Combination of Stable Labelled Antibodies and LC-MS to Accurately Quantify Therapeutic Monoclonal Antibodies in the Clinical Laboratory Ravindra Chaudhari (Presenter) PROMISE Advanced Proteomics

Authors: Dorothée LEBERT1, Guillaume PICARD1, Ravindra CHAUDHARI1, Aurélien MILLET2, Jérôme GUITTON2, Jean-François JOURDIL3, Françoise STANKE3.
1 Promise Advanced Proteomics, F-38040, Grenoble, France 2 Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, Laboratoire de pharmaco-toxicologie, F-69495, Pierre Bénite, France. 3 CHU Grenoble, Laboratoire de pharmaco-toxicologie, F-38000, Grenoble, France

We will present an innovative and generic approach involving the use of stable isotope labeled mAbs used as internal standards and LC-MS to allow accurate, sensitive and specific quantification, as required for therapeutic drug monitoring. Our approach was already tested and implemented with success in pharmacology laboratories on anti-TNF antibodies (Infliximab and Adalimumab) and was recently implemented for monitoring Cetuximab.

Introduction

Monoclonal antibodies offers unprecedent opportunity to drug development with remarkable efficacy and safety and have significantly improved the prognosis of more than 14 millions patients with new declared cancers and patients currently living with chronic inflammatory disease. Today, therapeutic monoclonal antibodies (mAb) are a very large market worldwide; this market is expanding, with increased applications and numerous clinical studies to investigate their utility, particularly in the treatment of immune disorders and cancers. Biosimilars are beginning to emerge on the market, and they will contribute to the increasing use of biotherapies. In the future, antibody-drug conjugates may also be included in the therapeutic arsenal. These novel drugs can be used either as a monotherapy or in combination, reinforcing the therapeutic Armamentarium of the clinicians.

Since mAb are increasingly promoted as major therapeutic agents in oncology, and because interindividual variability, survival and response have been linked to the doses administered, the ability to accurately quantify mAb in blood may be an important part of the development and use of these treatments and therapeutic monitoring of biotherapy has become a new preoccupation for clinicians.

Therapeutic monitoring consists in monitoring the clearance of a monoclonal antibody, injected alone or in combination, in the serum of a patient. It enables to determine precisely and objectively the patient response to a treatment and enables also to better control efficiency while anticipating therapeutic escape and side effects.

Methods

We will present an innovative and generic approach involving the use of stable isotope labeled mAbs used as internal standards and LC-MS to allow accurate, sensitive and specific quantification, as required for TDM. Our approach was already tested and implemented with success on anti-TNF antibodies (Infliximab and Adalimumab) and was recently implemented for monitoring Cetuximab.

Results

(i) For infliximab and adalimumab, samples were prepared using Mass Spectrometry Immuno Assay (MSIA™) followed by trypsin digestion and submitted to multiple reaction monitoring (MRM) for quantification of IFX. The chromatographic run lasted 13 min. The range of quantification was 1 to 26 mg/L. For two internal quality controls spiked with 6 and 12 mg/L of IFX, the method was reproducible (coefficients of variation (CV%): 12.7 and 2.1), repeatable (intra-day CV%: 5.5 and 5.0), and accurate (inter-day and intra-day deviations from nominal values: +6.4 to +3.7 % and 5.5 to 9.2 %, respectively). There was no cross - contamination effect. Samples from 45 patients treated with IFX were retrospectively analyzed by LC-MS/MS and results were compared to those obtained with an in-house ELISA assay and the commercial Lisa Tracker® method. Good agreement was found between LC-MS/MS and in-house ELISA (mean underestimation of 13 % for in-house ELISA), but a significant bias was found with commercial ELISA (mean underestimation of 136 % for commercial ELISA). This method will make it possible to standardize IFX quantification between laboratories.

(ii) For Cetuximab, the analysis was performed in positive mode using parallel reaction monitoring acquisition with resolution set at 70 000. We reported a generic, rapid and high-throughput sample preparation protocol based on IgG capture followed by trypsin digestion and an on-line SPE clean-up. The optimized method exhibited good analytical performances and linearity in a range from 5 to 150 µg/ml. The within-run and the between-run precision of the assay were equal or less than 10%, for six replicates at each of three concentrations and evaluated on three separated days. The plasma concentration of CTX from nineteen patients was also determined. Interestingly, this work shows that quantification of mAb in clinical samples is not limited to classical QqQ instruments and demonstrate that LC-HRMS is also a relevant approach for this purpose.

Conclusions & Discussion

We reported here simple, generic and high-throughput preparation protocols perfectly suited to the monitoring of monoclonal antibodies in clinical labs equipped with LC-MS instruments. Our work can now be extended to various mAbs, in a context of TDM in pharmacological labs but also to rapidly assess the PK properties of mAbs in development.

(1) Determination of cetuximab in plasma by LC-HRMS-orbitrap with a stable labeled 13C,15N-Cetuximab internal standard.

Millet A, Lebert D, Picard G, You B, Ceruse Ph, Guitton J, submitted.

(2) Infliximab quantitation in human plasma by liquid chromatography-tandem mass spectrometry: towards a standardization of the methods?

Jourdil JF, Lebert D, Gautier-Veyret E, Lemaitre F, Bonaz B, Picard G, Tonini J, Stanke-Labesque F.

(3) Absolute and multiplex quantification of antibodies in serum using PSAQ™ standards and LC-MS/MS.

Lebert D, Picard G, Beau-Larvor C, Troncy L, Lacheny C, Maynadier B, Low W, Mouz N, Brun V, Klinguer-Hamour C, Jaquinod M, Beck A.