An Interlaboratory Comparison on the Characterization of a Sub-micrometer Polydisperse Particle Dispersion.

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.

A Silicone Oil-Free Syringe Tailored for Intravitreal Injection of Biologics.

Intravitreal injections (IVI) of biologics targeting vascular endothelial growth factor (anti-VEGF) led to a paradigm shift in the management and prognosis of prevalent retinal conditions. Yet, IVI are typically performed with syringes that are neither developed nor approved for this purpose. Notably, syringes lubricated with silicone oil (SiO) are extensively used despite multiple reports showing that such syringes can cause deposition of SiO droplets in the vitreous body and patient discomfort. Thus, there is a need for SiO-free substitutes specifically tailored for IVI. Here, we report on the development and testing of such a syringe. This syringe has no dead volume, and its design allows for high-accuracy dosing. Also, it permits pharmaceutical compounding and storage of bevacizumab, ranibizumab, and aflibercept for up to 30 days without compromising their functional binding or transport properties. Finally, the new syringe demonstrated a favorable safety profile regarding release of SiO compared to SiO lubricated alternatives, including commercially prefilled syringes. Accordingly, the newly developed syringe is an appealing alternative for IVI.

In-vitro assessment of release of silicone oil droplets with the use of variety of syringes and needles used in intravitreal injections.

PURPOSE: To assess the variability of silicone oil (SO) particles released across syringes from the same lot and the role of different needle gauges. MATERIALS AND METHODS: Four syringe models and six needle models were assessed for SO release. About 50 microliters of a buffer solution were loaded into the syringe, needle or syringe/needle setup. The data were analyzed by imaging flow cytometry with fluorescently labeling for SO. RESULTS: All syringe models had a high coefficient of variation in SO release across syringes from the same lot. The amount of SO was significantly greater in the syringe when the needle was attached. SO particles with the BD 30G needle attached to the syringe were statistically greater than the 27G counterpart (p = 0.005). None of the other comparisons was statistically different. Finally, the number of SO particles was higher in the syringe/needle setup than in needles only (p = 0.0024). CONCLUSION: We found a high variability in SO content across syringes from the same lot. Additionally, there was no clear association between needle gauge and the number of SO particles, as well as their coefficient of variation. Finally, the needles accounted for a small number of SO particles in comparison to the combined syringe-needle setup.

The in vitro micronucleus assay using imaging flow cytometry and deep learning.

The in vitro micronucleus (MN) assay is a well-established assay for quantification of DNA damage, and is required by regulatory bodies worldwide to screen chemicals for genetic toxicity. The MN assay is performed in two variations: scoring MN in cytokinesis-blocked binucleated cells or directly in unblocked mononucleated cells. Several methods have been developed to score the MN assay, including manual and automated microscopy, and conventional flow cytometry, each with advantages and limitations. Previously, we applied imaging flow cytometry (IFC) using the ImageStream® to develop a rapid and automated MN assay based on high throughput image capture and feature-based image analysis in the IDEAS® software. However, the analysis strategy required rigorous optimization across chemicals and cell lines. To overcome the complexity and rigidity of feature-based image analysis, in this study we used the Amnis® AI software to develop a deep-learning method based on convolutional neural networks to score IFC data in both the cytokinesis-blocked and unblocked versions of the MN assay. We show that the use of the Amnis AI software to score imagery acquired using the ImageStream® compares well to manual microscopy and outperforms IDEAS® feature-based analysis, facilitating full automation of the MN assay.

Silicone oil-free syringes, siliconized syringes and needles: quantitative assessment of silicone oil release with drugs used for intravitreal injection.

PURPOSE: This study aimed to quantify the amount of silicone oil (SO) released across a variety of syringe and needle models routinely used for intravitreal injection. METHODS: The release of SO was assessed in eight models of syringes, two of which were reported to be 'SO-free', and eleven models of needles with unknown SO content. To evaluate SO release within the context of anti-VEGF therapeutics, syringes were evaluated using aflibercept, bevacizumab, buffer, ziv-aflibercept and formulation buffer. All syringe tests were performed with or without agitation by flicking for syringes. Needles were evaluated without agitation only. Samples were fluorescently labelled to identify SO, and triplicate measurements were collected using imaging flow cytometry. RESULTS: Seven out of 8 syringe models showed a statistically significant increase in the SO particle count after agitation. The two SO-free syringe models (HSW Norm-Ject, Daikyo Crystal Zenith) released the least SO particles, with or without agitation, whereas the BD Ultra-Fine and Saldanha-Rodrigues syringes released the most. More SO was released when the syringes were prefilled with formulation buffer than with ziv-aflibercept. Syringes filled with aflibercept and bevacizumab had intermediate levels. Agitation increased the release of SO into each of the drug solutions. Silicone oil (SO) was detected in all needles. CONCLUSIONS: Agitation of the syringe by flicking leads to a substantial increase in the number of SO particles. Silicone oil (SO)-free syringes had the best performance, but physicians must also be aware that needles are siliconized and also contribute to the injection of SO into the vitreous.

