Expediting online liquid chromatography for real-time monitoring of product attributes to advance process analytical technology in downstream processing of biopharmaceuticals.

The application of Process Analytical Technology (PAT) principles for manufacturing of biotherapeutics proffers the prospect of ensuring consistent product quality along with increased productivity as well as substantial cost and time savings. Although this paradigm shift from a traditional, rather rigid manufacturing model to a more scientific, risk-based approach has been advocated by health authorities for almost two decades, the practical implementation of PAT in the biopharmaceutical industry is still limited by the lack of fit-for-purpose analytical methods. In this regard, most of the proposed spectroscopic techniques are sufficiently fast but exhibit deficiencies in terms of selectivity and sensitivity, while well-established offline methods, such as (ultra-)high-performance liquid chromatography, are generally considered as too slow for this task. To address these reservations, we introduce here a novel online Liquid Chromatography (LC) setup that was specifically designed to enable real-time monitoring of critical product quality attributes during time-sensitive purification operations in downstream processing. Using this online LC solution in combination with fast, purpose-built analytical methods, sampling cycle times between 1.30 and 2.35 min were achieved, without compromising on the ability to resolve and quantify the product variants of interest. The capabilities of our approach are ultimately assessed in three case studies, involving various biotherapeutic modalities, downstream processes and analytical chromatographic separation modes. Altogether, our results highlight the expansive opportunities of online LC based applications to serve as a PAT tool for biopharmaceutical manufacturing.

Clinical performance and risk factors of all-ceramic screw-retained implant crowns in the posterior region based on a retrospective investigation.

OBJECTIVES: Clinical data on all-ceramic screw-retained implant crowns (SICs) luted on titanium base abutments (TBAs) over more than 3 years are sparse. This study aimed to evaluate the clinical performance and potential risk factors for these restorations. MATERIALS AND METHODS: Analysis took place based on the medical patient-records of three dental offices. Implant survival and prosthetic complications over time were evaluated. The study included SICs in premolar and molar regions made from monolithic lithium disilicate ceramic (M_LiDi) or veneered zirconia (V_ZiO) luted on a TBA documented over an observation time of at least 3 years. Survival and complication rates were calculated and compared by a log-rank test. Cox-Regressions were used to check potential predictors for the survival (p < .05). RESULTS: Six hundred and one crowns out of 371 patients met the inclusion criteria and follow-up period was between 3.0 and 12.9 (mean: 6.4 (SD: 2.1)) years. Over time, six implants had to be removed and 16 restorations had to be refabricated. The estimated survival rates over 10 years were 93.5% for M_LiDi and 95.9% for V_ZiO and did not differ significantly among each other (p = .80). However, V_ZiO showed significantly higher complication rates (p = .003). Material selection, sex, age, and implant diameter did not affect the survival of investigated SICs but crown height influences significantly the survival rate (hazard ratio, HR = 1.26 (95%CI: 1.08, 1.49); p = .043). CONCLUSIONS: Screw-retained SICs luted on TBAs that were fabricated from monolithic lithium disilicate ceramic or veneered zirconia showed reliable and similar survival rates. Increasing crown heights reduced survival over the years.

A contemporary training concept in critical care cardiology.

Critical care cardiology (CCC) in the modern era is shaped by a multitude of innovative treatment options and an increasingly complex, ageing patient population. Generating high-quality evidence for novel interventions and devices in an intensive care setting is exceptionally challenging. As a result, formulating the best possible therapeutic approach continues to rely predominantly on expert opinion and local standard operating procedures. Fostering the full potential of CCC and the maturation of the next generation of decision-makers in this field calls for an updated training concept, that encompasses the extensive knowledge and skills required to care for critically ill cardiac patients while remaining adaptable to the trainee's individual career planning and existing educational programs. In the present manuscript, we suggest a standardized training phase in preparation of the first ICU rotation, propose a modular CCC core curriculum, and outline how training components could be conceptualized within three sub-specialization tracks for aspiring cardiac intensivists.

Monolithic hybrid abutment crowns: Influence of crown height, crown morphology and material on the implant-abutment complex.

