Modeling study of long-term stability of the monoclonal antibody infliximab and biosimilars using liquid-chromatography-tandem mass spectrometry and size-exclusion chromatography-multi-angle light scattering.

Monoclonal antibodies (mAbs) represent a dynamic class of biopharmaceutical products, as evidenced by an increasing number of market authorizations for mAb innovator and biosimilar products. Stability studies are commonly performed during product development, for instance, to exclude unstable molecules, optimize the formulation or determine the storage limit. Such studies are time-consuming, especially for mAbs, because of their structural complexity which requires multiple analytical techniques to achieve a detailed characterization. We report the implementation of a novel methodology based on the accelerated stability assessment program (ASAP) in order to model the long-term stability of mAbs in relation to different structural aspects. Stability studies of innovator infliximab and two different biosimilars were performed using forced degradation conditions alongside in-use administration conditions in order to investigate their similarity regarding stability. Thus, characterization of post-translational modifications was achieved using liquid-chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the formation of aggregates and free chain fragments was characterized using size-exclusion chromatography-multi-angle light scattering (SEC-MALS-UV/RI) analysis. Consequently, ASAP models were investigated with regard to free chain fragmentation of mAbs concomitantly with N57 deamidation, located in the hypervariable region. Comparison of ASAP models and the long-term stability data from samples stored in intravenous bags demonstrated a relevant correlation, indicating the stability of the mAbs. The developed methodology highlighted the particularities of ASAP modeling for mAbs and demonstrated the possibility to independently consider the different types of degradation pathways in order to provide accurate and appropriate prediction of the long-term stability of this type of biomolecule.

Structural identification and absolute quantification of monoclonal antibodies in suspected counterfeits using capillary electrophoresis and liquid chromatography-tandem mass spectrometry.

Monoclonal antibodies (mAbs) represent a major category of biopharmaceutical products which due to their success as therapeutics have recently experienced the emergence of mAbs originating from different types of trafficking. We report the development of an analytical strategy which enables the structural identification of mAbs in addition to comprehensive characterization and quantification in samples in potentially counterfeit samples. The strategy is based on the concomitant use of capillary zone electrophoresis analysis (CZE-UV), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS). This analytical strategy was applied to the investigation of different samples having unknown origins seized by the authorities, and potentially incorporating an IgG 4 or an IgG 1. The results achieved from the different techniques demonstrated to provide orthogonal and complementary information regarding the nature and the structure of the different mAbs. Therefore, they allowed to conclude unequivocally on the identification of the mAbs in the potentially counterfeit samples. Finally, a LC-MS/MS quantification method was developed which specificity was to incorporate a different mAbs labeled with stable isotopes as internal standard. The LC-MS/MS quantification method was validated and thus demonstrated the possibility to use common peptides with the considered IgG in order to achieve limit of quantification as low as 41.4 nM. The quantification method was used to estimate the concentration in the investigated samples using a single type of internal standard and experimental conditions, even in the case of mAbs with no stable isotope labeled homologues available.

CEToolbox: Specialized calculator for capillary electrophoresis users as an android application.

CE has been demonstrated to be a useful and powerful separation method for the characterization of charged and neutral molecules. Since the end of the 1980s and the development of the first commercialized CE device, the use of this separation method has continued to grow for academic and industrial research involving inexorably increasing of the number of CE users. Whatever the application domain, each CE user is daily confronted to the same problems often based on basic calculations of separation properties. In order to help the community of CE users to get quickly and easily a lot of information, and desiring to provide a tool running on mobile platforms, CEToolbox has been developed as a free Android application. Within few clicks, CEToolbox offers extensive injection information as injected volume, total capillary volume, proportion and amount of injected sample, rinsing time, and electrical field. Moreover, three additional tabs allow to obtain the calculation of the viscosity and the conductivity of BGE, and the separation flow rates. Finally, a last tab is dedicated to the calculation of electroosmotic mobility and effective mobilities for a maximum of 20 compounds. CEToolbox, which can be downloaded for free on Google and F-Droid application stores, was developed to simplify the daily of CE users regardless of the CE devices.

Accelerated Stability Assessment Program to Predict Long-term Stability of Drugs: Application to Ascorbic Acid and to a Cyclic Hexapeptide.

