Characterization of Therapeutic Antibodies and Antibody Drug Conjugates by MS

HomeResearch TopicsBiopharma/pharma Companion Diagnostics and TDMCharacterization of Therapeutic Antibodies and Antibody Drug Conjugates by MS
Characterization of Therapeutic Antibodies and Antibody Drug Conjugates by MS 2016-11-10T20:12:55+00:00

Notable peer-reviewed articles—MS-based absolute quantitation of protein therapeutics–monoclonal antibodies (mAbs), antibody-drug conjugates (ADC), and bispecific antibodies (bsAb).

 

Articles of note

Absolute quantitation of protein therapeutics in biological matrices by enzymatic digestion and LC-MS

The advancement of biotechnology has led to an increase in biotherapeutic drugs, especially recombinant proteins and monoclonal antibodies. Ligand-binding assays or immunoassays are the standard methods of choice in pharmacokinetic studies in support of drug discovery and development for protein therapeutics. LC-MS-based methodologies are increasingly used as alternatives to immunoassays for absolute protein quantitation in biological samples. We review recent advancements in absolute quantitation of protein therapeutics in biological matrices by enzymatic digestion and LC-MS. Read more ›

Figure 7. Generation of seven fragments of near 25 kDa after IdeS digestion and DTT reduction of trastuzumab-mc_DSEA.

Antibody drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion (free full text)

Here we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA)-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms, electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR) for both monomeric and multimeric species. Read more ›

Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry (free full text)

This Feature will introduce the strategies of therapeutic antibodies (mAbs) in-depth characterization by mass spectrometry (MS) and discuss analytical comparison of biosimilar to originator mAbs, with the cases of trastuzumab and cetuximab. In addition, the structural and functional insights gained both by state-of-the art and emerging MS methods used for biobetters and next generation antibodies design and optimization will also be highlighted. Read more ›

Characterization of intact antibodyCharacterization of intact antibody-drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography-mass spectrometry

Antibody-drug conjugates (ADCs) are designed to facilitate the targeted delivery of cytotoxic drugs to improve their tumor fighting effects and minimize systemic toxicity. However, efficacy and safety can potentially be compromised due to the release of conjugated drugs from the ADC with time while in circulation, resulting in changes in the drug-to-antibody ratio (DAR). Current understanding of this process is limited because existing methods such as immunoassays fail to distinguish ADCs with different DARs. Here we demonstrate a novel method with bead-based affinity capture and capillary liquid chromatography-mass spectrometry to allow direct measurement of drug release by quantifying DAR distributions of the ADC in plasma/serum. This method successfully identified individual intact conjugated antibody species produced due to drug loss from ADCs (e.g., an engineered site-specific anti-MUC16 THIOMAB-drug conjugate) and measured the corresponding DAR distributions in vitro and in vivo. Read more ›

Characterization of the drug-to-antibody ratio distribution for antibody-drug conjugates in plasma/serum

BACKGROUND: Antibody-drug conjugates (ADCs) are a new class of cancer therapeutics that deliver potent cytotoxins specifically to tumors to minimize systemic toxicity. However, undesirable release of covalently linked drugs in circulation can affect safety and efficacy. The objective of this manuscript was to propose and assess the assays that allow for the characterization of the drug deconjugation in plasma/serum.
RESULTS: ADCs of three main drug conjugation platforms, linked via lysine, site-specific engineered cysteine or reduced interchain disulfide cysteine residues, were analyzed using affinity capture for sample enrichment coupled with LC-MS or hydrophobic interaction chromatography-UV for detection. These novel approaches enabled measurement of the relative abundance of individual ADC species with different drug-to-antibody ratios, while maintaining their structural integrity. Read more ›

Characterization of therapeutic antibodies and related products

This article is part of the Fundamental and Applied Reviews in Analytical Chemistry 2013 special issue. The Analytical Chemistry team is pleased to present the 2013 special issue of fundamental and applied reviews in analytical chemistry. The 19 topics in this issue provide critical reviews from pioneers in selected cutting edge topics and by experts in traditional core areas. These manuscripts highlight the innovations occurring in many of the subfields of analytical chemistry. Read more ›

Development of a native nanoelectrospray mass spectrometry method for determination of the drug-to-antibody ratio of antibody-drug conjugates

Antibody-drug conjugates (ADCs), an increasingly important therapeutic modality for targeted cancer treatment, have been studied using many analytical methods. A class of ADCs that utilize the reduced interchain disulfide cysteine residues for drug attachment has attracted particular interest in drug development. One challenge in analytical characterization of this class of ADCs is that the intact mass information of the ADC molecule is not attainable using conventional reversed-phase liquid chromatography-mass spectrometry methods. In this paper, we report a mass spectrometry (MS) method engaging enzymatic digestion, nanoelectrospray ionization (nano-ESI), and native MS to achieve direct determination of the intact mass and, furthermore, to calculate the average drug-to-antibody ratio (DAR) of the cysteine-linked ADCs. Read more ›

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap (free full text)

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. Read more ›

Mass spectrometric characterization of transglutaminase based site-specific antibody-drug conjugates

Antibody drug conjugates (ADCs) are becoming an important new class of therapeutic agents for the treatment of cancer. ADCs are produced through the linkage of a cytotoxic small molecule (drug) to monoclonal antibodies that target tumor cells. Traditionally, most ADCs rely on chemical conjugation methods that yield heterogeneous mixtures of varying number of drugs attached at different positions. The potential benefits of site-specific drug conjugation in terms of stability, manufacturing, and improved therapeutic index has recently led to the development of several new site-specific conjugation technologies. However, detailed characterization of the degree of site specificity is currently lacking. In this study we utilize mass spectrometry to characterize the extent of site-specificity of an enzyme-based site-specific antibody-drug conjugation technology that we recently developed. Read more ›

Modern chromatographic and mass specModern chromatographic and mass spectrometric techniques for protein biopharmaceutical characterization

Protein biopharmaceuticals such as monoclonal antibodies and therapeutic proteins are currently in widespread use for the treatment of various life-threatening diseases including cancer, autoimmune disorders, diabetes and anemia. The complexity of protein therapeutics is far exceeding that of small molecule drugs; hence, unraveling this complexity represents an analytical challenge. The current review provides the reader with state-of-the-art chromatographic and mass spectrometric tools available to dissect primary and higher order structures, post-translational modifications, purity and impurity profiles and pharmacokinetic properties of protein therapeutics. Read more ›

Time resolved native ion-mobility mass spectrometry to monitor dynamics of IgG4 Fab arm exchange and “bispecific” monoclonal antibody formation

Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half molecules (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissociate and recombine in bispecific antibodies both in vitro and in vivo. Read more ›