Vestibular Migraine and Recurrent Vertigo in Children: A Diagnostic Focus From a Tertiary Hospital Study.

BACKGROUND: To improve diagnostic precision in pediatric vertigo, particularly in Vestibular Migraine of Childhood (VMC), probable VMC (pVMC), Recurrent Vertigo of Childhood (RVC), and unspecified categories, by delineating clinical characteristics and prevalence to refine diagnostics and treatments. METHODS: Retrospective analysis of 102 pediatric patients (five to 18 years; 46 females, 56 males) at the Dizziness Center of the Otolaryngology Department in a tertiary-level hospital from January 2019 to December 2023. Patients were classified into VMC, pVMC, RVC, and indeterminate groups. Evaluations included audiometry and vestibular tests (video head impulse test [vHIT] or caloric testing), conducted in the audiology unit and vestibular testing laboratory. Data were analyzed using IBM SPSS 20.0. RESULTS: Diagnoses were 8.8% VMC, 31.4% pVMC, 51.0% RVC, and 8.8% indeterminate. Nausea and vomiting were common in VMC and pVMC; cochlear symptoms like tinnitus and hearing loss predominated in VMC. Although vestibular testing showed no significant group differences, VMC had more vHIT abnormalities and RVC had more caloric test anomalies. CONCLUSIONS: This study highlights the need for comprehensive diagnostics in pediatric vestibular disorders, revealing unique and overlapping traits across VMC, pVMC, and RVC. Insights call for further research to refine diagnostic criteria and improve treatment methods.

Chromosome-scale genome, together with transcriptome and metabolome, provides insights into the evolution and anthocyanin biosynthesis of Rubus rosaefolius Sm. (Rosaceae).

Rubus rosaefolius is a kind of red raspberry possessing high nutritional and pharmaceutical value. Here we present a chromosome-level draft genome of R. rosaefolius. Of the total 131 assembled scaffolds, 70 with a total size of 219.02 Mb, accounting for 99.33% of the estimated genome size, were anchored to seven pseudochromosomes. We traced a whole-genome duplication (WGD) event shared among members of the Rosaceae family, from which were derived 5090 currently detectable duplicated gene pairs (dgps). Of the WGD-dgps 75.09% underwent purifying selection, and approximately three-quarters of informative WGD-dgps expressed their two paralogs with significant differences. We detected a wide variety of anthocyanins in the berries of R. rosaefolius, and their total concentration remained relatively stable during berry development but increased rapidly during the ripening stage, mainly because of the contributions of pelargonidin-3-O-glucoside and pelargonidin-3-O-(6″-O-malonyl)glucoside. We identified many structural genes that encode enzymes, such as RrDFR, RrF3H, RrANS, and RrBZ1, and play key roles in anthocyanin biosynthesis. The expression of some of these genes significantly increased or decreased with the accumulation of pelargonidin-3-O-glucoside and pelargonidin-3-O-(6″-O-malonyl)glucoside. We also identified some transcription factors and specific methylase-encoding genes that may play a role in regulating anthocyanin biosynthesis by targeting structural genes. In conclusion, our findings provide deeper insights into the genomic evolution and molecular mechanisms underlying anthocyanin biosynthesis in berries of R. rosaefolius. This knowledge may significantly contribute to the targeted domestication and breeding of Rubus species.

Simulated seasonal diets alter yak rumen microbiota structure and metabolic function.

