2014 Future Science Ltd
Michael T Furlong*‡1,2, Craig Titsch‡1, Weifeng Xu1, Hao Jiang1, Mohammed Jemal3 & Jianing Zeng1 1 Bristol-Myers Squibb, R&D, Route 206 & Province Line Road, Princeton, NJ 08543, USA 2 Current: Molecular & Bioanalytical Pharmacology Amicus Therapeutics 3 931 Edinburg Road, Hamilton Square, NJ 08690, USA *Author for correspondence: Tel.: +1 609 662 2071 [email protected] ‡ These authors contributed equally
quantifying a diversity of human therapeutic mAbs in animal PK/TK studies: • Universality: the sequences are present in the constant regions of human therapeutic mAb candidates, thus enabling their general applicability across multiple mAb drug candidates and development programs (Figure 1A) ; • Selectivity: the sequences are not found in the antibody constant region or any other plasma/serum protein sequences present in any animal species commonly used in nonclinical drug development studies, thus ensuring quantification that is free of plasma protein-derived interferences (Figure 1A) ; • The sequences are reliably produced from trypsin digestion of human mAbs; • The sequences possess favorable LC–MS/MS characteristics, such as good chromatographic peak shape, adequate chromatographic retention and efficient ionization. The amino acid sequences of the universal surrogate peptides identified and used in
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Key Terms Surrogate peptides: Peptide derived from proteolytic digestion of a protein analyte. The peptide is quantified by LC–MS/MS as a ‘surrogate’ for the protein analyte. Antibody variable regions: These regions are found on the amino terminal portion of antibody light and heavy chains. The amino acid sequences of the variable regions vary greatly from antibody to antibody. Signature surrogate peptide: Surrogate peptide that is unique to the protein analyte of interest. In monoclonal antibodies, signature surrogate peptides can be found in the variable regions of either the light or heavy chains. Universal peptide LC–MS/MS assay: Single LC–MS/ MS assay capable of bioanalytical support of a diversity of proteins that contain a shared universal surrogate peptide. Antibody constant region: These regions are found on the carboxy terminal portion of antibody light and heavy chains. The amino acid sequences of the constant regions are identical across all antibodies within a species and subclass. Universal surrogate peptides: Single surrogate peptide that is found in many variants of a particular therapeutic protein class. In monoclonal antibodies, universal surrogate peptides can be found in the constant regions of either the light or heavy chains.
our laboratories are as follows: light chain (k class) – TVAAPSVFIFPPSDEQLK – and heavy chain (IgG1 and IgG4 subclasses) – VVSVLTVLHQDWLNGK [7,9] . Preliminary experiments indicate that the closely related IgG2 variant of the heavy chain universal peptide (VVSVLTVVHQDWLNGK) could be incorporated into a universal LC–MS/MS assay to support bioanalysis of human IgG2-based therapeutic mAbs [7] . Currently, the universal peptide LC–MS/MS assay is applicable only to animal studies. Herein, we report the extension of the universal assay concept to potentially enable bioanalytical support of clinical studies. The new exploratory LC–MS/MS assay is based upon a single tryptic peptide that is located in the constant region of hinge region-stabilized IgG4-based human therapeutic mAb drugs and drug candidates, and is not present in the constant regions of endogenous antibodies present in the human plasma/serum samples. This single LC– MS/MS assay, upon further refinement and full validation, is predicted to enable reliable quantification of all hinge region-stabilized human IgG4 therapeutic mAb drug candidates in clinical PK studies. Experimental In silico analyses
Antibody sequences were downloaded from the Immunogenetics information system website [10] . In silico trypsin digests were carried out using the ExPASy Bioinfomatics Resource Portal [11] .
