Exposing the molecular heterogeneity of glycosylated biotherapeutics.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

Modified stochastic diffusion particle tracking model driven by skew Brownian motion: Analysis of sediment particle motion in turbulent flow under the effects of ejection and sweep.

Turbulent bursting events have been classified into outward interactions (Q1), ejections (Q2), inward interactions (Q3), and sweeps (Q4) in various studies. Ejections (Q2) and sweeps (Q4) have been identified as significant contributors to time consumption, momentum flux, and sediment flux. Additionally, research has shown that the distribution of these events varies nonuniformly at different bed elevations. Despite extensive investigations into the nonuniform distribution of turbulent bursting events, their impact on sediment transport has been rarely explored. In this work, we developed a modified stochastic diffusion particle tracking model (SD-PTM) driven by skew Brownian motion (SBM) using the stochastic Lagrangian approach to scrutinize sediment particle movement in turbulent flows. The model incorporates turbulent characteristics derived from a direct numerical simulation dataset, allowing for a comprehensive analysis of sediment particle dynamics. Moreover, the proposed model accounts for the nonuniform spatial distribution of ejection and sweep events, as well as the particle movement direction during these events. Numerical simulations of the model were conducted to trace sediment particle trajectories in the streamwise and vertical directions. The analysis of sediment transport involved calculating the variance of particle trajectories to examine anomalous diffusion. The model's performance was evaluated by comparing it with flow velocity and sediment concentration profiles obtained from measurements in previous studies. In conclusion, our study suggests that the motion of sediment particles in turbulent flow can be thoroughly investigated under extreme flow conditions using the modified SD-PTM driven by SBM.

Poorer outcomes associated with more invasive lead management strategies for Abbott Riata® leads: a large, multicenter experience.

BACKGROUND: Over 100,000 Abbott Riata® were implanted in the United States before they were recalled in 2010. There are still a significant number of Abbott Riata® leads in use, and it is unclear how these leads should be managed at the time of generator change or lead malfunction. Although data comparing both Sprint Fidelis® and Abbott Riata® leads in this setting is available, there are no multicenter comparative studies of outcomes for various lead management strategies, including lead extraction (LE), lead abandonment/revision (LA), and generator change (GC) only at the time of device at elective replacement interval (ERI) for Abbott Riata® leads. METHODS: A retrospective, multicenter study was undertaken to compare short-term outcomes (major complications-MC, death, extended or re-hospitalizations within 60 days-RH, lead malfunction-LM) and total outcomes (short-term outcomes & lead malfunction during follow-up) of patients with Riata® leads undergoing LE, LA, or GC. RESULTS: 152 patients (65 ± 13 years, 68% male) were followed for a mean 33 ± 30 months following intervention. Out of 166 procedures, 13 patients underwent LE, 16 patients underwent LA, and 137 patients underwent GC. There was 1 major complication in each group, yielding an event rate of 7.7% for LE, 6.3% for LA, and 0.7% for GC cohorts. There were significantly more short-term and total adverse outcomes in the group of patients getting LE and LA versus GC only (38.5% & 31.3% vs 7.3%, P < 0.001). Total Riata® lead dwell time follow-up was 17,067 months. A total of 3 Riata® lead malfunctions were noted during long-term follow-up. Inappropriate shocks were similar between LE 7.7% (1/13), LA 6.3% (1/16). and GC 11.0% (4/136); P = 0.57. CONCLUSIONS: There were more short-term and total adverse outcomes in more invasive management strategies (LE and LA) versus GC alone. The failure rate of Riata® leads was substantially lower compared to previous reports. Therefore, we recommend only performing battery exchange when a device with an active Riata® lead is at ERI, unless there is malfunction of the Riata® lead noted on testing. There were significantly more short-term adverse outcomes in the lead extraction (5/13) and lead abandonment/revision (5/16) groups than the generator only (8/137) group (P < 0.001). GIB - Gastrointestinal bleed, CHF - congestive heart failure, NSTEMI - non-ST elevation MI.

Semi-analytical modeling of sediment-laden open-channel flows with the effects of stratification, hindered settling, and eddy viscosities.

