Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies.

Despite the popularity of therapeutic monoclonal antibodies (mAbs), data relative to their ionic physico-chemical properties are very scarce in the literature. In this work, isoelectric points (pIs) of 23 Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAbs were determined by imaged capillary isoelectric focusing (icIEF), and ranged from 6.1 to 9.4. The obtained values were in good agreement with those calculated by both Vector NTI and MassLynx softwares. icIEF can therefore be considered as a reference technique for such a determination. The relative percentages of acidic and basic variants determined by cation exchange chromatography (CEX) using both salt- and pH-gradients were comprised between 15% and 30% for most mAbs and were in good agreement with each other, whereas generic icIEF seems to overestimate the amount of acidic charge variants in mAb products. To our knowledge, this is the first study focusing on the ionic properties of a wide range of FDA and EMA approved reference mAbs, using both generic chromatographic and electrophoretic methodologies. To illustrate the interest of the study for mAb developability purposes, ionic properties of a clinical mAb candidate (dalotuzumab) were also investigated.

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. II- Identification of sub-units for the characterization of even and odd load drug species.

This paper is the second part of a two-part series dedicated to the development of an on-line comprehensive HICxRPLC-UV/MS method for the characterization of a commercial inter-chain cysteine-linked ADC (brentuximab vedotin, Adcetris(®)). The first part focused on the optimization of the chromatographic conditions. In the second part of this series of papers, the structural characterization of the Brentuximab Vedotin was extensively discussed. With the combination of HIC and RPLC-MS data, the average DAR was easily measured in HIC and, at the same time, the predominant positional isomers were identified in RPLC-MS in one single injection. It was also demonstrated that the retention data obtained in the first and second dimensions was particularly useful to assist ADC characterization through the identification of sub-units. Using this methodology, the presence of odd DARs (1, 3 and 5) and their relative abundance was assessed by a systematic evaluation of HIC x RPLC-UV/MS data for both commercial and stressed ADC samples. Finally, once the exhaustive characterization of ADC was completed, MS could be conveniently replaced by UV detection to quickly assess the conformity of different ADCs batches.

Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I - Optimization of separation conditions.

Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be characterized during development and for further quality control. The coupling of on-line HIC x RPLC to high resolution mass spectrometry can be considered as a very efficient analytical method, providing extensive information on ADC sample, within a reduced time scale. Our intention in this first paper is to present the approach used to rationally optimize the numerous conditions that can affect the quality of the 2D-separation. HIC and RPLC conditions were therefore optimized to prevent salt precipitation due to solvent mixing and to enhance sensitivity, while limiting the total analysis time. We demonstrated that adding salt in the sample solvent before HIC injection allows a significant peak shape improvement. The gradient profile was also carefully optimized in both dimensions, leading to a two-step gradient in HIC and bracketed gradient in RPLC. This study shows that on-line HIC x RPLC hyphenated to high resolution mass spectrometry is a useful method to obtain rapid and extensive structural information on the peaks observed in the first HIC dimension, thereby leading, in a single step requiring 75min, to the precise determination of the average drug-to-antibody ratio (DAR) by HIC as well as the knowledge of the drug load distribution for a particular DAR. The structural characterization of ADC fragments by RPLC-QTOF will be discussed in the second part of this two-part series.

A new anti-human Fc method to capture and analyze ADCs for characterization of drug distribution and the drug-to-antibody ratio in serum from pre-clinical species.

Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. They combine monoclonal antibody specificity for over-expressed tumor antigens and the high cytoxicity of small molecular drugs (SMDs) and can therefore selectively kill tumor cells while minimizing toxicity to normal cells. Nevertheless, the premature deconjugation of ADCs in the circulation may trigger off target toxicity in patients. The released free drug level must be low in circulation for an extended period of time as well as the de-conjugation rate to ensure an acceptable therapeutic window. As a result, the assessment of the stability of the linker between payload and mAb in the systemic circulation is of paramount importance before entering in clinical trial. Here we report a new universal method to immunocapture and analyze by LC-MS the stability and distribution of ADCs in sera from relevant preclinical species (mouse, rat and cynomolgus monkey). Furthermore we demonstrated that this workflow can be applied to both ADCs with cleavable and non cleavable linkers. Last but not least, the results obtained in cynomolgus serum using immunoprecipitation and LC-MS analysis were cross validated using an ELISA orthogonal method. As the ligand used for immunoprecipitation is targeting the Fc part of mAb (CaptureSelect™ Human IgG-Fc PK Biotin), this protocol can be applied to analyze the stability of virtually all ADCs in sera for preclinical studies without the need to prepare specific molecular tools.

A sensitive multidimensional method for the detection, characterization, and quantification of trace free drug species in antibody-drug conjugate samples using mass spectral detection.

