Large-scale map of RNA-binding protein interactomes across the mRNA life cycle.

mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 8,742 known and 20,802 unreported interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life-cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing the splicing and export functions of enhancer of rudimentary homolog (ERH), and by showing that small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200) interacts with stress granule proteins and binds cytoplasmic RNA differently during stress.

Structural Characterization of BIAN Type Molecules Used in Viscosity Reduction of Alkyl Magnesium Solutions.

N1,N2-diphenylacenaphthylene-1,2-diimines (BIANs) have been used to reduce the undesired high viscosity of alkyl magnesium solutions, which are known to form polymeric structures. In order to understand the mechanisms, analyses of the BIAN alkyl magnesium solutions have been carried out under inert conditions with SEC-MS, NMR, and FTIR and were compared to the structures obtained from HPLC-MS, FTIR, and NMR after aqueous workup. While viscosity reduction was shown for all BIAN derivatives used, only the bis (diisopropyl)-substituted BIAN could be clearly assigned to a single reaction product, which also could be reused without loss of efficiency or decomposition. All other derivatives have been shown to behave differently, even under inert conditions, and decompose upon contact with acidic solvents. While the chemical reactions observed after the workup of the used BIANs are dominated by (multiple) alkylation, mainly on the C = N double bond, the observation of viscosity reduction cannot be assigned to this reaction alone, but to the interaction of the nitrogen atoms of BIANs with the Mg of the alkyl magnesium polymers, as could be shown by FTIR and NMR measurements under inert conditions.

A native SEC-MS workflow and validation for analyzing drug-to-antibody ratio and drug load distribution in cysteine-linked antibody-drug conjugates.

The development and optimization of Antibody-Drug Conjugates (ADCs) hinge on enhanced analytical and bioanalytical characterization, particularly in assessing critical quality attributes (CQAs). The ADC's potency is largely determined by the average number of drugs attached to the monoclonal antibody (mAb), known as the drug-to-antibody ratio (DAR). Furthermore, the drug load distribution (DLD) influences the therapeutic window of the ADC, defining the range of dosages effective in treating diseases without causing toxic effects. Among CQAs, DAR and DLD are vital; their control is essential for ensuring manufacturing consistency and product quality. Typically, hydrophobic interaction chromatography (HIC) or reversed-phase liquid chromatography (RPLC) with UV detector have been used to quantitate DAR and DLD in quality control (QC) environment. Recently, Native size-exclusion chromatography-mass spectrometry (nSEC-MS) proves the potential as a platformable quantitative method for characterizing DAR and DLD across various cysteine-linked ADCs in research or early preclinical development. In this work, we established and assessed a streamlined nSEC-MS workflow with a benchtop LC-MS platform, to quantitatively monitor DAR and DLD of different chemotype and drug load level cysteine-linked ADCs. Moreover, to deploy this workflow in QC environment, complete method validation was conducted in three independent laboratories, adhering to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2(R1) guidelines. The results met the predefined analytical target profile (ATP) and performance criteria, encompassing specificity/selectivity, accuracy, precision, linearity, range, quantification/detection limit, and robustness. Finally, the method validation design offers a reference for other nSEC-MS methods that are potentially used to determine the DAR and DLD on cysteine-linker ADCs. To the best of our knowledge, this study is the first reported systematic validation of the nSEC-MS method for detecting DAR and DLD. The results indicated that the co-validated nSEC-MS workflow is suitable for DAR and DLD routine analysis in ADC quality control, release, and stability testing.

Bispecific antibody characterization by a combination of intact and site-specific/chain-specific LC/MS techniques.

Bispecific antibodies (bsAbs) are considered as an important class of biopharmaceutical drugs, with about 160 products in clinical trials. From an analytical point of view, the correct chain-association is one of the most critical challenge to monitor during bsAbs development and production. In the present study, a full analytical workflow has been developed based on the use of various chromatographic modes: size exclusion chromatography (SEC), ion exchange chromatography (IEX), reversed phase liquid chromatography (RPLC), and hydrophilic interaction chromatography (HILIC), all combined with high resolution mass spectrometry (MS). This analytical strategy was applied to Hemlibra® (emicizumab), which is certainly the most successful commercial bsAb to date. Using this strategy, it was possible to monitor the presence of mispaired bsAb species and detect and identify additional post-translational modifications (PTMs).

Characterizing Soluble Protein Aggregates Using Native Mass Spectrometry Coupled with Temperature-Controlled Electrospray Ionization and Size-Excl usion Chromatography.