During pharmaceutical development, the stability of the product is assessed during long-term study. If any stability issues are discovered at this point of the process, it will result in re-formulation and important loss of time and cost. Therefore, important efforts are made in order to select the most stable product. Nevertheless, predicting the stability of the developed product at early stage of the development is challenging. Accelerated stability assessment program (ASAP), based on modified Arrhenius equation and isoconversion approach, appears as an interesting tool allowing to evaluate stability and shelf-life of pharmaceutical product in a short period of time. Nevertheless, few studies using these approaches are published in the literature, and the majority concern small drug molecules. Here, this approach was applied on a small drug molecule, ascorbic acid (AA), and on a cyclic hexapeptide named cFEE. AA and cFEE have been exposed to various temperatures for a maximum of 3 weeks, and then analyzed by capillary electrophoresis coupled to UV detection (CZE-UV) for AA or LC-MS for cFEE. The level of major degradation products was used to build ASAP models and predict the stability of both compounds. Comparison between predicted and long-term data were found accurate for both compounds undergoing two different degradation pathways (oxidation and hydrolysis), confirming the real interest of accelerated predicting stability approach for consistent determination of long-term stability shelf-life of pharmaceutical products.

Comprehensive and quantitative stability study of ascorbic acid using capillary zone electrophoresis with ultraviolet detection and high-resolution tandem mass spectrometry.

Ascorbic acid is a powerful antioxidant compound involved in many biological functions, and a chronic deficiency is at the origin of scurvy disease. A simple, rapid, and cost-effective capillary electrophoresis method was developed for the separation and simultaneous quantification of ascorbic acid and the major degradation products: dehydroascorbic acid, furfural, and furoic acid. Systematic optimization of the conditions was performed that enabled baseline separation of the compounds in less than 10 min. In addition to simultaneous quantification of ascorbic acid alongside to the degradation products, stability studies demonstrated the possibility using capillary electrophoresis to separate and identify the major degradation products. Thus, high-resolution tandem mass spectrometry experiments were conducted in order to identify an unknown degradation product separated by capillary electrophoresis and significantly present in degraded samples. Comparison of mass spectrometry data and capillary electrophoresis electropherograms allowed to identify unambiguously trihydroxy-keto-valeraldehyde. Finally, capillary electrophoresis was successfully applied to evaluate the composition of different pharmaceutical preparation of ascorbic acid. Results showed the excellent performance of the capillary electrophoresis method due to the separation of excipients from the compounds of interest, which demonstrated the relevance of using an electrophoretic separation in order to perform comprehensive stability studies of ascorbic acid.

Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future.

INTRODUCTION: The development and optimization of antibody drug conjugates (ADCs) rely on improving their analytical and bioanalytical characterization, by assessing critical quality attributes (CQAs). Among the CQAs, the glycoprofile, drug load distribution (DLD), the amount of unconjugated antibody (D0), the average drug-to-antibody ratio (DAR), the drug conjugation sites and the residual drug-linker and related product proportions (SMDs) in addition to high and low molecular weight species (H/LMWS), and charge variants are the most important ones. Areas covered: The analytical and structural toolbox for the characterization of 1st, 2d and 3d generation ADCs was significantly extended in the last 3 years. Here, we reviewed state-of-the-art techniques, such as liquid chromatography, high resolution native and ion mobility mass spectrometry, multidimensional liquid chromatography and capillary electrophoresis hyphenated to mass spectrometry, reported mainly since 2016. Expert commentary: These emerging techniques allow a deep insight into important CQAs that are related to ADC Chemistry Manufacturing and Control (CMC) as well as an improved understanding of in vitro and in vivo ADC biotransformations. This knowledge and the development of quantitative bioanalytical assays will continue to contribute to early-developability assessment for the optimization of all the ADC components (i.e. antibody, drug, and linker) and help to bring next-generation ADCs into late clinical development and to the market.

Revealing the potential of capillary electrophoresis/mass spectrometry: the tipping point.

The hyphenation of capillary electrophoresis and mass spectrometry (CE/MS) remains a minor technique compared with liquid chromatography/mass spectrometry (LC/MS), which represents nowadays the standard instrumentation, regardless of its introduction thirty years ago. However, from a theoretical point of view, CE coupling should be quite favorable especially with electrospray ionization mass spectrometry (ESI-MS). At the time, the sensitivity provided by CE/MS was often limited, due to hyphenation requirements, which at some point appeared to disqualify CE/MS from benefiting from the performance gain driving the evolution of MS instruments. However, this context has been significantly modified in a matter of a few years. The development of innovative CE/MS interfacing systems has enabled an important improvement regarding sensitivity and reinforced robustness in order to provide an instrumentation accessible to the largest scientific community. Because of the unique selectivity delivered by the electrophoretic separation, CE/MS has proved to be particularly relevant for the analysis of biological molecules. The conjunction of these aspects is motivating the interest in CE/MS analysis and shows that CE/MS is mature enough to enrich the toolbox of analytical techniques for the analysis of complex biological samples. Here we discuss the characteristics of the major types of high-sensitivity CE/ESI-MS instrumentation and emphasize the late evolution and future positioning of CE/MS analysis for the characterization of biological molecules like peptides and proteins, through some pertinent applications.