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mAbs & peptides
mAbs 1, 2 and 3 were produced at Bristol-Myers Squibb. mAb 1 is comprised of a human IgG4 subclass heavy chain and a human k-light chain. The heavy chain of mAb 1 contains a serine-to-proline amino acid substitution at position 241 (Kabat numbering [12]). mAb 2 is a stable isotope-labeled (SIL) version of mAb 1, at all arginine (13C615N4) and lysine (13C615N2) positions. mAb 2 served as the analytical IS for quantification of mAb 1. mAb 3 is a mouse mAb that binds to the variable region of mAb 1 and interferes with the ability of mAb 1 to bind to its pharmacological target. Peptides 1 and 2 used for method development were custom-synthesized at Bristol-Myers Squibb. The amino acid sequences of the peptides are shown in Table 1. Chemicals & reagents
Trypsin from bovine pancreas, iodoacetamide, dithiothreitol, HPLC grade acetonitrile, HPLC grade methanol and PBS were purchased from Sigma-Aldrich (St. Louis, MO, USA). Formic acid was purchased from EMD Chemicals (Billerica, MA, USA). Ammonium bicarbonate was purchased from MP Biomedicals (Solon, OH, USA). Calibration standards & QC samples
The mAb 1 calibration curve and QC samples were prepared by dilution of the mAb 1 stock solution (10 mg/ml) into blank human serum followed by further serial dilution in blank serum to obtain the desired final concentrations. Calibration standard concentrations were 5.00, 10.0, 25.0, 50.0, 100, 200, 450 and 500 µg/ml. QC concentrations were 5.00, 15.0, 25.0, 60.0, 125, 250 and 400 µg/ml. Sample preparation
Samples were processed using our previously reported ‘pellet digestion’ methodology in 96-well plates as follows: serum samples (25 µl) were fortified with 25 µl of IS working solution (mAb 2 at 200 µg/ml in PBS) [8,13] . Matrix blank samples were fortified with PBS instead of IS working solution. The mixtures were briefly vortexed and then centrifuged for 1 min at 200 g. Methanol (200 µl) was added to each well followed by vigorous vortexing for 2 min and centrifugation at 200 g for 2 min. Following removal of the supernatant, the pellets were suspended in 100 mM aqueous ammonium bicarbonate (50 µl) by vigorous vortexing for 2 min. The resulting suspensions were further fortified with 100 mM dithiothreitol (10 µl), followed by centrifugation at 200 g for 1 min, vigorous vortexing for 2 min and incubation for 1 h at 60°C in an Eppendorf Thermomixer® (NY, USA) at 1000 rpm.
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An exploratory universal LC–MS/MS assay
Human therapeutic mAb analyte
A
Variable region
Constant region
Constant region
Endogenous animal plasma/serum antibodies
{
{
Non-identical constant regions
Human therapeutic mAb analyte
B
Variable region
Preliminary Communication
Endogenous human plasma/serum antibodies
{
{
Identical constant regions
Figure 1. Surrogate peptide options for bioanalysis of human therapeutic monoclonal antibodies are based in part on the species of the matrix sample. (A) For LC–MS/MS quantification of human therapeutic mAb analytes in animal samples, universal surrogate peptides, located in the constant regions of the human mAb light and heavy chains, may be used as alternatives to signature surrogate peptides from the variable regions. The locations of universal surrogate peptides in human mAb analytes are depicted as green bars. (B) For LC–MS/MS quantification of human mAb analytes in human samples, signature surrogate peptides, located in the variable regions of the human mAb light and heavy chains, must be used. The locations of the signature surrogate peptides in human mAb analytes are depicted as yellow bars. mAb: Monoclonal antibody. Please see colour figure at www.future-science.com/doi/full/10.4155/BIO.14.64
Cysteine thiol alkylation was carried out by addition of 100 mM iodoacetamide (25 µl), centrifugation at 200 g for 1 min, vigorous vortexing for 2 min and incubation in the dark for 30 min at 30°C in a thermomixer (1000 rpm). Trypsin solution (25 µl of an 8.0 mg/ml solution in 0.1% aqueous formic acid) was added, followed by centrifugation at 200 g for 1 min, vigorous vortexing for 1 min and incubation in the dark for 30 min at 50°C in a thermomixer (1000 rpm). The digested samples were treated with 10% aqueous formic acid (25 µl), followed by centrifugation at 200 g for 1 min and vigorous vortexing for 2 min. A final
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high speed centrifugation (5000 g; 5 min) was carried out to pellet residual particulates. The supernatants (120 µl) were transferred to 96-well collection plates for LC–MS/MS analysis. Chromatographic & mass spectrometric conditions
Experiments were conducted using a Shimadzu Nexera HPLC system and SIL-30AC autosampler (Columbia, MD, USA) coupled to an AB Sciex API 5500 triple quadrupole mass spectrometer equipped with a TurboIonspray™ source (Concord, Ontario, Canada).
