Subunit Level Research Articles

Development of a Novel Label-Free Subunit HILIC-MS Method for Domain-Specific Free Thiol Identification and Quantitation in Therapeutic Monoclonal Antibodies.

Cysteine residues are crucial for the formation of conserved disulfide bonds in therapeutic monoclonal antibodies (mAbs), which are essential for their folding and structural stability. The presence of free thiols in mAbs can indicate incomplete disulfide bond formation, potentially impacting the molecule's conformational stability. Free thiol quantitation has been achieved using labeling-based strategies such as maleimide and haloalkyl derivatives at both intact and peptide levels. However, intact-level measurement only provides total free thiol levels, while peptide-level measurement is time-consuming and more prone to assay-induced artifacts. In this study, we present a novel label-free HILIC-MS method that separates free thiol species at the subunit level, followed by free thiol localization by the MS2 fragmentation pattern. This allows for facile identification and quantitation of intrachain free thiols at domain-specific resolution. Compared to bottom-up approaches, this subunit HILIC-MS method excels in simpler sample preparation and higher throughput and enables chain-specific free thiol analysis for bispecific mAbs. This method can be readily applied for screening mAb candidates with elevated levels of free thiols in early-stage developability assessment and facilitating an effective comparability evaluation of mAb samples during process development.

CE-MS and CE-MS/MS for the multiattribute analysis of monoclonal antibody variants at the subunit level

The analysis of product-related substances and impurities is a critical step in the biopharmaceutical quality control of multiattribute monoclonal antibodies (mAbs), as posttranslational modifications or other variants can influence the product's biological activity. Many approaches are available for variant analysis; however, they are either variant-specific, mAb-specific, time-consuming, or require expensive equipment. Here, we present a generic capillary electrophoretic method based on a neutral-coated capillary which was coupled to mass spectrometry (MS) via the nanoCEasy interface for mAb variant analysis at the subunit level (enzymatically digested and reduced mAb). The method enabled the separation of several (i) size variants (e.g. glycosylation variants) and (ii) charge variants (e.g. c-terminal lysin clipping) as well as (iii) multiple other proteoforms (e.g. additional glycation) and (iv) incompletely reduced subunits. Separated variants were confirmed by MS/MS fragmentation even for small mass deviations like deamidation or open disulfide bridges. The system, initially developed for one mAb, was tested with nine other IgG1s to show the general applicability of the system. The presented multiattribute method enables fast and detailed characterization of mAb variants with little sample preparation and relatively simple separation equipment enabling the separation of a large set of mAb variants.

Label-Free Mapping of Multivalent Binding Pathways with Ligand-Receptor-Anchored Nanopores.

Understanding single-molecule multivalent ligand-receptor interactions is crucial for comprehending molecular recognition at biological interfaces. However, label-free identifications of these transient interactions during multistep binding processes remains challenging. Herein, we introduce a ligand-receptor-anchored nanopore that allows the protein to maintain structural flexibility and favorable orientations in native states, mapping dynamic multivalent interactions. Using a four-state Markov chain model, we clarify two concentration-dependent binding pathways for the Omicron spike protein (Omicron S) and soluble angiotensin-converting enzyme 2 (sACE2): sequential and concurrent. Real-time kinetic analysis at the single-monomeric subunit level reveals that three S1 monomers of Omicron S exhibit a consistent and robust binding affinity toward sACE2 (-13.1 ± 0.2 kcal/mol). These results highlight the enhanced infectivity of Omicron S compared to other homologous spike proteins (WT S and Delta S). Notably, the preceding binding of sACE2 to Omicron S facilitates the subsequent binding steps, which was previously obscured in bulk measurements. Our single-molecule studies resolve the controversy over the disparity between the measured spike protein binding affinity with sACE2 and the viral infectivity, offering valuable insights for drug design and therapies.

