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As reported in the previous commentary (Ishii-Watabe et al., J Pharm Sci 2017), the Japanese biopharmaceutical research group is promoting collaborative multilaboratory studies to evaluate and standardize new methodologies for biopharmaceutical characterization and quality control. We have conducted the studies and held 2 annual meetings in 2018 and 2019. At the 2018 meeting, Dr. Rukman DeSilva of the U.S. Food and Drug Administration and Dr. Srivalli Telikepalli of the National Institute of Standards and Technology participated as guest speakers. At the 2019 meeting, we invited Prof. John Carpenter of the University of Colorado, Prof. Gerhard Winter and Prof. Wolfgang Friess of Ludwig Maximilian University of Munich, and Dr. Tim Menzen of Coriolis Pharma Research, as guest commentators. In both meetings, the main research topic was strategies for the characterization and control of protein aggregates/subvisible particles in drug products. Specifically, the use of the light obscuration method for insoluble particulate matter testing with reduced injection volumes, and a comparison of analytical performance between flow imaging and light obscuration were discussed. Other topics addressed included host cell protein analysis, bioassay, and quality control strategies. In this commentary, the recent achievements of the research group, meeting discussions, and future perspectives are summarized. [ABSTRACT FROM AUTHOR]

The research and development of next-generation innovative medicines is a prominent interest of both the government and industries in Japan. On June 29, 2017, a kickoff meeting of a new research group focused on the quality issues of biopharmaceuticals was held in Tokyo with Prof. John Carpenter as an invited guest. The group's research focuses mainly on the evaluation and control of protein aggregates/subvisible particles in drug products, but the research topics also include glycan analysis, host-cell protein evaluation, bioassay validation, and analytical quality by design. The purpose of the group's activities is to resolve the critical and fundamental quality issues important to pharmaceutical companies through the collaboration of industries, academia, and regulatory agencies. In this commentary, our current plan to address these issues and the discussion at the kickoff meeting are described. [ABSTRACT FROM AUTHOR]

T cell-redirecting bispecific antibodies (bsAbs) have been under development as a new class of biotherapeutics for cancer immunotherapy. T cell-redirecting bsAbs simultaneously bind tumor-associated antigens on tumor cells and CD3 on T cells, resulting in T cell-mediated cytotoxicity against tumor cells. In this study, we prepared a tandem scFv-typed bsAb targeting HER2 and CD3 (HER2-CD3), and evaluated the impact of aggregation of HER2-CD3 on the in vitro immunotoxicity. A cell-based assay using CD3-expressing reporter cells revealed that the aggregates of HER2-CD3 directly activated CD3-expressing immune cells in the absence of target antigen (HER2)-expressing cells. Comparison of the aggregates generated under various stress conditions indicated the possibility that insoluble protein particles, which were detected by qLD analysis and contained non-denatured functional domains, contributed to the activation of CD3-expressing immune cells. In addition, HER2-CD3 aggregates stimulated hPBMCs and strongly induced the secretion of inflammatory cytokines and chemokines. The cytokine/chemokine-release profiles suggested that the aggregates could induce inflammatory responses not only by CD3-mediated T cell activation but also by other immune cell activations. These results indicated the potential risk of aggregation of T cell-redirecting bsAbs, which could induce unwanted immune cell activation and inflammation and thereby immune-mediated adverse reactions. [ABSTRACT FROM AUTHOR]