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Table 1. Amino acid sequences, locations and SRM transitions/charge states for surrogate peptide analytes and IS. Peptide
Sequence
Universal IgG4 peptide from heavy YGPPCPPCPAPEFLGGPSVFLFPPKPK chain constant region (Peptide 1) YGPPCPPCPAPEFLGGPSVFLFPPK‡PK‡ (unlabeled and labeled)† Confirmatory peptide from heavy chain variable region (Peptide 2) (unlabeled and labeled)
ASGITFSNSGMHWVR ASGITFSNSGMHWVR
§
Precursor ion mass (m/z units) and charge state
Product ion mass (m/z units) and charge state
985.3 [M+3H] 3+
912.1 [M+3H] 3+
990.6 [M+3H] 3+
917.4 [M+3H] 3+
825.5 [M+2H] 2+
1073.5 [MH] +
830.5 [M+2H]
1083.5 [MH] +
2+
The thiol groups on all cysteine residues in the Universal IgG4 peptide and IS were carboxamidated with iodoacetamide during sample preparation (see ‘Experimental’ section). ‡ Denotes lysine labeled with 13C615N2; used as IS. § Denotes arginine labeled with 13C615N4; used as IS. †
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Chromatographic separation was performed at 60°C on a 1.7 µm, 2.1 × 100 mm, Acquity UPLC™ BEH C18 column (Waters Corporation, Milford, MA, USA). Mobile phase A consisted of 0.1% formic acid in water; mobile phase B consisted of 0.1% formic acid in acetonitrile. Samples were separated by gradient elution using the following program: 0.0–0.2 min, 5% B; 0.2–9.5 min, 5–35% B; 9.5–9.6 min, 35–95% B; 9.6–10.6 min, 95% B; 10.6–10.7 min, 95–5% B; 12.0 min, stop. The flow rate was 600 µl/min. The sample injection volume was 2 µl. Mass spectrometer parameters employed for detection of all analytes and IS detection were as follows: ionspray voltage (IS) +4000 V, temperature 600°C, nebulizer gas (GS1) 40, TurboIonSpray gas (GS2) 50, collision-activated dissociation gas 8, curtain gas 30, dwell time 50 ms and entrance potential 10 V. Analyte-specific mass spectrometer parameters were as follows: Peptide 1 analyte and SIL IS: declustering potential +50 V, collision energy +38 V and collision exit potential +5 V; Peptide 2 analyte and SIL IS: declustering potential +100 V, collision energy +40 V and collision exit potential +20 V. The SRM transitions used to monitor Peptides 1, 2 and the corresponding SIL IS peptides are shown in Table 1.
and precision statistics for QC samples were calculated using Watson LIMS.
LC–MS/MS data acquisition, processing & sample quantification
QCs
Analyst software (Version 1.5.1) was used for raw LC–MS/MS data acquisition and chromatogram processing. Data regression using peak area ratios of the analyte to the IS was carried out using Watson LIMS (version 7.3; Thermo Fisher Scientific Inc., MA, USA). Calibration curves were constructed using peak area ratios of the calibration standards by applying a linear, 1/x² weighted, least-squares regression algorithm. All calibration curves, QCs and study sample concentrations were then calculated from their peak area ratios against the calibration line. Mean accuracy
The predicted concentrations of at least two-thirds of all analytical QC samples (i.e., all QCs except for the LLOQ QC, 5 µg/ml) had to be within ±15.0% of their nominal concentrations. At least 50% of the analytical QC samples and their mean had to be within ±15.0% of their nominal concentrations at each level. The predicted concentrations of at least 50% of the LLOQ QC samples and their mean had to be within ±20.0% of the nominal concentration. Additionally, %CV values for all analytical QC levels could not exceed 15.0% (20.0% for the LLOQ QC level).