This study proposes semi-analytical models for simultaneous distribution of fluid velocity and suspended sediment concentration in an open-channel turbulent flow using three kinds of eddy viscosities. Apart from the classical parabolic eddy viscosity which is based on a log-law velocity profile, we consider two recently proposed eddy viscosities based on the concept of velocity and length scales. To deal with the flows with high sediment concentration, several turbulent features such as the hindered settling mechanism and the stratification effect are incorporated in the model. The governing system of highly nonlinear differential equations is solved using the homotopy analysis method (HAM), which produces solutions in the form of convergent series. Numerical and theoretical convergence analyses are provided for all three types of eddy viscosities. The effects of parameters on the derived models are discussed physically. Experimental data on both dilute and non-dilute flows are considered to verify the HAM-based solutions. The effects of the stratification correction factor (ß) and the turbulent Schmidt number (α) reveal that they should be determined optimally for applicability of the proposed models in terms of accurate prediction with data. This optimal procedure required further investigation of these parameters, and, thus, an analysis of ß and α is carried out, which linked them with the particle diameter through particle settling velocity, reference fluid velocity, and reference sediment concentration by proposing regression equations. Furthermore, using the optimal values of the parameters, the proposed models corresponding to the eddy viscosities based on the exponentially decreasing turbulent kinetic energy function and von Karman's similarity hypothesis are seen to be superior to the model corresponding to a parabolic eddy viscosity. Finally, a comment on the HAM is made where it is observed that the method can remove the numerical singularity of the governing equations at the water surface, which arises because of the consideration of vanishing eddy viscosity thereat.

Comparison of quantifiable electrocardiographic changes associated with severe hyperkalemia.

BACKGROUND: Hyperkalemia (HK) is a life-threatening condition that is frequently evaluated by electrocardiogram (ECG). ECG changes in severe HK (≥ 6.3 mEq/L) are not well-characterized. This study sought to compare and correlate ECG metrics in severe HK to baseline normokalemic ECGs and serum potassium. METHODS: A retrospective analysis of 340 severe HK encounters with corresponding normokalemic ECGs was performed. RESULTS: Various ECG metrics were analyzed. P wave amplitude in lead II, QRS duration, T wave slope, ratio of T wave amplitude: duration, and ratios of T wave: QRS amplitudes were significantly different between normokalemic and HK ECGs. P wave amplitude attenuation in lead II correlated better with serum potassium than in V1. T wave metrics that incorporated both T wave and QRS amplitudes correlated better than metrics utilizing T wave metrics alone. CONCLUSION: Multiple statistically significant and quantifiable differences among ECG metrics were observed between normokalemic and HK ECGs and correlated with increasing degrees of serum potassium and along the continuum of serum potassium. When incorporated into a logistic regression model, the ability to distinguish HK versus normokalemia on ECG improved significantly. These findings could be integrated into an ECG acquisition system that can more accurately identify severe HK.

Electrophysiological evaluation following development of new and persistent left bundle branch block after transcatheter aortic valve replacement: A single center pilot study.

INTRODUCTION: New and persistent left bundle branch block (NP-LBBB) following Transcatheter Aortic Valve Replacement (TAVR) is an ongoing concern with incidence ranging from as low as 4% to up to 65% (varying for different types of valves). Such patients are at risk of developing high-grade atrioventricular block (HAVB) warranting permanent pacemaker (PPM) implantation. However, currently, there are no consensus guidelines or large prospective studies to risk stratify these patients for safer discharge after TAVR. OBJECTIVES: To provide insight from a single center study on using modified electrophysiology (EP) study to risk stratify post-TAVR patients to outpatient monitoring for low-risk versus pacemaker implantation for high-risk patients. METHODS AND RESULTS: Between June 2020 and March 2023, all patients who underwent a TAVR procedure (324 patients) at our institution were screened for development of NP-LBBB post-operatively. Out of 26 patients who developed NP-LBBB, after a pre-specified period of observation, 18 patients were deemed eligible for a modified EP study to assess His-Ventricular (HV) interval. 11 out of 18 patients (61.1%) had normal HV interval (HV < 55 ms). Three out of 18 patients (16.7%) had HV prolongation (55 ms < HV < 70 ms) without significant HV prolongation (defined as an increase in HV interval > 30%) with intra-procedural procainamide challenge. Four out of 18 patients (22.2%) had significant HV prolongation (HV > 70 ms) warranting PPM implantation based on a multidisciplinary approach and shared decision-making with the patients. Total of 50% of patients discharged with PPM (two out of four patients) were noted to be pacemaker dependent based on serial device interrogations. All patients who did not receive PPM were discharged with ambulatory monitoring with 30-day event monitor and did not develop HAVB on serial follow-up. CONCLUSION: Normal HV interval up to 55 ms on modified EP study after TAVR and development of NP-LBBB can be utilized as a threshold for risk stratification to facilitate safe discharge. The optimal upper limit of HV interval threshold remains unclear in determining appropriate candidacy for PPM.

