Patient-centric Comparability Assessment of Biopharmaceuticals.

The comparability assessment of a biological product after implementing a manufacturing process change should involve a risk-based approach. Process changes may occur at any stage of the product lifecycle: early development, clinical manufacture for pivotal trials, or post-approval. The risk of the change to impact product quality varies. The design of the comparability assessment should be adapted accordingly. A working group reviewed and consolidated industry approaches to assess comparability of traditional protein-based biological products during clinical development and post-approval. The insights compiled in this review article encompass topics such as a risk-evaluation strategy, the design of comparability studies, definition of assessment criteria for comparability, holistic evaluation of data, and the regulatory submission strategy. These practices can be leveraged across the industry to help companies in design and execution of comparability assessments, and to inform discussions with global regulators.

Using enhanced development tools offered by analytical Quality by Design to support switching of a quality control method.

Quality by Design (QbD) principles play an increasingly important role in the pharmaceutical industry. Here, we used an analytical QbD (AQbD) approach to develop a capillary electrophoresis sodium dodecyl sulfate under reducing conditions (rCE-SDS), with the aim of replacing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as release and stability test method for a commercialized monoclonal antibody product. Method development started with defining analytical method performance requirements as part of an analytical target profile, followed by a systematic risk assessment of method input parameters and their relation to defined method outputs. Based on this, design of experiments studies were performed to identify a method operable design region (MODR). The MODR could be leveraged to improve method robustness. In a bridging study, it was demonstrated that the rCE-SDS method is more sensitive than the legacy SDS-PAGE method, and a conversion factor could be established to compensate for an off-set due to the higher sensitivity, without losing the correlation to the historical data acquired with the former method. Overall, systematic application of analytical Quality by Design principles for designing and developing a new analytical method helped to elucidate the complex dependency of method outputs on its input parameters. The link of the method to product quality attributes and the definition of method performance requirements were found to be most relevant for derisking the analytical method switch, regarding impact on the control strategy.

Compliance and regulatory considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under the good manufacturing practice (GMP) regime, due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. This article is the second part of a two-tiered publication aiming at providing guidance for implementation of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) in a QC laboratory. The first part [1] focuses on technical considerations, while this second part provides considerations related to GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Technical considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under good manufacturing practice (GMP) due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. Here, current literature related to the development and application of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) is compiled with the aim of providing guidance for the implementation of MAM in a QC laboratory. This article, focusing on technical considerations, is the first part of a two-tiered publication, whereby the second part will focus on GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

An Intra-Company Analysis of Inherent Particles in Biologicals Shapes the Protein Particle Mitigation Strategy Across Development Stages.

To better understand protein aggregation and inherent particle formation in the biologics pipeline at Novartis, a cross-functional team collected and analyzed historical protein particle issues. Inherent particle occurrences from the past 10 years were systematically captured in a protein particle database. Where the root cause was identified, a number of product attributes (such as development stage, process step, or protein format) were trended. Several key themes were revealed: 1) there was a higher propensity for inherent particle formation with non-mAbs than with mAbs; 2) the majority of particles were detected following manufacturing at scale, and were not predicted by the small-scale studies; 3) most issues were related to visible particles, followed by subvisible particles; 4) 50% of the issues were manufacturing related. These learnings became the foundation of a particle mitigation strategy across development and technical transfer, and resulted in a set of preventive actions. Overall, this study provides further insight into a recognized industry challenge and hopes to inspire the biopharmaceutical industry to transparently share their experiences with inherent particles formation.

Deterministic Photon Sorting in Waveguide QED Systems.

Sorting quantum fields into different modes according to their Fock-space quantum numbers is a highly desirable quantum operation. In this Letter, we show that a pair of two-level emitters, chirally coupled to a waveguide, may scatter single- and two-photon components of an input pulse into orthogonal temporal modes with a fidelity ≳0.9997. We develop a general theory to characterize and optimize this process and reveal that such a high fidelity is enabled by an interesting two-photon scattering dynamics: while the first emitter gives rise to a complex multimode field, the second emitter recombines the field amplitudes, and the net two-photon scattering induces a self-time reversal of the input pulse mode. The presented scheme can be employed to construct logic elements for propagating photons, such as a deterministic nonlinear-sign gate with a fidelity ≳0.9995.

Rydberg exciton-polaritons in a Cu2O microcavity.

