The effect of comb length on the in vitro and in vivo properties of self-assembled poly(oligoethylene glycol methacrylate)-based block copolymer nanoparticles.

Comb copolymer analogues of poly(lactic acid)-polyethylene glycol block copolymers (PLA-b-PEG) offer potential to overcome the inherent chemistry and stability limitations of their linear block copolymer counterparts. Herein, we examine the differences between P(L)LA10K-b-PEG10K and linear-comb copolymer analogues thereof in which the linear PEG block is replaced by poly(oligo(ethylene glycol) methacrylate) (POEGMA) blocks with different side chain (comb) lengths but the same overall molecular weight. P(L)LA10K-b-POEGMA47510K and P(L)LA10K-b-POEGMA200010K block copolymers were synthesized via activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) and fabricated into self-assembled nanoparticles using flash nanoprecipitation via confined impinging jet mixing. Linear-comb copolymer analogues based on PLA-b-POEGMA yielded smaller but still well-controlled nanoparticle sizes (88 ± 2 nm and 114 ± 1 nm respectively compared to 159 ± 2 nm for P(L)LA10K-b-PEG10K nanoparticles) that exhibited improved colloidal stability relative to linear copolymer-based nanoparticles over a 15 day incubation period while maintaining comparably high cytocompatibility, although the comb copolymer analogues had somewhat lower loading capacity for doxorubicin hydrochloride. Cell spheroid studies showed that the linear-comb copolymers promoted enhanced tumor transport and thus cell killing compared to conventional linear block copolymers. In vivo studies showed all NP types could passively accumulate within implanted CT26 tumors but with different accumulation profiles, with P(L)LA10K-b-POEGMA200010K NPs showing continuous accumulation throughout the full 24 h monitoring period whereas tumor accumulation of P(L)LA10K-b-POEGMA47510K NPs was significant only between 8 h and 24 h. Overall, the linear-comb copolymer analogues exhibited superior stability, biodistribution, spheroid penetration, and inherent tunability over linear NP counterparts.

Development of a capillary zone electrophoresis method to monitor magnesium ion consumption during in vitro transcription for mRNA production.

Messenger RNA (mRNA) vaccines represent a landmark in vaccinology, especially with their success in COVID-19 vaccines, which have shown great promise for future vaccine development and disease prevention. As a platform technology, synthetic mRNA can be produced with high fidelity using in vitro transcription (IVT). Magnesium plays a vital role in the IVT process, facilitating the phosphodiester bond formation between adjacent nucleotides and ensuring accurate transcription to produce high-quality mRNA. The development of the IVT process has prompted key inquiries about in-process characterization of magnesium ion (Mg++) consumption, relating to the RNA polymerase (RNAP) activation, fed-batch mode production yield, and mRNA quality. Hence, it becomes crucial to monitor the free Mg++ concentration throughout the IVT process. However, no free Mg++ analysis method has been reported for complex IVT reactions. Here we report a robust capillary zone electrophoresis (CZE) method with indirect UV detection. The assay allows accurate quantitation of free Mg++ for the complex IVT reaction where it is essential to preserve IVT samples in their native-like state during analysis to avoid dissociation of bound Mg complexes. By applying this CZE method, the relationships between free Mg++ concentration, the mRNA yield, and dsRNA impurity level were investigated. Such mechanistic understanding facilitates informed decisions regarding the quantity and timing of feeding starting materials to increase the yield. Furthermore, this approach can serve as a platform method for analyzing the free Mg++ in complex sample matrices where preserving the native-like state of Mg++ binding is key for accurate quantitation.

Improved assay development of pharmaceutical modalities using feedback-controlled liquid chromatography optimization.

