LC-MS Bioanalysis Research Articles

Multiplexed monitoring of therapeutic antibodies for inflammatory diseases using Fab-selective proteolysis nSMOL coupled with LC-MS

Monoclonal antibodies have accelerated the availability of treatment options for many diseases in which the molecular mechanism has been elucidated in detail. Therefore, an assay that can universally analyze antibodies for clinical pharmacokinetics and cross-sectional studies would be indispensable.We have developed a universal antibody bioanalysis with a Fab-selective tryptic reaction, named nano-surface and molecular-orientation limited (nSMOL) proteolysis, that collects the specific antibody signature peptides in biological samples. Using the nSMOL method, we have fully validated the bioanalysis of many antibodies, Fc-fusion proteins, and their biosimilars.Inflammatory immune diseases often require long-term clinical management because of the remission and relapse observed. Accurate antibody monitoring in systemic circulation could contribute to the improvement of clinical outcomes. Because several biopharmaceuticals can be selected as practical treatment options, the assay development that quantitates many antibodies simultaneously would be applicable in many theraprutic monitoring.In this study, we have validated the LC-MS bioanalysis method for seven-mixed antibodies (Infliximab, Adalimumab, Ustekinumab, Golimumab, Eculizumab, Etanercept, and Abatacept) using the nSMOL normal reaction condition and two-mixed antibodies (Tocilizumab and Mepolizumab) using the acidified reduction acceleration condition, as reported in our previous papers. Moreover, this multiplexed assay has been verified using clinical patient samples. The nSMOL approach enables the quantitation of several immunosuppressive antibodies simultaneously in human serum, and nSMOL can potentially be applicable to the drug-drug interaction assays or therapeutic antibody monitoring of several inflammatory immune diseases to optimize administration.

Analytical convergence in surrogate control matrices.

Analytical convergence in surrogate control matrices.

Critical considerations for immunocapture enrichment LC-MS bioanalysis of protein therapeutics and biomarkers.

In recent years, immunocapture enrichment coupled with LC-MS technology has seen more applications for the measurement of low abundant protein therapeutics and biomarkers in biological matrices. In this article, several critical considerations for the application of immunocapture enrichment to LC-MS bioanalysis of protein therapeutics and biomarkers, including reagent selection, reagent characterization, designing of capture format, etc. are discussed. All these considerations are critical in developing reliable and robust bioanalytical assays with high assay specificity and sensitivity. Successful examples using the immunocapture LC-MS approach in the quantification of biotherapeutic and low abundant protein biomarkers will also be discussed.

Application of nSMOL coupled with LC-MS bioanalysis for monitoring the Fc-fusion biopharmaceuticals Etanercept and Abatacept in human serum.

The principle of nano‐surface and molecular‐orientation limited (nSMOL) proteolysis has a unique characteristic Fab‐selective proteolysis for antibody bioanalysis that is independent of a variety of monoclonal antibodies by the binding antibody Fc via Protein A/G in a pore with 100 nm diameter and modified trypsin immobilization on the surface of nanoparticles with 200 nm diameter. Since minimizing peptide complexity and protease contamination while maintaining antibody sequence specificity enables a rapid and broad development of optimized methods for liquid chromatography‐mass spectrometry (LC‐MS) bioanalysis, the application of regulatory LC‐MS for monitoring antibody biopharmaceuticals is expected. nSMOL is theoretically anticipated to be applicable for representative Fc‐fusion biopharmaceuticals, because Protein A/G‐binding site Fc exists on the C‐terminus, and its functional domain is available to orient and interact with the reaction solution. In this report, we describe the validated LC‐MS bioanalysis for monitoring Ethanercept and Abatacept using nSMOL technology. The quantitation range of Ethanercept in human serum was from 0.195 to 100 μg/mL using the signature peptide VFCTK (aa.43‐47), and that of Abatacept was from 0.391 to 100 μg/mL using the signature peptide MHVAQPAVVLASSR (aa.1‐14). Both proteins fulfilled the guideline criteria for low‐molecular‐weight drug compounds. The results indicate that the clinical and therapeutic monitoring for antibody and Fc‐fusion biopharmaceuticals are adequately applicable using nSMOL proteolysis coupled with LC‐MS bioanalysis.

