A validated LC-MS/MS method for the quantitation of daratumumab in rat serum using rapid tryptic digestion without IgG purification and reduction.

Daratumumab, a humanized monoclonal antibody utilized in treating immunoglobulin light-chain amyloidosis and relapsed/refractory multiple myeloma, was quantified in rat serum through a simple, economical and effective liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. A surrogate peptide, LLIYDASNR, derived from trypsin hydrolysis, was quantitatively analyzed with LLIYDASN [13C6, 15N4] RAT as an internal standard. This corrected variations from sample pretreatment and mass spectrometry response, involving denaturation and trypsin hydrolysis in a two-step process lasting approximately 1 hour. Methodological validation demonstrated a linear range of 1 µg/mL to 1000 µg/mL in rat serum. Precision, accuracy, matrix effect, sensitivity, stability, selectivity, carryover, and interference met acceptance criteria. The validated LC-MS/MS approach was successfully applied to a pharmacokinetic study of daratumumab in rats at an intravenous dose of 15 mg/kg.

A Comprehensive Immunocapture-LC-MS/MS Bioanalytical Approach in Support of a Biotherapeutic Ocular PK Study.

BI-X, a therapeutic protein under development for the treatment of human ocular disease via intravitreal administration, binds to its therapeutic targets and endogenous albumin in the vitreous humor. A monkey ocular pharmacokinetic (PK) study following BI-X administration was conducted to measure drug and albumin levels in plasma, the vitreous humor, the aqueous humor, and retina tissue at various timepoints post-dose. A comprehensive bioanalytical approach was implemented in support of this study. Five immunocapture-LC-MS/MS assays were developed and qualified for quantitating BI-X in different matrices, while ELISA was used for albumin measurement. Immunocapture at the protein or peptide level was evaluated to achieve adequate assay sensitivity. Drug and albumin assays were applied for the analysis of the monkey study samples.

A robust and validated LC-MS/MS method for the quantification of ramucirumab in rat and human serum using direct enzymatic digestion without immunoassay.

A new liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established to quantify the anti-gastric cancer fully human monoclonal antibody (ramucirumab) in rat and human serum. The surrogate peptide (GPSVLPLAPSSK) for ramucirumab was generated by trypsin hydrolysis and quantified using the isotopically labeled peptide GPSVLPLAPSSK[13C6, 15N2]ST containing two more amino acids at the carboxyl end as an internal standard to correct for variations introduced during the enzymatic hydrolysis process and any mass spectrometry changes. Additionally, the oxidation and deamidation of unstable peptides (VVSVLTVLHQDWLNGK and NSLYLQMNSLR) were detected. The quantitative range of the proposed method was 1-1000 µg/mL, and complete methodological validation was performed. The precision, accuracy, matrix effect, sensitivity, stability, selectivity, carryover, and interference of the measurements met the required standards. The validated LC-MS/MS method was applied to pharmacokinetic studies in rats administered ramucirumab at 15 mg/kg intravenously. Overall, a robust, efficient, and cost-effective LC-MS/MS method was successfully developed for quantifying ramucirumab in rat and human serum.

A validated LC-MS/MS method for the quantification of bevacizumab in rat, cynomolgus monkey, and human serum.

Bevacizumab is a humanized monoclonal antibody used in the treatment of advanced colorectal and non-small cell lung cancer. Our main aim was to establish a simple, economical, and high efficiency liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying the content of bevacizumab in various biological fluids (rat, cynomolgus monkey, and human serum). A surrogate peptide of bevacizumab, specifically FTFSLDTSK, was generated through trypsin hydrolysis, and quantified using an isotopically labeled peptide containing two amino acids, FTFSLDTSK[13C6, 15N2]ST, as an internal standard to correct for variations introduced during the enzymatic hydrolysis process and any mass spectrometry variabilities. The pre-treatment process included denaturation, disulfide bond reduction and alkylation, trypsin hydrolysis, and termination of the reaction, with a total duration of approximately 2.5-3 h. The results of the methodological validation showed that the linear range in three different biological matrices was 0.2 µg/mL to300 µg/mL, with an LLOQ of 0.2 µg/mL. The precision and accuracy of the measurements met the required standards. The validated LC-MS/MS method was used to conduct pharmacokinetic analysis in rats administered bevacizumab at a dose of 10 mg/kg intravenously.

