Monitoring of Visible Particles in Parenteral Products by Manual Visual Inspection-Reassessing Size Threshold and Other Particle Characteristics that Define Particle Visibility.

Visible particles are a critical quality attribute for parenteral products and must be monitored. A carefully designed, executed, and controlled drug product manufacturing process including a final 100 % visual inspection and appropriate end-product controls ensures that visible particles are consistently minimized and demonstrates that the injectable DP is practically free from visible particles. Visual inspection, albeit appearing as a simple analytical procedure, requires several technical and operational controls to ensure adequate performance. To gather new data on particle visibility and shed light on this decade-old challenge, a multi-company blinded visual inspection threshold study was conducted. A major goal of the study was visual assessment of several particle types of different sizes in small volume vials, as a challenging configuration for visual inspection, across 9 biopharmaceutical companies in order to determine the visibility limit. The study results provide key insights into limitations and challenges of visual inspection, namely, no universal visibility limit can be applied to all particle types as the detectability varies with particle type, number, and size. The study findings underscore the necessity of setting realistic expectations on size-based visibility limits in visual inspection, robust procedures for analyst training and qualification, and harmonization of guidelines globally.

Sub-Visible Particle Classification and Label Consistency Analysis for Flow-Imaging Microscopy Via Machine Learning Methods.

Sub-visible particles can be a quality concern in pharmaceutical products, especially parenteral preparations. To quantify and characterize these particles, liquid samples may be passed through a flow-imaging microscopy instrument that also generates images of each detected particle. Machine learning techniques have increasingly been applied to this kind of data to detect changes in experimental conditions or classify specific types of particles, primarily focusing on silicone oil. That technique generally requires manual labeling of particle images by subject matter experts, a time-consuming and complex task. In this study, we created artificial datasets of silicone oil, protein particles, and glass particles that mimicked complex datasets of particles found in biopharmaceutical products. We used unsupervised learning techniques to effectively describe particle composition by sample. We then trained independent one-class classifiers to detect specific particle populations: silicone oil and glass particles. We also studied the consistency of the particle labels used to evaluate these models. Our results show that one-class classifiers are a reasonable choice for handling heterogeneous flow-imaging microscopy data and that unsupervised learning can aid in the labeling process. However, we found agreement among experts to be rather low, especially for smaller particles (< 8 µm for our Micro-Flow Imaging data). Given the fact that particle label confidence is not usually reported in the literature, we recommend more careful assessment of this topic in the future.

Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product.

Characterization of particulate impurities such as aggregates is necessary to develop safe and efficacious adeno-associated virus (AAV) drug products. Although aggregation of AAVs can reduce the bioavailability of the virus, only a limited number of studies focus on the analysis of aggregates. We explored three technologies for their capability to characterize AAV monomers and aggregates in the submicron (<1 µm) size range: (i) mass photometry (MP), (ii) asymmetric flow field flow fractionation coupled to a UV-detector (AF4-UV/Vis) and (iii) microfluidic resistive pulse sensing (MRPS). Although low counts for aggregates impeded a quantitative analysis, MP was affirmed as an accurate and rapid method for quantifying the genome content of empty/filled/double-filled capsids, consistent with sedimentation velocity analytical ultracentrifugation results. MRPS and AF4-UV/Vis enabled the detection and quantification of aggregate content. The developed AF4-UV/Vis method separated AAV monomers from smaller aggregates, thereby enabling a quantification of aggregates <200 nm. MRPS was experienced as a straightforward method to determine the particle concentration and size distribution between 250-2000 nm, provided that the samples do not block the microfluidic cartridge. Overall, within this study we explored the benefits and limitations of the complementary technologies for assessing aggregate content in AAV samples.

An Interlaboratory Study to Identify Potential Visible Protein-Like Particle Standards.