Top-Down Lc–Ms Quantitation of Intact Denatured and Native Monoclonal Antibodies in Biological Samples

The requirements for developing antibody biotherapeutics benefit from understanding the nature and relevant aspects of the entire molecule. The method presented herein employs on-line multidimensional LC-quadrupole time-of-flight (QTOF)-MS for the quantitative determination of an antibody isolated from biological samples while maintaining the intact native biologically active conformation of the antibody. Following method optimization for a model antibody, an incurred biotherapeutic in cynomologus monkey was quantified in its intact top-down native conformation. A partial method validation demonstrated acceptable precision and accuracy although improved sensitivity requires further studies. An on-line multidimensional LC-MS approach presents a proof-of-principle example for quantifying an intact, native antibody isolated from an incurred biological sample via immunoaffinity techniques coupled with top-down QTOF LC-MS bioanalysis.

Verification between Original and Biosimilar Therapeutic Antibody Infliximab Using nSMOL Coupled LC-MS Bioanalysis in Human Serum

Background: Infliximab (IFX) is a chimeric therapeutic monoclonal antibody targeting tumor necrosis factor alpha (TNFα)-mediated inflammatory immune diseases. However, despite of an initial good clinical response, decrease in response to long-term treatment is a common observation.Objective: Recent studies suggest that IFX level in circulation has a correlation with clinical bioavailabil-ity. Therefore, the management of IFX dosage for individual manifestation by IFX monitoring may be valuable for the improvement of therapeutic response and outcomes.Method: In order to develop a broad IFX therapeutic monitoring in human serum, we have developed the validated IFX bioanalysis for RemicadeTM and its biosimilar product using our nano-surface and molecu-lar-orientation limited proteolysis (nSMOL) technology coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The nSMOL chemistry has a unique property of Fab-selective prote-olysis, and makes it possible a global bioanalysis for many monoclonal antibodies.Results: The quantitation range of IFX in serum was from 0.293 to 300 μg/ml with good linearity. Quan-titation verification at the concentrations of 0.293, 0.879, 14.1 and 240 μg/ml was within 1.56-7.53% of precision and 98.9-111% of accuracy using H-chain signature peptide SINSATHYAESVK. Moreover, cross-verified bioanalysis of Remicade quantitation using biosimilar standard, and its opposite combina-tion, obtained an identical and inter-comparative results.Conclusion: The nSMOL strategy has the potential as a practical therapeutic monitoring technology in IFX therapeutic applications.

The utility of qNMR to improve accuracy and precision of LC-MS bioanalysis

The utility of qNMR to improve accuracy and precision of LC-MS bioanalysis

Outsourcing in bioanalysis: a CRO perspective.

Steve Lowes from Q2 Solutions speaks to Sankeetha Nadarajah, Managing Commissioning Editor: about outsourcing strategy implementation. Steve started his industrial career at VG Biotech in the UK that became the LC-MS instrument entity of Waters Corporation. Since joining the CRO group that became Advion and then Q2 Solutions, his career has focused on regulated bioanalysis with particular emphasis on LC-MS. He is a founding member of the Global Bioanalysis Consortium and a past-chair of the AAPS Bioanalytical Focus Group. At Q2Solutions, Steve leads the scientific disciplines around LC-MS bioanalysis for both small molecule and biomolecule applications including biomarker assays. Steve has over 40 peer-reviewed publications on bioanalysis and is a frequent speaker at national and international conferences.

LC–MS bioanalysis of Trastuzumab and released emtansine using nano-surface and molecular-orientation limited (nSMOL) proteolysis and liquid–liquid partition in plasma of Trastuzumab emtansine-treated breast cancer patients

Antibody-drug conjugates (ADCs) consist of monoclonal antibody and cytotoxic drugs covalently attached via stable crosslinkers, and are prospective antibody drugs for cancer therapy. To cover the overall pharmacokinetic understanding of ADCs, both the antibody and the released drugs are necessary for practical clinical observation. The nano-surface and molecular-orientation limited (nSMOL) proteolysis is a universal approach for antibody bioanalysis that enable Fab-selective proteolysis, which maintains antibody sequence specificity while decreasing excess analyte peptides. In this study, we describe quantitative assays for ADC in human plasma using nSMOL for the antibody and polarity-selective liquid–liquid partition with a methanol/ethyl acetate mixed solvent for the cytotoxic drugs. This approach led to the successful development of LC–MS validated bioanalysis of the antibody and released drugs within 20% for lower limit of quantitation and 15% for another concentration setting of Trastuzumab emtansine (T-DM1), Trastuzumab antibody and emtansine conjugated with crosslinker (DM1-MCC). The validated concentration ranges in human plasma were 0.06–250μg/mL for T-DM1 and 0.39–200ng/mL for DM1-MCC. These results indicate that LC–MS method with a two-sided approach, using nSMOL and liquid–liquid partition, show potential for the precise pharmacokinetic study for ADC development and treatment.