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Conduct of & acceptance criteria for precision & accuracy runs
The exploratory universal IgG4 surrogate peptide assay was subjected to three precision and accuracy runs. Calibration curves were extracted and injected at the beginning and end of each run. Seven QC levels were evaluated (n = 6 replicates at each level). Acceptance criteria were as follows: Calibration standards
The predicted concentrations of at least three-quarters of all calibration standards had to be within ±15.0% of nominal values with the exception of the LLOQ, which had to be within ±20.0%. At least 50% of the calibration standards at all concentration levels had to meet the above criteria. Standards not meeting these criteria were excluded from the regression. At least one replicate of the lowest concentration in the standard curve had to meet the above criteria and be included within the final regression for that level to qualify as the LLOQ. If this criterion was not met, the next standard level was subjected to the same test and the LLOQ was raised accordingly.
Intra-run
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Evaluation of an exploratory universal LC–MS/MS assay
Inter-run
The mean predicted analytical QC concentrations across all three precision and accuracy runs had to be within ±15.0% of their nominal concentrations (±20.0% for the LLOQ QC) and the %CV of the mean predicted analytical QC concentrations could not exceed 15.0% (20.0% for the LLOQ QC). Conduct of & acceptance criteria for matrix sample stability experiments
Independent mAb 1 matrix QC samples sets (n = 3 replicates; 15.0, 60.0 and 400 µg/ml concentrations) were prepared and subjected to the following stresses: three freeze–thaw cycles, 26 h at room temperature and 29 days at -70°C. Stressed QCs were extracted and quantified against freshly prepared and extracted calibration curves. Stability for a given stress condition was established if the mean observed concentration of the stressed samples was within ±15.0% of the nominal concentration. Evaluation of assay performance in the presence of anti-mAb 1 antibodies
A set of blank human serum samples were spiked with mAb 1 (20 µg/ml), as well as 0–500 µg/ml of cynomolgus monkey affinity-purified polyclonal antiserum (anti-mAb1 antibodies). A second set of human serum samples was spiked with mAb 1 (40 µg/ml), as well as 0–800 µg/ml of mAb 3 (mouse monoclonal anti-mAb1 antibodies). Details of polyclonal and mAb preparation, purification and characterization will be reported separately. Samples were extracted in triplicate and quantified against a mAb 1 calibration curve prepared in human serum. Results & discussion Limitations of the existing universal LC–MS/MS assay approach to mAb quantification
The heretofore described universal LC–MS/MS assay approach to mAb quantification is limited to animal studies (Figure 1A) and is not applicable to human studies [7,9] . This limitation is related to assay specificity – the universal surrogate peptide sequences are present in both human therapeutic mAbs and endogenous human antibodies present in human matrix samples, thus disqualifying these universal peptides from use in quantification of human therapeutic mAbs in human samples. Based upon these assay selectivity considerations, signature surrogate peptides from the variable regions of the human mAb light and/or heavy chains must be used for LC–MS/MS quantification of human mAb analytes in human samples (Figure 1B) .
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Preliminary Communication
Key Terms Affinity-purified polyclonal antiserum: Highly purified preparation of antidrug antibodies harvested by affinity purification from the serum of immunized animals.