Europium labeled lactosylated albumin as a model workflow for the development of biotherapeutics.

Lactosylated albumin is currently used as a radiopharmaceutical agent to image the liver asialoglycoprotein receptors and quantify hepatic liver function in various diseases. A lactosylated protein (LACTAL) conjugate showed excellent liver uptake compared to non-lactosylated protein and a high signal to noise ratio, based on the biodistribution in mice using 99mTc-scintigraphy. However, in the laboratory, it is useful to have a method that can be used in daily practice to quantify cellular targeting or biodistribution. We propose a methodology from synthesis validation to pre-clinical demonstration and introduce a new practical detector (LACTAL.Eu) of the LACTAL molecule in biological media. We confirmed the purity and colloidal stability of the sample through physical analytical techniques, then showed the absence of in vitro toxicity of the agent and demonstrated in vitro targeting. Taking advantage of the fluorescence decay of the lanthanide, we performed measurements directly on the cell media without any further treatment. Finally, biodistribution in mice was confirmed by ex vivo measurements.

Affinity profiling of monoclonal antibody and antibody-drug-conjugate preparations by coupled liquid chromatography-surface plasmon resonance biosensing.

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (Rmax) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2. Graphical abstract ᅟ.

Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry.

We report on the online coupling of FcRn affinity liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) in native conditions to study the influence of modifications on the interaction of recombinant mAbs with the immobilized FcRn receptor domain. The analysis conditions were designed to fit the requirements of both affinity LC and ESI-MS. The mobile phase composition was optimized to maintain the proteins studied in native conditions and enable sharp pH changes in order to mimic properly IgGs Fc domain/FcRn receptor interaction. Mobile phase components needed to be sufficiently volatile to achieve native MS analysis. MS data demonstrated the conservation of the pseudonative form of IgGs and allowed identification of the separated variants. Native FcRn affinity LC-ESI-MS was performed on a therapeutic mAb undergoing various oxidation stress. Native MS detection was used to determine the sample oxidation level. Lower retention was observed for mAbs oxidized variants compared to their intact counterparts indicating decreased affinities for the receptor. This methodology proved to be suitable to identify and quantify post-translational modifications at native protein level in order to correlate their influence on the binding to the FcRn receptor. Native FcRn affinity LC-ESI-MS can tremendously reduce the time required to assess the biological relevance of the IgG microheterogeneities thus providing valuable information for biopharmaceutical research and development.

Glycoform Separation and Characterization of Cetuximab Variants by Middle-up Off-Line Capillary Zone Electrophoresis-UV/Electrospray Ionization-MS.

Monoclonal antibodies (mAbs) are highly complex glycoproteins that present a wide range of microheterogeneities that requires multiple analytical methods for full structure assessment and quality control. Capillary zone electrophoresis-mass spectrometry (CZE-MS) couplings, especially by electrospray ionization (ESI), appear to be really attractive methods for the characterization of biological samples. However, due to the presence of non- or medium volatile salts in the background electrolyte (BGE), online CZE-ESI-MS coupling is difficult to implement for mAbs isoforms separation. Here, we report an original strategy to perform off-line CZE-ESI-MS using CZE-UV/fraction collection technology to perform CZE separation, followed by ESI-MS infusion of the different fractions using the capillary electrophoresis-electrospray ionization (CESI) interface as the nanoESI infusion platform. As the aim is to conserve electrophoretic resolution and complete compatibility with ESI-MS without sample treatment, hydroxypropylcellulose (HPC) coated capillary was used to prevent analyte adsorption and asymmetric CZE conditions involving different BGE at both ends of the capillary have been developed. The efficiency of our strategy was validated with the separation of Cetuximab charge variant by the middle-up approach. Molecular weights were measured for six charge variants detected in the CZE separation of Cetuximab subunits. The first three peaks correspond to Fc/2 variants with electrophoretic resolution up to 2.10, and the last three peaks correspond to F(ab')2 variants with average electrophoretic resolution of 1.05. Two Fc/2 C-terminal lysine variants were identified and separated. Moreover, separation of Fc/2 fragments allowed the glycoprofiling of the variants with the characterization of 7 different glycoforms. Regarding the F(ab')2 domain, 8 glycoforms were detected and separated in three different peaks following the presence of N-glycolyl neuraminic acid residues in some glycan structures. This work highlights the potential of CZE technology to perform separation of mAbs especially when they carry sialic acid carbohydrates.