Correction: Normalization strategy for the LC-MS bioanalysis of protein kinetics assays via internal proteolytic analyte utilized as control standard: application in studies of HIV-1 protease cleavage of HIV-1 Gag polyprotein in HIV maturation inhibition research

Correction for ‘Normalization strategy for the LC-MS bioanalysis of protein kinetics assays via internal proteolytic analyte utilized as control standard: application in studies of HIV-1 protease cleavage of HIV-1 Gag polyprotein in HIV maturation inhibition research’ by Joseph L. Cantone et al., Anal. Methods, 2017, DOI: 10.1039/c7ay01666b.

Normalization strategy for the LC-MS bioanalysis of protein kinetics assays via internal proteolytic analyte utilized as control standard: application in studies of HIV-1 protease cleavage of HIV-1 Gag polyprotein in HIV maturation inhibition research

The LC-MS bioanalysis of protein kinetics assays is simplified by a data normalization strategy via internal proteolytic analyte utilized as a control standard.

The use of a restricted access volatile supramolecular solvent for the LC/MS-MS assay of bisphenol A in urine with a significant reduction of phospholipid-based matrix effects

Restricted access-volatile supramolecular solvents (RAM-VOL-SUPRAS) are here proposed as a new strategy for the quick removal of protein and phospholipids and efficient analyte extraction in LC-MS bioanalysis. Quantification of bisphenol A in urine was selected to prove the suitability of this approach for the intended purpose. RAM-VOL-SUPRAS were spontaneously synthesized in urine by addition of hexanol (83 μL) dissolved in THF (150 μL). SUPRAS composition was environment-dependent and an equation for prediction of SUPRAS volume under given experimental conditions was proposed. Urinary proteins were removed by flocculation by the combined action of THF and hexanol. Phospholipids were extracted in the SUPRAS by the formation of mixed aggregates with hexanol and precipitated as the SUPRAS extract (75 μL) was evaporated to dryness. BPA, re-extracted from the residue, was analysed by LC-(ESI)-MS/MS. Removal of phospholipids by precipitation was proved by monitoring them in both evaporated and unevaporated urine SUPRAS extracts by LC-MS. This removal led to significant reduction in matrix-effects in the determination of BPA. The method quantification limit in urine was 0.025 ng mL−1 and the repeatability for 0.4 ng mL−1 of BPA, expressed as relative standard deviation, was 4.5%. Concentrations of BPA in the urine samples analysed were in the range 0.357–1.58 ng mL−1. Recoveries were within the range 96–107%. This new approach for sample treatment in bioanalysis, based on the simplicity of dual precipitation of proteins and phospholipids, allows obtaining much cleaner extracts than conventional procedures.

LC-MS quantification of protein drugs: validating protein LC-MS methods with predigestion immunocapture.

A refinement of protein LC-MS bioanalysis is to use predigestion immunoaffinity capture to extract the drug from matrix prior to digestion. Because of their increased sensitivity, such hybrid assays have been successfully validated and applied to a number of clinical studies; however, they can also be subject to potential interferences from antidrug antibodies, circulating ligands or other matrix components specific to patient populations and/or dosed subjects. The purpose of this paper is to describe validation experiments that measure immunocapture efficiency, digestion efficiency, matrix effect and selectivity/specificity that can be used during method optimization and validation to test the resistance of the method to these potential interferences. The designs and benefits of these experiments are discussed in this report using an actual assay case study.

Abstract 2962: Development of the LCMS bioanalysis for clinical pharmacokinetics of antibody drugs using Fab-selective limited proteolysis (nSMOL)