Developing Analytical ion Exchange Chromatography Methods for Antibody Drug Conjugates Containing the Hydrolysis-Prone Succinimide-Thioether Conjugation Chemistry

Charge variants are one of the most important quality attributes for protein therapeutics, including antibody drug conjugates (ADCs). ADCs are conjugation products between monoclonal antibodies (mAbs) and highly potent payloads. After attaching a payload, the charge profile of a mAb can be modified due to the change in net charge or surface charge. In this study, we present a unique challenge of charge assay development that arises from a desirable engineering of ADCs that incorporates the hydrolysis-prone succinimide-thioether conjugation chemistry. This engineered hydrolysis at conjugation sites is usually not complete during conjugation process and continuously progressing during mild stress. This hydrolysis also creates a carboxylic functional group, which manifests as acidic peaks in the ADC charge profiles. As a result, ion exchange chromatograms become sensitive measurements of this hydrolysis, which often masks the charge profile change due to other important post-translational modifications. In this study, two approaches were explored to address this unique challenge: to remove the hydrolysis heterogeneity by incubating ADCs under high pH conditions to drive complete hydrolysis; and to analyze charge variants at the subunit level after IdeS digestion. Acceptable charge profiles and quantitative integration results were successfully obtained by both approaches.

Genetic and environmental variation in protein composition of Belgian soy determined with a novel size-exclusion chromatography method

To allow the determination of soy protein composition on protein subunit level in a quantitative manner, a novel method combining size-exclusion high-performance liquid chromatography (SE-HPLC) and peak deconvolution was developed. Peaks corresponding to the different soy protein (subunits) were identified based on the analysis of (i) marker proteins, (ii) 7S and 11S globulin-enriched fractions, and (iii) cross-validation with SDS-PAGE analysis. Given the recent introduction of soybean cultivation in Belgium, the optimized SE-HPLC method was here applied to study the impact of genotype, growing location, harvest year and inoculation strategy on the protein composition of Belgian soy. Especially genotype and harvest year were key to determining protein composition, while all factors influenced the chemical composition of soy flour. The developed SE-HPLC method can be used to further understand the impact of soybean cultivation practices in Belgium and elsewhere on protein composition, which is highly relevant for novel applications of soy. In addition, this SE-HPLC method can be used to determine how various soy processing methods may affect protein composition.

Roles of soybean β-conglycinin subunit fractions in fibril formation and the effects of glycinin on them

This research is aimed at exploring the fibrillation of soybean β-conglycinin (7S) subunits (αα′ and β) at 90 °C and pH 2.0, as well as the effects of glycinin (11S) on them. Fibril formation kinetics was monitored by measuring changes in thioflavin T (Th T) fluorescence intensity of the proteins. This analysis showed that αα′ subunits had a higher fibrillation capacity but slower fibril formation rate than the β subunit. Glycinin accelerated the rate of fibrillation of the αα′ and β subunits but weakened the fibril structure. The size of the fibrils first decreased and then increased with increasing heating time. Circular dichroism and intrinsic fluorescence spectroscopy analysis indicated that the β-sheet structure and hydrophobic interactions were important for the generation of ordered fibrils. Shear viscosity and transmission electron microscopy analysis suggested that the αα′ subunits formed relatively rigid fibrils that led to a high solution viscosity. In contrast, β subunit fibrils became unstable after prolonged heating, forming shorter fibrils (fragments) that tended to aggregate, which led to a lower solution viscosity. This research also suggested that the extension region of αα′ subunits played an important role in fibrillation, and the presence of the highly hydrophobic basic polypeptides of glycinin might be the main reason for the inhibition of soy protein fibrillation. Our analysis of soy protein fibrillization at the subunit level provides insights that might enhance the future application of soy protein fibrils as functional ingredients in foods.