PURPOSE: This in vitro study investigated the influence of material selection, crown morphology, and vertical crown height on the biomechanical behavior of monolithic hybrid abutment crowns (HACs). METHODS: Ninety implants were embedded in accordance with ISO standard 14801; ninety HACs were mounted (N=90). Monolithic crowns with varying group-specific designs were luted using titanium bases. HACs were fabricated from monolithic lithium disilicate ceramic (LD) or zirconia-reinforced lithium silicate ceramic (ZLS). The crown morphology was either maxillary premolar (LD_PM, ZLS_PM) or molar (LD_MO). The three groups were further divided into three subgroups of ten specimens, each designed with a small (7.5 mm), middle (10.5 mm), and high (13.5 mm) configuration of crown heights (N=10). A load-to-failure test at 30° off-axis was conducted using a universal testing machine until failure. For statistical analysis, Kolmogorov-Smirnov and Mann-Whitney U tests were conducted (P < 0.05). RESULTS: All LD_MO groups presented the highest failure values (808.7 to 947.9 N), followed by the LD_PM (525.8 to 722.8 N) and ZLS_PM groups (312.6 to 478.8N). A comparison between LD and ZLS materials (P < 0.001) as well as the crown morphology (P < 0.001) showed significant differences in failure values. The values in the subgroups of ZLS_PM (low, middle, high) decreased as the crown height increased. The fracture modes showed no consistent patterns across the test groups. CONCLUSIONS: Material selection, crown morphology, and vertical crown height appear to be important factors that may influence the clinical failure values and patterns of HACs.

Sex-related differences in patients presenting with heart failure-related cardiogenic shock.

BACKGROUND: Heart failure-related cardiogenic shock (HF-CS) accounts for a significant proportion of all CS cases. Nevertheless, there is a lack of evidence on sex-related differences in HF-CS, especially regarding use of treatment and mortality risk in women vs. men. This study aimed to investigate potential differences in clinical presentation, use of treatments, and mortality between women and men with HF-CS. METHODS: In this international observational study, patients with HF-CS (without acute myocardial infarction) from 16 tertiary-care centers in five countries were enrolled between 2010 and 2021. Logistic and Cox regression models were used to assess differences in clinical presentation, use of treatments, and 30-day mortality in women vs. men with HF-CS. RESULTS: N = 1030 patients with HF-CS were analyzed, of whom 290 (28.2%) were women. Compared to men, women were more likely to be older, less likely to have a known history of heart failure or cardiovascular risk factors, and lower rates of highly depressed left ventricular ejection fraction and renal dysfunction. Nevertheless, CS severity as well as use of treatments were comparable, and female sex was not independently associated with 30-day mortality (53.0% vs. 50.8%; adjusted HR 0.94, 95% CI 0.75-1.19). CONCLUSIONS: In this large HF-CS registry, sex disparities in risk factors and clinical presentation were observed. Despite these differences, the use of treatments was comparable, and both sexes exhibited similarly high mortality rates. Further research is necessary to evaluate if sex-tailored treatment, accounting for the differences in cardiovascular risk factors and clinical presentation, might improve outcomes in HF-CS.

Clinical performance of monolithic lithium disilicate hybrid abutment crowns over at least 3.5 years.

PURPOSE: Hybrid abutment crowns (HACs) made from monolithic ceramics represent an efficient option for single restorations on implants. However, long-term data are scarce. The purpose of this clinical trial was to evaluate the survival and complication rates of CAD-CAM fabricated HACs over a time period of at least 3.5 years. MATERIALS AND METHODS: Twenty-five patients with a total of 40 HACs made of monolithic lithium disilicate ceramic bonded to a titanium base CAD-CAM abutment were retrospectively evaluated. All implants and screw-retained restorations were placed and manufactured in the same department of a university hospital. Only crowns that had been in service for more than 3.5 years were included in the study. HACs were evaluated regarding technical and biological complications. Functional Implant Prosthodontic Scores (FIPS) were obtained. RESULTS: The mean observation time was 5.9 ± 1.4 years. Implant survival was 100%, and HAC survival was 97.5%. Over the observation period, one crown fracture was observed, necessitating refabricating of the restoration. Three minor biological complications were found. The overall mean FIPS score was 8.69 ± 1.12 points. CONCLUSIONS: Within the limitations of this study, monolithic screw-retained HACs milled from lithium disilicate ceramics and bonded to titanium bases appeared to be a reliable treatment option over more than 3.5 years due to their low biological and technical complication rates.

Clinical presentation, shock severity and mortality in patients with de novo versus acute-on-chronic heart failure-related cardiogenic shock.