Giant Rydberg excitons with principal quantum numbers as high as n = 25 have been observed in cuprous oxide (Cu2O), a semiconductor in which the exciton diameter can become as large as ∼1 µm. The giant dimension of these excitons results in excitonic interaction enhancements of orders of magnitude. Rydberg exciton-polaritons, formed by the strong coupling of Rydberg excitons to cavity photons, are a promising route to exploit these interactions and achieve a scalable, strongly correlated solid-state platform. However, the strong coupling of these excitons to cavity photons has remained elusive. Here, by embedding a thin Cu2O crystal into a Fabry-Pérot microcavity, we achieve strong coupling of light to Cu2O Rydberg excitons up to n = 6 and demonstrate the formation of Cu2O Rydberg exciton-polaritons. These results pave the way towards realizing strongly interacting exciton-polaritons and exploring strongly correlated phases of matter using light on a chip.

A Doppler ultrasound self-monitoring approach for detection of relevant individual decompression stress in scuba diving.

Observing modern decompression protocols alone cannot fully prevent diving injuries especially in repetitive diving. Professional audio Doppler bubble measurements are not available to sports scuba divers. If those non-professionals were able to learn audio Doppler self-assessment for bubble grading, such skill could provide significant information on individual decisions with respect to diving safety. We taught audio Doppler self-assessment of subclavian and precordial probe position to 41 divers in a 45-min standardized, didactically optimized training. Assessment before and after air dives within sports diving limits was made through 684 audio Doppler measurements in dive-site conditions by both trained divers and a medical professional, plus additional 2D-echocardiography reference. In all dives (average maximum depth 22 m; dive time 44 min), 33% of all echocardiography measurements revealed bubbles. The specificity of audio bubble detection in combination of both detection sites was 95%, and sensitivity over all grades was 40%, increasing with higher bubble grades. Dive-site audio-Doppler-grading underestimated echo-derived bubble grades. Bubble detection sensitivity of audio Doppler self-assessments, compared to an experienced examiner, was 62% at subclavian and 73% at precordial position. 6 months after the training and 4.5 months after the last measurement, the achieved Doppler skill level remained stable. Audio Doppler self-assessment can be learned by non-professionals in a single teaching intervention. Despite accurate bubble grading is impossible in dive-site conditions, relevant high bubble grades can be detected by non-professionals. This qualitative information can be important in self-evaluating decompression stress and assessing measures for increased diving safety.

Sonographic Estimation Rather Than Quantification of Fluid Status Using Inferior Vena Cava and Other Major Vessel Parameters in a Non-Bleeding Fluid Loss and Centralization Model.

OBJECTIVE: A well-accepted step in emergency sonography is the estimation of a fluid deficit through Inferior Vena Cava (IVC) diameter variability with known cut-offs especially in bleeding. We sought to answer, whether a non-bleeding fluid deficit can be quantified through sonographic assessment of IVC diameter variability and related aortic parameters. Sport divers were used as human hypovolemic vasoconstriction models since immersion is known to cause relevant volume depletion through vasoconstriction and induced diuresis. MATERIALS AND METHODS: Forty-one sport divers performed 342 single and repetitive dives to account for intra- and interindividual variability and were assessed for inferior Vena Cava and neighboring aortic diameters as well as their cardiac/respiratory variations. Dive-related weight loss was measured together with sonographic vessel diameter changes inferior to the right atrium. RESULTS: Highest correlation with dive-related weight loss of max. 2.9 kg per an average 47 minutes dive was found with r=0.34 for the difference of IVC maximum diameter related to minimum Aortic diameter. Single or combined parameters, as well as Collapsibility Index, showed lower or no correlations. Vascular parameters were able to explain 7.5% of the variance of fluid losses, whereas interindividual effects explained 10%. The remaining 82.5% is of mixed intraindividual counterregulatory effects. CONCLUSION: IVC diameter changes in immersion-induced hypovolemic centralization provides qualitative information on relevant fluid loss only. Confounding factors like inter and intraindividual variability prevent a sufficient correlation for useful quantification of the experienced non-bleeding fluid deficit in the clinical setting.

Asymmetric Rydberg blockade of giant excitons in Cuprous Oxide.

The ability to generate and control strong long-range interactions via highly excited electronic states has been the foundation for recent breakthroughs in a host of areas, from atomic and molecular physics to quantum optics and technology. Rydberg excitons provide a promising solid-state realization of such highly excited states, for which record-breaking orbital sizes of up to a micrometer have indeed been observed in cuprous oxide semiconductors. Here, we demonstrate the generation and control of strong exciton interactions in this material by optically producing two distinct quantum states of Rydberg excitons. This is made possible by two-color pump-probe experiments that allow for a detailed probing of the interactions. Our experiments reveal the emergence of strong spatial correlations and an inter-state Rydberg blockade that extends over remarkably large distances of several micrometers. The generated many-body states of semiconductor excitons exhibit universal properties that only depend on the shape of the interaction potential and yield clear evidence for its vastly extended-range and power-law character.

Enhanced nonlinear interaction of polaritons via excitonic Rydberg states in monolayer WSe2.