CE-MS/MS and CE-timsTOF to separate and characterize intramolecular disulfide bridges of monoclonal antibody subunits and their application for the assessment of subunit reduction protocols

Characterization at the subunit level enables detailed mass spectrometric characterization of posttranslational modifications (PTMs) of monoclonal antibodies (mAbs). The implemented reduction often leaves the intramolecular disulfide bridges intact. Here, we present a capillary electrophoretic (CE) method based on a neutral-coated capillary for the separation of immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) digested and reduced mAb subunits followed by mass spectrometry (MS), MS/MS identification, and trapped ion mobility mass spectrometry (timsTOF). Our CE approach enables the separation of (i) different subunit moieties, (ii) various reduction states, and (iii) positional isomers of these partly reduced subunit moieties. The location of the remaining disulfide bridges can be determined by middle-down electron transfer higher energy collisional dissociation (EThcD) experiments. All these CE-separated variants show differences in ion mobility in the timsTOF measurements. Applying the presented CE-MS/MS method, reduction parameters such as the use of chaotropic salts were studied. For the investigated antibodies, urea improved the subunit reduction significantly, whereas guanidine hydrochloride (GuHCl) leads to multiple signals of the same subunit in the CE separation. The presented CE-MS method is a powerful tool for the disulfide-variant characterization of mAbs on the subunit level. It enables understanding disulfide bridge reduction processes in antibodies and potentially other proteins.

Effects of soybean β-conglycinin subunits composition on its emulsifying properties and the mechanism

In the soybean β-conglycinin system, subunit composition has an important influence on its emulsification properties. To elucidate the mechanism of subunit action in the emulsification process, the separated and purified β-subunit and the mixture α- and α′-subunits (referred to αα′-subunit) were remixed in varying proportions, and then the physicochemical properties and structural properties of the subunit emulsifying system were characterized. The results demonstrated that when the ratio of αα'-subunit to β-subunit was 5:5, the subunit aqueous dispersion system exhibited a minimum particle size of 47.58 nm, a maximum solubility of 87.21%, and the emulsification stability and emulsification activity were also optimal. Scanning electron microscope (SEM) images revealed excellent cross-linking between αα′-subunit and β-subunit, forming a protein network structure that provided a structural basis for its emulsification. The emulsion with a subunit ratio of 5:5 exhibited the highest fraction of interfacial protein adsorption of 23.43% and the largest interfacial protein concentration of 11.79 mg/m2. Inter-subunit adsorption and emulsion formation were primarily driven by hydrophobic interactions, with the β-subunit exhibiting a higher affinity for adsorption than the αα′-subunit. The action mechanism of the subunits in the emulsion formation was proposed as a “three-stage” process, including establishing an initial weak protein network structure within the aqueous dispersion, followed by developing an emulsion interface layer and ultimately culminating in forming an emulsion gel network structure. Elucidating the emulsifying mechanism at the subunit level is crucial for enhancing the theoretical framework of soybean protein emulsification.

Associations of genetically predicted circulating levels of cytokines with telomere length: a Mendelian randomization study.