Full antibody primary structure and microvariant characterization in a single injection using transient isotachophoresis and sheathless capillary electrophoresis-tandem mass spectrometry.

Here we report the complete characterization of the primary structure of a multimeric glycoprotein in a single analysis by capillary electrophoresis (CE) coupled to mass spectrometry (MS). CE was coupled to electrospray ionization tandem MS by means of a sheathless interface. Transient isotachophoresis (t-ITP) was introduced in this work as an electrokinetically based preconcentration technique, allowing injection of up to 25% of the total capillary volume. Characterization was based on an adapted bottom-up proteomic strategy. Using trypsin as the sole proteolytic enzyme and data from a single injection per considered protein, 100% of the amino acid sequences of four different monoclonal antibodies could be achieved. Furthermore, illustrating the effectiveness and overall capabilities of the technique, the results were possible through identification of peptides without tryptic miscleavages or posttranslational modifications, demonstrating the potency of the technique. In addition to full sequence coverages, posttranslational modifications (PTMs) were simultaneously identified, further demonstrating the capacity of this strategy to structurally characterize glycosylations as well as faint modifications such as asparagine deamidation or aspartic acid isomerization. Together with the exquisite detection sensitivity observed, the contributions of both the CE separation mechanism and selectivity were essential to the result of the characterization with regard to that achieved with conventional MS strategies. The quality of the results indicates that recent improvements in interfacing CE-MS coupling, leading to a considerably improved sensitivity, allows characterization of the primary structure of proteins in a robust and faster manner. Taken together, these results open new research avenues for characterization of proteins through MS.

Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes.

Development of nano-electrospray (nanoESI) sources allowed to increase significantly the sensitivity which is often lacking when studying biological noncovalent assemblies. However, the flow rate used to infuse the sample into the mass spectrometer cannot be precisely controlled with nanoESI and the robustness of the system could represent an issue. In this study, we have used a sheathless capillary electrophoresis-mass spectrometry (CESI) prototype as a nanoESI infusion device. The hydrodynamic mobilization of the capillary content was characterized and the ability of the system to generate a stable electrospray under controlled flow rate conditions ranging from 4 up to 900 nL/min was demonstrated. The effect of the infusing flow rate on the detection of an intact model protein analyzed under native conditions was investigated. Results demonstrated a significant increase in sensitivity of 46-fold and a signal-to-noise ratio improvement of nearly 5-fold when using an infusing flow rate from 456.9 down to 13.7 nL/min. The CESI prototype was further used to detect successfully the ß ring homodimer in its native conformation. Obtained results were compared with those achieved with conventional ESI. Intensity signals were increased by a factor of 5, while sample consumption decreased 80 times. ß ring complexed with the P14 peptide was also studied. Finally, the CESI interface was used to observe the quaternary structure of native hemocyanins from Carcinus maenas crabs; this high molecular complex coexisting under various degrees of complexation and resulting in masses ranging from 445 kDa to 1.34 MDa.

Enhancing affinity purification of monoclonal antibodies from human serum for subsequent CZE-MS analysis.

Due to the separation technique employed, capillary electrophoresis coupled to mass spectrometry (CE-MS) analysis performances are significantly influenced by the chemical composition and the complexity of the sample. In various applications, that impact has prevented the use of CE-MS for the characterization and quantification of proteins in biological samples. Here we present the development and evaluation and a sample preparation procedure, based on affinity purification, for the specific extraction of the monoclonal antibody (mAbs) infliximab from human serum in order to perform subsequent proteolytic digestion and CE-MS/MS analysis. Three distinctive sample preparation strategies were envisaged. In each case, the different steps composing the protocol were thoroughly optimized and evaluated in order to provide a sample preparation addressing the important complexity of serums samples while providing an optimal compatibility with CE-MS/MS analysis. The different sample preparation strategies were assessed concerning the possibility to achieve an appropriate absolute quantification of the mAbs using CE-MS/MS for samples mimicking patient serum samples. Also, the possibility to perform the characterization of several types of post-translational modifications (PTMs) was evaluated. The sample preparation protocols allowed the quantification of the mAbs in serums samples for concentration as low as 0.2 µg·mL-1 (2.03 nM) using CE-MS/MS analysis, also the possibility to characterize and estimate the modification level of PTMs hotspots in a consistent manner. Results allowed to attribute the effect on the electrophoretic separation of the different steps composing sample preparation. Finally, they demonstrated that sample preparation for CE-MS/MS analysis could benefit greatly for the extended applicability of this type of analysis for complex biological matrices.