Potential expansion of the universal LC–MS/MS assay concept into human studies
Human IgG4 subclass antibodies are distinct from other subclasses in that they exhibit the inherit capacity to exchange half-molecules (Figure 2) [14] . This phenomenon, commonly called ‘Fab-arm exchange’, occurs both in vitro [14,15] and in vivo [16,17] . The exchange can occur between: two human IgG4-based therapeutic mAb molecules; two endogenous human IgG4 antibody molecules; or between human IgG4-based therapeutic mAb molecules and endogenous human IgG4 antibody molecules upon administration of the therapeutic mAb to human subjects. Fab-arm interchange is potentially undesirable from a drug-stability and an efficacy perspective. Angal et al. [18] and others [16,17] have shown that incorporation of a single amino substitution – serine to proline at position 241 (Kabat numbering [12]) – into the hinge region of human IgG4 mAbs substantially reduces Fab-arm exchange. This structure-stabilizing substitution has subsequently been incorporated into many human IgG4-based therapeutic mAb drugs and drug candidates [10,16] . The S241P amino acid substitution creates a predicted tryptic peptide with an amino acid sequence and mass that is distinct from the corresponding sequence in the endogenous human IgG4 antibodies present in clinical plasma/serum samples (Figure 3) . We reasoned that a single LC–MS/MS assay based upon a tryptic peptide that contains the S241P substitution as the surrogate peptide (Peptide 1; Figure 3 and Table 1) should in theory enable quantification of all hinge region-stabilized (S241P-containing) human IgG4 mAbs, free from interference from the large excess of endogenous IgG4 antibodies that would be present in human matrix samples, thus rendering the assay ‘universal’ for this class of human therapeutic mAb drugs and drug candidates. Development & evaluation of an exploratory universal IgG4 clinical LC–MS/MS assay
An exploratory universal IgG4 LC–MS/MS assay based upon Peptide 1 was developed in human serum using mAb 1 as a model hinge region-stabilized human IgG4 therapeutic mAb. The assay was subjected to three method performance evaluation runs. As shown in Table 2, acceptance criteria were met for precision and accuracy. Representative chromatograms for Peptide 1 are shown in Figure 4. Calibration curve
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Human IgG4 therapeutic mAb
Fab-arm exchange
Hybrid IgG4 antibodies Endogenous human IgG4 antibodies Figure 2. IgG4-based human therapeutic monoclonal antibodies and endogenous IgG4 human antibodies undergo ‘Fab-arm exchange’ in vitro and in vivo. Shown is an exchange between an IgG4-based therapeutic mAb and an endogenous IgG4 human antibody; this exchange occurs in the bloodstream after administration of human IgG4-based mAbs to human subjects. mAb: Monoclonal antibody.
data and representative calibration curves are provided in Supplementary Figure 1 & Supplementary Table 1. mAb 1 matrix stability experiments were also performed using Peptide 1 as the surrogate peptide. Matrix stability was demonstrated for three freeze–thaw cycles, 26 h at room temperature and 29 days at -70°C. Reliability evaluation of the exploratory universal IgG4 LC–MS/MS assay: quantitative comparison with a confirmatory surrogate peptide
To further evaluate the reliability of the universal IgG4 assay, a second confirmatory ‘signature’ surrogate peptide (Peptide 2; Table 1), located in the variable Endogenous human plasma/serum IgG4
Human IgG4 therapeutic mAb analyte
Serine-Proline substitution
YGPPCPSCPAPEFLGGPSVFLFPPKPK (average mass: 2830.4) YGPPCPPCPAPEFLGGPSVFLFPPKPK (average mass: 2840.4) Figure 3. A single amino substitution (S241P) is deployed in the hinge region of many human IgG4-based therapeutic monoclonal antibodies. Distinct predicted peptide sequences arising from trypsin digestion of S241P-stabilized human IgG4 therapeutic mAbs and endogenous human IgG4 antibodies are also shown. mAb: Monoclonal antibody. Please see colour figure at www.future-science.com/doi/full/10.4155/ BIO.14.64