ProteoExcelTP: Development of a simple excel-based tool for surrogate peptide selection in mass spectrometry based targeted proteomics.

Selection of surrogate peptides plays a major role to achieve reproducible and accurate quantification of desired proteins in targeted proteomics. Currently, available peptide selection tools suffer from the limitation of not covering entire proteins including all the species and inflexibility in applying the exclusion criteria. Here, we have developed an excel-based ProteoExcelTP tool which can automatically select the most appropriate surrogate peptides with high flexibility in terms of addition, deletion or changing the exclusion criteria. The developed ProteoExcelTP tool has also been validated by comparison of obtained peptides from the tool with those selected in previously reported works. This is the first time to develop an excel based tool for quick and accurate selection of surrogate peptides for entire protein family of all the species. The tool is having the unique advantage of a highly user-friendly nature. It can be customized according to the specific need of the researchers. ProteoExcelTP tool will significantly enhance the throughput of the quantitative proteomic analysis. The tool can immensely help the scientists working in the field of proteomics by significantly minimizing their effort in accurate selection of surrogate peptides for quantification of endogenous proteins.

Surrogate peptide selection and internal standardization for accurate quantification of endogenous proteins.

Relative quantification techniques have dominated the field of proteomics. However, biomarker discovery, mathematical model development and studies on transporter-mediated drug disposition still need absolute quantification of proteins. The quality of data of trace-level protein quantification is solely dependent on the specific selection of surrogate peptides. Selection of surrogate peptides has a major impact on the accuracy of the method. In this article, the advanced approaches for selection of surrogate peptides, which can provide absolute quantification of the proteins are discussed. In addition, internal standardization, which accounts for variations in the quantitation process to achieve absolute protein quantification is discussed.

Development and validation of a UPLC-MS/MS method to quantitate anti-PD1 monoclonal antibody (Toripalimab), and comparison with electrochemiluminescence immunoassay.

Toripalimab, a humanized IgG4 monoclonal antibody (mAb) against programmed death receptor-1, is being extensively studied to treat various malignancies. At present, there is no complete methodology reported for quantifying toripalimab, except for an electrochemiluminescence immunoassay (ECLIA) mentioned in several clinical studies. Therefore, a sensitive and robust ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to accurately detect toripalimab levels, compared with the ECLIA. Plasma samples were pretreated by a five-step process, encompassing denaturation, reduction, alkylation, enzymatic hydrolysis and quenching. And a unique, sensitive and stable enzymatic peptide (ASGYTFTDYEMHWVR) selected as surrogate of toripalimab was eluted and monitored by UPLC-MS/MS system with the linear range of 5.0375-201.5 µg/mL. After fully validated, the UPLC-MS/MS method was applied to determine 77 plasma samples from 29 patients in a phase I clinical trial, and compared with ECLIA based on 56 samples. Wilcoxon paired samples test showed toripalimab levels by UPLC-MS/MS were significantly higher than that by ECLIA (p < 0.001), though a strong correlation was observed (r = 0.96). Moreover, Passing-Bablok regression analysis exhibited constant and proportional biases: UPLC-MS/MS = 2.25 + 1.21 * ECLIA. This discrepancy could be mainly attributed to different forms determined: total mAb for UPLC-MS/MS and free mAb for ECLIA, respectively. As a result, this UPLC-MS/MS method may be complementary to ECLIA to monitor different forms of toripalimab. Beyond that, it can be easily modified to simultaneously quantitate multiple-analyte with a small volume of plasma.

A Diaminodiacid (DADA) Strategy for the Development of Disulfide Surrogate Peptides.

Disulfide bond-containing peptides are useful molecular scaffolds with diagnostic and therapeutic applications due to their good biological activity and good target selectivity, but their utility is sometimes limited by the lability of the disulfide moiety under reducing conditions and in the presence of disulfide bond isomerase. The development of disulfide surrogates with improved redox stability has been an area of ongoing research; and one possible strategy is based on a diaminodiacid (DADA) moiety, which can be used to synthesize the disulfide bond replacement peptides with precise structures and enhanced stability through automated solid-phase peptide synthesis (SPPS). This review summarizes recent developments in the DADA-based SPPS of peptide disulfide surrogates. Some representative applications and structural studies on the DADA-based disulfide surrogates are described.