Post-translational modifications comparative identification and kinetic study of infliximab innovator and biosimilars in serum using capillary electrophoresis-tandem mass spectrometry.

Despite reports indicating the potential impact of post-translational modifications on the activity of a monoclonal antibody, their prediction or monitoring post-administration remains a challenge. In addition, with the expiration of patents concerning the early generation of mAbs, the production of biosimilars is constantly increasing. Structural differences of biosimilars compared to the innovator product are commonly evaluated for the formulated product in the context of biosimilarity assessment. However, estimating their structural outcome after administration is particularly difficult. Due to the complexity of in vivo studies, there is a need to develop analytical strategies to predict PTMs consequently to their administration and their impact on mAbs potency. Here, we identified and evaluated the modification kinetics of 4 asparagine deamidations and 2 aspartate isomerizations of infliximab innovator product (Remicade®) and two biosimilars (Inflectra® and Remsima®) in vitro using serum incubation at 37 °C. The methodology was based on a bottom-up approach with capillary electrophoresis hyphenated with mass spectrometry analysis for an unequivocal assignment of modified and unmodified forms. 2 asparagines demonstrated a gradual deamidation correlated with incubation time. The specific extraction efficiency was evaluated to determine possible changes in the antigen binding affinity of infliximab with the incubation. Results showed the possibility to achieve an additional aspect concerning biosimilarity assessment, oriented on the study of the structural stability after administration.

Simultaneous quantification and structural characterization of monoclonal antibodies after administration using capillary zone electrophoresis-tandem mass spectrometry.

Monoclonal antibodies (mAbs) are demonstrating major success in various therapeutic areas such as oncology and the treatment of immune disorders. Over the past two decades, novel analytical methodologies allowed to address the challenges of mAbs characterization in the context of their production. However, after administration only their quantification is performed and insights regarding their structural evolution remain limited. For instance, clinical practice has recently highlighted significant inter-patient differences in mAb clearance and unexpected clinical responses, without providing alternative interpretations. Here, we report the development of a novel analytical strategy based on capillary zone electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) for the simultaneous absolute quantification and structural characterization of infliximab (IFX) in human serum. CE-MS/MS quantification was validated over the range 0.4-25 µg·mL-1 corresponding to the IFX therapeutic window and achieved a LOQ of 0.22 µg·mL-1 (1.5 nM) while demonstrating outstanding specificity compared to the ELISA assay. CE-MS/MS allowed structural characterization and estimation of the relative abundance of the six major N-glycosylations expressed by IFX. In addition, the results allowed characterization and determination of the level of modification of post-translational modifications (PTMs) hotspots including deamidation of 4 asparagine and isomerization of 2 aspartate. Concerning N-glycosylation and PTMs, a new normalization strategy was developed to measure the variation of modification levels that occur strictly during the residence time of IFX in the patient's system, overcoming artefactual modifications induced by sample treatment and/or storage. The CE-MS/MS methodology was applied to the analysis of samples from patients with Crohn's disease. The data identified a gradual deamidation of a particular asparagine residue located in the complementary determining region that correlated with IFX residence time, while the evolution of IFX concentration showed significant variability among patients.

Bothrops atrox and Bothrops lanceolatus Venoms In Vitro Investigation: Composition, Procoagulant Effects, Co-Factor Dependency, and Correction Using Antivenoms.