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region of the heavy chain of mAb 1, was incorporated into the universal IgG4 LC–MS/MS assay. Representative chromatograms for Peptide 2 are shown in Figure 5. Precision and accuracy acceptance criteria were met for Peptide 2 (Table 2) . Peptide 1 and Peptide 2 QC datasets were compared using an incurred samples reanalysis-based calculation [9,19] . Very good quantitative agreement between the two peptides was observed in all three precision and accuracy runs, Table 3 and Supplementary Tables 2A–C . These data indicate that quantitative data obtained with the universal IgG4 LC–MS/MS assay would be reliable, even though the universal assay relies upon a single surrogate peptide (Peptide 1). The confirmatory peptide contains a methionine residue, which could potentially undergo oxidation during calibration curve and QC samples preparation, extraction and analysis. This possibility was not directly assessed. However, a SIL mAb protein was deployed as the analytical IS in these studies. Any confirmatory peptide methionine oxidation that may occur in each sample would be compensated for by oxidation to an equivalent extent in the corresponding labeled methionine of the mAb protein IS. Therefore, quantitative data from the confirmatory peptide can be considered reliable in the context of these studies. Evaluation of the exploratory universal IgG4 LC–MS/MS assay with simulated human serum study samples: anti-mAb 1 antibodies do not interfere with the universal IgG4 LC–MS/MS assay
The administration of protein drugs to humans or animals can elicit the production of antidrug
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Evaluation of an exploratory universal LC–MS/MS assay
Preliminary Communication
Table 2. Intra- and inter-day QC accuracy and precision summary for surrogate peptides. Surrogate peptide
Nominal mAb concentration (µg/ml)
Intra-day (n = 6)
Inter-day (n = 18: 3 days × 6 replicates)
Mean (µg/ml) %DIFF
CV (%)
Mean (µg/ml) %DIFF
CV (%)
Universal IgG4 peptide (Peptide 1)
5.00
5.31
6.2
4.7
5.29
5.8
5.3
15.0
15.3
2.0
2.3
15.7
4.7
3.8
25.0
26.5
6.2
2.1
26.8
7.2
3.3
60.0
63.9
6.5
2.1
64.2
6.9
3.2
125
125
0.0
2.4
129
3.0
4.3
250
246
-1.6
3.0
248
-0.7
5.1
400
363
-9.3
4.3
369
-7.8
7.3
Confirmatory peptide 5.00 (Peptide 2) 15.0
5.04
0.8
3.4
5.11
2.2
6.8
14.4
-4.1
6.7
15.2
1.0
7.6
25.0
25.3
1.0
4.2
25.5
2.1
4.2
60.0
63.8
6.3
4.7
62.7
4.6
4.8
125
125
-0.3
1.7
126
0.8
3.4
250
252
0.9
3.6
257
2.7
3.3
400
410
2.6
4.6
405
1.3
3.5
DIFF: Percentage difference from the nominal concentration.
antibodies (ADA). ADA are typically polyclonal (heterogeneous) in nature, that is, they can bind to multiple unique regions of the protein drug (Figure 6) . The binding of ADA to protein drugs in study samples can potentially interfere with accurate bioana lysis of the protein drug, particularly if ligand binding assays (LBA) are deployed [6,20,21] . The susceptibility of LBA formats to ADA interference is due to their reliance upon binding interactions between the protein analyte and LBA reagents. ADA present in study samples can potentially prevent LBA reagents from binding to the protein analyte, thus impacting LBA reliability (Figure 6) . In contrast, typical LC–MS ana lysis-enabling sample-processing procedures can disrupt ADA–protein interactions, rendering LC–MS assays potentially less susceptible to ADA-mediated assay interference (Figure 6) [22] . In order to evaluate potential ADA impacts on quantification of clinical samples using the universal IgG4 LC–MS/MS assay, blank human serum samples were co-spiked with mAb 1 and either affinity-purified polyclonal antisera raised in monkeys against mAb 1 or mAb 3, a mAb that binds to the target binding region of mAb 1 and interferes with the ability of mAb 1 to bind to its drug target. As shown in Figure 7A & B, the presence of up to 25-fold molar excess of ADA did not adversely impact the quantification of mAb 1 in the samples. These results suggest that a fully validated version of this universal IgG4 LC–MS/MS assay should be able to support reliable
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quantification of hinge region-stabilized human IgG4 therapeutic mAbs in human samples, even if ADA are present. The LLOQ for the exploratory universal IgG4 assay described herein was 5 µg/ml, which may be of insufficient sensitivity to support clinical studies in many cases. Preliminary experiments indicate the LLOQ of this assay could be lowered by approximately tenfold if the serum tryptic digests quantified in these preliminary studies were subjected to solid-phase extraction prior to LC–MS/MS analysis. Conclusion We have developed and evaluated a single exploratory LC–MS/MS assay that is potentially capable of quantifying all hinge region-stabilized human IgG4 therapeutic mAb drugs and drug candidates in human plasma/serum samples. The assay uses a single ‘universal IgG4’ surrogate peptide that was chosen based upon the incorporation of a single amino acid substitution into the hinge region of many human IgG4-based therapeutic mAbs. The assay was developed and evaluated using a model hinge region-staKey Terms Antidrug antibodies: Antibodies often produced by the immune systems of humans or animals in response to dosing of drugs or drug candidates. Antidrug antibodies are typically polyclonal (heterogeneous) in nature; they can bind to multiple unique regions of the drug.
