Mass Spectrometry Approaches for Identification and Quantitation of Therapeutic Monoclonal Antibodies in the Clinical Laboratory

Ladwig, Paula M.; Barnidge, David R.; Willrich, Maria Alice V.

doi:10.1128/cvi.00545-16

Cited by 61 publications

(42 citation statements)

References 54 publications

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“…Mass spectrometry (MS) has been applied to the analysis of therapeutic mAbs over the past decades and has become the predominant tool in the characterization of their structures.- [11][12][13] Currently, top-down, middle-down and bottom-up MS are three main approaches applied in the characterization of PTMs, localization of disulfide bonds, and verification of protein sequences in analyzing therapeutic mAbs. [14][15][16] Among these three approaches, a bottom-up approach combining liquid chromatography (LC) and tandem MS (MS/MS) is routinely used in the pharmaceutical industry owing to its standardized workflow and availability of instrument choice and software.…”

Section: Introductionmentioning

confidence: 99%

Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

Jin

Lin

et al. 2018

mAbs

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The pharmaceutical industry's interest in monoclonal antibodies (mAbs) and their derivatives has spurred rapid growth in the commercial clinical pipeline of these effective therapeutics. The complex micro-heterogeneity of mAbs requires in-depth structural characterization for critical quality attribute assessment and quality assurance. Currently, mass spectrometry (MS)-based methods are the gold standard in mAb analysis, primarily with a bottom-up approach in which immunoglobulins G (IgGs) and their variants are digested into peptides to facilitate the analysis. Comprehensive characterization of IgGs and the micro-variants remains challenging at the proteoform level. Here, we used both top-down and middle-down MS for in-depth characterization of a human IgG1 using ultra-high resolution Fourier transform MS. Our top-down MS analysis provided characteristic fingerprinting of the IgG1 proteoforms at unit mass resolution. Subsequently, the tandem MS analysis of intact IgG1 enabled the detailed sequence characterization of a representative IgG1 proteoform at the intact protein level. Moreover, we used the middle-down MS analysis to characterize the primary glycoforms and micro-variants. Micro-variants such as low-abundance glycoforms, C-terminal glycine clipping, and C-terminal proline amidation were characterized with bond cleavages higher than 44% at the subunit level. By combining top-down and middle-down analysis, 76% of bond cleavage (509/666 amino acid bond cleaved) of IgG1 was achieved. Taken together, we demonstrated the combination of top-down and middle-down MS as powerful tools in the comprehensive characterization of mAbs.

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Section: Introductionmentioning

confidence: 99%

Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

Jin

Lin

et al. 2018

mAbs

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“…Mass spectrometry is an alternative methodology in the detection of M-proteins as well as t-mAbs [11]. DARA and other t-mAbs have been detected and distinguished to some extent using molecular mass as a measurement tool, as it provides increased specificity based on unique masses to each monoclonal antibody [12,13].…”

Section: Discussionmentioning

confidence: 99%

Complete Depletion of Daratumumab Interference in Serum Samples from Plasma Cell Myeloma Patients Improves the Detection of Endogenous M-Proteins in a Preliminary Study