Yak is the only ruminant on the Qinghai-Tibetan Plateau that grazes year-round. Although previous research has shown that yak rumen microbiota fluctuates in robust patterns with seasonal foraging, it remains unclear whether these dynamic shifts are driven by changes in environment or nutrient availability. The study examines the response of yak rumen microbiota (bacteria, fungi, and archaea) to simulated seasonal diets, excluding the contribution of environmental factors. A total of 18 adult male yaks were randomly divided into three groups, including a nutrition stress group (NSG, simulating winter pasture), a grazing simulation group (GSG, simulating warm season pasture), and a supplementation group (SG, simulating winter pasture supplemented with feed concentrates). Volatile fatty acids (VFAs) profiling showed that ruminal acetate, propionate and total VFA contents were significantly higher (p < 0.05) in GSG rumen. Metagenomic analysis showed that Bacteroidetes (53.9%) and Firmicutes (37.1%) were the dominant bacterial phyla in yak rumen across dietary treatments. In GSG samples, Actinobacteriota, Succinivibrionaceae_UCG-002, and Ruminococcus albus were the most abundant, while Bacteroides was significantly more abundant in NSG samples (p < 0.05) than that in GSG. The known fiber-degrading fungus, Neocallimastix, was significantly more abundant in NSG and SG samples, while Cyllamyces were more prevalent in NSG rumen than in the SG rumen. These findings imply that a diverse consortium of microbes may cooperate in response to fluctuating nutrient availability, with depletion of known rumen taxa under nutrient deficiency. Archaeal community composition showed less variation between treatments than bacterial and fungal communities. Additionally, Orpinomyces was significantly positively correlated with acetate levels, both of which are prevalent in GSG compared with other groups. Correlation analysis between microbial taxa and VFA production or between specific rumen microbes further illustrated a collective response to nutrient availability by gut microbiota and rumen VFA metabolism. PICRUSt and FUNGuild functional prediction analysis indicated fluctuation response of the function of microbial communities among groups. These results provide a framework for understanding how microbiota participate in seasonal adaptations to forage availability in high-altitude ruminants, and form a basis for future development of probiotic supplements to enhance nutrient utilization in livestock.

Targeted brain delivery of RVG29-modified rifampicin-loaded nanoparticles for Alzheimer's disease treatment and diagnosis.

Alzheimer's disease (AD) is an aging-related neurodegenerative disease. The main pathological features of AD are ß-amyloid protein (Aß) deposition and tau protein hyperphosphorylation. Currently, there are no effective drugs for the etiological treatment of AD. Rifampicin (RIF) is a semi-synthetic broad-spectrum antibiotic with anti-ß-amyloid deposition, anti-inflammatory, anti-apoptosis, and neuroprotective effects, but its application in AD treatment has been limited for its strong hydrophobicity, high toxicity, short half-life, low bioavailability, and blood-brain barrier hindrance. We designed a novel brain-targeted and MRI-characteristic nanomedicine via loading rabies virus protein 29 (RVG29), rifampicin, and Gd on poly (l-lactide) nanoparticles (RIF@PLA-PEG-Gd/Mal-RVG29). The cytotoxicity assay demonstrated that RIF@PLA-PEG-Gd/Mal-RVG29 had favorable biocompatibility and security. Fluorescence imaging in vivo showed that PLA-PEG-Gd/Mal-RVG29 could deliver rifampicin into the brain by enhancing cellular uptake and brain targeting performance, leading to improvement of the bioavailability of rifampicin. In in vivo study, RIF@PLA-PEG-Gd/Mal-RVG29 improved the spatial learning and memory capability of APP/PS1 mice in the Morris water maze, as compared to rifampicin. Immunofluorescence, TEM, immunoblotting, and H&E staining revealed that RIF@PLA-PEG-Gd/Mal-RVG29 reduced Aß deposition in hippocampal and cortex of APP/PS1 mice, improved the damage of synaptic ultrastructure, increased the expression level of PSD95 and SYP, as well as reduced the necrosis of neurons. These findings suggest that RIF@PLA-PEG-Gd/Mal-RVG29 may be an effective strategy for the treatment of AD.

Association Between Long-Term Visit-to-Visit Hemoglobin A1c and Cardiovascular Risk in Type 2 Diabetes: The ACCORD Trial.