AIMS: Heart failure-related cardiogenic shock (HF-CS) accounts for a significant proportion of CS cases. Whether patients with de novo HF and those with acute-on-chronic HF in CS differ in clinical characteristics and outcome remains unclear. The aim of this study was to evaluate differences in clinical presentation and mortality between patients with de novo and acute-on-chronic HF-CS. METHODS AND RESULTS: In this international observational study, patients with HF-CS from 16 tertiary care centres in five countries were enrolled between 2010 and 2021. To investigate differences in clinical presentation and 30-day mortality, adjusted logistic/Cox regression models were fitted. Patients (n = 1030) with HF-CS were analysed, of whom 486 (47.2%) presented with de novo HF-CS and 544 (52.8%) with acute-on-chronic HF-CS. Traditional markers of CS severity (e.g. blood pressure, heart rate and lactate) as well as use of treatments were comparable between groups. However, patients with acute-on-chronic HF-CS were more likely to have a higher CS severity and also a higher mortality risk, after adjusting for relevant confounders (de novo HF 45.5%, acute-on-chronic HF 55.9%, adjusted hazard ratio 1.38, 95% confidence interval 1.10-1.72, p = 0.005). CONCLUSION: In this large HF-CS cohort, acute-on-chronic HF-CS was associated with more severe CS and higher mortality risk compared to de novo HF-CS, although traditional markers of CS severity and use of treatments were comparable. These findings highlight the vast heterogeneity of patients with HF-CS, emphasize that HF chronicity is a relevant disease modifier in CS, and indicate that future clinical trials should account for this.

Complications in patients with cardiogenic shock on veno-arterial extracorporeal membrane oxygenation therapy: distribution and relevance. Results from an international, multicentre cohort study.

AIMS: Veno-arterial extracorporeal membrane oxygenation therapy (VA-ECMO) restores circulation and tissue oxygenation in cardiogenic shock (CS) patients, but can also lead to complications. This study aimed to quantify VA-ECMO complications and analyse their association with overall survival as well as favourable neurological outcome (cerebral performance categories 1 + 2). METHODS AND RESULTS: All-comer patients with CS treated with VA-ECMO were retrospectively enrolled from 16 centres in four countries (2005-2019). Neurological, bleeding, and ischaemic adverse events (AEs) were considered. From these, typical VA-ECMO complications were identified and analysed separately as device-related complications. n = 501. Overall, 118 were women (24%), median age was 56.0 years, median lactate was 8.1 mmol/L. Acute myocardial infarction caused CS in 289 patients (58%). Thirty-days mortality was 40% (198/501 patients). At least one device-related complication occurred in 252/486 (52%) patients, neurological AEs in 108/469 (23%), bleeding in 192/480 (40%), ischaemic AEs in 123/478 (26%). The 22% of patients with the most AEs accounted for 50% of all AEs. All types of AEs were associated with a worse prognosis. Aside from neurological ones, all AEs and device-related complications were more likely to occur in women; although prediction of AEs outside of neurological AEs was generally poor. CONCLUSION: Therapy and device-related complications occur in half of all patients treated with VA-ECMO and are associated with a worse prognosis. They accumulate in some patients, especially in women. Aside from neurological events, identification of patients at risk is difficult, highlighting the need to establish additional quantitative markers of complication risk to guide VA-ECMO treatment in CS.

Association between left ventricular ejection fraction, mortality and use of mechanical circulatory support in patients with non-ischaemic cardiogenic shock.

BACKGROUND: Currently, use of mechanical circulatory support (MCS) in non-ischaemic cardiogenic shock (CS) is predominantly guided by shock-specific markers, and not by markers of cardiac function. We hypothesise that left ventricular ejection fraction (LVEF) can identify patients with a higher likelihood to benefit from MCS and thus help to optimise their expected benefit. METHODS: Patients with non-ischaemic CS and available data on LVEF from 16 tertiary-care centres in five countries were analysed. Cox regression models were fitted to evaluate the association between LVEF and mortality, as well as the interaction between LVEF, MCS use and mortality. RESULTS: N = 807 patients were analysed: mean age 63 [interquartile range (IQR) 51.5-72.0] years, 601 (74.5%) male, lactate 4.9 (IQR 2.6-8.5) mmol/l, LVEF 20 (IQR 15-30) %. Lower LVEF was more frequent amongst patients with more severe CS, and MCS was more likely used in patients with lower LVEF. There was no association between LVEF and 30-day mortality risk in the overall study cohort. However, there was a significant interaction between MCS use and LVEF, indicating a lower 30-day mortality risk with MCS use in patients with LVEF ≤ 20% (hazard ratio 0.72, 95% confidence interval 0.51-1.02 for LVEF ≤ 20% vs. hazard ratio 1.31, 95% confidence interval 0.85-2.01 for LVEF > 20%, interaction-p = 0.017). CONCLUSION: This retrospective study may indicate a lower mortality risk with MCS use only in patients with severely reduced LVEF. This may propose the inclusion of LVEF as an adjunctive parameter for MCS decision-making in non-ischaemic CS, aiming to optimise the benefit-risk ratio.