Development of meaningful and reliable analytical assays in the (bio)pharmaceutical industry can often be challenging, involving tedious trial and error experimentation. In this work, an automated analytical workflow using an AI-based algorithm for streamlined method development and optimization is presented. Chromatographic methods are developed and optimized from start to finish by a feedback-controlled modeling approach using readily available LC instrumentation and software technologies, bypassing manual user intervention. With the use of such tools, the time requirement of the analyst is drastically minimized in the development of a method. Herein key insights on chromatography system control, automatic optimization of mobile phase conditions, and final separation landscape for challenging multicomponent mixtures are presented (e.g., small molecules drug, peptides, proteins, and vaccine products) showcased by a detailed comparison of a chiral method development process. The work presented here illustrates the power of modern chromatography instrumentation and AI-based software to accelerate the development and deployment of new separation assays across (bio)pharmaceutical modalities while yielding substantial cost-savings, method robustness, and fast analytical turnaround.

Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.

Accelerating Pharmaceutical Process Development with an Acoustic Droplet Ejection-Multiple Reaction Monitoring-Mass Spectrometry Workflow.

Fast-paced pharmaceutical process developments (e.g., high-throughput experimentation, directed evolution, and machine learning) involve the introduction of fast, sensitive, and accurate analytical assays using limited sample volumes. In recent years, acoustic droplet ejection (ADE) coupled with an open port interface has been invented as a sampling technology for mass spectrometry, providing high-throughput nanoliter analytical measurements directly from the standard microplates. Herein, we introduce an ADE-multiple reaction monitoring-mass spectrometry (ADE-MRM-MS) workflow to accelerate pharmaceutical process research and development (PR&D). This systematic workflow outlines the selection of MRM transitions and optimization of assay parameters in a data-driven manner using rapid measurements (1 sample/s). The synergy between ADE sampling and MRM analysis enables analytical assays with excellent sensitivity, selectivity, and speed for PR&D reaction screenings. This workflow was utilized to develop new ADE-MRM-MS assays guiding a variety of industrial processes, including (1) screening of Ni-based catalysts for C-N cross-coupling reaction at 1 Hz and (2) high-throughput regioisomer analysis-enabled enzyme library screening for peptide ligation reaction. ADE-MRM-MS assays were demonstrated to deliver accurate results that are comparable to conventional liquid chromatography (LC) experiments while providing >100-fold throughput enhancement.

An improved workflow for the development of MS-compatible liquid chromatography assay purity and purification methods by using automated LC Screening instrumentation and in silico modeling.

The development of liquid chromatography UV and mass spectrometry (LC-UV-MS) assays in pharmaceutical analysis is pivotal to improve quality control by providing critical information about drug purity, stability, and presence and identity of byproducts and impurities. Analytical method development of these assays is time-consuming, which often causes it to become a bottle neck in drug development and poses a challenge for process chemists to quickly improve the chemistry. In this study, a systematic and efficient workflow was designed to develop purity assay and purification methods for a wide range of compounds including peptides, proteins, and small molecules with MS-compatible mobile phases (MP) by using automated LC screening instrumentation and in silico modeling tools. Initial LC MPs and chromatography column screening experiments enabled quick identification of conditions which provided the best resolution in the vicinity of the target compounds, which is further optimized using computer-assisted modeling (LC Simulator from ACD/Labs). The experimental retention times were in good agreement with the predicted retention times from LC Simulator (ΔtR < 7%). This workflow presents a practical workflow to significantly expedite the time needed to develop optimized LC-UV-MS methods, allowing for a facile, automatic method optimization and reducing the amount of manual work involved in developing new methods during drug development.

Post-ERCP cholecystitis: Incidence, characteristics, and outcomes from a prospective multicenter biliary endoscopy registry.