Background: To explore the association between visit-to-visit variability of glycated hemoglobin (HbA1c) and cardiovascular outcomes in the patients with type 2 diabetes mellitus (T2DM) of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study. Methods: We conducted a post-hoc analysis on the ACCORD population including 9,544 participants with T2DM. Visit-to-visit variability of HbA1c was defined as the individual SD, coefficient of variation (CV), and variability independent of the mean (VIM) across HbA1c measurements. The clinical measurements included primary outcome [the first occurrence of non-fatal myocardial infarction (MI), non-fatal stroke or cardiovascular death], total mortality, cardiovascular death, non-fatal MI event, non-fatal stroke, total stroke, heart failure, macrovascular events, and major coronary events (CHD). Results: Over a median follow-up of 4.85 years, 594 and 268 participants experienced all-cause mortality and cardiovascular mortality, respectively. After adjusting for baseline HbA1c levels and confounding factors, the adjusted hazard ratio (HR) comparing patients in the highest vs. the lowest quartile CV of HbA1c variability was 1.61 (95% CI 1.29-2.00) for the primary outcome. Similar trends for secondary outcome were also observed. There was no association between HbA1c fluctuation and non-fatal stroke. Noticeably, there was 66% greater risk for the all-cause mortality among patients in the highest vs. the lowest quartile (HR 1.66, 95% CI 1.27-2.17). Conclusions: Greater variability of HbA1c is associated with higher risk for cardiovascular complications and all-cause death in T2DM. Our study stresses the significance of well-controlled glycemic levels for improving cardiovascular outcomes. Further randomized clinical trials are required to confirm these findings.

High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry.

Antibody-drug conjugates (ADCs) are one of the fastest growing classes of anticancer therapies. Combining the high targeting specificity of monoclonal antibodies (mAbs) with cytotoxic small molecule drugs, ADCs are complex molecular entities that are intrinsically heterogeneous. Primary sequence variants, varied drug-to-antibody ratio (DAR) species, and conformational changes in the starting mAb structure upon drug conjugation must be monitored to ensure the safety and efficacy of ADCs. Herein, we have developed a high-throughput method for the analysis of cysteine-linked ADCs using trapped ion mobility spectrometry (TIMS) combined with top-down mass spectrometry (MS) on a Bruker timsTOF Pro. This method can analyze ADCs (∼150 kDa) by TIMS followed by a three-tiered top-down MS characterization strategy for multi-attribute analysis. First, the charge state distribution and DAR value of the ADC are monitored (MS1). Second, the intact mass of subunits dissociated from the ADC by low-energy collision-induced dissociation (CID) is determined (MS2). Third, the primary sequence for the dissociated subunits is characterized by CID fragmentation using elevated collisional energies (MS3). We further automate this workflow by directly injecting the ADC and using MS segmentation to obtain all three tiers of MS information in a single 3-min run. Overall, this work highlights a multi-attribute top-down MS characterization method that possesses unparalleled speed for high-throughput characterization of ADCs.

An increase in dietary lipid content from different forms of double-low rapeseed reduces enteric methane emission in Datong yaks on the Qinghai-Tibetan Plateau.

Enteric methane (CH4 ) emission in cattle generally decreases by approximately 1 g/g dry matter intake (DMI) with an increase in dietary lipids of 10 g/kg dry matter (DM). The effect of dietary lipids on CH4 emission in yaks has not been reported and is the subject of this study. Four Datong yaks were used in a 4 × 4 Latin-square design in which the four treatments included restricted intakes of double-low rapeseed differing in form and lipid (ether extract-EE) content: (a) rapeseed meal (EE 32.6 g/kg DM); (b) rapeseed meal and rapeseed cake (EE 45.8 g/kg DM); (c) rapeseed meal and whole cracked rapeseed (EE 54.5 g/kg DM) and (d) rapeseed meal and rapeseed oil (EE 62.7 g/kg DM). The digestibility of feed components did not differ among treatments. The ruminal total volatile fatty acids (p = .082) and acetic acid (p = .062) concentrations tended to be lowest in yaks consuming the diet with highest lipid content. In addition, CH4 production was lowest in this group (p = .004), and declined by 1.75 g/g DMI per 10 g/kg DM reduction in dietary lipid content, a rate substantially faster than in cattle.