Enzymatic degradation pattern of polysorbate 20 impacts interfacial properties of monoclonal antibody formulations.

Polysorbate 20 (PS20) is widely used to maintain protein stability in biopharmaceutical formulations. However, PS20 is susceptible to hydrolytic degradation catalyzed by trace amounts of residual host cell proteins present in monoclonal antibody (mAb) formulations. The resulting loss of intact surfactant and the presence of PS20 degradation products, such as free fatty acids (FFAs), may impair protein stability. In this study, two hydrolytically-active immobilized lipases, which primarily targeted either monoester or higher-order ester species in PS20, were used to generate partially-degraded PS20. The impact of PS20 degradation pattern on critical micelle concentration (CMC), surface tension, interfacial rheology parameters and agitation protection was assessed. CMC was slightly increased upon monoester degradation, but significantly increased upon higher-order ester degradation. The PS20 degradation pattern also significantly impacted the dynamic surface tension of a mAb formulation, whereas changes in the equilibrium surface tension were mainly caused by the adsorption of FFAs onto the air-water interface. In an agitation protection study, monoester degradation resulted in the formation of soluble mAb aggregates and proteinaceous particles, suggesting that preferential degradation of PS20 monoester species can significantly impair mAb stability. Additional mAbs should be tested in the future to assess the impact of the protein format.

Ubiquitin: Characterization of a Host Cell Protein Covalently Attached to a Monoclonal Antibody Product by LC-MS/MS.

Host cell protein (HCP) characterization is a crucial quality parameter for biotherapeutic drug safety and stability. With a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified ubiquitin in ultrafiltration/diafiltration (UF/DF) pools of one of our monoclonal antibody (mAb) products. Since ubiquitin occurs physiologically as a post-translational modification (PTM) involved in many cellular functions, we suspected the possibility that if identified as an HCP, it may occur as a covalent modification on the mAb. In fact, in this study we characterized and quantified the ubiquitin modification on the Fc domain of mAbX by data dependent acquisition (DDA) and data independent acquisition (DIA) - MS workflows. Covalent binding and site localization were confirmed by identifying a characteristic diglycine motif on the modified peptide. Initially observed reduced detectability of ubiquitin in samples prepared with native digestion was attributed to impaired digestion and subsequent removal along with the mAb in the precipitation step. Our work has contributed to a better understanding of ubiquitin as an HCP considering its specific features such as occurrence in different topologies and provided insight into how covalent binding to a drug product can affect its identification by MS when native digestion conditions are used.

Fast HPLC-based affinity method to determine capsid titer and full/empty ratio of adeno-associated viral vectors.

Recombinant adeno-associated viruses (rAAVs) are promising gene delivery vectors in the emerging field of in vivo gene therapies. To ensure their consistent quality during manufacturing and process development, multiple analytical techniques have been proposed for the characterization and quantification of rAAV capsids. Despite their indisputable capabilities for performing this task, current analytical methods are rather time-consuming, material intensive, complicated, and costly, restricting their suitability for process development in which time and sample throughput are severe constraints. To eliminate this bottleneck, we introduce here an affinity-based high-performance liquid chromatography method that allows the determination of the capsid titer and the full/empty ratio of rAAVs within less than 5 min. By packing the commercially available AAVX affinity resin into small analytical columns, the rAAV fraction of diverse serotypes can be isolated from process-related impurities and analyzed by UV and fluorescence detection. As demonstrated by both method qualification data and side-by-side comparison with AAV enzyme-linked immunosorbent assay results for rAAV8 samples as well as by experiments using additional rAAV2, rAAV8, and rAAV9 constructs, our approach showed good performance, indicating its potential as a fast, simple and efficient tool for supporting the development of rAAV gene therapies.