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Preliminary Communication Furlong, Titsch, Xu, Jiang, Jemal & Zeng
A 2.5e2
Intensity (cps)
Intensity (cps)
6.9e2
8.2 B
8.4 Time (min)
8.6
2.9e2
8.4 Time (min)
8.6
8.2
8.4 Time (min)
8.6
8.2
8.4 Time (min)
8.6
5.4e4 Intensity (cps)
Intensity (cps) C
8.2
8.2
8.4 Time (min)
8.6
3.0e3
Intensity (cps)
Intensity (cps)
5.6e4
8.2
8.4 Time (min)
8.6
Figure 4. Representative Peptide 1 (Universal IgG4 peptide) LC–MS/MS chromatograms. (A) Blank human serum; (B) blank human serum spiked with monoclonal antibody 2-IS; and (C) serum spiked with monoclonal antibody 1 at the LLOQ (5 µg/ml) and monoclonal antibody 2-IS. Left panels: analytes; right panels: IS. The SRM transitions monitored were 985.3→912.1 and 990.6→917.4 for Peptide 1 analyte and IS, respectively.
bilized human IgG4 therapeutic mAb. Precision and accuracy of the assay was demonstrated using three performance evaluation runs. Reliability of the assay was further demonstrated by quantitative comparison between the universal IgG4 surrogate peptide and an independent surrogate peptide in the same mAb analyte. The assay was successfully applied to the quantification of the model human IgG4 therapeutic mAb in simulated human serum study samples that contained ADA. Based on the results of these exploratory studies, the assay is expected to be capable, after refinement as needed and full validation, of providing reliable hinge region-stabilized human IgG4 mAb concentrations, with little or no anticipated interferences from ADA that may be present in clinical study samples.
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Bioanalysis (2014) 6(13)
Future perspective LC–MS/MS is a useful platform for the bioanalysis of therapeutic mAbs. LC–MS/MS assays used for bioanalysis of human therapeutic mAbs frequently rely upon quantification of a ‘signature’ surrogate peptide whose amino acid sequence is unique to the protein analyte of interest. The signature peptide approach can be time-, labor- and cost-intensive due to the fact that a new LC–MS/MS assay must be developed for each new mAb that requires bioanalytical support during drug development. These challenges can, in principle, be substantially overcome by the deployment of universal assays capable of quantifying a variety of human therapeutic mAb candidates. We have recently developed and reported a single universal assay that is capable of
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Evaluation of an exploratory universal LC–MS/MS assay
Preliminary Communication
A 2.6e2
Intensity (cps)
Intensity (cps)
6.9e2
5.2 B
5.4 Time (min)
5.6
5.2
5.4 Time (min)
5.6
5.2
5.4 Time (min)
5.6
5.2
5.4 Time (min)
5.6
3.8e4
Intensity (cps)
Intensity (cps)
2.1e2
5.2 C
5.4 Time (min)
5.6
1.3e3
Intensity (cps)
Intensity (cps)
4.4e4
5.2
5.4 Time (min)
5.6
Figure 5. Representative Peptide 2 (confirmatory peptide) LC–MS/MS chromatograms. (A) Blank human serum; (B) blank human serum spiked with monoclonal antibody 2-IS; and (C) serum spiked with monoclonal antibody 1 at the LLOQ (5 µg/ml) and monoclonal antibody 2-IS. Left panels: analytes; right panels: IS. The SRM transitions monitored were 825.5→1073.5 and 830.5→1083.5 for Peptide 2 analyte and IS, respectively.
quantifying a variety of human therapeutic mAbs in animal studies [7,9] . In this report, we have carried out exploratory studies based upon a novel universal IgG4based peptide, which supports the possible extension
of the universal assay concept into the clinical realm. As a consequence of their applicability to variety of mAbs, these two universal assays will be very useful to bioanalytical scientists supporting both nonclinical
Table 3. Quantitative comparison of universal IgG4 peptide and confirmatory peptide. Analytical run
Percentage of individual QC replicates with percentage difference ≤20%†‡
Mean percentage difference §
1
95.2
-0.337
2
97.6
-1.19
3
97.6
-0.0294
See Supplementary Table 2A–C for individual QC replicate percentage difference values. N = 42 QC samples from each run (6 replicates × 7 QC concentration levels) were compared. ‡ Percentage difference = (confirmatory peptide observed concentration - universal IgG4 peptide observed concentration)/(mean of the two values) × 100. § Mean percentage difference computed as the overall mean of the individual percentage difference values from each run.