Rapid Analysis of Reduced Antibody Drug Conjugate by Online LC-MS/MS with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Antibody drug conjugates (ADCs), which harness the high targeting specificity of monoclonal antibodies (mAb) with the potency of small molecule therapeutics, are one of the fastest growing pharmaceutical classes. Nevertheless, ADC conjugation techniques and processes may introduce intrinsic heterogeneity including primary sequence variants, varied drug-to-antibody ratio (DAR) species, and drug positional isomers, which must be monitored to ensure the safety and efficacy of ADCs. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for characterization of ADCs. However, the conventional bottom-up MS analysis workflows require an enzymatic digestion step which can be time consuming and may introduce artifactual modifications. Herein, we develop an online LC-MS/MS method for rapid analysis of reduced ADCs without digestion, enabling determination of DAR, characterization of the primary sequence, and localization of the drug conjugation site of the ADC using high-resolution Fourier transform ion cyclotron resonance (FTICR) MS. Specifically, a model cysteine-linked ADC was reduced to generate six unique subunits: light chain (Lc) without drug (Lc0), Lc with 1 drug (Lc1), heavy chain (Hc) without drug (Hc0), and Hc with 1-3 drugs (Hc1-3, respectively). A concurrent reduction strategy is applied to assess ADC subunits in both the partially reduced (intrachain disulfide bonds remain intact) and fully reduced (all disulfide bonds are cleaved) forms. The entire procedure including the sample preparation and LC-MS/MS takes less than 55 min, enabling rapid multiattribute analysis of ADCs.

Electromyographic Comparison of the Efficacy of Ultrasound-guided Suprainguinal and Infrainguinal Fascia Iliaca Compartment Block for Blockade of the Obturator Nerve in Total Knee Arthroplasty: A Prospective Randomized Controlled Trial.

BACKGROUND AND OBJECTIVES: The knee is innervated by the femoral, obturator, and sciatic nerves. An infrainguinal fascia iliaca compartment block (FICB) is often used as a technique for pain management after hip and knee arthroplasty. This approach blocks the femoral nerve, lateral femoral cutaneous nerve, and obturator nerve. Previous studies show suprainguinal FICB achieves improved postoperative analgesia compared with infrainguinal FICB after hip fracture. However, the analgesic effects of suprainguinal or infrainguinal FICB on the obturator nerve after total knee arthroplasty (TKA) remain to be established. This study compared the efficacy of suprainguinal versus infrainguinal FICB for the blockade of the obturator nerve using electromyography and quantification of total opioid consumption during the 24 hours after TKA. METHODS: This prospective, randomized controlled clinical study enrolled 74 patients scheduled to undergo TKA. Patients were randomized 1:1 to receive suprainguinal FICB (group S) or infrainguinal FICB (group I) with 30 mL of 0.375% ropivacaine. The primary endpoint was the mean amplitude of the adductor longus compound muscle action potential (CMAP) at 0 (before the block), 10, 20, and 30 minutes after FICB. The secondary endpoint was total opioid consumption during the 24 hours after TKA. RESULTS: Data from 62 patients were included in the analysis. The mean amplitude of the adductor longus CMAP was significantly lower in group S compared with group I (repeated-measures analysis of variance; F=4.73, P=0.034). At 24 hours after TKA, mean (SD) total opioid consumption was significantly lower in group S, compared with group I (131.5±76.8 vs. 201.5±85.1 µg) (P=0.001). CONCLUSIONS: Suprainguinal FICB significantly increased the incidence of successful obturator nerve block and significantly decreased fentanyl consumption 24 hours after TKA compared with infrainguinal FICB.

Assessing localized conformational stability of antibody-drug conjugate by protein conformation assay.

Antibody-drug conjugates (ADCs) are a class of attractive therapeutic agents to fight cancer with conjugation of potent chemical agents on target-selective antibodies. The conceptually elegant approach has encountered mounting practical challenges in combining the mAb and potent drug while maintaining the conformational and physiochemical stability of the bioconjugates. The attachment of hydrophobic drug-linker with antibody could potentially alter the antibody conformational scaffold, locally or globally. Here we propose to use a protein conformation assay (PCA) to measure the higher-order structure of antibodies upon drug-linker conjugation. The PCA analysis provides insights into the formation of partially unfolded ADCs, which may correlate with protein stability and aggregation propensity. To further elucidate the cause of the unfolding events, in-depth peptide mapping combined with the PCA conformational footprints were performed on a commercial ADC trastuzumab emtansine in this study. The locally altered conformational hot-spots observed in PCA matched with conjugation sites with high occupancy rate identified in peptide mapping. In summary, by combining PCA and in-depth peptide mapping, a snapshot of ADC structural conformation and stability profile could be obtained and provide a swift and convenient measurement of the 'fitness' of ADC to facilitate payload selection, conjugation process development and early predictive developability assessment.