Immunoaffinity LC-MS/MS to quantify a PEGylated anti-Factor D Fab biotherapeutic in cynomolgus monkey serum.

Aim: To develop a sensitive hybrid immunoaffinity LC-MS/MS monkey serum assay to quantify multiple components of anti-Factor D; a complex PEGylated Fab biotherapeutic explored as a therapy for age-related macular degeneration. Materials & methods: Immunoaffinity enrichment of PEGylated anti-Factor D Fab, including fully conjugated, partially conjugated and unconjugated (i.e., free) Fab species, using a capture reagent coupled to magnetic beads was performed. The surrogate peptides derived from the therapeutic Fab via trypsin digestion were measured to obtain the total Fab concentrations. Results & conclusion: The method demonstrated the ability to accurately quantify both PEGylated and unconjugated Fab species. It was successfully validated with a LLOQ at 25.0 ng/ml.

An LC-MS/MS assay with a label-free internal standard for quantitation of serum cystatin C.

Cystatin C is considered as an alternative to the evaluation of glomerular filtration rate. In this study, we highlighted an LC-MS/MS approach for the absolute quantitation of serum cystatin C based on label-free internal standards. A tryptic peptide (ALDFAVGEYNK) was selected as the surrogate whilst analogue (ALDFAVGEYQK) served as an internal standard. After denaturation, reduction, alkylation, digestion and concentration, the target peptides were separated on an LC column and monitored under MRM. The calibration range was from 0.25 mg/L to 15 mg/L with LLOQ of 0.05 mg/L and LOD of 0.03 mg/L, respectively. The certified reference material (ERM-DA471) was determined at 5.12 mg/L with bias of 6.57%. The recovery was between 89.68% and 92.43%. The RSD of intra- and inter-assay imprecision were both <10%. Good stability was also observed. The assay also demonstrated that the quantification of native cystatin C in human serum could be achieved using label-free internal standards. The assay was robust, cheap and sensitive.

Critical reagent screening and characterization: benefits and approaches for protein biomarker assays by hybrid LC-MS.

In recent years, hybrid ligand-binding assays (LBAs)/LC-MS assays have been increasingly used for quantitation of protein biomarkers in biological matrices. However, unlike in LBAs where the importance of critical reagent screening and characterization is well understood and widely reported, benefits of well-characterized hybrid LC-MS assay reagents are frequently underestimated. Two groups of analyte-specific reagents, binding reagents and assay calibrators, are considered the critical reagents for biomarker assays. In this article, we summarize the similarities and differences of critical reagents used in LBAs and hybrid LC-MS assays, overview the benefits and approaches of critical reagent screening, characterization, antibody conjugation and discuss bioanalytical considerations in hybrid LC-MS assay development for robust measurements of protein biomarkers in biological matrices.

Highly selective and sensitive LC-MS/MS quantification of a therapeutic protein in human serum using immunoaffinity capture enrichment.

We report the development, validation and application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method for the determination of a recombinant protein drug candidate, NVS001, in human serum. A unique surrogate peptide, IPAETTIYNR (IPA), was identified to distinguish NVS001 from its endogenous counterpart, i.e., the full length and the C-terminal of protein X in LC-MS/MS. The selection of IPA for the LC-MS/MS determination of NVS001 was supported by the absence of peak responses due to endogenous components in the LC-MS/MS chromatograms of the extracted blank human serum samples. The optimal chromatographic separation of IPA from the extracted matrix components was achieved on a Waters Cortecs C18 (100 × 2.1 mm, 2.7 µm) column using gradient elution with a run cycle time of approximately 7.5 min. The mobile phases were water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, and stability. The validated assay dynamic range was 10.0 to 1000 ng/mL using a 50 µL sample volume. The accuracy and precision for the LLOQ (10.0 ng/mL) sample results were within ±9.2% bias and ≤6.0% CV, respectively. From the intra-day and inter-day assay performance evaluations, the precision of the QC sample (30, 500 and 750 ng/mL) results were ≤3.5% CV and the accuracy within ±3.3% bias, respectively. An additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented in support of a first-in-human study.

Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum using an immobilized trypsin.

Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) without an immunopurification technique have not been applied to the determination of serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-alkylated serum cetuximab without immunopurification was digested for 20minutes using immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the determination of serum samples in head and neck cancer patients treated with cetuximab. The chromatographic run time was 10minutes and no peaks interfering with surrogate peptides in serum digestion products were observed. The calibration curve of absolute cetuximab in serum was linear over the concentration range of 4-200µg/mL. The lower limit of quantification of cetuximab in human serum was 4µg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and 88.0-100.7%, respectively. The serum concentration range of cetuximab was 19-140µg/mL in patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r=0.899, P <0.01) and the mean bias was 1.5% in cancer patients. In conclusion, the present simple and rapid method with acceptable analytical performance can be helpful for evaluating the absolute concentration of serum cetuximab in clinical settings.

Measurement of lipid transfer proteins in genetically engineered maize using liquid chromatography with tandem mass spectrometry (LC-MS/MS).

Endogenous allergenicity evaluation is a required part of the risk assessment for genetically engineered (GE) crops. Although maize is not considered a major allergenic food, a lipid transfer protein (Zea m 14) in maize grain has been identified as a potential IgE-mediated food allergen. Currently, the relationship between allergen exposure and risk of sensitization is not well understood. Hence, reliable quantitative methods are useful for determining the natural range and variability of allergen levels across multiple geographies and genetic backgrounds. A LC-MS/MS analytical method was developed and validated in our laboratory to quantify Zea m 14 in grain from 2 GE maize hybrids and 20 non-GE maize hybrids. The measured Zea m 14 levels in GE maize grain and conventional non-GE maize grain ranged from 146.87 to 574.93 ng/mg across 16 field sites located in the United States and Argentina. The method accurately quantified endogenous Zea m 14 from maize grain and results show Zea m 14 levels in the GE maize varieties were within the natural variation observed in traditionally bred non-GE maize.

Transgenesis affects endogenous soybean allergen levels less than traditional breeding.

The regulatory body that oversees the safety assessment of genetically modified (GM) crops in the European Union, the European Food Safety Authority (EFSA), uniquely requires that endogenous allergen levels be quantified as part of the compositional characterization of GM versions of crops, such as soybean, that are considered to be major allergenic foods. The value of this requirement for assessing food safety has been challenged for multiple reasons including negligible risk of altering allergen levels compared with traditional non-GM breeding. Scatter plots comparing the mean endogenous allergen levels in non-GM soybean isoline grain with the respective levels in GM grain or concurrently grown non-GM commercial reference varieties clearly show that transgenesis causes less change compared with traditional breeding. This visual assessment is confirmed by the quantitative fit of the line of identity (y = x) to the datasets. The current science on allergy does not support the requirement for quantifying allergen levels in GM crops to support safety assessment.

Development, Validation, and Interlaboratory Evaluation of a Quantitative Multiplexing Method To Assess Levels of Ten Endogenous Allergens in Soybean Seed and Its Application to Field Trials Spanning Three Growing Seasons.

As part of the regulatory approval process in Europe, comparison of endogenous soybean allergen levels between genetically engineered (GE) and non-GE plants has been requested. A quantitative multiplex analytical method using tandem mass spectrometry was developed and validated to measure 10 potential soybean allergens from soybean seed. The analytical method was implemented at six laboratories to demonstrate the robustness of the method and further applied to three soybean field studies across multiple growing seasons (including 21 non-GE soybean varieties) to assess the natural variation of allergen levels. The results show environmental factors contribute more than genetic factors to the large variation in allergen abundance (2- to 50-fold between environmental replicates) as well as a large contribution of Gly m 5 and Gly m 6 to the total allergen profile, calling into question the scientific rational for measurement of endogenous allergen levels between GE and non-GE varieties in the safety assessment.

A multiplexed immunocapture liquid chromatography tandem mass spectrometry assay for the simultaneous measurement of myostatin and GDF-11 in rat serum using an automated sample preparation platform.