Conjugation processes and stability studies associated with the production and shelf life of antibody-drug conjugates (ADCs) can result in free (non-conjugated) drug species. These free drug species can increase the risk to patients and reduce the efficacy of the ADC. Despite stringent purification steps, trace levels of free drug species may be present in formulated ADCs, reducing the therapeutic window. The reduction of sample preparation steps through the incorporation of multidimensional techniques has afforded analysts more efficient methods to assess trace drug species. Multidimensional methods coupling size-exclusion and reversed phase liquid chromatography with ultra-violet detection (SEC-RPLC/UV) have been reported, but offer limited sensitivity and can limit method optimization. The current study addresses these challenges with a multidimensional method that is specific, sensitive, and enables method control in both dimensions via coupling of an on-line solid phase extraction column to RPLC with mass spectral detection (SPE-RPLC/MS). The proposed method was evaluated using an antibody-fluorophore conjugate (AFC) as an ADC surrogate to brentuximab vedotin and its associated parent maleimide-val-cit-DSEA payload and the derived N-acetylcysteine adduct formed during the conjugation process. Assay sensitivity was found to be 2 orders more sensitive using MS detection in comparison to UV-based detection with a nominal limit of quantitation of 0.30 ng/mL (1.5 pg on-column). Free-drug species were present in an unadulterated ADC surrogate sample at concentrations below 7 ng/mL, levels not detectable by UV alone. The proposed SPE-RPLC/MS method provides a high degree of specificity and sensitivity in the assessment of trace free drug species and offers improved control over each dimension, enabling straightforward integration into existing or novel workflows.

Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates.

Antibody drug conjugates (ADCs) are highly cytotoxic drugs covalently attached via conditionally stable linkers to monoclonal antibodies (mAbs) and are among the most promising next-generation empowered biologics for cancer treatment. ADCs are more complex than naked mAbs, as the heterogeneity of the conjugates adds to the inherent microvariability of the biomolecules. The development and optimization of ADCs rely on improving their analytical and bioanalytical characterization by assessing several critical quality attributes, namely the distribution and position of the drug, the amount of naked antibody, the average drug to antibody ratio, and the residual drug-linker and related product proportions. Here brentuximab vedotin (Adcetris) and trastuzumab emtansine (Kadcyla), the first and gold-standard hinge-cysteine and lysine drug conjugates, respectively, were chosen to develop new mass spectrometry (MS) methods and to improve multiple-level structural assessment protocols.

Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis.

Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. Because ADCs combine the monoclonal antibody specificity with the high toxicity of a drug, they can selectively kill tumor cells while minimizing toxicity to normal cells. Most of the current ADCs in clinical trials are controlled, but heterogeneous mixtures of isomers and isoforms. Very few protocols on ADC characterization at the peptide level have been published to date. Here, we report on the improvement of an ADC peptide mapping protocol to characterize the drug-loaded peptides by LC-MS analysis. These methods were developed on brentuximab vedotin (Adcetris), a commercial ADC with an average of four drugs linked to interchain cysteine residues of its antibody component. Because of the drug hydrophobicity, all the steps of this protocol including enzymatic digestion were improved to maintain the hydrophobic drug-loaded peptides in solution, allowing their unambiguous identification by LC-MS. For the first time, the payloads positional isomers observed by RP-HPLC after IdeS-digestion and reduction of the ADC were also characterized.

Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion.

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. In addition, by analyzing the Fc fragment in the same run, full glycoprofiling and demonstration of the absence of additional conjugation was easily achieved. As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.

Cetuximab Fab and Fc N-glycan fast characterization using IdeS digestion and liquid chromatography coupled to electrospray ionization mass spectrometry.

Antibodies and related products represent one of the fastest growing areas of new drug development within the pharmaceutical industry. Monoclonal antibodies (mAbs) undergo many posttranslational modifications (PTMs) that must be extensively characterized. Here we described a rapid mass spectrometry (MS) method for the characterization of cetuximab glycosylation. The reported analytical technique is based on the use of a cystein protease, immunoglobulin-degrading enzyme of Streptococcus pyogenes that allows a fast limited proteolysis of the mAb with low material consumption. The resulting large fragments are analyzed by ultrahigh-performance liquid chromatography combined to an electrospray ionization mass spectrometer and a time-of-flight analyzer (ESI-TOF). Cetuximab is a potent chimeric mouse/human antibody worldwide approved for the treatment of colon and head and neck cancers. This antibody, produced by SP2/0 murine myeloma cells, is N-glycosylated both in the Fc and Fab moieties, which have been shown to impact on safety and PK/PD and considered as a critical quality attribute. The method can also be applied for biosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.