Assessing Particle Formation of Biotherapeutics in Biological Fluids.

The stability of therapeutic proteins can be impacted in vivo after administration, which may affect patient safety or treatment efficacy, or both. Stability testing of therapeutic proteins using models representing physiologic conditions may guide preclinical development strategy; however, to date only a few studies assessing the physical stability are available in the public domain. In this manuscript, the stability of seven fluorescently labeled monoclonal antibodies (mAbs) was evaluated in human serum and phosphate-buffered saline, two models often discussed to be representative of the situation in humans after intravenous administration. Subvisible particles were analyzed using light obscuration, flow imaging, and imaging flow cytometry. All methods showed that serum itself formed particles under in vitro conditions. Imaging flow cytometry demonstrated that mean particle size and counts of mAbs increased substantially in serum over five days; however, particle formation in phosphate-buffered saline was comparably low. Stability differences were observed across the mAbs evaluated, and imaging flow cytometry data indicated that fluorescently labeled mAbs primarily interacted with serum components. The results indicate that serum may be more suitable as in vitro model to simulate physiologic intravenous conditions in patients closely and evaluate the in vivo stability of therapeutic proteins. Fluorescence labeling and detection methods may be applied to differentiate particles containing therapeutic protein from high amounts of serum particles that form over time.

The Impact of Formulation Composition and Process Settings of Traditional Batch Versus Continuous Freeze-Drying On Protein Aggregation.

The long-term stability of therapeutic protein products can be extended by freeze-drying. However, the freeze-drying process itself has several harmful stresses. A rationalized formulation design can significantly mitigate protein damage caused by freezing, dehydration and interfacial stresses of lyophilization and reconstitution. Recently, a continuous spin-freeze-drying concept was proposed as a more economical, controllable, flexible and qualitative alternative to batch freeze-drying. The purpose of this work is to compare spin-freeze-drying to traditional batch freeze-drying with regard to protein physical stability. The impacts of spinning, freezing and drying were investigated for both processing methods. Herewith, the interaction between these process phases and two common rational formulation strategies, (i.e. adding a disaccharide and a surfactant) was examined. Protein aggregates formed due to the process phase stresses were characterized with particle counting techniques and size exclusion chromatography. It was found that spin-freeze-drying exhibited essentially identical stresses causing comparable aggregation in all the process phases as compared to batch freeze-drying. Moreover, there were also analogous impacts of the formulation excipients. These observations led to the conclusion that similar freeze-drying formulation excipients and strategies tested for decades in batch freeze-drying of proteins can be utilized for spin-freeze-drying; in order to maintain protein stability during processing.

Advanced Characterization of Silicone Oil Droplets in Protein Therapeutics Using Artificial Intelligence Analysis of Imaging Flow Cytometry Data.

Monitoring protein particles is increasingly emphasized in the development of biopharmaceuticals due to potential immunogenicity. Accurate quantitation of protein particles is complicated by silicone oil droplets, a common pharmaceutical component in pre-filled syringes. Though silicone oil is typically regarded as harmless, numerous reports have indicated protein adsorption may render these particles with immunostimulatory properties. Imaging flow cytometry (IFC) is an emerging pharmaceutical method capable of capturing high-resolution brightfield and fluorescence imagery from samples in suspension. In this study, we created a data analysis strategy using artificial intelligence (AI) software to classify brightfield images collected with IFC as protein or silicone oil. The AI software performs image classification using deep learning with a convolutional neural network architecture, for identification of subtle morphological phenotypes. The AI model yielded robust classification of particles >2 µm across various sources of protein and silicone oil particles and over the instrument life cycle. Next, the AI model was combined with IFC fluorescence images to differentiate potentially immunogenic protein-adsorbed silicone and innocuous native silicone. The methods reported herein provide added analytical capability for characterization of particulate matter in therapeutic formulations, and may be applied for optimization of protein formulations and evaluation of product consistency.