Abstract: Background: Amino acids (AAs) play important roles in maintaining an optimal hydration state of stratum corneum (SC) as a natural moisturizing factor (NMF). Recently, however, we have reported that lactate and potassium significantly affect the hydration state of SC. Objective: To explore the source of lactate and potassium in SC, we compared the concentration of various NMFs such as AAs, pyrrolidone carbonic acid (PCA), lactate, sodium, and potassium in SC between anhidrotic and adjacent hidrotic areas of patients with acquired idiopathic generalized anhidrosis or segmental anhidrosis. Methods: We examined 13 anhidrotic areas and the adjacent hidrotic skin of 10 different patients. We first determined anhidrotic and hidrotic areas of each patient by the iodine starch method and examined the hydration state of SC by measuring the high-frequency conductance. Then we obtained SC by tape stripping and measured the content of AAs, PCA, lactate, urea, sodium, and potassium in SC obtained from the anhidrotic and hidrotic areas. We examined the effect of increased insensible perspiration on the SC hydration and the concentrations of NMFs. Results: The SC of anhidrotic areas showed significantly low hydration. Among NMFs, lactate, urea, sodium, and potassium were significantly decreased in the SC of anhidrotic areas, while AAs and PCA were not significantly different between hidrotic and anhidrotic areas. Increased insensible perspiration increased SC hydration as well as NMFs other than AAs and PCA. Conclusion: Sweat constitutes lactate, urea, sodium, and potassium in NMFs and plays a crucial role in maintaining the physiological hydration state of SC. [Copyright &y& Elsevier]

We previously reported that CD31bright cells, which were sorted from cultured AC133+ cells of adult peripheral blood cells, differentiated more efficiently into endothelial cells than CD31+ cells or CD31- cells, suggesting that CD31bright cells may be endothelial precursor cells. In this study, we found that CD31bright cells have a strong ability to release cytokines. The mixture of vascular endothelial growth factor (VEGF), thrombopoietin (TPO), and stem cell factor stimulated ex vivo expansion of the total cell number from cultured AC133+ cells of adult peripheral blood cells and cord blood cells, resulting in incrementation of the adhesion cells, in which endothelial nitric oxide synthase and kinase insert domain-containing receptor were positive. Moreover, the mixture of VEGF and TPO increased the CD31bright cell population when compared with VEGF alone or the mixture of VEGF and stem cell factor. These data suggest that TPO is an important growth factor that can promote endothelial precursor cells expansion ex vivo. [ABSTRACT FROM AUTHOR]

The presence of replication-competent adenovirus (RCA) in clinical lots of adenovirus vectors raises a variety of safety concerns. To detect RCA in adenovirus vector products, the cell culture/cytopathic effect (CPE) method has generally been preferred. However, it is difficult to evaluate the amount of RCA clearly and quantitatively by this method. In addition, the cell culture/CPE method requires large-scale cell culturing and a substantial amount of time. For the purpose of establishing a method to detect RCA more sensitively and rapidly, we developed the infectivity PCR, a hybrid method that combines the infectivity assay and quantitative PCR. This method allows RCA to be quantified by real-time quantitative PCR using primers and a probe designed for E1 DNA. By infectivity PCR, 1 pfu of RCA spiked into 109 particles of adenovirus vectors could be detected. In contrast, CPE was observed in the cells infected with 104 pfu of RCA spiked into 109 particles of adenovirus vectors. The glass-beads method was suitable for extracting DNA rapidly from the RCA-infected cells. These results showed that infectivity PCR combined with the glass-beads-based DNA extraction method was useful for the detection of RCA in adenovirus vector products. [Copyright &y& Elsevier]

Protein aggregates are a potential risk factor for immunogenicity. The measurement, characterization, and control of protein aggregates in drug products are indispensable for the development of biopharmaceuticals, including therapeutic mAbs. In this study, Fcγ receptor (FcγR)-expressing reporter cell lines were used to analyze the FcγR-activation properties of mAb aggregates. Comparison of aggregates of mAbs harboring different IgG subclasses revealed that the FcγR-activation profiles of the mAb aggregates were dependent on IgG subclass. In addition, aggregates of Fc-engineered mAb with enhanced FcγR-activation properties exhibited stronger activation of FcγRs than was observed in the wild-type aggregates, whereas aggregates of Fc-engineered mAb with decreased FcγR-activation properties showed reduced activation. These results suggest that FcγR activation by mAb aggregates depends greatly on the Fc functions of the native (nonaggregated) mAbs. We also showed that aggregates of mAbs smaller than 1 μm in size have the potential to directly activate FcγRs. Unintended immune cell activation can be induced on account of FcγR activation by mAb aggregates and such FcγR activation may contribute to immunogenicity, and therefore, analysis of the FcγR-activation properties of mAb aggregates using FcγR-expressing reporter cell lines is a promising approach for the characterization of mAb aggregates. [ABSTRACT FROM AUTHOR]