Echocardiographic changes and heart failure hospitalizations following rhythm control for arrhythmia-induced cardiomyopathy: results from a multicenter, retrospective study.

BACKGROUND: The incidence and prevalence of arrhythmia-induced cardiomyopathy (AIC) are unclear but likely underrecognized. LV dysfunction is common among patients with atrial fibrillation (AF), atrial flutter (AFL), and frequent premature ventricular contractions (PVC). The hallmark of AIC is the improvement of left ventricular ejection fraction (LVEF) following arrhythmia treatment. Changes in echocardiographic parameters and their effect on outcomes after rhythm control for AIC are not well understood. We aimed to study echocardiographic parameters and outcomes following rhythm control for AIC. METHODS: A multicenter, retrospective study was conducted at 4 different medical centers involving patients with AIC. Clinical, echocardiographic, and outcome (mortality and heart failure hospitalizations [HFH]) parameters were extracted from the medical record. RESULTS: Two hundred fifty-five patients (age 66 ± 11 years, 73% male) with AIC caused by AF (51%), atrial tachycardia/AFL (20%), and PVCs (29%) were included and followed for a median period of 6 months after successful rhythm control. Significant improvements in left ventricular (LV) ejection fraction (P < 0.0001), LV end-systolic volume (ml) (90 ± 48 to 58 ± 30; P < 0.0001), LV internal diameter end diastole (cm) (5.5 ± 0.78 to 5.3 ± 0.64; P = 0.0001) and end systole (4.7 ± 0.95 to 4.3 ± 1.02; P < 0.0001), right atrial pressure (mmHg) (11.3 ± 5.0 to 7.4 ± 3.2; P = 0.0001), and right ventricular function (n (%)) (42 (44) to 9 (11); P < 0.0001) were noted following arrhythmia treatment. No deaths occurred during follow-up. HFH occurred in 7 patients. Arrhythmia recurrence rate was 50.5%. Neither echocardiographic parameters nor recurrence of arrhythmia correlated with HFH. CONCLUSION: Arrhythmia treatment significantly improved echocardiographic LV dimensions, LVEF, and RAP in this multicenter AIC cohort, underscoring the need for early recognition and aggressive rhythm control in suspected AIC patients. The event rate was too low to assess for outcome predictors.

Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli.

BACKGROUND: Escherichia coli (E. coli) is a promising host for production of recombinant proteins (including antibodies and antibody fragments) that don't require complex post-translational modifications such as glycosylation. During manufacturing-scale production of a one-armed antibody in E. coli (periplasmic production), variability in the degree of reduction of the antibody's disulfide bonds was observed. This resulted in variability in the free thiol content, a potential critical product quality attribute. This work was initiated to understand and prevent the variability in the total free thiol content during manufacturing. RESULTS: In this study, we found that the reduction in antibody's disulfide bonds was observed to occur during homogenization and the ensuing homogenate hold step where in the antibody is exposed to redox enzymes and small molecule reductants present in homogenate. Variability in the downstream processing time between the start of homogenization and end of the homogenate hold step resulted in variability in the degree of antibody disulfide bond reduction and free thiol content. The disulfide bond reduction in the homogenate is catalyzed by the enzyme disulfide bond isomerase C (DsbC) and is highly site-specific and occurred predominantly in the intra-chain disulfide bonds present in the Fc CH2 region. Our results also imply that lack of glycans in E. coli produced antibodies may facilitate DsbC accessibility to the disulfide bond in the Fc CH2 region, resulting in its reduction. CONCLUSIONS: During E. coli antibody manufacturing processes, downstream processing steps such as homogenization and subsequent processing of the homogenate can impact degree of disulfide bond reduction in the antibody and consequently product quality attributes such as total free thiol content. Duration of the homogenate hold step should be minimized as much as possible to prevent disulfide bond reduction and free thiol formation. Other approaches such as reducing homogenate temperature, adding flocculants prior to homogenization, using enzyme inhibitors, or modulating redox environments in the homogenate should be considered to prevent antibody disulfide bond reduction during homogenization and homogenate processing steps in E. coli antibody manufacturing processes.