Characterization of Protein Aggregates, Silicone Oil Droplets, and Protein-Silicone Interactions Using Imaging Flow Cytometry.

Protein therapeutic exposure to siliconized surfaces during manufacturing and storage has potential to induce protein aggregation or generate protein-silicone complexes that are potentially immunogenic. Consequently, assessing the stability and safety of protein therapeutics requires discrimination of protein and silicone oil particles and evaluation of protein-silicone oil interactions. Industry-established methods are challenged to distinguish protein aggregates from silicone oil droplets for particles smaller than 5 µm. In addition, emerging techniques for accessing particles in the sub-5 µm range are limited by low sampling volumes, narrow size ranges, complications such as clogging, and an inability to evaluate protein-silicone interactions. In this study, imaging flow cytometry was evaluated as a new method for discrimination of protein aggregates and silicone oil droplets, as well as for quantification of protein-silicone interactions. A simple and fast fluorescence labeling protocol using low concentrations of extrinsic dyes was developed. The results demonstrate that imaging flow cytometry can be used to discriminate fluorescently labeled protein aggregates and silicone oil droplets with greater than 95% accuracy, regardless of size, and protein-silicone oil interactions can be assessed qualitatively through inspection of image data or quantitatively using features extracted from the image data.

Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material.

Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.

MicroRNA-mediated disruption of dendritogenesis during a critical period of development influences cognitive capacity later in life.

The prenatal period of cortical development is important for the establishment of neural circuitry and functional connectivity of the brain; however, the molecular mechanisms underlying this process remain unclear. Here we report that disruption of the actin-cytoskeletal network in the developing mouse prefrontal cortex alters dendritic morphogenesis and synapse formation, leading to enhanced formation of fear-related memory in adulthood. These effects are mediated by a brain-enriched microRNA, miR-9, through its negative regulation of diaphanous homologous protein 1 (Diap1), a key organizer of the actin cytoskeletal assembly. Our findings not only revealed important regulation of dendritogenesis and synaptogenesis during early brain development but also demonstrated a tight link between these early developmental events and cognitive functions later in life.

Characterization of Protein Particles in Therapeutic Formulations Using Imaging Flow Cytometry.

Quantitation of particles >10 µm in therapeutic protein formulations is required by pharmacopeia guidelines, and characterization of particles <10 µm is increasingly expected. Established methods offer limited ability to detect or characterize small particles; consequently, new methods are needed to measure the sub-10 µm size range. Here, we evaluate imaging flow cytometry (IFC) as a new method for detection of protein aggregates, taking advantage of key enabling attributes including rapid multi-modal high-resolution imaging of individual particles, low sample volume, high sampling efficiency, wide dynamic size and concentration range, and low clog risk. IFC sensitivity was compared with dynamic imaging, a "gold standard" technique for analysis of particles in protein formulations. Both techniques yielded similar results for polystyrene beads ≥2 µm. However, IFC demonstrated greater protein particle detection sensitivity, especially for the sub-10 µm size range. Interestingly, for an aggregated lysozyme sample, IFC detected protein particles using fluorescence images, whereas dynamic imaging failed to detect even large particles >25 µm due to high transparency. The results corroborate implementation of IFC as an advanced technique for protein particle analysis, offering in-depth characterization of particle physical and chemical properties, and enhanced sensitivity for sub-10 µm and transparent particles.

Technology-Enabled Remote Monitoring and Self-Management - Vision for Patient Empowerment Following Cardiac and Vascular Surgery: User Testing and Randomized Controlled Trial Protocol.