Myostatin, also known as growth differentiation factor 8 (GDF-8), is a protein acting as a negative regulator in skeletal muscle growth. Inhibition of myostatin by therapeutic agents provides opportunities for current unmet medical needs. In order to better understand drug engagement to aid the drug development, we have developed a hybrid LC-MS/MS method which can differentially measure myostatin and another protein from the same GDF family, GDF-11. Although the two proteins share high homology, the LC-MS/MS assay provided the specificity based on monitoring of unique surrogate peptide generated from enzymatic digestion. An automated sample preparation platform, Agilent AssayMap Bravo, was used for automated immunocapture. Capture antibody that is non-competing with our investigational drug and has similar binding affinity to both myostatin and GDF-11 was used. Therefore, total myostatin and GDF-11 including both free form and drug-bound form were captured and measured. The enriched sample was digested after reduction and alkylation. Two surrogate peptides (IPAMVVDR for myostatin and IPGMVVDR for GDF-11) were monitored and the lower limit of quantitation (LLOQ) was established at 1.0 ng/mL for myostatin and 0.1 ng/mL for GDF-11. The accuracy was demonstrated with recovery for IPAMVVDR between 99.2% and 103.1% and for IPGMVVDR between 90.3% and 114.5%. The developed hybrid assay exhibits sufficient sensitivity, accuracy and specificity to differentiate between the highly structurally similar myostatin and GDF-11. This analytical approach was successfully applied to a rat toxicology study, and was demonstrated to be a powerful tool for biomarker measurement in the present of a therapeutic agent.

LC-MS/MS determination of a human mAb drug candidate in rat serum using an isotopically labeled universal mAb internal standard.

We report the application of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical method for the determination of a recombinant human immunoglobulin G1 (hIgG1), NVSMAb-1, in rat serum. A stable isotopically labeled universal monoclonal antibody (SILuMab), instead of stable isotopically labeled surrogate peptide, was employed as the internal standard. The internal standard was added to the sample matrix in the first step of the sample preparation process, which involved protein precipitation and pellet digestion. The digestion of the resulting pellet with trypsin was performed prior to analysis of surrogate peptides of both NVSMAb-1 and SILuMab using LC-MS/MS. Precipitation reagents (1% TCA in IPA, 75% MeOH and 14% PEG) and digestion conditions (50°C for 2h and 60°C for 0.5h) were evaluated by monitoring LC-MS/MS responses of GPS and VVS in the resulting sample extracts. Overall, the use of 1% TCA in IPA appeared to be more effective as compared to 75% methanol in protein precipitation and removal of unwanted matrix components, e.g., albumin, and more appealing than 14% PEG as it avoided additional steps that are necessary to remove PEG or reduce PEG to a negligible level. The yield (LC-MS/MS response) of GPS is less sensitive than VVS to the changes of digestion conditions (time and temperature). The results obtained using SILuMab over SIL surrogate peptide as the internal standard appeared unaffected by the suboptimal sample processing method. For the current assay, surrogate peptide GPSVFPLAPSSK (GPS) was selected as surrogate peptide over VVSVLTVLHQDWLNGK (VVS) for quantitative analysis of NVSMAb-1. The optimal chromatographic separation was achieved on a Waters Cortecs C18 (100×2.1mm, 2.7µm) column using gradient elution with a total cycle time of approximately 8min. The mobile phases were water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The current method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, and stability. The validated assay dynamic range was 10-5000µg/mL using 20µL of rat serum. The accuracy and precision for the LLOQs (10µg/mL) were within ±6.0% bias and ≤6.5% CV, respectively. From the intra-day and inter-day assay performance evaluations, the precision of the other QC sample (30, 300, 2500 and 3750µg/mL) results were ≤6.8% CV and the accuracy within ±4.8% bias, respectively. Additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented in support of a toxicity study in rats administered 30, 150 and 750mg/kg/week intravenous infusion and 150mg/kg/week subcutaneous injection of NVSMAb-1.

Quantitative analysis of hIgG1 in monkey serum by LC-MS/MS using mass spectrometric immunoassay.

AIM: A sensitive generic LC-MS/MS method for hIgG1 quantification in cynomolgus monkey serum using mass spectrometric immunoassay disposable automation research tips (MSIA-D.A.R.T.'S™) is reported. RESULTS: The hIgG1 was captured with a biotinylated mouse anti-hIgG antibody (50.0 µg/ml) targeting the fragment crystallizable (Fc) region. Elution from the streptavidin-coated MSIA-D.A.R.T.'s was conducted with 0.4% trifluoroacetic acid in water. The method was selective and linear from 10.0 to 1000 ng/ml using 100 µl of serum. The method was evaluated regarding accuracy, precision, carry-over, dilution, auto-sampler stability and applied for the determination of hIgG1 concentration in monkey serum after intravitreal administration. CONCLUSION: The present assay is suitable for quantitative analysis of hIgG1-based therapeutic proteins in monkey serum at low levels.