Strong optical nonlinearities play a central role in realizing quantum photonic technologies. Exciton-polaritons, which result from the hybridization of material excitations and cavity photons, are an attractive candidate to realize such nonlinearities. While the interaction between ground state excitons generates a notable optical nonlinearity, the strength of such interactions is generally not sufficient to reach the regime of quantum nonlinear optics. Excited states, however, feature enhanced interactions and therefore hold promise for accessing the quantum domain of single-photon nonlinearities. Here we demonstrate the formation of exciton-polaritons using excited excitonic states in monolayer tungsten diselenide (WSe2) embedded in a microcavity. The realized excited-state polaritons exhibit an enhanced nonlinear response ∼[Formula: see text] which is ∼4.6 times that for the ground-state exciton. The demonstration of enhanced nonlinear response from excited exciton-polaritons presents the potential of generating strong exciton-polariton interactions, a necessary building block for solid-state quantum photonic technologies.

Strongly Correlated States of Light and Repulsive Photons in Chiral Chains of Three-Level Quantum Emitters.

We study the correlated transport of photons through a chain of three-level emitters that are coupled chirally to a photonic mode of a waveguide. It is found that this system can transfer a weak classical input into a strongly correlated state of light in a unitary manner. Our analysis reveals two-photon scattering eigenstates, that are akin to Fano resonances or shape resonances in particle collisions and facilitate the emergence of antibunched light with long-range correlations upon crossing a critical length of the chain. By operating close to conditions of electromagnetically induced transparency of the three-level medium, a high degree of antibunching and photon transmission can be maintained in the presence of moderate losses. These features suggest a promising mechanism for single-photon generation and may open the door to exploring correlated quantum many-body states of light with repulsively interacting photons.

Estimating Inert Gas Bubbling from Simple SCUBA Diving Parameters.

Inert gas bubbles frequently occur in SCUBA divers' vascular systems, eventually leading to decompression accidents. Only in professional settings, dive profiles can be adjusted on individual basis depending on bubble grades detected through ultrasonography. A total of 342 open-circuit air dives following sports diving profiles were assessed using echocardiography. Subsequently, (Eftedal-Brubakk) bubble grades were correlated with dive and individual parameters. Post-dive cardiac bubbles were observed in 47% of all dives and bubble grades were significantly correlated with depth (r=0.46), air consumption (r=0.41), age (r=0.25), dive time (r=0.23), decompression diving (r=0.19), surface time (r=- 0.12). Eftedal-Brubakk categorical bubble grades for sports diving with compressed air can be approximated by bubble grade = (age*50-1 - surface time*150-1+maximum depth*45-1+air consumption*4500-1)2 (units in years, hours, meter, and bar*liter; R2=0.31). Thus, simple dive and individual parameters allow reasonable estimation of especially relevant medium to higher bubble grades for information on relevant decompression stress after ascent. Echo bubble grade 0 is overestimated by the formula derived. However, echo might fail to detect minor bubbling only. The categorical prediction of individual decompression stress with simple bio and dive data should be evaluated further to be developed towards dive computer included automatic ex-post information for decision-making on individual safety measures.

Plasma-Enhanced Interaction and Optical Nonlinearities of Cu_{2}O Rydberg Excitons.

We theoretically investigate the nonlinear optical transmission through a cuprous oxide crystal for wavelengths that cover the series of highly excited excitons, observed in recent experiments. Since such Rydberg excitons have strong van der Waals interactions, they can dynamically break the conditions for resonant exciton creation and dramatically modify the refractive index of the material in a nonlinear manner. We explore this mechanism theoretically and determine its effects on the optical properties of a semiconductor for the case of degenerate pair-state asymptotes of Rydberg excitons in Cu_{2}O. Upon analyzing the additional effects of a dilute residual electron-hole plasma, we find quantitative agreement with previous transmission measurements, which provides strong indications for the enhancement of Rydberg-induced nonlinearities by surrounding free charges.

Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology.

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that 'everything is everywhere'. With the discovery of lateral budding in 'Kolteria novifilia' and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how 'microbial dark matter' can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection.

Polariton Exchange Interactions in Multichannel Optical Networks.

We examine the dynamics of Rydberg polaritons with dipolar interactions that propagate in multiple spatial modes. The dipolar excitation exchange between different Rydberg states mediates an effective exchange between polaritons that enables photons to hop across different spatial channels. Remarkably, the efficiency of this photon exchange process can increase with the channel distance and becomes optimal at a finite rail separation. Based on this mechanism, we design a simple photonic network that realizes a two photon quantum gate with a robust π phase, protected by the symmetries of the underlying photon interaction and the geometry of the network. These capabilities expand the scope of Rydberg electromagnetically induced transparency towards multidimensional geometries for nonlinear optical networks and explorations of photonic many-body physics.

