Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model.

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Mechanistic modeling explains the production dynamics of recombinant adeno-associated virus with the baculovirus expression vector system.

Current manufacturing processes for recombinant adeno-associated viruses (rAAVs) have less-than-desired yields and produce significant amounts of empty capsids. The increasing demand and the high cost of goods for rAAV-based gene therapies motivate development of more efficient manufacturing processes. Recently, the US Food and Drug Administration (FDA) approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS), a technology that demonstrated production of high titers of full capsids. This work presents a first mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV maturation in the BEVS. The model predictions are successfully validated for in-house and literature experimental measurements of the vector genome and of structural and non-structural proteins collected during rAAV manufacturing in the BEVS with the TwoBac and ThreeBac constructs. A model-based analysis of the process is carried out to identify the bottlenecks that limit full capsid formation. Vector genome amplification is found to be the limiting step for rAAV production in Sf9 cells using either the TwoBac or ThreeBac system. In turn, vector genome amplification is hindered by limiting Rep78 levels. Transgene and non-essential baculovirus protein expression in the insect cell during rAAV manufacturing also negatively influences the rAAV production yields.

Hemodynamic nature of black-blood enhancement in long-term coiled cerebral aneurysms.

PURPOSE: Vessel wall imaging (VWI) with black-blood (BB) technique can demonstrate aneurysmal enhancement preluding to growth/rupture in treatment-naive cerebral aneurysms. Interestingly, recent works showed that BB enhancement may also occur in endovascularly treated aneurysms, though its meaning is controversial. Hypothesizing a flow-related mechanism of BB enhancement, we explored its relationship with incomplete occlusion status and coil packing density at DSA. METHODS: We analyzed the subjects undergoing 3T MRI between January 2017 and October 2020 for a previous aneurysmal coiling. All the MRI studies included pre- and post-contrast 3D BB sequences. The presence of intra-aneurysmal pre-contrast BB signal was assessed. BB enhancement (when present) was classified as follows: (1) enhancement at the neck, (2) intrasaccular/intra-coil enhancement, and (3) peripheral enhancement. Coil packing density and aneurysmal occlusion status (according to the modified Raymond-Roy classification, MRRC) were determined on post-treatment DSA and compared with BB findings using generalized linear mixed-effect model and ANOVA. Significant p values were <0.05. RESULTS: Forty-eight aneurysms from 44 patients were eligible for analysis. Pre-contrast BB signal was observed in 50% of the aneurysms and showed a relationship with baseline aneurysmal size. BB enhancement was detectable in 31 aneurysms (65%), being significantly associated with incomplete aneurysmal occlusion and reduced coil packing density at DSA. CONCLUSION: BB enhancement of coiled aneurysms is related with increasing degrees of post-coiling aneurysmal remnants and with loose coil packing density at DSA. This supports a hemodynamic interpretation of BB enhancement in long-term coiled aneurysms.

Quality-by-control of intensified continuous filtration-drying of active pharmaceutical ingredients.

Continuous manufacturing and closed-loop quality control are emerging technologies that are pivotal for next-generation pharmaceutical modernization. We develop a process control framework for a continuous carousel for integrated filtration-drying of crystallization slurries. The proposed control system includes model-based monitoring and control routines, such as state estimation and real-time optimization, implemented in a hierarchical, three-layer quality-by-control (QbC) framework. We implement the control system in ContCarSim, a publicly available carousel simulator. We benchmark the proposed control system against simpler methods, comprising a reduced subset of the elements of the overall control system, and against open-loop operation (the current standard in pharmaceutical manufacturing). The proposed control system demonstrates superior performance in terms of higher consistency in product quality and increased productivity, proving the benefits of closed-loop control and of model-based techniques in pharmaceutical manufacturing. This study represents a step forward toward end-to-end continuous pharmaceutical processing, and in the evolution of quality-by-design toward quality-by-control.

A review on the modernization of pharmaceutical development and manufacturing - Trends, perspectives, and the role of mathematical modeling.