Telomere length (TL) has been regarded as a biomarker of aging, and TL shortening is associated with numerous chronic illnesses. The mounting evidence has shown that inflammatory cytokines are involved in maintaining or shortening TL, the causality of cytokines with TL remains unknown. Therefore, we performed a two-sample Mendelian randomization (MR) analysis to estimate the underlying correlations of circulating inflammatory cytokines with TL. Genetic instrumental variables for inflammatory cytokines were identified through a genome-wide association study (GWAS) involving 8,293 European individuals. Summary statistics of TL were derived from a UK Bio-bank cohort comprising 472,174 samples of individuals with European descent. We employed the inverse-variance weighted (IVW) approach as our main analysis, and to ensure the reliability of our findings, we also conducted additional analyses including the weighted median, MR-Egger, MR pleiotropy residual sum and outlier test, and weighted model. Lastly, the reverse MR analyses were performed to estimate the likelihood of inverse causality between TL and the cytokines identified in the forward MR analysis. Cochran's Q test were employed to quantify the degree of heterogeneity. After applying Bonferroni correction, a higher circulating level of Interleukin-7 (IL-7) was suggestively associated with TL maintaining (OR:1.01, 95%CI:1.00-1.02, P=0.032 by IVW method). The study also revealed suggestive evidence indicating the involvement of Interleukin-2 receptor, alpha subunit (IL-2Rα) level was negatively associated with TL maintaining (OR:0.98, 95%CI:0.96-1.00, P=0.045 by IVW method), and the weighted median approach was consistent (OR:0.99, 95%CI:0.97-1.00, P=0.035). According to the findings of reverse MR analysis, no significant causal relationship between TL and cytokines was explored. Our analysis did not reveal any substantial heterogeneity in the Single nucleotide polymorphisms or horizontal pleiotropy. Our MR analysis yielded suggestive evidence supporting the causality between circulating IL-7 and IL-2Rα and telomere length, necessitating further investigations to elucidate the mechanisms by which these inflammatory cytokines may impact the progression of telomeres.

FRI532 Thyroid Hormone Resistance: A Possible New Variant In The THRB Gene

Abstract Disclosure: A. Siddiqui: None. Z. Khan: None. T. Ahmed: None. K. Selk: None. Background: Resistance to the Thyroid Hormone (RTH) is caused by reduced target tissue response to thyroid hormone, resulting in the loss of the normal feedback mechanism that suppresses secretion of thyrotropin (TSH). Most cases of RTH are familial, usually inherited in an autosomal dominant manner, with over 100 different pathogenic variants having been described. Clinical Case: A 45-year-old male with history of attention deficit disorder presented for management of hypothyroidism. The patient was Anti-TPO positive, consistent with Hashimoto's thyroiditis and he was treated with varying doses of levothyroxine. Labs on 100 mcg levothyroxine showed a TSH of 7.07 (0.35-4.94 uIU/mL), free t4 1.86 (0.7-1.48 ng/dL), and free t3 4.41 (2.3-4.2 pg/mL). This initially prompted concern for a TSHoma. An alpha subunit level was found to be 0.3 ng/dL and pituitary MRI was within normal limits. Given continued persistent thyroid hormone elevations, further testing for human anti-mouse antibody interference was performed and also unrevealing. The patient continued to report feeling tired and fatigued, with multiple joint pains and inability to focus or concentrate. His levothyroxine was increased to 125 mcg per day. However, even after the adjustment, he still did not notice any change in his symptoms. A trial of addition of liothyronine 5mcg daily was also performed but he self-discontinued this as he did note any improvement. Overall, his findings were highly suspicious for thyroid hormone resistance. Genetic testing for a thyroid hormone resistance beta (THRB) mutation was performed. Direct sequence analysis of the coding region and splice-sites of the THRB gene identified one copy of the c.1368G>T (p.Leu456Phe) variant of uncertain significance. Analysis of this variant using bioinformatics tools for the prediction of the effect of amino acid changes on protein structure and function yielded predictions that this variant is damaging. However, the THRB c.1368G>T (p.Leu456Phe) variant has not been described in online databases or published literature. It also has not been reported in large, multi-ethnic general populations. Conclusion: This is the first case to our knowledge demonstrating the THRB c.1368G>T (p.Leu456Phe) variant gene as likely giving rise to RTH. Further testing of family members has been recommended to help clarify the clinical significance of this finding. Presentation: Friday, June 16, 2023

Influence of pH and ionic strength on the physicochemical and structural properties of soybean β-conglycinin subunits in aqueous dispersions