BACKGROUND AND AIMS: The incidence, risk factors, and outcomes of post-ERCP cholecystitis are poorly described. We aimed to describe cases of post-ERCP cholecystitis from a prospective multicenter registry with protocolized 30-day follow-up. METHODS: Patient- and procedure-related data from 7 centers were obtained. The primary outcome was post-ERCP cholecystitis, defined according to a Delphi-based criteria and causal attribution system. Risk factors and outcomes were described for all cases. RESULTS: Seventeen cases of post-ERCP cholecystitis were identified among 4428 patients with gallbladders undergoing ERCP between 2018 and 2023 (incidence, 0.38%; 95% confidence interval, 0.20-0.57). In ERCPs with covered metal stenting, 7 of 467 resulted in cholecystitis (incidence, 1.50%; 95% confidence interval, 0.40-2.60). Patients had symptoms at a median of 5 days (interquartile range, 5) after ERCP. Management strategies included cholecystectomy, percutaneous cholecystostomy, and endoscopic stent removal/exchange. CONCLUSIONS: Estimates of post-ERCP cholecystitis incidence can inform discussions around procedural risk.

Automated Hydrophobic Interaction Chromatography Screening Combined with In Silico Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

A rare case of a congenital pancreatic duplication cyst in an infant complicated by an upper GI bleed, pancreatitis, cyst infection and gastric outlet obstruction.

Enteric duplication cysts are rare congenital entities most commonly found in the esophagus, ileum or colon but can be in remote locations such as the biliary tree, liver or pancreas. Pancreatic duplication cysts are very uncommon and usually present in adulthood with pancreatitis or abdominal pain. Here, we present a unique and complex case of an infant with a pancreatic duplication cyst initially presenting with an upper gastrointestinal bleed followed by pancreatitis, cyst infection and gastric outlet obstruction.

In Silico Method Development of Achiral and Chiral Tandem Column Reversed-phase Liquid Chromatography for Multicomponent Pharmaceutical Mixtures.

Tandem column liquid chromatography (LC) is a convenient, cost-effective approach to resolve multicomponent mixtures by serially coupling columns on readily available one-dimensional separation systems without specialized user training. Yet, adoption of this technique remains limited, mainly due to the difficulty in identifying optimal selectivity out of many possible tandem column combinations. At this point, method development and optimization require laborious "hit-or-miss" experimentation and "blind" screening when investigating different column selectivity without standard analytes. As a result, many chromatography practitioners end up combining two columns of similar selectivity, limiting the scope and potential of tandem column LC as a mainstay for industrial applications. To circumvent this challenge, we herein introduce a straightforward in silico multifactorial approach as a framework to expediently map the separation landscape across multiple tandem columns (achiral and chiral) and eluent combinations (isocratic and gradient elution) under reversed-phase LC conditions. Retention models were built using commercially available LC simulator software showcasing less than 2% difference between experimental and simulated retention times for analytes of interest in multicomponent pharmaceutical mixtures (e.g., metabolites and cyclic peptides).

In Silico Multifactorial Modeling for Streamlined Development and Optimization of Two-Dimensional Liquid Chromatography.

Continued adoption of two-dimensional liquid chromatography (2D-LC) in industrial laboratories will depend on the development of approaches to make method development for 2D-LC more systematic, less tedious, and less reliant on user expertise. In this paper, we build on previous efforts in these directions by describing the use of multifactorial modeling software that can help streamline and simplify the method development process for 2D-LC. Specifically, we have focused on building retention models for second dimension (2D) separations involving variables including gradient time, temperature, organic modifier blending, and buffer concentration using LC simulator (ACD/Labs) software. Multifactorial retention modeling outcomes are illustrated as resolution map planes or cubes that enable straightforward location of 2D conditions that maximize resolution while minimizing analysis time. We also illustrate the practicality of this approach by identifying conditions that yield baseline separation of all compounds co-eluting from a first dimension (1D) separation using a single combination of 2D stationary phase and elution conditions. The multifactorial retention models were found to be very accurate for both the 1D and 2D separations, with differences between experimental and simulated retention times of less than 0.5%. Pharmaceutical applications of this approach for multiple heartcutting 2D-LC were demonstrated using IEC-IEC or achiral RPLC-chiral RPLC for 2D separations of multicomponent mixtures. The framework outlined here should help make 2D-LC method development more systematic and streamline development and optimization for a variety of 2D-LC applications in both industry and academia.