Conjugation Site Analysis by MS/MS Protein Sequencing.

In-depth knowledge about the site of drug-linker conjugation is important for the understanding of the conjugation efficiency and the exact locations of payloads for antibody-drug conjugates (ADCs). Here we describe a peptide mapping-based protocol, covering sample preparation procedure, LC-MS/MS setup, and data processing (auto and manual), to determine the locations of drug-linker attachment on mAbs. In comparison with classical mAb peptide mapping, some improvements will be highlighted for maintaining hydrophobic drug-loaded peptides in solution, enabling efficient chromatographic separation and mass spectrometric detection, and allowing for their unambiguous identification in LC-MS/MS map by using diagnostic fragmentation ions of the payload.

Declined circulating Elabela levels in patients with essential hypertension and its association with impaired vascular function: A preliminary study.

Background: Elabela (ELA) is a newly identified endogenous ligand of apelin receptor (APJ) which has been confirmed to be implicated in the pathogenesis of hypertension. Previous experiments have revealed the critical role of ELA in eliciting vasodilation and lowering blood pressure. However, the role of plasma ELA levels in hypertensive patients and its relationship with vascular function have not been investigated.Method: Thirty-one patients with essential hypertension (EH) and 31 age-matched healthy subjects as controls were recruited in the study. Plasma ELA concentration and vascular function parameters including brachial artery flow-mediated dilation (FMD) and brachial-ankle pulse wave velocity (baPWV) were measured.Results: We observed remarkably lower plasma ELA concentration in hypertensive patients as compared with controls (1.29 ± 0.56 ng/ml vs. 1.79 ± 0.55 ng/ml; P = 0.001). Linear correlation analysis showed that ELA was negatively correlated with systolic blood pressure (r = -0.388, P = 0.002) and diastolic blood pressure (r = -0.321, P = 0.011) and positively correlated with FMD (r = 0.319, P = 0.011). There was no statistically significant relationship between ELA and baPWV (r = 0.234, P = 0.067). Stepwise multiple linear analysis also identified a close association of plasma ELA levels with endothelial function.Conclusion: The present study demonstrates for the first time that circulating ELA levels are reduced in patients with EH. The fall in endogenous ELA levels may be involved in the pathogenesis of hypertension-related vascular damage.

Middle-Down Multi-Attribute Analysis of Antibody-Drug Conjugates with Electron Transfer Dissociation.

Antibody-drug conjugates (ADCs) are designed to combine the target specificity of monoclonal antibodies and potent cytotoxin drugs to achieve better therapeutic outcomes. Comprehensive evaluation of the quality attributes of ADCs is critical for drug development but remains challenging due to heterogeneity of the construct. Currently, peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the predominant approach to characterize ADCs. However, it is suboptimal for sequence characterization and quantification of ADCs because it lacks a comprehensive view of coexisting variants and suffers from varying ionization effects of drug-conjugated peptides compared to unconjugated counterparts. Here, we present the first middle-down RPLC-MS analysis of both cysteine (Adcetris; BV) and lysine (Kadcyla; T-DM1) conjugated ADCs at the subunit level (∼25 kDa) with electron transfer dissociation (ETD). We successfully achieved high-resolution separation of subunit isomers arising from different drug conjugation and subsequently localized the conjugation sites. Moreover, we obtained a comprehensive overview of the microvariants associated with each subunits and characterized them such as oxidized variants with different sites. Furthermore, we observed relatively high levels of conjugation near complementarity-determining regions (CDRs) from the heavy chain but no drug conjugation near CDRs of light chain (Lc) from lysine conjugated T-DM1. Based on the extracted ion chromatograms, we accurately measured average drug to antibody ratio (DAR) values and relative occupancy of drug-conjugated subunits. Overall, the middle-down MS approach enables the evaluation of multiple quality attributes including DAR, positional isomers, conjugation sites, occupancy, and microvariants, which potentially opens up a new avenue to characterize ADCs.