Recently, the pharmaceutical industry has been facing several challenges associated to the use of outdated development and manufacturing technologies. The return on investment on research and development has been shrinking, and, at the same time, an alarming number of shortages and recalls for quality concerns has been registered. The pharmaceutical industry has been responding to these issues through a technological modernization of development and manufacturing, under the support of initiatives and activities such as quality-by-design (QbD), process analytical technology, and pharmaceutical emerging technology. In this review, we analyze this modernization trend, with emphasis on the role that mathematical modeling plays within it. We begin by outlining the main socio-economic trends of the pharmaceutical industry, and by highlighting the life-cycle stages of a pharmaceutical product in which technological modernization can help both achieve consistently high product quality and increase return on investment. Then, we review the historical evolution of the pharmaceutical regulatory framework, and we discuss the current state of implementation and future trends of QbD. The pharmaceutical emerging technology is reviewed afterwards, and a discussion on the evolution of QbD into the more effective quality-by-control (QbC) paradigm is presented. Further, we illustrate how mathematical modeling can support the implementation of QbD and QbC across all stages of the pharmaceutical life-cycle. In this respect, we review academic and industrial applications demonstrating the impact of mathematical modeling on three key activities within pharmaceutical development and manufacturing, namely design space description, process monitoring, and active process control. Finally, we discuss some future research opportunities on the use of mathematical modeling in industrial pharmaceutical environments.

A benchmark simulator for quality-by-design and quality-by-control studies in continuous pharmaceutical manufacturing - Intensified filtration-drying of crystallization slurries.

This article introduces ContCarSim, a benchmark simulator for the development and testing of quality-by-design and quality-by-control strategies in the continuous intensified filtration-drying of paracetamol/ethanol slurries on a novel carousel technology, developed by Alconbury Weston Ltd (United Kingdom). The simulator is based on a detailed mechanistic mathematical modeling framework, and has been validated with filtration and drying experiments on a prototype equipment. A set of design- and control-relevant challenges to be addressed through ContCarSim are proposed. A case study is developed, to demonstrate the features of the simulator and its suitability to design, test and optimize the unit operation. ContCarSim is expected to promote the transition to end-to-end continuous pharmaceutical manufacturing and the adoption of closed-loop quality control by the pharmaceutical industry. The simulator can also be employed as a benchmark for data analytics and process monitoring studies.

Powder composition monitoring in continuous pharmaceutical solid-dosage form manufacturing using state estimation - Proof of concept.

In continuous solid-dosage form manufacturing, the powder feeding system is responsible for supplying downstream the correct formulation of the drug product ingredients. The composition of the powder delivered by the feeding system is inferred from the measurements of powder mass flow from the system feeders. The mass flows are, in turn, inferred from the loss in weight measured in the feeder hoppers. Most loss-in-weight feeders post-process the mass flow signal to deliver a smoothed value to the user. However, such estimated mass flows can exhibit a low signal-to-noise ratio. As the feeders are critical elements of the control strategy of the manufacturing line, better instantaneous estimates of mass flow are desirable for improving the quality assurance. In this study, we propose a model-based approach for monitoring the composition of the powder fed to a continuous solid-dosage line. The monitoring system is based on a moving-horizon state estimator, which carries out model-based reconciliation of the feeder mass measurements, thus enabling accurate composition estimation of the powder mixture. Experimental datasets from a direct compression line are used to validate the methodology. Results demonstrate improvement with respect to current industrial solutions.

Mathematical modeling and digital design of an intensified filtration-washing-drying unit for pharmaceutical continuous manufacturing.

This paper introduces a comprehensive mathematical model of a novel integrated filter-dryer carousel system, designed for continuously filtering, washing and drying a slurry stream into a crystals cake. The digital twin includes models for dead-end filtration, cake washing and convective cake drying, based on dynamic multi-component mass, energy and momentum balances. For set of feed conditions and control inputs, the model allows tracking the solvents and impurities content in the cake (critical quality attributes, CQAs) throughout the whole process. The model parameters were identified for the isolation of paracetamol from a multi-component slurry, containing a non-volatile impurity. The calibrated model was used for identifying the probabilistic design space and maximum throughput for the process, expressing the combinations of the carousel feed conditions and control inputs for which the probability of meeting the target CQAs is acceptable.