In Vitro Survival and Fracture Force of Lithium-Disilicate Hybrid Abutment Crowns.

PURPOSE: To evaluate the behavior of hybrid abutment crowns fabricated from monolithic lithium disilicate ceramic (LDC) and to compare the influence of different in-vitro artificial aging protocols. MATERIAL AND METHODS: 32 monolithic hybrid abutment crowns of monolithic LDC were fabricated. 24 were artificially aged applying 3 different protocols up to a 20 year-simulation (1.2 × 106, 2.4 × 106, 4.8 × 106 chewing cycles, thermocycling), one control group underwent no artificial aging (N=32, n=8). Load-to-failure tests were conducted for all specimens and failure values were compared (p<0.05). RESULTS/CONCLUSIONS: All specimens passed in-vitro aging. Mean failure load values between 532.6 and 562.8 N were found but did neither differ significantly among the test groups nor from the control group. Within the limitations of this in-vitro pilot study, hybrid abutment crowns manufactured from monolithic LDC seem to offer appropriate long-lasting mechanical stability over a simulation period up to 20 years. The failure values and complication pattern seem to be independent of several aging protocols in this test set-up.

Biomechanical behavior of implants with different diameters in relation to simulated bone loss- an in vitro study.

OBJECTIVES: Bone resorption around implants could influence the resistance of the implant abutment complex (IAC). The present in vitro study aimed to assess the stability to static fatigue of implants presenting different levels of bone losses and diameters. MATERIALS AND METHODS: Ninety implants with an internal conical connection with 3 different implant diameters (3.3 mm (I33), 3.8 mm (I38), and 4.3 mm (I43)) and 3 simulated bone loss settings (1.5 mm (I_15), 3.0 mm (I_30), and 4.5 mm (I_45) (n = 10)) were embedded and standard abutments were mounted. All specimens were artificially aged (1,200,000 cycles, 50 N, simultaneous thermocycling) and underwent subsequently load-to-fracture test. For statistical analysis, Kolmogorov-Smirnov test, Kruskal-Wallis test, and Mann-Whitney U test (p < 0.05) were applied. RESULTS: All test specimens withstood the artificial aging without damage. The mean failure values were 382.1 (± 59.2) N (I3315), 347.0 (± 35.7) N (I3330), 315.9 N (± 30.9) (I3345), 531.4 (± 36.2) N (I3815), 514.5 (± 40.8) N (I3830), 477.9 (± 26.3) N (I3845), 710.1 (± 38.2) N (I4315), 697.9 (± 65.2) N (I4330), and 662.2 N (± 45.9) (I4345). The stability of the IACs decreased in all groups when bone loss inclined. Merely, the failure load values did not significantly differ among subgroups of I43. CONCLUSIONS: Larger implant diameters and minor circular bone loss around the implant lead to a higher stability of the IAC. The smaller the implant diameter was, the more the stability was affected by the circumferential bone level. CLINICAL RELEVANCE: Preserving crestal bone level is important to ensure biomechanical sustainability at implant systems with a conical interface. It seems sensible to take the effect of eventual bone loss around implants into account during implant planning processes and restorative considerations.

Extracorporeal Life Support in Infarct-Related Cardiogenic Shock.

BACKGROUND: Extracorporeal life support (ECLS) is increasingly used in the treatment of infarct-related cardiogenic shock despite a lack of evidence regarding its effect on mortality. METHODS: In this multicenter trial, patients with acute myocardial infarction complicated by cardiogenic shock for whom early revascularization was planned were randomly assigned to receive early ECLS plus usual medical treatment (ECLS group) or usual medical treatment alone (control group). The primary outcome was death from any cause at 30 days. Safety outcomes included bleeding, stroke, and peripheral vascular complications warranting interventional or surgical therapy. RESULTS: A total of 420 patients underwent randomization, and 417 patients were included in final analyses. At 30 days, death from any cause had occurred in 100 of 209 patients (47.8%) in the ECLS group and in 102 of 208 patients (49.0%) in the control group (relative risk, 0.98; 95% confidence interval [CI], 0.80 to 1.19; P = 0.81). The median duration of mechanical ventilation was 7 days (interquartile range, 4 to 12) in the ECLS group and 5 days (interquartile range, 3 to 9) in the control group (median difference, 1 day; 95% CI, 0 to 2). The safety outcome consisting of moderate or severe bleeding occurred in 23.4% of the patients in the ECLS group and in 9.6% of those in the control group (relative risk, 2.44; 95% CI, 1.50 to 3.95); peripheral vascular complications warranting intervention occurred in 11.0% and 3.8%, respectively (relative risk, 2.86; 95% CI, 1.31 to 6.25). CONCLUSIONS: In patients with acute myocardial infarction complicated by cardiogenic shock with planned early revascularization, the risk of death from any cause at the 30-day follow-up was not lower among the patients who received ECLS therapy than among those who received medical therapy alone. (Funded by the Else Kröner Fresenius Foundation and others; ECLS-SHOCK ClinicalTrials.gov number, NCT03637205.).