Saccharomyces cerevisiae Centromere RNA Is Negatively Regulated by Cbf1 and Its Unscheduled Synthesis Impacts CenH3 Binding.

Two common features of centromeres are their transcription into noncoding centromere RNAs (cen-RNAs) and their assembly into nucleosomes that contain a centromere-specific histone H3 (cenH3). Here, we show that Saccharomyces cerevisiae cen-RNA was present in low amounts in wild-type (WT) cells, and that its appearance was tightly cell cycle-regulated, appearing and disappearing in a narrow window in S phase after centromere replication. In cells lacking Cbf1, a centromere-binding protein, cen-RNA was 5-12 times more abundant throughout the cell cycle. In WT cells, cen-RNA appearance occurred at the same time as loss of Cbf1's centromere binding, arguing that the physical presence of Cbf1 inhibits cen-RNA production. Binding of the Pif1 DNA helicase, which happens in mid-late S phase, occurred at about the same time as Cbf1 loss from the centromere, suggesting that Pif1 may facilitate this loss by its known ability to displace proteins from DNA. Cen-RNAs were more abundant in rnh1Δ cells but only in mid-late S phase. However, fork pausing at centromeres was not elevated in rnh1Δ cells but rather was due to centromere-binding proteins, including Cbf1 Strains with increased cen-RNA lost centromere plasmids at elevated rates. In cbf1Δ cells, where both the levels and the cell cycle-regulated appearance of cen-RNA were disrupted, the timing and levels of cenH3 centromere binding were perturbed. Thus, cen-RNAs are highly regulated, and disruption of this regulation correlates with changes in centromere structure and function.

Supersolidity around a Critical Point in Dipolar Bose-Einstein Condensates.

We explore spatial symmetry breaking of a dipolar Bose-Einstein condensate in the thermodynamic limit and reveal a critical point in the phase diagram at which crystallization occurs via a second-order phase transition. This behavior is traced back to the significant effects of quantum fluctuations in dipolar condensates, which moreover stabilize a new supersolid phase, namely a regular honeycomb pattern with high modulational contrast and near-perfect superfluidity.

Evaluation of a real-time magnetic resonance imaging-guided electrophysiology system for structural and electrophysiological ventricular tachycardia substrate assessment.

AIMS: Potential advantages of real-time magnetic resonance imaging (MRI)-guided electrophysiology (MR-EP) include contemporaneous three-dimensional substrate assessment at the time of intervention, improved procedural guidance, and ablation lesion assessment. We evaluated a novel real-time MR-EP system to perform endocardial voltage mapping and assessment of delayed conduction in a porcine ischaemia-reperfusion model. METHODS AND RESULTS: Sites of low voltage and slow conduction identified using the system were registered and compared to regions of late gadolinium enhancement (LGE) on MRI. The Sorensen-Dice similarity coefficient (DSC) between LGE scar maps and voltage maps was computed on a nodal basis. A total of 445 electrograms were recorded in sinus rhythm (range: 30-186) using the MR-EP system including 138 electrograms from LGE regions. Pacing captured at 103 sites; 47 (45.6%) sites had a stimulus-to-QRS (S-QRS) delay of ≥40 ms. Using conventional (0.5-1.5 mV) bipolar voltage thresholds, the sensitivity and specificity of voltage mapping using the MR-EP system to identify MR-derived LGE was 57% and 96%, respectively. Voltage mapping had a better predictive ability in detecting LGE compared to S-QRS measurements using this system (area under curve: 0.907 vs. 0.840). Using an electrical threshold of 1.5 mV to define abnormal myocardium, the total DSC, scar DSC, and normal myocardium DSC between voltage maps and LGE scar maps was 79.0 ± 6.0%, 35.0 ± 10.1%, and 90.4 ± 8.6%, respectively. CONCLUSION: Low-voltage zones and regions of delayed conduction determined using a real-time MR-EP system are moderately associated with LGE areas identified on MRI.

Pif1 family DNA helicases: A helpmate to RNase H?

An R-loop is a structure that forms when an RNA transcript stays bound to the DNA strand that encodes it and leaves the complementary strand exposed as a loop of single stranded DNA. R-loops accumulate when the processing of RNA transcripts is impaired. The failure to remove these RNA-DNA hybrids can lead to replication fork stalling and genome instability. Resolution of R-loops is thought to be mediated mainly by RNase H enzymes through the removal and degradation of the RNA in the hybrid. However, DNA helicases can also dismantle R-loops by displacing the bound RNA. In particular, the Pif1 family DNA helicases have been shown to regulate R-loop formation at specific genomic loci, such as tRNA genes and centromeres. Here we review the roles of Pif1 family helicases in vivo and in vitro and discuss evidence that Pif1 family helicases act on RNA-DNA hybrids and highlight their potential roles in complementing RNase H for R-loop resolution.