Abstract Monoclonal antibody drugs (mAbs) have become a major therapeutic strategy in cancer care. The approved mAbs have been about 40 items, and a lot of clinical trials have been now performing in the world. For precise cancer therapy, overall pharmacokinetic (PK) information in blood or tumor tissue will become the mainstream indicators for many clinicians. Therefore, versatile and robust analytical technologies for the direct detection of antibody will be important outcome in clinical applications. The bioanalysis of mAbs using LCMS is a significant platform for the selective quantitation or the feasibility of the multiplex assays for combination therapy. For regulated LCMS bioanalysis independent of a variety of mAbs or biological taxonomy sources, we have focused on the two features: antibody structure-indicated analysis, and complementarity-determining region (CDR)-targeting quantitation. Using the previous LCMS bioanalysis by general proteomic method, the signature proteolytic peptides of the mAb should be quantified from the massive analyte and residual protease because immunoglobulin molecules are second major protein in plasma proteome. The excess peptides will be cause to decrease the selectivity and sensitivity. To address this issues, we have recently reported the novel method for the selective proteolysis on the Fab by limiting protease access to antibodies, which have named nano-surface and molecular-orientation limited (nSMOL) proteolysis. nSMOL chemistry is designed based on the unique features: (1) protease reaction on nanoparticle surface; (2) Fc immobilization onto the resin, such that the Fab is oriented outward to the solution; and (3) Fab-selective limited proteolysis by making use the difference of the protease nanoparticle diameter (200 nm) and the Ab resin pore size (100 nm). nSMOL has made it possible to minimize the sample complexity while the maintaining the sequence specificity in CDR. We are now performing the bioanalytical validation for several mAbs in accordance with the Guideline on Bioanalytical Method Validation in Pharmaceutical Development. The results of validation study show that nSMOL is possible to cover the clinical dose, and more accurate and sensitive than ELISA and conventional proteomic approaches. For clinical feasibility test, we measured the plasma concentration of Bevacizumab in patients with recurrent non-small cell lung cancer according to nSMOL protocols. nSMOL proteolysis coupled with LCMS bioanalysis is a novel method for the clinical PK independent of a variety of monoclonal antibodies, and have confirmed the clinical usability by the fully validated bioanalysis. This analytical approach will be applicable for many new mAbs and biosimilar drugs. Furthermore, the clinical trials based on LCMS may be expected to aid acceleration of the development of many biopharmaceuticals. Citation Format: Takashi Shimada, Noriko Iwamoto, Sho Saeki, Ji-ichiro Sasaki, Taka-Aki Sato, Akinobu Hamada. Development of the LCMS bioanalysis for clinical pharmacokinetics of antibody drugs using Fab-selective limited proteolysis (nSMOL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2962.

Application of nano-surface and molecular-orientation limited proteolysis to LC-MS bioanalysis of cetuximab.

We recently reported the principle of nano-surface and molecular-orientation limited (nSMOL) proteolysis, which is useful for LC-MS bioanalysis of antibody drugs. The nSMOL is a Fab-selective limited proteolysis which utilizes the difference of protease-immobilized nanoparticle diameter (200 nm) and antibody collection resin pore (100 nm). We have demonstrated the full validation for chimeric antibody cetuximab bioanalysis in human plasma using nSMOL. Signature peptides (SQVFFK, ASQSIGTNIHWYQQR and YASESISGIPSR) in cetuximab complementarity-determining region were simultaneously quantitated by LC-MS multiple reaction monitoring. This nSMOL quantification showed sensitivity of 0.586 µg/ml and linearity of 0.586 to 300 µg/ml. Full validation study archived the guideline criteria of low Mw drug compounds. These results indicate that nSMOL is also significant method for cetuximab bioanalysis.

Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL−1. The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.

QbD-Driven Development and Validation of a Bioanalytical LC-MS Method for Quantification of Fluoxetine in Human Plasma.

The current studies describe the Quality by Design (QbD)-based development and validation of a LC-MS-MS method for quantification of fluoxetine in human plasma using fluoxetine-D5 as an internal standard (IS). Solid-phase extraction was employed for sample preparation, and linearity was observed for drug concentrations ranging between 2 and 30 ng/mL. Systematic optimization of the method was carried out by employing Box-Behnken design with mobile phase flow rate (X1), pH (X2) and mobile phase composition (X3) as the method variables, followed by evaluating retention time (Rt) (Y1) and peak area (Y2) as the responses. The optimization studies revealed reduction in the variability associated with the method variables for improving the method robustness. Validation studies of the developed method revealed good linearity, accuracy, precision, selectivity and sensitivity of fluoxetine in human plasma. Stability studies performed in human plasma through freeze-thaw, bench-top, short-term and long-term cycles, and autosampler stability revealed lack of any change in the percent recovery of the drug. In a nutshell, the developed method demonstrated satisfactory results for analysis of fluoxetine in human plasma with plausible utility in pharmacokinetic and bioequivalence studies.