Vedolizumab and Anti-Tumour Necrosis Factor α Real-World Outcomes in Biologic-Naïve Inflammatory Bowel Disease Patients: Results from the EVOLVE Study.

BACKGROUND AND AIMS: This study aimed to compare real-world clinical effectiveness and safety of vedolizumab, an α4ß7-integrin inhibitor, and anti-tumour necrosis factor-α [anti-TNFα] agents in biologic-naïve ulcerative colitis [UC] and Crohn's disease [CD] patients. METHODS: This was a 24-month retrospective medical chart study in adult UC and CD patients treated with vedolizumab or anti-TNFα in Canada, Greece and the USA. Inverse probability weighting was used to account for differences between groups. Primary outcomes were cumulative rates of clinical effectiveness [clinical response, clinical remission, mucosal healing] and incidence rates of serious adverse events [SAEs] and serious infections [SIs]. Secondary outcomes included cumulative rates of treatment persistence [patients who did not discontinue index treatment during follow-up] and dose escalation and incidence rates of disease exacerbations and disease-related surgeries. Adjusted analyses were performed using inverse probability weighting. RESULTS: A total of 1095 patients [604 UC, 491 CD] were included. By 24 months, rates of clinical effectiveness were similar between groups, but incidence rates of SAEs (hazard ratio [HR] = 0.42 [0.28-0.62]) and SIs (HR = 0.40 [0.19-0.85]) were significantly lower in vedolizumab vs anti-TNFα patients. Rates of treatment persistence [p < 0.01] by 24 months were higher in vedolizumab patients with UC. Incidence rates of disease exacerbations were lower in vedolizumab patients with UC (HR = 0.58 [0.45-0.76]). Other outcomes did not significantly differ between groups. CONCLUSION: In this real-world setting, first-line biologic therapy in biologic-naïve patients with UC and CD demonstrated that vedolizumab and anti-TNFα treatments were equally effective at controlling disease symptoms, but vedolizumab has a more favourable safety profile.

Molecular Nuances Governing the Self-Assembly of 1,3:2,4-Dibenzylidene-d-sorbitol.

1,3:2,4-Dibenzylidene-d-sorbitol (DBS) is the gold-standard for low-molecular-weight organogelators (LMOGs). DBS gels a wide array of solvents, as illustrated by the large Hansen sphere representing gels (2δd = 33.5 MPa1/2, δp = 7.5 MPa1/2, and δh = 8.7 MPa1/2; radius = 11.2 MPa1/2). Derivatives of DBS have been synthesized to isolate and determine molecular features essential for organogelation. In this work, π-π stacking and hydrogen bonding are the major noncovalent interactions examined. The importance of π-π stacking was studied using 1,3:2,4 dicyclohexanecarboxylidene-d-sorbitol (DCHS), which eliminates possible π-π stacking while still conserving the other structural aspects of DBS. The replacement of the benzyl groups with cyclohexyl groups led to a very a poor gelator; only one of the several solvents examined, carbon tetrachloride, formed a gel. 1,3:2,4-Diethylidene-d-sorbitol (DES), another DBS analogue incapable of π-π stacking but with very different polarity, gelated a large Hansen space (2δd = 34.0 MPa1/2, δp = 10.9 MPa1/2, and δh = 10.8 MPa1/2; radius = 9.2 MPa1/2). DES gels solvents with higher δp and δh values than DBS. To assess the role of hydrogen bonding, DBS was acetalated (A-DBS), and it was found that the Hansen space gelated by A-DBS shifted to less polar solvents with higher hydrogen-bonding Hansen solubility parameters (HSPs) (2δd = 33.8 MPa1/2, δp = 6.3 MPa1/2, and δh = 9.6 MPa1/2; radius = 11.1 MPa1/2) than for DBS. These systematic structural modifications are the first step in exploring how specific intermolecular features alter aspects of Hansen space corresponding to positive gelation outcomes.

Genetic requirement for Mycl and efficacy of RNA Pol I inhibition in mouse models of small cell lung cancer.