Understanding the impact of pH and ionic strength on the physicochemical and structural properties of soy proteins at subunit level is essential for design and fabrication of many plant-based foods. In this study, soybean β-conglycinin and its subunit fractions αα′ and β were dispersed in solutions with different pH values (3.7, 7.6, and 9.0) at low (5 mM NaCl) and high (400 mM NaCl) ionic strengths, respectively. The solubility, rheology, particle size, zeta potential, microstructure, secondary structure, and tertiary structure of the different dispersions were analyzed using a range of analytical methods. The β-conglycinin, αα′- and β-subunits aggregated near the isoelectric point (pH 3.7). Increasing the ionic strength led to the assembly of more homogeneous units. An increase in ionic strength at pH 7.6 and pH 9.0 led to electrostatic screening, which promoted dissociation of the aggregates. The β-subunit showed a greater sensitivity to pH and ionic strength than the αα′-subunits. Based on the evidence from a range of analytical methods, the highly hydrophilic extension region of the αα′-subunits played an important role in determining the stability of the β-conglycinin dispersions under different environmental conditions. Moreover, the N-linked glycans appeared to impact the conformation and aggregation state of the β-conglycinin.

Mechanisms of heat-mediated aggregation behavior of water-soluble cod protein.

Cod proteins (CPs) are considered potential functional ingredients for developing gel-based foods, but present studies on the aggregation behavior of CPs upon heating remain limited. With this respect, the heat-induced aggregation kinetics of CPs at a subunit level was investigated. Based on different centrifugal forces, CPs aggregates were divided into three fractions: large-sized, intermediary-sized, and small-sized aggregates. SDS-PAGE and diagonal SDS-PAGE indicated that myosin heavy chains exhibited a higher affinity with actin to form intermediary-sized and large-sized aggregates; tropomyosin and myosin light chains were hardly engaged in the thermal aggregation and formed small-sized aggregates. The highly-polymerized aggregates adopted considerable transitions of helix-to-sheet in protein structures, whereas the structure of small-sized aggregates featured substantial helix-coil transitions. Furthermore, molecular interactions at different heating stages were revealed. These novel insights might advance our knowledge on the heat-induced aggregation behavior of CPs and provide fundamental information for the application of CPs in gel-based foods.

Monitoring oxidation in recombinant monoclonal antibodies at subunit level through two-dimensional liquid chromatography coupled with mass spectrometry

Monoclonal antibodies (mAbs) are highly susceptible to chemical/post-translational modifications (PTMs), a crucial concern for chemical and biological safety during mAb production and development. Modifications in different mAb domains have different consequences on mAb stability and activity. Hence, characterization and monitoring of domain-specific modification in mAbs is of significant interest so as to be able to assure consistency in product quality. Generally, mAb samples are enzymatically digested and analyzed with traditional reversed-phase chromatography for domain-specific characterization. However, conventional analytical methods do not provide complete resolution and identification of mAb modifications. Here, a novel workflow of two-dimensional liquid chromatography with mass spectrometry interfacing has been proposed to overcome the limitations of conventional one-dimensional analytical methods and provide complete information on oxidized mAb domains. The proposed method successfully analysed the oxidized mAb samples and the control sample at the subunit level, simultaneously separating oxidized variants that are otherwise not detected by one-dimensional methods. The proposed 2D WCX-RPC-MS setup uses 2D multiple heart-cut method that identified 10 oxidation variants in mAb A, 6 in the Fab fragment and 4 in the Fc fragment, of which 3 were exclusive and not identified by the conventional 1D analysis. Further the 2D method takes only 7–13 h in sample preparation and analysis to anticipate oxidation in mAb fragments. Hence, the proposed workflow offers a faster and more convenient platform for routine monitoring and domain-specific mAb characterization with mass spectrometry coupling.