Multiplex LCMS Bioanalysis of Brentuximab Vedotin, Rituximab and Cetuximab towards Therapeutic Drug Monitoring Application by Combined Calibration Curve Using Fab-Selective Limited Proteolysis nSMOL

Background: Recently, monoclonal antibody (mAb) bioanalysis using mass spectrometry has begun to be recognized as useful technology for mAbs measurement other than ELISA. We have recently exploited a high-precision method for bioanalysis of monoclonal antibody (mAb) using mass spectrometry. The method is nano-surface and molecular-orientation limited (nSMOL) proteolysis, which is useful for LCMS bioanalysis of many kinds of antibody drugs. Methods: nSMOL is Fab-selective limited proteolysis which consists of the difference of protease nanoparticle diameter (200 nm) and antibody resin pore diameter (100 nm). For limited proteolysis of antibody, Protein A resin (pore: 100 nm) slurry was added to plasma including monoclonal antibody, and the antibody Fc region was immobilized to the resin at 25°C for 10 min with gentle vortexing. Antibody-immobilized resin was washed with PBS, and limited proteolysis was performed with trypsin-conjugated FG beads (diameter: 200 nm). Limited proteolysis of Fab region on antibody was achieved by these two diameter difference. After nSMOL proteolysis, the generated peptides were collected by only simple filtration. Results: In this study, we have demonstrated that the first full validation dataset for bioanalysis using nSMOL of antibody-drug conjugate (ADC), Brentuximab vedotin, in human plasma using nSMOL proteolysis. Full validation using nSMOL proteolysis fulfilled criteria of guideline on bioanalytical method validation in pharmaceutical development for small molecule drug compounds. Conclusions: These results indicate that nSMOL is also significant method for precise quantification of ADC in plasma, such as Brentuximab vedotin. Furthermore, we report that nSMOL proteolysis is able to apply for not only single-analyte but also multi-analyte bioanalysis of each mAbs in plasma, so that, nSMOL proteolysis is feasible multiplex bioanalysis for many clinical pharmacokinetic study and therapeutic drug monitoring.

Fully validated LCMS bioanalysis of Bevacizumab in human plasma using nano-surface and molecular-orientation limited (nSMOL) proteolysis

The chemistry of nano-surface and molecular-orientation limited (nSMOL) proteolysis is the Fab-selective limited proteolysis by making use the difference of protease nanoparticle diameter (200 nm) and antibody resin pore diameter (100 nm). In this report, we have demonstrated that the full validation for Bevacizumab bioanalysis in human plasma using nSMOL. The immunoglobulin fraction was collected by Protein A resin from plasma, then nSMOL reaction was performed using the FG nanoparticle-immobilized trypsin under the nondenaturing physiological condition at 50 °C for 6 h. After removal of resin and nanoparticles, the signature peptide of Bevacizumab complementarity-determining region (CDR) and internal standard P14R were simultaneously quantified by LCMS multiple reaction monitoring (MRM). This nSMOL method quantification of Bevacizumab showed sensitivity of 0.146 μg/ml and linearity of 0.146–300 μg/ml. The intra- and inter-assay precision of lower limit of quantification (LLOQ), low quality control (LQC), middle quality control (MQC), and high quality control (HQC) was 7.94–15.2% and 14.6%, 7.15–13.5% and 11.7%, 2.63–6.47% and 5.83%, and 3.09–4.35% and 4.45%, respectively. These results indicate that nSMOL is also significant method for Bevacizumab bioanalysis in human plasma.

The blood-brain barrier permeability properties of plant N-alkylamides

Background: N-alkylamides (NAAs), a large group of secondary metabolites occurring in more than 25 plant families, are potential drug candidates as several biological and medical functions are known such as central nervous activities [1]. Objective: Our general goal was to investigate if NAAs with a different structure, e.g. spilanthol and pellitorine, are able to pass the blood-brain barrier (BBB) and if so, to what extent. Methods: A blood to brain (multiple time regression (MTR)), as well as a brain to blood (efflux) transport experiment were conducted in an in vivo mice model to investigate the initial BBB rate kinetics. The mice were anesthetized, after which the NAA dose solution was injected. Blood was obtained at regular time points after injection, and thereafter, the mice were decapitated and the brains collected. Quantification of the NAAs was done using a bio-analytical LC-MS method. Results: The MTR experiment indicated that spilanthol was able to cross the BBB in mice. A rapid but highly significant influx of spilanthol into the brains was observed with an unidirectional influx rate of 217.0 µl/(g×min). The curve reached a plateau-phase after about 10 minutes exposure time and can be explained by efflux of spilanthol out of the brain or distribution or elimination of spilanthol. The efflux transfer constant calculated for spilanthol was 0.1 min-1. This equals a brain half-time disappearance of 6.4 min. The comparative results of on-going BBB studies, including the permeability properties of pellitorine, will be reported as well. Seen the different possible pharmacological targets of these plant NAAs and their pharmaco-ethnological use, our BBB results may trigger further exploration into the medicinal use of these important plant metabolites.