Small cell lung cancer (SCLC) is a devastating neuroendocrine carcinoma. MYCL (L-Myc) is frequently amplified in human SCLC, but its roles in SCLC progression are poorly understood. We isolated preneoplastic neuroendocrine cells from a mouse model of SCLC and found that ectopic expression of L-Myc, c-Myc, or N-Myc conferred tumor-forming capacity. We focused on L-Myc, which promoted pre-rRNA synthesis and transcriptional programs associated with ribosomal biogenesis. Deletion of Mycl in two genetically engineered models of SCLC resulted in strong suppression of SCLC. The high degree of suppression suggested that L-Myc may constitute a therapeutic target for a broad subset of SCLC. We then used an RNA polymerase I inhibitor to target rRNA synthesis in an autochthonous Rb/p53-deleted mouse SCLC model and found significant tumor inhibition. These data reveal that activation of RNA polymerase I by L-Myc and other MYC family proteins provides an axis of vulnerability for this recalcitrant cancer.

Human secreted tau increases amyloid-beta production.

The interaction of amyloid-beta (Aß) and tau in the pathogenesis of Alzheimer's disease is a subject of intense inquiry, with the bulk of evidence indicating that changes in tau are downstream of Aß. It has been shown however, that human tau overexpression in amyloid precursor protein transgenic mice increases Aß plaque deposition. Here, we confirm that human tau increases Aß levels. To determine if the observed changes in Aß levels were because of intracellular or extracellular secreted tau (eTau for extracellular tau), we affinity purified secreted tau from Alzheimer's disease patient-derived cortical neuron conditioned media and analyzed it by liquid chromatography-mass spectrometry. We found the extracellular species to be composed predominantly of a series of N-terminal fragments of tau, with no evidence of C-terminal tau fragments. We characterized a subset of high affinity tau antibodies, each capable of engaging and neutralizing eTau. We found that neutralizing eTau reduces Aß levels in vitro in primary human cortical neurons where exogenously adding eTau increases Aß levels. In vivo, neutralizing human tau in 2 human tau transgenic models also reduced Aß levels. We show that the human tau insert sequence is sufficient to cause the observed increase in Aß levels. Our data furthermore suggest that neuronal hyperactivity may be the mechanism by which this regulation occurs. We show that neuronal hyperactivity regulates both eTau secretion and Aß production. Electrophysiological analysis shows for the first time that secreted eTau causes neuronal hyperactivity. Its induction of hyperactivity may be the mechanism by which eTau regulates Aß production. Together with previous findings, these data posit a novel connection between tau and Aß, suggesting a dynamic mechanism of positive feed forward regulation. Aß drives the disease pathway through tau, with eTau further increasing Aß levels, perpetuating a destructive cycle.

TNT003, an inhibitor of the serine protease C1s, prevents complement activation induced by cold agglutinins.

Activation of the classical pathway (CP) of complement is often associated with autoimmune disorders in which disease pathology is linked to the presence of an autoantibody. One such disorder is cold agglutinin disease (CAD), an autoimmune hemolytic anemia in which autoantibodies (cold agglutinins) bind to red blood cells (RBCs) at low temperatures. Anemia occurs as a result of autoantibody-mediated CP activation on the surface of the erythrocyte, leading to the deposition of complement opsonins that drive extravascular hemolysis in the liver. Here we test the effects of TNT003, a mouse monoclonal antibody targeting the CP-specific serine protease C1s, on CP activity induced by cold agglutinins on human RBCs. We collected 40 individual CAD patient samples and showed that TNT003 prevented cold agglutinin-mediated deposition of complement opsonins that promote phagocytosis of RBCs. Furthermore, we show that by preventing CP activation, TNT003 also prevents cold agglutinin-driven generation of anaphylatoxins. Finally, we provide evidence that CP activity in CAD patients terminates prior to activation of the terminal cascade, supporting the hypothesis that the primary route of RBC destruction in these patients occurs via extravascular hemolysis. Our results support the development of a CP inhibitor for the treatment of CAD.