et al. 2020

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Background: Therapeutic humanized IgG1 kappa monoclonal antibody (t-mAb), daratumumab (DARA) is a Food and Drug Administration approved drug for the treatment of relapsed/refractory plasma cell myeloma (PCM). DARA appears on serum protein electrophoresis (SPEP) and on serum immunofixation (sIFE) as an IgG kappa monoclonal immunoglobulin protein (M-protein), complicating the assessment of the patients’ response to therapy. A more ominous threat to patient safety can occur with the misinterpretation of the presence of a small t-mAb spike as being the residual product of the patient’s neoplastic clone, presented either as oligoclonality or new clonality, which could result in incorrect interpretation of failure to achieve remission. Methods: In this report, we describe a novel and cost-effective technique based on biotinylated recombinant CD38 and streptavidin-coated magnetic beads to capture and remove residual DARA present in PCM patient serum samples. The treated samples are then run like regular samples on SPEP and sIFE. We validated this simple technique in DARA-spiked PCM samples and patient samples on DARA treatment. Results: Our simple capture technique completely extracted DARA in all of the tested serum specimens and allowed the assessment of residual M-protein without DARA interference. The results were reproducible and highly specific for DARA, and did not have any impact on endogenous M-protein migration and quantification by SPEP and sIFE. The cost of this technique is much lower and it can be performed in-house with a very short turnaround time compared to the currently available alternative methods. There is a great need for such reflex technologies to avoid interpretation errors. Conclusions: This method is an effective way to eliminate DARA interference in SPEP and sIFE, and can be easily implemented in any clinical laboratory without any patent restriction. This simple technique can be adopted for other t-mAbs using their respective ligands and will help to reduce additional doses of toxic treatment and further testing in patients on t-mAbs with a false positive M-protein spike.

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“…Today, MS is the reference analytical strategy for PK investigations of small molecule drugs and becomes important in PK investigations of biologicals (e.g. antibody-based drugs [21][22][23] ). More particularly, liquid chromatography (LC) coupled to tandem MS (LC-MS/MS) is the gold standard for MS-based drug monitoring from any liquid sample.…”

Section: Current Measurement Methods Applied To Clinical Pharmacologymentioning

confidence: 99%

Approaching sites of action of drugs in clinical pharmacology: New analytical options and their challenges

Longuespée

Theile

Fresnais

et al. 2020

Brit J Clinical Pharma

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Clinical pharmacology is an important discipline for drug development aiming to define pharmacokinetics (PK), pharmacodynamics (PD) and optimum exposure to drugs, i.e. the concentration-response relationship and its modulators. For this purpose, information on drug concentrations at the anatomical, cellular and molecular sites of action is particularly valuable. In pharmacological assays, the limited accessibility of target cells in readily available samples (i.e. blood) often hampers mass spectrometry-based monitoring of the absolute quantity of a compound and the determination of its molecular action at the cellular level. Recently, new sample collection methods have been developed for the specific capture of rare circulating cells, especially for the diagnosis of circulating tumour cells. In parallel, new advances and developments in mass spectrometric instrumentation now allow analyses to be scaled down to the cellular level. Together, these developments may permit the monitoring of minute drug quantities and show their effect at the cellular level. In turn, such PK/PD associations on a cellular level would not only enrich our pharmacological knowledge of a given compound but also expand the basis for PK/PD simulations. In this review, we describe novel concepts supporting clinical pharmacology at the anatomical, cellular and molecular sites of action, and highlight the new challenges in mass spectrometry-based monitoring. Moreover, we present methods to tackle these challenges and define future needs. K E Y W O R D S clinical pharmacology, mass spectrometry, personalized medicine, review, sites of action 1 | INTRODUCTION Clinical pharmacology studies the relevant parameters of drug fate (pharmacokinetics, PK) and efficacy (pharmacodynamics, PD) in humans in order to establish the dose-response and concentrationresponse relationship. It also defines intraindividual and interindividual modulators of these relationships, leading to different effectiveness, safety and dose requirements. Monitoring drug concentrations and effects is therefore of considerable importance during drug development, starting with the very first clinical trials. 1 Clinical pharmacology clarifies mechanisms of both beneficial and adverse drug effects in order to get closer to drug effectiveness monitoring. A drug's site of action can be defined at different scales: anatomical (the compartment the drug has to reach, e.g. tissues), cellular (the

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Mass Spectrometry Approaches for Identification and Quantitation of Therapeutic Monoclonal Antibodies in the Clinical Laboratory

Cited by 61 publications

References 54 publications

Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

Complete Depletion of Daratumumab Interference in Serum Samples from Plasma Cell Myeloma Patients Improves the Detection of Endogenous M-Proteins in a Preliminary Study

Approaching sites of action of drugs in clinical pharmacology: New analytical options and their challenges

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