Visible protein-like particle standards may improve visual inspection and/or appearance testing practices used in the biotechnology industry. They may improve assay performance resulting in better alignment and more standardized training among different companies. The National Institute of Standards and Technology (NIST) has conducted an interlaboratory study to test whether the standards under development mimic typical proteinaceous particles found in biotherapeutics and if they can be implemented during the visual inspection process. Fourteen organizations from industry and government have participated. A total of 20 labs from these 14 organizations participated with analysts from 6 formulation, 7 analytical, 4 quality control, and 3 manufacturing labs. The circulated samples consisted of abraded ethylene tetrafluoroethylene (ETFE) particles or photolithographic particles. The results consist of qualitative ratings, which varied substantially among organizations and within labs. Polydisperse ETFE particle suspensions, containing particles enriched in greater than 150 µm in size, were rated more favorably than the photolithographic particles by formulation and analytical scientists. The largest monodisperse photolithographic particles (approximately 300 µm in size) were favored equally compared to ETFE by all scientists. Solution modifications to decrease the settling rate or to alter optical properties of the ETFE solutions yielded lower ratings by the analysts. Both particle types received mixed ratings for their usability and for their application for visual inspection and for training purposes. Industry feedback will assist NIST in developing reference material(s) for visible protein-like particles.

Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future.

Polysorbate (PS) 20 and 80 are the main surfactants used to stabilize biopharmaceutical products. Industry practices on various aspects of PS based on a confidential survey and following discussions by 16 globally acting major biotechnology companies is presented in two publications. Part 1 summarizes the current practice and use of PS during manufacture in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS.1 The current part 2 of the survey focusses on understanding, monitoring, prediction, and mitigation of PS degradation pathways in order to propose an effective control strategy. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature.

Industry Perspective on the use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates.

Polysorbates (PS) are widely used as a stabilizer in biopharmaceutical products. Industry practices on various aspects of PS are presented in this part 1 survey report based on a confidential survey and following discussions by 16 globally acting major biotechnology companies. The current practice and use of PS during manufacture across their global manufacturing sites are covered in addition to aspects like current understanding of the (in)stability of PS, the routine QC testing and control of PS, and selected regulatory aspects of PS. The results of the survey and extensive cross-company discussions are put into relation with currently available scientific literature. Part 2 of the survey report (upcoming) will focus on understanding, monitoring, prediction, and mitigation of PS degradation pathways to develop an effective control strategy.

Oil-Immersion Flow Imaging Microscopy for Quantification and Morphological Characterization of Submicron Particles in Biopharmaceuticals.

Flow imaging microscopy (FIM) is widely used to analyze subvisible particles starting from 2 µm in biopharmaceuticals. Recently, an oil-immersion FIM system emerged, the FlowCam Nano, designed to enable the characterization of particle sizes even below 2 µm. The aim of our study was to evaluate oil-immersion FIM (by using FlowCam Nano) in comparison to microfluidic resistive pulse sensing and resonant mass measurement for sizing and counting of particles in the submicron range. Polystyrene beads, a heat-stressed monoclonal antibody formulation and a silicone oil emulsion, were measured to assess the performance on biopharmaceutical relevant samples, as well as the ability to distinguish particle types based on instrument-derived morphological parameters. The determination of particle sizes and morphologies suffers from inaccuracies due to a low image contrast of small particles and light-scattering effects. The ill-defined measured volume impairs an accurate concentration determination. Nevertheless, FlowCam Nano in its current design complements the limited toolbox of submicron particle analysis of biopharmaceuticals by providing particle images in a size range that was previously not accessible with commercial FIM instruments.

Taking Subvisible Particle Quantitation to the Limit: Uncertainties and Statistical Challenges With Ophthalmic Products for Intravitreal Injection.

Subvisible particles are a critical quality attribute of pharmaceutical products. The reliability of particle quantitation increases with the number of particles in the analyzed sample volume. However, for analyses of low-volume drug products, such as ophthalmic products for intravitreal injection or biopharmaceuticals in general, sample volumes as small as possible should be used to avoid pooling and consequently, the contamination with foreign particles. The aim of our study was to evaluate the variability of particle concentrations obtained by light obscuration measurements to define the minimum required analyzed sample volume to achieve statistically meaningful results by using conditions that are practically feasible. Statistical evaluation suggests that for particle concentrations close to a predefined limit, large sample volumes (a multiple of typical intravitreal product volumes) would be required for a high probability to correctly classify samples with respect to the predefined limit. Below a minimum analyzed volume, even a measurement result of 0 particles does not allow to conclude compliance with the respective particle concentration limit with sufficient certainty. A small analyzed volume could be justified as long as the measurement uncertainty remains acceptable compared with the predefined limit.

Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy.

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells.

Synthesis and biological evaluation of C(5)-substituted derivatives of leukotriene biosynthesis inhibitor BRP-7.

Pharmacological intervention with 5-lipoxygenase (5-LO) pathway leading to suppression of leukotriene (LT) biosynthesis is a clinically validated strategy for treatment of respiratory and cardiovascular diseases such as asthma and atherosclerosis. Here we describe the synthesis of a series of C(5)-substituted analogues of the previously described 5-LO-activating protein (FLAP) inhibitor BRP-7 (IC50 = 0.31 µM) to explore the effects of substitution at the C(5)-benzimidazole (BI) ring as a strategy to increase the potency against FLAP-mediated 5-LO product formation. Incorporation of polar substituents on the C(5) position of the BI core, exemplified by compound 11 with a C(5)-nitrile substituent, significantly enhances the potency for suppression of 5-LO product synthesis in human neutrophils (IC50 = 0.07 µM) and monocytes (IC50 = 0.026 µM).

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes.

5-Lipoxygenase (5-LO), the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) from arachidonic acid, is regulated by androgens in human neutrophils and monocytes accounting for sex differences in LT formation. Here we show that progesterone suppresses the synthesis of 5-LO metabolites in human primary monocytes. 5-LO product formation in monocytes stimulated with Ca(2+)-ionophore A23187 or with lipopolysaccharide/formyl peptide was suppressed by progesterone at concentrations of 10-100 nM in cells from females and at 1 µM in cells from males. Progesterone down-regulated 5-LO product formation in a rapid and reversible manner, but did not significantly inhibit 5-LO activity in cell-free assays using monocyte homogenates. Also, arachidonic acid release and its metabolism to other eicosanoids in monocytes were not significantly reduced by progesterone. The inhibitory effect of progesterone on LTs was still observed when mitogen-activated protein kinases were pharmacologically blocked, stimulatory 1-oleoyl-2-acetyl-sn-glycerol was exogenously supplied, or extracellular Ca(2+) was removed by chelation. Instead, suppression of PKA by means of two different pharmacological approaches (i.e. H89 and a cell-permeable PKA inhibitor peptide) prevented inhibition of 5-LO product generation by progesterone, to a similar extent as observed for the PKA activators prostaglandin E2 and 8-Br-cAMP, suggesting the involvement of PKA. In summary, progesterone affects the capacity of human primary monocytes to generate 5-LO products and, in addition to androgens, may account for sex-specific effects on pro-inflammatory LTs.

Indirubin-3'-monoxime exerts a dual mode of inhibition towards leukotriene-mediated vascular smooth muscle cell migration.

AIMS: The small molecule indirubin-3'-monoxime (I3MO) has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and neointima formation in vivo. The influence of I3MO on VSMC migration and vascular inflammation, two additional key players during the onset of atherosclerosis and restenosis, should be investigated. METHODS AND RESULTS: We examined the influence of I3MO on VSMC migration, with focus on monocyte-derived leukotrienes (LTs) and platelet-derived growth factors (PDGFs) as elicitors. Exogenous LTB4 and cysteinyl leukotrienes as well as LT-enriched conditioned medium of activated primary human monocytes induced VSMC migration, which was inhibited by I3MO. I3MO also blunted migration of VSMC stimulated with the PDGF, the strongest motogen tested in this study. Induction of haem oxygenase 1 accounted for this anti-migratory activity of I3MO in VSMC. Notably, I3MO not only interfered with the migratory response in VSMC, but also suppressed the production of pro-migratory LT in monocytes. Conditioned media from monocytes that were activated in the presence of I3MO failed to induce VSMC migration. In cell-based and cell-free assays, I3MO selectively inhibited 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, with an IC50 in the low micromolar range. CONCLUSION: Our study reveals a novel dual inhibitory mode of I3MO on LT-mediated VSMC migration: (i) I3MO interferes with pro-migratory signalling in VSMC and (ii) I3MO suppresses LT biosynthesis in monocytes by direct inhibition of 5-LO. These inhibitory actions on both migratory stimulus and response complement the previously demonstrated anti-proliferative properties of I3MO and may further promote I3MO as promising vasoprotective compound.