Coronary Angiography After Out-of-Hospital Cardiac Arrest Without ST-Segment Elevation: One-Year Outcomes of a Randomized Clinical Trial.

Importance: Myocardial infarction is a frequent cause of out-of-hospital cardiac arrest (OHCA). The long-term effect of early coronary angiography on patients with OHCA with possible coronary trigger but no ST-segment elevation remains unclear. Objective: To compare the clinical outcomes of early unselective angiography with the clinical outcomes of a delayed or selective approach for successfully resuscitated patients with OHCA of presumed cardiac origin without ST-segment elevation at 1-year follow-up. Design, Setting, and Participants: The TOMAHAWK trial was a multicenter, international (Germany and Denmark), investigator-initiated, open-label, randomized clinical trial enrolling 554 patients between November 23, 2016, to September 20, 2019. Patients with stable return of spontaneous circulation after OHCA of presumed cardiac origin but without ST-segment elevation on the postresuscitation electrocardiogram were eligible for inclusion. A total of 554 patients were randomized to either immediate coronary angiography after hospital admission or an initial intensive care assessment with delayed or selective angiography after a minimum of 24 hours. All 554 patients were included in survival analyses during the follow-up period of 1 year. Secondary clinical outcomes were assessed only for participants alive at 1 year to account for the competing risk of death. Interventions: Early vs delayed or selective coronary angiography and revascularization if indicated. Main Outcomes and Measures: Evaluations in this secondary analysis included all-cause mortality after 1 year, as well as severe neurologic deficit, myocardial infarction, and rehospitalization for congestive heart failure in survivors at 1 year. Results: A total of 281 patients were randomized to the immediate angiography group and 273 to the delayed or selective group, with a median age of 70 years (IQR, 60-78 years). A total of 369 of 530 patients (69.6%) were male, and 268 of 483 patients (55.5%) had a shockable arrest rhythm. At 1 year, all-cause mortality was 60.8% (161 of 265) in the immediate angiography group and 54.3% (144 of 265) in the delayed or selective angiography group without significant difference between the treatment strategies, trending toward an increase in mortality with immediate angiography (hazard ratio, 1.25; 95% CI, 0.99-1.57; P = .05). For patients surviving until 1 year, the rates of severe neurologic deficit, myocardial infarction, and rehospitalization for congestive heart failure were similar between the groups. Conclusions and Relevance: This study found that a strategy of immediate coronary angiography does not provide clinical benefit compared with a delayed or selective invasive approach for patients 1 year after resuscitated OHCA of presumed coronary cause and without ST-segment elevation. Trial Registration: ClinicalTrials.gov Identifier: NCT02750462.

Towards a Precise NMR Quantification of Acute Phase Inflammation Proteins from Human Serum.

Nuclear Magnetic Resonance (NMR) spectra of human serum and plasma show, besides metabolites and lipoproteins, two characteristic signals termed GlycA and B arising from the acetyl groups of glycoprotein glycans from acute phase proteins, which constitute good markers for inflammatory processes. Here, we report a comprehensive assignment of glycoprotein glycan NMR signals observed in human serum, showing that GlycA and GlycB signals originate from Neu5Ac and GlcNAc moieties from N-glycans, respectively. Diffusion-edited NMR experiments demonstrate that signal components can be associated with specific acute phase proteins. Conventionally determined concentrations of acute phase glycoproteins correlate well with distinct features in NMR spectra (R2 up to 0.9422, p-value <0.001), allowing the simultaneous quantification of several acute phase inflammation proteins. Overall, a proteo-metabolomics NMR signature of significant diagnostic potential is obtained within 10-20 min acquisition time. This is exemplified in serum samples from COVID-19 and cardiogenic shock patients showing significant changes in several acute phase proteins compared to healthy controls.