Bioconjugation with polyethylene glycol (PEG) is important for protein drug development as it has improved biological stability. In contrast, proteins including PEGylated ones are susceptible to physicochemical stresses. Particularly, protein drugs in solution may form aggregates or subvisible particles if they are exposed to dropping stress during transportation. However, many PEGylation studies have focused on its usefulness, such as the extension of half-life in blood, and changes in the physical properties or biological responses of PEGylated proteins under dropping stress remain unexplored. Here, we prepared four PEGylated ovalbumin (PEG-OVA) molecules conjugated with different lengths (5 or 20 kDa) and numbers (large [L] or small [S]) of PEG, analyzed the formation of subvisible particles under dropping stress, and examined their impact on antibody production and clearance. Under dropping stress, the aggregated particle concentration of 20 kDa PEG-OVA (S) and (L) solutions was approximately 3-fold that of the OVA solution. Moreover, administration of 20 kDa PEG-OVA with dropping stress induced anti-PEG antibody production and clearance of PEG-OVA. As a mechanism, dropping stress could enhance the uptake of 20 kDa PEG-OVA (L) by macrophages. These findings could provide insights into proper transportation conditions to ensure the quality of PEGylated protein drugs. [ABSTRACT FROM AUTHOR]

Flow imaging (FI) has emerged as a powerful tool to evaluate insoluble particles derived from protein aggregates as an orthogonal method to light obscuration (LO). However, few reports directly compare the FI and LO method in the size and number of protein particles in commercially available therapeutic protein injections. In this study, we measured the number of insoluble particles in several therapeutic protein injections using both FI and LO, and characterized these particles to compare the analytical performance of the methods. The particle counts measured using FI were much higher than those measured using LO, and the difference depended on the products or features of particles. Some products contained a large number of transparent and elongated particles, which could escape detection using LO. Our results also suggested that the LO method underestimates the size and number of silicone oil droplets in prefilled syringe products compared to the FI method. The count of particles ≥10 μm in size in one product measured using FI exceeded the criteria (6000 counts per container) defined in the compendial particulate matter test using the LO method. Thus precaution should be taken when setting the acceptance criteria of specification tests using the FI method. [ABSTRACT FROM AUTHOR]

Pre-filled syringes (PFS) have been in widespread use as an administration device for therapeutic antibodies in recent decades. Generally, the inner barrel and syringe of PFS are coated with silicone oil (SO) for lubrication. Multiple studies have focused on the fact that the SO adsorbs denatured antibody molecules, and induces antibody aggregation. Aggregated antibodies are recognized as a potential risk for evoking immunogenic responses in patients. The characteristics of the aggregated antibody-SO complexes, including their concentration, population, shape, three-dimensional (3D) image, and Fcγ Receptors (FcγRs) activation have been obscurely acknowledged so far. In the present work, we prepared aggregated antibody-SO complexes by agitation and analyzed using multifaceted techniques such as flow imaging, confocal fluorescence microscopy, and cell-based assays for FcγRs activation. The results emphasized that the SO accelerates the increase in sub-visible particles and antibody aggregation. The confocal fluorescence microscopy analysis revealed the high-resolution 3D images of aggregated antibody-SO complexes. The FcγRs reporter cell assay clarified that the pre-mixed and agitated Ab + SO have higher FcγRs activation capability compared to the agitated Ab. Overall, this study advances the view that SO has an effect to increase the risk of agitation-induced aggregated antibody particles. [ABSTRACT FROM AUTHOR]