Abstract #1413579: Rathke Cleft Cyst Unveiling Incidental Prolactin Producing Adenoma

Rathke cleft cysts (RCCs) are benign sellar and or/suprasellar lesions that arise from remnants of Rathke's pouch. Pituitary hormonal dysfunction associated with RCCs most often present as hormonal deficiencies due to compression. However pituitary hyper-secretion may coexist with RCC challenging diagnosis and management. A 37-year-old female presented with worsening headache, bilateral blurry vision and pressure-like eye pain for two weeks. She denied any galactorrhea or irregular periods. Her medications included as-needed butalbital-acetaminophen-caffeine and alprazolam. CT Head without IV contrast revealed a pituitary lesion measuring 1.9 cm extending near the inferior margin of the optic chiasm. Subsequent MRI Pituitary with and without IV contrast revealed an enhancing sellar/suprasellar solid lesion measuring 1.6 x 2.7 x 1.9 cm with small hypoenhancing components associated with expansion of the sella, rightward infundibulum deviation, mass effect on the optic chiasm and left cavernous sinus invasion. Pituitary secretion workup revealed an elevated serum prolactin level of 50.4 ng/mL (range 4.8 - 23.3 ng/mL). Repeat prolactin level was 75.7 mg/ml. Dilution studies 1:10 and 1:100 revealed prolactin levels of 78.3 ng/mL and 75.9 ng/mL respectively; absence of hook effect. Her elevated prolactin was attributed to stalk effect given the large size of her sellar mass and relatively minimally elevated prolactin level. Serum Alpha Subunit, ILGF-1, TSH, FT4, LF, FSH, AM cortisol and ACTH level were all normal. She underwent endoscopic transsphenoidal pituitary mass resection with pathology results revealing prolactin producing adenoma and RCC. Immunostains were strongly positive for chromogranin A and prolactin while negative for FSH, ACTH, TSH, beta hCG, GH. Prolactin levels 11 days post-resection remained elevated at 50.7 ng/mL. She continued to have regular periods without galactorrhea. Additionally, she expressed desire to attain pregnancy. This case illustrates the presence of a secreting pituitary prolactinoma in the setting of an RCC. Whether these two entities share a common pathophysiology or simply co-exist is unknown. In one study of 33 surgically treated RCC at presentation, 60% had gonadotroph deficiency, 36% had ACTH deficiency, 36% had TSH deficiency, 18% had ADH deficiency and 31% had hyperprolactinemia (1). It is possible that the majority of this patient 's sellar mass was comprised of RCC as a much higher prolactin level would have been expect for the size of her mass. Interesting, her sellar mass exhibited enhancement which is uncharacteristic for RCC, classical described as cystic lesions without calcification or enhancement. Given that her post-resection prolactin level remained elevated and she desires pregnancy, she may require dopamine agonist therapy.

Abstract #1402838: Thyroid Stimulating Hormone-Secreting Adenoma: A Rare Case of Hyperthyroidism