BACKGROUND: Tens of thousands of cardiac and vascular surgeries (CaVS) are performed on seniors in Canada and the United Kingdom each year to improve survival, relieve disease symptoms, and improve health-related quality of life (HRQL). However, chronic postsurgical pain (CPSP), undetected or delayed detection of hemodynamic compromise, complications, and related poor functional status are major problems for substantial numbers of patients during the recovery process. To tackle this problem, we aim to refine and test the effectiveness of an eHealth-enabled service delivery intervention, TecHnology-Enabled remote monitoring and Self-MAnagemenT-VIsion for patient EmpoWerment following Cardiac and VasculaR surgery (THE SMArTVIEW, CoVeRed), which combines remote monitoring, education, and self-management training to optimize recovery outcomes and experience of seniors undergoing CaVS in Canada and the United Kingdom. OBJECTIVE: Our objectives are to (1) refine SMArTVIEW via high-fidelity user testing and (2) examine the effectiveness of SMArTVIEW via a randomized controlled trial (RCT). METHODS: CaVS patients and clinicians will engage in two cycles of focus groups and usability testing at each site; feedback will be elicited about expectations and experience of SMArTVIEW, in context. The data will be used to refine the SMArTVIEW eHealth delivery program. Upon transfer to the surgical ward (ie, post-intensive care unit [ICU]), 256 CaVS patients will be reassessed postoperatively and randomly allocated via an interactive Web randomization system to the intervention group or usual care. The SMArTVIEW intervention will run from surgical ward day 2 until 8 weeks following surgery. Outcome assessments will occur on postoperative day 30; at week 8; and at 3, 6, 9, and 12 months. The primary outcome is worst postop pain intensity upon movement in the previous 24 hours (Brief Pain Inventory-Short Form), averaged across the previous 14 days. Secondary outcomes include a composite of postoperative complications related to hemodynamic compromise-death, myocardial infarction, and nonfatal stroke- all-cause mortality and surgical site infections, functional status (Medical Outcomes Study Short Form-12), depressive symptoms (Geriatric Depression Scale), health service utilization-related costs (health service utilization data from the Institute for Clinical Evaluative Sciences data repository), and patient-level cost of recovery (Ambulatory Home Care Record). A linear mixed model will be used to assess the effects of the intervention on the primary outcome, with an a priori contrast of weekly average worst pain intensity upon movement to evaluate the primary endpoint of pain at 8 weeks postoperation. We will also examine the incremental cost of the intervention compared to usual care using a regression model to estimate the difference in expected health care costs between groups. RESULTS: Study start-up is underway and usability testing is scheduled to begin in the fall of 2016. CONCLUSIONS: Given our experience, dedicated industry partners, and related RCT infrastructure, we are confident we can make a lasting contribution to improving the care of seniors who undergo CaVS.

Quantum dots as a platform for nanoparticle drug delivery vehicle design.

Nanoparticle-based drug delivery (NDD) has emerged as a promising approach to improving upon the efficacy of existing drugs and enabling the development of new therapies. Proof-of-concept studies have demonstrated the potential for NDD systems to simultaneously achieve reduced drug toxicity, improved bio-availability, increased circulation times, controlled drug release, and targeting. However, clinical translation of NDD vehicles with the goal of treating particularly challenging diseases, such as cancer, will require a thorough understanding of how nanoparticle properties influence their fate in biological systems, especially in vivo. Consequently, a model system for systematic evaluation of all stages of NDD with high sensitivity, high resolution, and low cost is highly desirable. In theory, this system should maintain the properties and behavior of the original NDD vehicle, while providing mechanisms for monitoring intracellular and systemic nanocarrier distribution, degradation, drug release, and clearance. For such a model system, quantum dots (QDots) offer great potential. QDots feature small size and versatile surface chemistry, allowing their incorporation within virtually any NDD vehicle with minimal effect on overall characteristics, and offer superb optical properties for real-time monitoring of NDD vehicle transport and drug release at both cellular and systemic levels. Though the direct use of QDots for drug delivery remains questionable due to their potential long-term toxicity, the QDot core can be easily replaced with other organic drug carriers or more biocompatible inorganic contrast agents (such as gold and magnetic nanoparticles) by their similar size and surface properties, facilitating translation of well characterized NDD vehicles to the clinic, maintaining NDD imaging capabilities, and potentially providing additional therapeutic functionalities such as photothermal therapy and magneto-transfection. In this review we outline unique features that make QDots an ideal platform for nanocarrier design and discuss how this model has been applied to study NDD vehicle behavior for diverse drug delivery applications.

Rapid multitarget immunomagnetic separation through programmable DNA linker displacement.

Immunomagnetic separation has become an essential tool for high-throughput and low-cost isolation of biomolecules and cells from heterogeneous samples. However, as magnetic selection is essentially a "black-and-white" assay, its application has been largely restricted to single-target and single-parameter studies. To address this issue, we have developed an immunomagnetic separation technology that can quickly sort multiple targets in high yield and purity using selectively displaceable DNA linkers. We envision that this technology will be readily adopted for experiments requiring high-throughput selection of multiple targets or further adapted for selection of a single target based on multiple surface epitopes.