2-Propenyl tin species, prepared from 3-halopropenes or 2-propenyl mesylate with tin(IV) chloride and tetrabutylammonium iodide in dichloromethane, causes nucleophilic addition to aldehydes to produce the corresponding homoallylic alcohols. [Copyright &y& Elsevier]

Development of an appropriate assay to detect anti-drug antibody (ADA) is important for assessing immunogenicity to therapeutic protein products. However, characterizing ADA assay methods is difficult because human ADA as a reference standard is not available in most cases. We compared the analytical performance of three ligand-binding assay methods for ADA, namely, surface plasmon resonance (SPR), electrochemiluminescence (ECL), and biolayer interferometry (BLI) methods, by using the anti-erythropoietin (EPO) monoclonal antibody reference panel developed by the World Health Organization (WHO) in 2015. Dose-dependent binding responses were observed for all nine anti-EPO antibodies in the anti-EPO panel by the SPR and BLI methods. In contrast, the ECL method did not clearly detect binding of low-affinity anti-EPO antibodies. Regarding IgG2 and IgM antibodies derived from the same clone, IgG2 exhibited a higher binding response in the SPR assay, whereas the IgM binding response was higher than that of IgG2 in the ECL assay. In the case of the BLI method, there was no consistent pattern observed in the binding responses of IgG2 or IgM. Results of the anti-EPO antibody reference panel, which contains a variety of monoclonal antibodies, indicated that the ability to detect ADAs differed among these assay methods. Therefore, with ligand-binding assays, differences in assay platforms can affect the sensitivity and other characteristics of assays to detect ADAs. These results show that understanding the analytical performance of ADA assays is important for an appropriate assessment of immunogenicity. Our study also indicated the benefits of using the established human ADA reference panel to assess the assay methods for ADA detection. [ABSTRACT FROM AUTHOR]

[Display omitted] Wnt/β-catenin pathway triggers the formation of a complex between β-catenin and T cell-specific transcription factor (TCF), which induces transcriptional activation. Excessive transcriptional activation of this pathway is associated with the development, cause, and deterioration of various cancers. Therefore, the Wnt/β-catenin pathway is an attractive drug target for cancer therapeutics and small molecule- and peptide-based protein–protein interaction (PPI) inhibitors have been developed. However, peptide-based PPI inhibitors generally have low cell-membrane permeability because of their large molecular size. To improve cell-membrane permeability, conjugating cell-penetrating peptides (CPPs) to PPI-inhibiting peptides is a useful method for developing intracellularly targeted PPI inhibitors. In this study, we focused on the interaction between β-catenin and liver receptor homologue-1 (LRH-1) and designed and synthesized a series of LRH-1-derived peptides to develop inhibitors against Wnt/β-catenin signaling. The results showed that a penetratin-conjugated LRH-1-derived peptide (Penetratin-st7) predominantly inhibited DLD-1 cell growth at 20 μM treatment via inhibition of the Wnt signaling pathway. This result suggests that Penetratin-st7 is one of promising PPI inhibitors between TCF and β-catenin. [ABSTRACT FROM AUTHOR]

Abstract: New technologies are needed to deliver medicines safely and effectively. Polymeric nanoparticulate carriers are one such technology under investigation. We examined the intracellular trafficking of doxorubicin-bound block copolymers quantitatively and by imaging doxorubicin-derived fluorescence using confocal microscopy. The polymers were internalized by endocytosis and distributed in endosomal/lysosomal compartments and the endoplasmic reticulum; unlike free doxorubicin, the polymers were not found in the nucleus. Moreover, the ATP-binding cassette protein B1 (ABCB1) transporter may be involved in the efflux of the polymer from cells. This drug delivery system is attractive because the endogenous transport system is used for the uptake and delivery of the artificial drug carrier to the target as well as for its efflux from cells to medium. Our results show that a drug delivery system strategy targeting this endogenous transport pathway may be useful for affecting specific molecular targets. [Copyright &y& Elsevier]