External validation of a novel signature of illness in continuous cardiorespiratory monitoring to detect early respiratory deterioration of ICU patients.

OBJECTIVE: The goal of predictive analytics monitoring is the early detection of patients at high risk of subacute potentially catastrophic illnesses. An excellent example of a targeted illness is respiratory failure leading to urgent unplanned intubation, where early detection might lead to interventions that improve patient outcomes. Previously, we identified signatures of this illness in the continuous cardiorespiratory monitoring data of intensive care unit (ICU) patients and devised algorithms to identify patients at rising risk. Here, we externally validated three logistic regression models to estimate the risk of emergency intubation developed in Medical and Surgical ICUs at the University of Virginia. APPROACH: We calculated the model outputs for more than 8000 patients in the University of California-San Francisco ICUs, 240 of whom underwent emergency intubation as determined by individual chart review. MAIN RESULTS: We found that the AUC of the models exceeded 0.75 in this external population, and that the risk rose appreciably over the 12 h before the event. SIGNIFICANCE: We conclude that there are generalizable physiological signatures of impending respiratory failure in the continuous cardiorespiratory monitoring data.

Development of a noise-assisted multivariate empirical mode decomposition framework for characterizing PM 2.5 air pollution in Taiwan and its relation to hydro-meteorological factors.

To better understand air pollution problems, the relationships between PM2.5 and hydro-meteorological variables are studied using a state-of-the-art multivariate nonlinear and non-stationary filtering method, noise-assisted multivariate empirical mode decomposition (NAMEMD), and the time-dependent intrinsic correlation (TDIC) algorithm. Three characteristic scales (annual, diurnal and semi-diurnal) are shown to be significant to PM2.5 characterization, based on using NAMEMD filtering. Temporal fluctuations of local correlations among PM2.5 and hydro-meteorological variables are presented. On diurnal and semi-diurnal scales, seasonal variation of the local correlation between temperature and humidity is observed. A combined wind speed and direction analysis can be conducted using the NAMEMD-based algorithm. The pollutant roses that are generated from the reconstructed wind directions reveal the sources of PM2.5 on different scales. PM2.5 is found to be related to land breeze at the diurnal scale and to winter monsoons at the annual scale. The scale-dependent wind direction that contributes to the increase of PM2.5 can be identified.

Effect of Floodplain Restoration on Photolytic Removal of Pharmaceuticals.

Floodplain restoration is popular to address excess nutrients, but its ability to enhance photolysis of emerging contaminants has not been evaluated. We used the numerical model MIKE-21 to simulate photolysis reactions within the inundated surface water of restored floodplains along a mid-size river. We examined both "high" and "low" floodplain scenarios where inundation occurs 5% (storms) and 50% (baseflow) of the year, respectively. We simulated photolysis of the pharmaceuticals morphine, codeine, and methamphetamine and, for context, compared it with nitrate removal (denitrification and plant uptake). Pollutant removal due to floodplain restoration was greater for the low floodplain (e.g., 18.8% for morphine) than for the high floodplain (5.6% for morphine) due to greater water exchange relative to channel flow. The fastest- and slowest-reacting pollutants (morphine and methamphetamine, respectively) were always transport- and reaction/kinetics-limited within floodplain surface water, respectively. Yet, those with intermediate decay-rate constants switched from reaction limitation to transport limitation as the floodplain length increased, and removal leveled off at an optimum length of ∼1000 m. However, as the floodplain width increased, the required floodplain length for 30% removal decreased. Optimal restored floodplain conditions for photolysis would maximize light exposure, which may differ from those for nutrients.

Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization.