Characterization of soluble protein aggregates provides valuable information for revealing mechanisms of protein aggregation process and assessing the activity and safety of protein therapeutics. However, the noncovalent interaction, the transient nature and higher degree of structural heterogeneity of the soluble aggregation system hinders precise characterization at the molecular level. Here, we describe methods using native mass spectrometry coupled with temperature-control electrospray ionization and size-exclusion chromatography to monitor the aggregation process and profile the aggregates in detail.

Simultaneous characterization of insulin HMWP and protamine sulphate in complex formulations through SEC-coupled mass spectrometry.

High molecular weight protein aggregates present in a recombinant human insulin and analogs are conventionally quantified by SEC-HPLC and identified by SEC-MALS as oligomeric population which lacks precise identification of species. The limitation of these two techniques is vanquished though simultaneous separation and identification by SEC coupled with MS. The identification was established with organic solvent based isocratic elution followed by MS for parallel separation and identification of HMWP species. The developed SEC-MS method is validated to establish the method capability and variability. Further investigations under stress conditions of Insulin analogues revealed the capability of the method to separate higher order oligomeric (Trimeric, and Tetrameric) species alongside covalent dimeric species. Additionally, the method holds good in separating and sequencing protamine peptides used in suspension (Neutral Protamine Hagedorn) and biphasic/mixed (70/30) formulations of Human insulin using ETD-MSMS. The data presented here shows insight towards utilization of state-of-the-art SEC-MS technique in the biopharmaceutical field as a tool to establish the structural comparability of higher order species in biosimilars and to investigate the lot to lot batch variability for protamine sulphate in-terms of sequence confirmation.

Simultaneous monitoring of multiple attributes of pyrrolobenzodiazepine antibody-drug conjugates by size exclusion chromatography - high resolution mass spectrometry.

Antibody-drug conjugates (ADCs) are an emerging class of oncology treatments combining the unique specificity of monoclonal antibodies with the highly cytotoxic properties of small molecule compounds. Pyrrolobenzodiazepines (PBDs) are highly potent agents capable of inhibiting cellular DNA replication which leads to apoptosis. To ensure efficacy and patient safety upon administration of such toxic and heterogeneous molecules, their structure and quality attributes must be closely monitored. Size exclusion chromatography (SEC) is a powerful, fast and robust tool for the separation of compounds varying in molecular weight. When using volatile components in the chromatographic mobile phase, SEC has also been shown to be amenable for interfacing to mass spectrometry, providing potential for reliable identification of protein isoforms across the size variants present. Here, we present a SEC-MS method developed for the characterisation of PBD-based ADCs on the intact molecular level. We demonstrate that information on ADC monomers such as the glycoform distribution and the average drug-antibody ratio (DAR) can be obtained in 15 minutes of analysis time. Qualitative and quantitative information on low and high molecular weight impurities such as aggregates and fragments, fundamental for critical quality attribute analysis of biopharmaceuticals, can be generated simultaneously. SEC-MS enables the characterisation of multiple product quality attributes of complex biotherapeutics at the same time.

System-Wide Profiling of Protein Complexes Via Size Exclusion Chromatography-Mass Spectrometry (SEC-MS).

In living cells, most proteins are organized in stable or transient functional assemblies, protein complexes, which control a multitude of vital cellular processes such as cell cycle progression, metabolism, and signal transduction. Over several decades, specific protein complexes have been analyzed by structural biology methods, initially X-ray crystallography and more recently single particle cryoEM. In parallel, mass spectrometry (MS)-based methods including in vitro affinity-purification coupled to MS or in vivo protein proximity-dependent labeling methods have proven particularly effective to detect complexes, thus nominating new assemblies for structural analysis. Those approaches, however, are either of limited in throughput or require specifically engineered protein systems.In this chapter, we present protocols for a workflow that supports the parallel analysis of multiple complexes from the same biological sample with respect to abundance, subunit composition, and stoichiometry. It consists of the separation of native complexes by size-exclusion chromatography (SEC) and the subsequent mass spectrometric analysis of the proteins in consecutive SEC fractions. In particular, we describe (1) optimized conditions to achieve native protein complex separation by SEC, (2) the preparation of the SEC fractions for MS analysis, (3) the acquisition of the MS data at high throughput via SWATH/DIA (data-independent analysis) mass spectrometry and short chromatographic gradients, and (4) a set of bioinformatic tools for the targeted analysis of protein complexes. Altogether, the parallel measurement of a high number of complexes from a single biological sample results in unprecedented system-level insights into the remodeling of cellular protein complexes in response to perturbations of a broad range of cellular systems.

The importance of being metal-free: The critical choice of column hardware for size exclusion chromatography coupled to high resolution mass spectrometry.