†
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Preliminary Communication Furlong, Titsch, Xu, Jiang, Jemal & Zeng
mAb
1. Protein(s) denaturation 2. Disulfide bond reduction/alkylation 3. Trypsin digestion
Tryptic peptides
ADA
LC–MS/MS quantification of surrogate peptide
X LBA assay reagent
Figure 6. Antidrug antibody binding to protein drugs (e.g., monoclonal antibodies) in vivo – potential impact on bioanalysis. ADA can potentially prevent ligand binding assay reagents from binding to the protein drug analyte, thus potentially impacting ligand binding assay performance. In contrast, typical LC–MS analysis-enabling sample processing procedures can disrupt ADA–protein drug interactions, rendering the LC–MS assay less susceptible to ADA-mediated assay interference. ADA: Antidrug antibody; LBA: Ligand binding assays; mAb: Monoclonal antibody.
and clinical studies involving a diversity of therapeutic mAb drug candidates.
Observed mAb 1 concentration (µg/ml)
A 20
10 Spiked polyclonal ADA (µg/ml)
Observed mAb 1 concentration (µg/ml)
0
500
40
The authors acknowledge Martin Corbett, Mian Gao, Lin Cheng and Yongmi An (Bristol-Myers Squibb, NJ, USA) for production of the stable-isotope labeled antibody mAb 2. Kimberly Voronin (Bristol-Myers Squibb) is acknowledged for production of peptide used for method development. Roger Demers (Tandem Labs, a Labcorp Company, NJ, USA) is acknowledged for preliminary method development experiments. Qin Ji and Mark E Arnold (Bristol-Myers Squibb) are acknowledged for careful review of and comments on this manuscript.
Financial & competing interests disclosure
20 Spiked monoclonal ADA (µg/ml) 0
0
800
Figure 7. Impact assessment of antidrug antibodies on the performance of the exploratory universal IgG4 LC–MS/MS assay. (A) Blank human serum was spiked with mAb 1 (20 µg/ml) along with a series of concentrations (0–500 µg/ml) of affinity purified cynomolgus monkey polyclonal anti-mAb 1 antiserum. (B) Blank human serum was spiked with mAb 1 (40 µg/ml) along with a series of concentrations (0–800 µg/ml) mAb 3, a mouse monoclonal antibody that binds to the target binding region of mAb 1. Samples were extracted and quantified in triplicate by LC–MS/MS. Data plotted are mean concentrations. CV values for all replicate determinations were under 3%. ADA: Antidrug antibody; mAb: Monoclonal antibody.
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To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.com/doi/full/10.4155/BIO.14.64
Acknowledgements
0 B
Supplementary data
Bioanalysis (2014) 6(13)
All of the authors are current or former employees of Bristol-Myers Squibb. The authors have no other 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 apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations.
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Evaluation of an exploratory universal LC–MS/MS assay
Preliminary Communication
Executive summary Development & evaluation of an exploratory universal IgG4 clinical LC–MS/MS assay • An exploratory universal LC–MS/MS assay has been developed to potentially enable reliable quantification of many human IgG4-based therapeutic monoclonal antibodies (mAbs) in clinical studies. • The exploratory universal IgG4 assay is based upon quantification of a mAb-specific universal surrogate tryptic peptide found in the constant region of IgG4-based therapeutic mAbs that contain a structurally stabilizing serine to proline substitution in the heavy chain constant region.
Reliability evaluation of the exploratory universal IgG4 LC–MS/MS assay: quantitative comparison with a confirmatory surrogate peptide • Three method performance evaluation runs in human serum matrix demonstrated the accuracy and precision of the exploratory assay. • Reliability of the exploratory assay was confirmed by quantitative comparison with data from an independent confirmatory peptide in the same mAb analyte.
Evaluation of the exploratory universal IgG4 LC–MS/MS assay with simulated human serum study samples: anti-mAb 1 antibodies do not interfere with the assay • The exploratory assay was shown to perform well even in the presence of antidrug antibodies, which can often be present in clinical study samples.
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