Free thiol content, and its consistency, is one of the product quality attributes of interest during technical development of manufactured recombinant monoclonal antibodies (mAbs). We describe a new, mid/high-throughput reversed-phase-high performance liquid chromatography (RP-HPLC) method coupled with derivatization of free thiols, for the determination of total free thiol content in an E. coli-expressed therapeutic monovalent monoclonal antibody mAb1. Initial selection of the derivatization reagent used an hydrophobicity-tailored approach. Maleimide-based thiol-reactive reagents with varying degrees of hydrophobicity were assessed to identify and select one that provided adequate chromatographic resolution and robust quantitation of free thiol-containing mAb1 forms. The method relies on covalent derivatization of free thiols in denatured mAb1 with N-tert-butylmaleimide (NtBM) label, followed by RP-HPLC separation with UV-based quantitation of native (disulfide containing) and labeled (free thiol containing) forms. The method demonstrated good specificity, precision, linearity, accuracy and robustness. Accuracy of the method, for samples with a wide range of free thiol content, was demonstrated using admixtures as well as by comparison to an orthogonal LC-MS peptide mapping method with isotope tagging of free thiols. The developed method has a facile workflow which fits well into both R&D characterization and quality control (QC) testing environments. The hydrophobicity-tailored approach to the selection of free thiol derivatization reagent is easily applied to the rapid development of free thiol quantitation methods for full-length recombinant antibodies.

A sensitive assay reveals structural requirements for α-synuclein fibril growth.

The accumulation of α-synuclein (α-syn) fibrils in neuronal inclusions is the defining pathological process in Parkinson's disease (PD). A pathogenic role for α-syn fibril accumulation is supported by the identification of dominantly inherited α-syn (SNCA) gene mutations in rare cases of familial PD. Fibril formation involves a spontaneous nucleation event in which soluble α-syn monomers associate to form seeds, followed by fibril growth during which monomeric α-syn molecules sequentially associate with existing seeds. To better investigate this process, we developed sensitive assays that use the fluorescein arsenical dye FlAsH (fluorescein arsenical hairpin binder) to detect soluble oligomers and mature fibrils formed from recombinant α-syn protein containing an N-terminal bicysteine tag (C2-α-syn). Using seed growth by monomer association (SeGMA) assays to measure fibril growth over 3 h in the presence of C2-α-syn monomer, we observed that some familial PD-associated α-syn mutations (i.e. H50Q and A53T) greatly increased growth rates, whereas others (E46K, A30P, and G51D) decreased growth rates. Experiments with wild-type seeds extended by mutant monomer and vice versa revealed that single-amino acid differences between seed and monomer proteins consistently decreased growth rates. These results demonstrate that α-syn monomer association during fibril growth is a highly ordered process that can be disrupted by misalignment of individual amino acids and that only a subset of familial-PD mutations causes fibril accumulation through increased fibril growth rates. The SeGMA assays reported herein can be utilized to further elucidate structural requirements of α-syn fibril growth and to identify growth inhibitors as a potential therapeutic approach in PD.

Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies.

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily alpha1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a alpha1,6-fucosyltransferase. FUT8(-/-) CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8(-/-) cells as hosts for antibody production. We have created zinc-finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8(-/-) CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN-treated cells can be directly selected to give FUT8(-/-) cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN-derived FUT8(-/-) cell lines were as transfectable as wild-type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild-type and ZFN-generated FUT8(-/-) cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN-generated FUT8(-/-) cell lines was fourfold higher than in the best-producing clones of FUT8(-/-) cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN-mediated transfer of the Fut8- phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies.

Determination of potential sources of PCBs and PBDEs in sediments of the Niagara River.

Sediments from Niagara River, an important waterway connecting two of the Great Lakes (Lake Erie to Lake Ontario), were analyzed for 14 congeners of polychlorinated biphenyls (PCBs) and 9 congeners of polybrominated diphenyl ethers (PBDEs) using accelerated solvent extraction and gas chromatography/mass spectrometry. Total concentrations of PCBs ranged from 1.7 to 124.6 ng/g were PCBs 138 and 153 were found in all samples. All sites but one showed PBDE in sediments with total concentrations as high as 148 ng/g, suggesting that PBDE is becoming an important class of POP. A land-use and coverage map was used to trace potential localized sources of PCB and PBDE contamination. Results indicate that the highest levels of PCBs and PBDEs were found in sediments collected from areas closest to the discharge locations of municipal wastewater treatment plants (WWTP) and local industries. This is the first study that suggests the importance of WWTP discharges as a potential source of PBDE contamination in the Great Lakes.