Impact of linker-drug on ion exchange chromatography separation of antibody-drug conjugates.

Charge variants are important attributes of monoclonal antibodies, including antibody-drug conjugates (ADCs), because charge variants can potentially influence the stability and biological activity of these molecules. Ion exchange chromatography (IEX) is widely used for charge variants analysis of mAbs and offers the feasibility of fractionation for in-depth characterization. However, the conjugated linker-drug on ADCs could potentially affect the separation performance of IEX, considering IEX separation relies on surface charge distribution of analyte and involves the interaction between analyte surface and IEX stationary phase. Here, we investigated weak cation exchange chromatography (WCX) for its application in analyzing three ADCs (two broad distribution ADCs and an ADC with controlled conjugation sites) and the 2-drug/4-drug loaded species isolated from the two broad distribution ADCs using hydrophobic interaction chromatography. The major peaks in WCX profile were characterized via fraction collection followed by capillary electrophoresis-sodium dodecyl sulfate or peptide mapping. Results suggested that both the number of drug loads and conjugation sites could impact WCX separation of an ADC. The hypothesis was that the linker drugs could interfere with the ionic interaction between its surrounding amino acids on the mAb surface and column resin, which reduced the retention of ADCs on WCX column in this study. Our results further revealed that WCX brings good selectivity towards positional isomers, but limited resolution for different drug load, which causes the peak compositions of the two broad-distribution ADCs to be highly complex. We also compared results from WCX and imaged capillary isoelectric focusing (icIEF). Results showed that separation in icIEF was less influenced by conjugated linker drugs for the ADCs studied in this work, and better alignment was found between the two techniques for the ADC with controlled conjugate sites. Overall, this work provides insights into the complexity of WCX analysis of ADCs, which should be considered during method development and sample characterization.

Effect of local anesthetic volume (20 mL vs 30 mL ropivacaine) on electromyography of the diaphragm and pulmonary function after ultrasound-guided supraclavicular brachial plexus block: a randomized controlled trial.

BACKGROUND AND OBJECTIVES: Diaphragmatic paralysis following supraclavicular brachial plexus block (SCBPB) is ascribed to phrenic nerve palsy. This study investigated the effect of 2 volumes of 0.375% ropivacaine on efficacy of block as a surgical anesthetic and as an analgesic and examined diaphragm compound muscle action potentials (CMAPs) and pulmonary function before and after SCBPB. METHODS: Eighty patients scheduled for removal of hardware for internal fixation after healing of an upper limb fracture distal to the shoulder were randomized to receive ultrasound-guided SCBPC for surgical anesthesia with 20 mL (Group A) or 30 mL (Group B) 0.375% ropivacaine. The latency and amplitude of diaphragm CMAPs and forced vital capacity (FVC), FVC% predicted, and forced expiratory volume in 1 s (FEV1) were measured before and 30 min after SCBPB. RESULTS: Block success as primary anesthetic in addition to analgesia was 81% in Group A and 91% in Group B. There were no obvious differences in the effectiveness of analgesia between the two groups. The mean time to onset of motor block was significantly longer in Group A (8.1±2.7 min) than in Group B (5.4 ± 2.8 min; p<0.05). The mean amplitude of the diaphragm CMAP was significantly lower in Group B than in Group A (p=0.03). The changes in FVC (Group A, - 8.1% vs Group B, -16.5%), FVC% (Group A, -8.0% vs Group B, -17.1%), and FEV1 (Group A, -9.5% vs Group B, -15.2%) from pre-SCBPB to post-SCBPB were significantly less in Group A than in Group B (all p=0.03). CONCLUSIONS: The incidence rates of phrenic nerve palsy and diaphragm paralysis were reduced, and lung function was less impaired in patients who received 20 mL vs 30 mL of 0.375% ropivacaine without any differences in block success. Selecting a lower volume of anesthetic for nerve block may be especially beneficial in obese patients or patients with cardiopulmonary disease. TRIAL REGISTRATION NUMBER: ChiCTR-IND-17012166.