The goal of the study was to evaluate the possibilities offered by a new generation of metal-free SEC column to perform direct SEC-MS of protein biopharmaceuticals using ammonium acetate as the main mobile phase additive. The prototype metal-free SEC column hardware used in this work was a polyether ether ketone (PEEK) infused stainless steel tube including PEEK frits. This PEEK-lined column provides a fully bioinert and metal-free fluidic path, while maintaining the stability of the metal hardware, and could be a good solution to limit possible undesired interactions between proteins and column wall/frits. This prototype metal-free SEC column was systematically compared with a conventional stainless-steel SEC column hardware packed with the same stationary phase material. Four different mAb products, namely trastuzumab, palivizumab, bevacizumab and NISTmAb, and one antibody drug conjugate (ADC), trastuzumab emtansine, were selected as test samples. It appears that peak symmetry, separation of low molecular weight species (LMWS), and the recovery of high molecular weight species (HMWS) were significantly improved for the different biopharmaceutical products on the metal-free SEC column. It has also been demonstrated that the largest differences between standard and metal-free SEC columns were observed for the most basic mAbs (high pI), which confirms that electrostatic interactions between the mAb and the metallic parts of the column (frits and inlet tube) could be responsible for the issues observed when performing SEC analysis with volatile mobile phase. Finally, it was feasible to perform SEC-MS analysis for a wide range of biopharmaceutical products using volatile mobile phase. Our results also highlight that an inappropriate column could bias the quantification of size variants when using MS-compatible mobile phases. Therefore, metal-free column, such as the PEEK-lined column, should be preferentially selected for SEC-MS analysis.

Investigation of monoclonal antibody dimers in a final formulated drug by separation techniques coupled to native mass spectrometry.

Therapeutic monoclonal antibodies (mAbs) are highly complex proteins that must be exhaustively characterized according to the regulatory authorities' recommendations. MAbs display micro-heterogeneity mainly due to their post-translational modifications, but also to their susceptibility to chemical and physical degradations. Among these degradations, aggregation is quite frequent, initiated by protein denaturation and then dimer formation. Here, we investigated the nature and structure of the high molecular weight species (HMW) present at less than 1% in an unstressed formulated roledumab biopharmaceutical, as a model of high purity mAb. HMW species were first purified through preparative size-exclusion chromatography (SEC) and then analyzed by a combination of chromatographic methods (ion-exchange chromatography (IEX), SEC) coupled to native mass spectrometry (MS), as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and capillary gel electrophoresis under non-reducing conditions. Both covalently and non-covalently bound dimers were identified at a proportion of 50/50. In-depth characterization of the HMW fraction by SEC and IEX hyphenated to native MS revealed the presence of three mAb dimer forms having the same mass, but differing by their charge and size. They were attributed to different compact and elongated dimers. Finally, high-resolution middle-up approaches using different enzymes (IdeS and IgdE) were performed to determine the mAb domains implicated in the dimerization. Our results revealed that the roledumab dimers were associated mainly by a single Fab-to-Fab arm-bound association.

Characterization of 30 therapeutic antibodies and related products by size exclusion chromatography: Feasibility assessment for future mass spectrometry hyphenation.

Despite the popularity of targeted and immune therapies, the number of studies dealing with the quantitation of aggregates for Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAb and related products are still very scarce in literature. In this work, 30 therapeutic proteins including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), Fc-fusion proteins and a bi-specific antibody (bsAb) were investigated using size exclusion chromatography (SEC). Their levels of high molecular weight species (HMWS) were experimentally estimated between 0.1% and 13.1%. Except for blinatumomab, etanercept and pembrolizumab, the HMWS amount for the other antibodies was well below the limit of 5% usually set a specification for therapeutic mAbs in the biopharmaceutical industry. The main chromatographic peak shape of 24 therapeutic antibodies and the NIST mAb [1] was found suitable (0.8

Determination of modification degree in BDDE-modified hyaluronic acid hydrogel by SEC/MS.

Determination of modification degree in BDDE-modified hyaluronic acid (HA) hydrogel is of particular interest. In this paper, three crosslinking parameters (degree of total modification, t-MOD; degree of cross-link modification, c-MOD; degree of pendent modification, p-MOD) are defined and determined by quantification of the modified fragments in hydrogel digestion by size exclusion chromatography combined with mass spectrometry (SEC-MS). The digestion products of a novel hyaluronidase HAase-B produced by Bacillus sp. A50 are studied and only a few modified fragments are identified by (1)H NMR and MS. As a result, Three HA hydrogels prepared in lab have different t-MOD, c-MOD and p-MOD, but the ratio of c-MOD to p-MOD result in the almost same value of 75%. Hydrogel products from Q-Med have nearly same t-MOD about 0.8% and c-MOD about 0.1%, the ratio of c-MOD to p-MOD is about 13%. Hydrogels from ANTEIS S.A all have much higher t-MOD values, the ratio of c-MOD and p-MOD is about 8%.