TSH-secreting adenomas account for only 0.5 to 3 % of all functioning pituitary tumors. We present this case report to highlight the importance of early diagnosis and prevention of severe complications. A 48-year-old postmenopausal female with a history of hypothyroidism was referred to our clinic for a second opinion. The patient had been stable on levothyroxine for over 10 years. Routine blood work was significant for elevated TSH and free T4. Despite adjustments to her levothyroxine, TSH and free T4 remained elevated. Her prior endocrinologist discontinued the levothyroxine and ordered an MRI brain, which showed an 8mm pituitary adenoma. The patient was referred to a neurosurgeon for evaluation, which then prompted the second endocrinology evaluation. Patient denied any palpitations, weight loss, heat intolerance, headaches, vision changes, or galactorrhea. Lab work repeated off of thyroid supplementation showed persistent elevated TSH at 6.5 mIU/L and free T4 at 2.1 ng/dL. Differential diagnosis included thyroid hormone resistance versus interference to thyroid assays versus TSHoma. The patient tested negative for the thyroid hormone resistance beta mutation, deeming thyroid hormone resistance unlikely. She tested negative for the human anti-mouse antibody, making interference to thyroid assays unlikely. Her alpha subunit level was normal, after which TSHoma was not a certainty. Since the diagnosis was unclear, patient was advised to follow up closely. Repeat MRI showed the tumor was now 1.2cm. Due to the increase in size, she was advised to proceed with trans-sphenoidal surgery (TSS). Pathology stained for TSH, confirming it was a TSH-secreting adenoma. After TSS, TSH normalized and free T4 was low, requiring the patient to be restarted on levothyroxine. TSH-secreting adenomas are quite rare, accounting for less than 1% of all cases of hyperthyroidism. Common clinical features include goiter, visual field defects, headache, menstrual disturbances, and galactorrhea. The presence of high free T4 and T3 and measurable serum TSH in the presence of a macroadenoma on MRI present strong evidence of a TSHoma. Definitive therapy includes transsphenoidal resection. Long-acting somatostatin analogs or dopamine agonists are used to restore euthyroidism prior to surgery. Long-term antithyroid drug therapy or thyroid ablation is not recommended because prolonged decreases in thyroid hormone secretion can stimulate TSH secretion and tumor growth. If TSHomas are left untreated, they can grow aggressively and impinge on nearby structures. Complications include visual field defects due to optic nerve compression, galactorrhea due to pituitary stalk compression, hypopituitarism leading to blood pressure dysregulation and decreased sexual function, and cardiac arrythmias leading to blood clots, stroke, and heart failure. Therefore, when hyperthyroxinemia is present with an elevated TSH, TSHoma should be considered. Even when alpha subunit levels are low or normal, TSHoma is still a possibility. Further investigation and close monitoring should be done to confirm diagnosis and prevent negative outcomes.

Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding. Bottom-up HDX-MS experiments including online pepsin digestion revealed that the binding sites of the affimer reagents were mainly located in the complementarity-determining region (CDR) 2 of the heavy chain of the respective antibodies. Three-dimensional models of the binding interaction between the affimer reagents and the antibodies were built by homology modeling and molecular docking based on the HDX data.

EFSA info‐session on Avian Influenza surveillance data in the context of the SIGMA Project

This report describes the content and the outcomes of the info-session held by EFSA in videoconference on 17 November 2022. The info-session aimed at informing the country-representatives on the new Avian influenza (AI) data collection based on the approach developed by the SIGMA project. EFSA is in charge of collating, validating, analysing, and summarising in an annual report the data resulting from the AI surveillance activities in poultry and wild birds carried out by the European Union (EU) Member States (MSs), upon mandate M-2017-0221, received from the European Commission. During the info-session, benefits of the SIGMA approach were shown, and the EFSA data models were presented together with the tools provided by EFSA to support data providers and validators. The goal of the SIGMA project is the increase of standardisation, harmonisation and interoperability of data on animal diseases and domestic animal populations. The SIGMA approach entails the application of the SIGMA animal population data model, designed to collect information at establishment and subunit (farm/house) level, and of the laboratory data model, the latter based on Standard Sample Description version 2 (SSD2), but further tailored to the animal health needs. As a major novelty, countries will be asked to submit poultry population data at subunit level and positive and negative laboratory test results at sample level, both for domestic and wild birds. With this level of detail, EFSA will be able to perform more informative statistical analysis to be included in the annual report on AI. Thanks to the automation of the process (under development) EFSA aims at reducing the time between data submission and report publication, making the scientific outputs more timely and relevant. Moreover, un-aggregated data can be more easily used for other risk assessment purposes by EFSA (e.g., welfare, biosecurity, etc.), avoiding double requests for data submission in the future. EFSA calls for the MSs’ collaboration during the pilot phase in 2023 as a crucial element for the success of the initiative.

Online Collision-Induced Unfolding of Therapeutic Monoclonal Antibody Glyco-Variants through Direct Hyphenation of Cation Exchange Chromatography with Native Ion Mobility-Mass Spectrometry.