Bothrops venoms are rich in enzymes acting on platelets and coagulation. This action is dependent on two major co-factors, i.e., calcium and phospholipids, while antivenoms variably neutralize venom-related coagulopathy effects. Our aims were (i) to describe the composition of B. atrox and B. lanceolatus venoms; (ii) to study their activity on the whole blood using rotational thromboelastometry (ROTEM); (iii) to evaluate the contribution of calcium and phospholipids in their activity; and (iv) to compare the effectiveness of four antivenoms (Bothrofav™, Inoserp™ South America, Antivipmyn™ TRI, and PoliVal-ICP™) on the procoagulant activity of these two venoms. Venom composition was comparable. Both venoms exhibited hypercoagulant effects. B. lanceolatus venom was completely dependent on calcium but less dependent on phospholipids than B. atrox venom to induce in vitro coagulation. The four antivenoms neutralized the procoagulant activity of the two venoms; however, with quantitative differences. Bothrofav™ was more effective against both venoms than the three other antivenoms. The relatively similar venom-induced effects in vitro were unexpected considering the opposite clinical manifestations resulting from envenomation (i.e., systemic bleeding with B. atrox and thrombosis with B. lanceolatus). In vivo studies are warranted to better understand the pathophysiology of systemic bleeding and thrombosis associated with Bothrops bites.

Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy.

We report the formulation, characterization, colloidal stability, and in vitro efficiency of Fisetin nanocrystals stabilized by poloxamer P407. Such nanocrystals present a nanometer scale (148.6 ± 1.1 nm) and a high homogeneity (polydispersity index of 0.17 ± 0.01), with a production yield of 97.0 ± 2.5%. The engineered formulations of nanocrystals suspension (pH of 7.4 ± 0.1), stabilized via steric repulsion, are stable for several days in aqueous environment (Milli Q water, NaCl 10 mM or mannitol 5% w/v), for few days in HEPES buffered saline (HBS) (20 / 150 mM) under sink conditions, and in culture medium. After freeze drying in 5% w/v mannitol, the nanocrystal formulations can be stored at -80 °C for at least 120 days. Drug release experiments displayed a 98.7 ± 5.1% cumulative release over 3 days in HBS. Compared to the free drug, the nanocrystal formulations showed an improved cytotoxicity highlighted by the decrease of the half maximal inhibitory concentration for both murine Lewis lung carcinoma (3LL) and human endothelial (EA.hy926) cell lines. In addition, after incubation with Fisetin nanosuspensions, significant changes in the cell morphology for both cell lines were observed, showing an improved anti-angiogenic effect of nanocrystals formulation compared to the free drug. Overall, Fisetin formulated as nanocrystals showed enhanced biopharmaceutical properties and in vitro activity, offering a wide range of indications for challenging applications in the clinic.

Characterization of the Primary Structure of Cysteine-Linked Antibody-Drug Conjugates Using Capillary Electrophoresis with Mass Spectrometry.

Capillary electrophoresis-mass spectrometry (CE-MS) enables the characterization of the primary structure of ADCs. An analytical method based on a derived bottom-up proteomic workflow is designed to provide detailed information about the amino acid sequence, the glycosylation profiling, and the location on the peptide backbone of the conjugated drugs. Here we describe the experimental protocol applied on the characterization of cysteine-linked brentuximab vedotin (Adcetris®).

Kinetic and structural characterization of therapeutic albumin chemical functionalization using complementary mass spectrometry techniques.

Protein conjugates such as antibody-drugs conjugates (ADCs) represents the next generation of therapeutic proteins. They allow to combine the biological properties of the protein format with the characteristics of the conjugated ligands. The reaction implemented to couple ligands to the peptide backbone represents a crucial aspect of the production of protein conjugates, influencing the nature and the heterogeneity of the conjugates obtained. Here, we report the concomitant use of MALDI-TOF MS and LC-MS/MS analysis to investigate the chemical functionalization of human serum albumin (HSA) by the intermediate of lysine residues, previously used to generate biopharmaceutical agents for medical imaging. A kinetic was performed by collecting samples after different reaction times and analyzing them using the two techniques. MALDI-TOF MS analyses allowed estimating the number of conjugated ligands in a robust manner and assess the global functionalization kinetic on the intact protein level. Results demonstrated a maximum of 38 modified residues out of the 59 lysines available showing the limitation of the chemical functionalization. Consequently, LC-MS/MS analysis provided a site-specific characterization of the residues undergoing chemical modification. Data exhibited unique properties due to the presence of the ligands which allowed to identify without ambiguity the residues exhibiting different modification rate and enabled the identification of the unmodified lysine. Results were compared to the structure of HSA described from crystallography data. The comparison strongly suggested that accessibility is influencing the residues respective reactivity. The relevant complementarity of the different techniques could be emphasized in order to perform an extensive characterization concerning the evolution of the primary structure of the protein during the chemical reaction, providing an improved insight on the conjugation process and offering the potentiality to tune the reaction.