A fast and sensitive high-throughput assay to assess polysorbate-degrading hydrolytic activity in biopharmaceuticals.

Hydrolysis of polysorbate in biopharmaceutical products has been ascribed to the enzymatic activity from trace levels of residual host cell proteins. In recent years, significant efforts to identify the causative enzymes typically used elaborate, material-intensive and time-consuming approaches. Therefore, the lack of fast and sensitive assays to monitor their activity remains a major bottleneck for supporting process optimization and troubleshooting activities where time and sample throughput are crucial constraints. To address this bottleneck, we developed a novel Electrochemiluminescence-based Polysorbase Activity (EPA) assay to measure hydrolytic activities in biotherapeutics throughout the drug substance manufacturing process. By combining the favorable features of an in-house designed surrogate substrate with a well-established detection platform, the method yields fast (∼36 h turnaround time) and highly sensitive readouts compatible with high-throughput testing. The assay capability for detecting substrate conversion in a precise and reliable manner was demonstrated by extensive qualification studies and by employing a number of recombinant hydrolases associated with polysorbate hydrolysis. In addition, high assay sensitivity and wide applicability were confirmed for in-process pool samples of three different antibody products by performing a head-to-head comparison between this method and an established liquid chromatography - mass spectrometry based assay for the quantification of free fatty acids. Overall, our results suggest that this new approach is well-suited to resolve differences in hydrolytic activity through all stages of purification.

Forced surgical extrusion using an axial tooth extraction system as a practicable technique for preserving severely destroyed teeth? - Clinical outcomes up to 4.8 years.

OBJECTIVE: Several extrusion techniques have been described to restore teeth with insufficient coronal tooth structure and to avoid their extraction. Still, there is little evidence for a treatment concept combining surgical extrusion using an atraumatic axial extraction system. MATERIALS AND METHODS: A total of nine patients, each with an iso- or subgingival fractured tooth, were retrospectively examined. Treatment of the damaged tooth comprised an atraumatic forced surgical extrusion performed with an axial tooth extraction system and a more coronal positioning within the socket. The teeth were initially splinted and subsequently restored. The follow-up period was up to 57.1 months and averaged 36.5 (SD: ±13.5) months. RESULTS: All nine teeth were still in situ, without signs of inflammation. During the period of the provisional restoration, six prosthetic complications occurred, which were resolved with little effort, whereas, success rate for the definitive restoration was 100%. No biological complications were observed concerning the root apex or soft tissue. The radiographically measured mean extrusion distance was 3.4 (SD: ±1.0) mm, so that a sufficient prosthetic ferrule could be reestablished. CONCLUSIONS: Surgical extrusions using an axial tooth extraction system demonstrate low biological and prosthetic complications rates over observation time. CLINICAL SIGNIFICANCE: The presented extrusion approach preserves soft and hard tissue and is an efficient treatment option for severely destroyed teeth. Saving hopeless teeth by this relatively predictable and feasible procedure has hardly any disadvantages for patients, and in case of failure, an implant or fixed partial denture are still an option.

The Fracture Load as a Function of the Material Thickness: The Key to Computing the Strength of Monolithic All-Ceramic Materials?

The thickness of a material has a significant impact on its fracture load. The aim of the study was to find and describe a mathematical relationship between the material thickness and the fracture load for dental all-ceramics. In total, 180 specimens were prepared from a leucite silicate ceramic (ESS), a lithium disilicate ceramic (EMX), and a 3Y-TZP zirconia ceramic (LP) in five thicknesses (0.4, 0.7, 1.0, 1.3, and 1.6 mm; n = 12). The fracture load of all specimens was determined using the biaxial bending test according to the DIN EN ISO 6872. The regression analyses for the linear, quadratic, and cubic curve characteristics of the materials were conducted, and the cubic regression curves showed the best correlation (coefficients of determination (R2): ESS R2 = 0.974, EMX R2 = 0.947, LP R2 = 0.969) for the fracture load values as a function of the material thickness. A cubic relationship could be described for the materials investigated. Applying the cubic function and material-specific fracture-load coefficients, the respective fracture load values can be calculated for the individual material thicknesses. These results help to improve and objectify the estimation of the fracture loads of restorations, to enable a more patient- and indication-centered situation-dependent material choice.