Post-translational modifications (PTMs) not only substantially increase structural heterogeneity of proteins but can also alter the conformation or even biological functions. Monitoring of these PTMs is particularly important for therapeutic products, including monoclonal antibodies (mAbs), since their efficacy and safety may depend on the PTM profile. Innovative analytical strategies should be developed to map these PTMs as well as explore possible induced conformational changes. Cation-exchange chromatography (CEX) coupled with native mass spectrometry has already emerged as a valuable asset for the characterization of mAb charge variants. Nevertheless, questions regarding protein conformation cannot be explored using this approach. Thus, we have combined CEX separation with collision-induced unfolding (CIU) experiments to monitor the unfolding pattern of separated mAbs and thereby pick up subtle conformational differences without impairing the CEX resolution. Using this novel strategy, only four CEX-CIU runs had to be recorded for a complete CIU fingerprint either at the intact mAb level or after enzymatic digestion at the mAb subunit level. As a proof of concept, CEX-CIU was first used for an isobaric mAb mixture to highlight the possibility to acquire individual CIU fingerprints of CEX-separated species without compromising CEX separation performances. CEX-CIU was next successfully applied to conformational characterization of mAb glyco-variants, in order to derive glycoform-specific information on the gas-phase unfolding, and CIU patterns of Fc fragments, revealing increased resistance of sialylated glycoforms against gas-phase unfolding. Altogether, we demonstrated the possibilities and benefits of combining CEX with CIU for in-depth characterization of mAb glycoforms, paving the way for linking conformational changes and resistance to gas-phase unfolding charge variants.

Revealing charge heterogeneity of stressed trastuzumab at the subunit level

Trastuzumab is known to be heterogeneous in terms of charge. Stressing trastuzumab under physiological conditions (pH 7.4 and 37 °C) increases charge heterogeneity further. Separation of charge variants of stressed trastuzumab at the intact protein level is challenging due to increasing complexity making it difficult to obtain pure charge variants for further characterization. Here we report an approach for revealing charge heterogeneity of stressed trastuzumab at the subunit level by pH gradient cation-exchange chromatography. Trastuzumab subunits were generated after limited proteolytic cleavage with papain, IdeS, and GingisKHAN®. The basic pI of Fab and F(ab)2 fragments allowed to use the same pH gradient for intact protein and subunit level analysis. Baseline separation of Fab subunits was obtained after GingisKHAN® and papain digestion and the corresponding modifications were determined by LC–MS/MS peptide mapping and middle-down MALDI-ISD FT-ICR MS. The described approach allows a comprehensive charge variant analysis of therapeutic antibodies that have two or more modification sites in the Fab region.

DiSiR: fast and robust method to identify ligand-receptor interactions at subunit level from single-cell RNA-sequencing data.

Most cell-cell interactions and crosstalks are mediated by ligand-receptor interactions. The advent of single-cell RNA-sequencing (scRNA-seq) techniques has enabled characterizing tissue heterogeneity at single-cell level. In the past few years, several methods have been developed to study ligand-receptor interactions at cell type level using scRNA-seq data. However, there is still no easy way to query the activity of a specific user-defined signaling pathway in a targeted way or to map the interactions of the same subunit with different ligands as part of different receptor complexes. Here, we present DiSiR, a fast and easy-to-use permutation-based software framework to investigate how individual cells are interacting with each other by analyzing signaling pathways of multi-subunit ligand-activated receptors from scRNA-seq data, not only for available curated databases of ligand-receptor interactions, but also for interactions that are not listed in these databases. We show that, when utilized to infer ligand-receptor interactions from both simulated and real datasets, DiSiR outperforms other well-known permutation-based methods, e.g. CellPhoneDB and ICELLNET. Finally, to demonstrate DiSiR's utility in exploring data and generating biologically relevant hypotheses, we apply it to COVID lung and rheumatoid arthritis (RA) synovium scRNA-seq datasets and highlight potential differences between inflammatory pathways at cell type level for control versus disease samples.