Native Reversed-Phase Liquid Chromatography: A Technique for LCMS of Intact Antibody-Drug Conjugates.

The synthesis of antibody-drug conjugates (ADCs) using the interchain cysteines of the antibody inherently gives a mixture of proteins with varying drug-to-antibody ratio. The drug distribution profiles of ADCs are routinely characterized by hydrophobic interaction chromatography (HIC). Because HIC is not in-line compatible with mass spectrometry (MS) due to the high salt levels, it is laborious to identify the constituents of HIC peaks. An MS-compatible alternative to HIC is reported here: native reversed phase liquid chromatography (nRPLC). This novel technique employs a mobile phase 50 mM ammonium acetate for high sensitivity in MS and elution with a gradient of water/isopropanol. The key to the enhancement is a bonded phase giving weaker drug-surface interactions compared to the noncovalent interactions holding the antibody-drug conjugates together. The hydrophobicity of the bonded phase is varied, and the least hydrophobic bonded phase in the series, poly(methyl methacrylate), is found to resolve the intact constituents of a model ADC (Ab095-PZ) and a commercial ADC (brentuximab vedotin) under the MS-compatible conditions. The nRPLC-MS data show that all species, ranging from drug-to-antibody ratios of 1 to 8, remained intact in the column. Another desired advantage of the nRPLC is the ability of resolving multiple positional isomers of ADC that are not well-resolved in other chromatographic modes. This supports the premise that lower hydrophobicity of the bonded phase is the key to enabling online nRPLC-MS analysis of antibody-drug conjugates.

Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry.

The pharmaceutical industry's interest in monoclonal antibodies (mAbs) and their derivatives has spurred rapid growth in the commercial and clinical pipeline of these effective therapeutics. The complex micro-heterogeneity of mAbs requires in-depth structural characterization for critical quality attribute assessment and quality assurance. Currently, mass spectrometry (MS)-based methods are the gold standard in mAb analysis, primarily with a bottom-up approach in which immunoglobulins G (IgGs) and their variants are digested into peptides to facilitate the analysis. Comprehensive characterization of IgGs and the micro-variants remains challenging at the proteoform level. Here, we used both top-down and middle-down MS for in-depth characterization of a human IgG1 using ultra-high resolution Fourier transform MS. Our top-down MS analysis provided characteristic fingerprinting of the IgG1 proteoforms at unit mass resolution. Subsequently, the tandem MS analysis of intact IgG1 enabled the detailed sequence characterization of a representative IgG1 proteoform at the intact protein level. Moreover, we used the middle-down MS analysis to characterize the primary glycoforms and micro-variants. Micro-variants such as low-abundance glycoforms, C-terminal glycine clipping, and C-terminal proline amidation were characterized with bond cleavages higher than 44% at the subunit level. By combining top-down and middle-down analysis, 76% of bond cleavage (509/666 amino acid bond cleaved) of IgG1 was achieved. Taken together, we demonstrated the combination of top-down and middle-down MS as powerful tools in the comprehensive characterization of mAbs.

Online Hydrophobic Interaction Chromatography-Mass Spectrometry for the Analysis of Intact Monoclonal Antibodies.

Therapeutic monoclonal antibodies (mAbs) are an important class of drugs for a wide spectrum of human diseases. Liquid chromatography (LC) coupled to mass spectrometry (MS) is one of the techniques in the forefront for comprehensive characterization of analytical attributes of mAbs. Among various protein chromatography modes, hydrophobic interaction chromatography (HIC) is a popular offline nondenaturing separation technique utilized to purify and analyze mAbs, typically with the use of non-MS-compatible mobile phases. Herein we demonstrate for the first time, the application of direct HIC-MS and HIC-tandem MS (MS/MS) with electron capture dissociation (ECD) for analyzing intact mAbs on quadrupole-time-of-flight (Q-TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, respectively. Our method allows for rapid determination of relative hydrophobicity, intact masses, and glycosylation profiles of mAbs as well as sequence and structural characterization of the complementarity-determining regions in an online configuration.