Effects of White Matter Hyperintensities on Brain Connectivity and Hippocampal Volume in Healthy Subjects According to Their Localization.

Purpose: To investigate the relationships between white matter hyperintensities (WMH) and hippocampal volume and their influence on brain networks by using resting-state functional connectivity (rs-fc) magnetic resonance (MR) according to their localization. Methods: In this exploratory cross-sectional study, 38 subjects from the public "Leipzig Study for Mind/Body/Emotion Interactions" (LEMON) data set were selected. Morphometric analyses of both WMH burden and the total hippocampal relative volume (tHRV) were performed for each subject with two automated software. The WMH were then categorized as total (tWMH), periventricular (pvWMH), deep (dWMH), and juxtacortical (jcWMH). Spearman's correlation analyses were performed to evaluate the relationships between the following variables: age, tWMH, pvWMH, dWMH, jcWMH, and tHRV. Subsequently, three different rs-fc MR group analyses were performed using a multiple regression model that included age, pvWMH, dWMH, and jcWMH as second-level covariates. The graph theoretical analysis was applied to evaluate the effects of pvWMH (analysis 1), jcWMH (analysis 2), and dWMH (analysis 3). Results: Spearman's correlation analysis revealed several statistically significant (p < 0.05) positive and negative correlations, in particular positive between age and tWMH, and negative between dWMH and tHRV. rs-fc MR analysis 1 and 2 did not reveal statistically significant results; analysis 3 revealed that dWMH influenced network properties of several cerebral regions, in particular global and local efficiency of both the hippocampi. Conclusion: The localization of WMH influences brain activity in healthy subjects. In particular, dWMH are inversely correlated with tHRV and influence several properties of different cerebral areas, included both the hippocampi. Impact statement In this exploratory research we evidenced how both the load and the localization of white matter hyperintensities influence brain activity; in particular, we evidenced an inverse correlation between the volume of the deep white matter hyperintensities and hippocampal volume, as well as a direct influence on the connectivity properties of this important cerebral region. This finding represent a new element for understanding the effects of white matter hyperintensities on brain networking, and a cue that could be taken into account for possible future studies investigating brain connectivity and cognitive functions in healthy and pathological conditions.

The impact of modifiable risk factors on lesion burden in patients with early multiple sclerosis.

INTRODUCTION: Some studies have indicated the importance of considering smoking, vitamin D deficiency and obesity as negative prognostic factors for clinical and MRI outcomes in multiple sclerosis (MS). This study aimed to evaluate the possible effects of these modifiable risk factors on brain MRI lesion burden of patients with early MS, also exploring the influence on initial clinical features. METHODS: MS patients were enrolled at diagnosis time and examined for smoking, body mass index (BMI), serum level of lipids and 25(OH) vitamin D. Brain MRIs' were acquired and lesion volume assessed by Jim software. Clinical data (disease course, disease duration, and EDSS score) were also collected. RESULTS: 64 patients were enrolled, of these 4 (6.2%) had a primary progressive course. Mean age was 39.8 ± 11.1 years and mean EDSS 1.5 ± 1.1. Forty (62.5%) patients were smokers and 40 (62.5%) were overweight (BMI>25). Insufficient levels of vitamin D (<20 ng/mL) were reported in 36 (56.2%) patients, while 24 (37.5%) patients had an altered lipid profile with total cholesterol >200 mg/dl and LDL >100 mg/dl. No association between early clinical features and modifiable risk factors were reported. Multiple regression analysis showed an association between lesion burden and smoking status (p 0.003), while no association was reported with BMI, altered lipid profile and vitamin D insufficiency. CONCLUSIONS: Several risk factors may play a role in evolution of MS. Our results show that smoking status, probably due to chronic vascular and neurotoxic effects of the cigarette components, can affect the brain damage from the early stages of MS. No association was observed with the other explored modifiable risk factors, although an effect due to the small sample size cannot be excluded.