Publications by authors named "Susumu Uchiyama"

186 Publications

A Multi-Method Approach to Assess the Self-Interaction Behavior of Infliximab.

J Pharm Sci 2021 Feb 5. Epub 2021 Feb 5.

Department of Pharmacy, Ludwig-Maximilians-Universität München, Pharmaceutical Technology and Biopharmaceutics, 81377 Munich, Germany. Electronic address:

Attractive self-interaction processes in antibody formulations increase the risk of aggregation and extraordinarily elevated viscosity at high protein concentrations. These challenges affect manufacturing and application. This study aimed to understand the self-interaction process of Infliximab as a model system with pronounced attractive self-interaction. The association mechanism was studied by a multi-method approach comprising analytical ultracentrifugation, dynamic light scattering, small angle X-ray scattering, self-interaction bio-layer interferometry and hydrogen-deuterium exchange mass spectrometry. Based on our results, both Fab and Fc regions of Infliximab are involved in self-interaction. We hypothesize a mechanism based on electrostatic interactions of polar and charged residues within the identified areas of the heavy and the light chain of the mAb. The combination of fast and reliable screening methods and low throughput but high resolution methods can contribute to detailed characterization and deeper understanding of specific self-interaction processes.
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http://dx.doi.org/10.1016/j.xphs.2021.02.002DOI Listing
February 2021

An influenza HA stalk reactive polymeric IgA antibody exhibits anti-viral function regulated by binary interaction between HA and the antibody.

PLoS One 2021 7;16(1):e0245244. Epub 2021 Jan 7.

Department of Pathology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0245244PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790537PMC
January 2021

Physicochemical Characterization of Sabin Inactivated Poliovirus Vaccine for Process Development.

J Pharm Sci 2020 Dec 17. Epub 2020 Dec 17.

Vaccine Operations, Global Vaccine Business Unit, Takeda Pharmaceutical Company Limited, 4720 Takeda, Mitsui, Hikari, Yamaguchi 743-0011, Japan.

Upscaling the production capacity of inactivated poliovirus vaccines (IPV) is urgently needed to eradicate polio worldwide. For the development of a robust manufacturing process for IPV, the impact of stresses on the properties of the poliovirus during manufacturing needs to be carefully evaluated. In this study, the physicochemical properties of Sabin poliovirus after low pH exposure were analyzed by asymmetrical flow field-flow fractionation coupled to multi-angle laser light scattering (AF4-MALS), sedimentation velocity analytical ultracentrifugation (SV-AUC), transmission electron microscopy (TEM), dynamic light scattering (DLS) and surface plasmon resonance (SPR). Low pH stress caused structural changes and aggregation of inactivated poliovirus virions, whereas degraded virion particles would not revert to native virions even after neutralization. Importantly, a complete loss of the D-antigenicity of IPV by low pH stress, followed by neutralization, was observed in SPR. These results suggest that the exposure of poliovirus particle to low pH stress would induce irreversible denaturation and aggregation of virus particles and lead to the loss of D-antigenicity; thus, low pH stress during the manufacturing of poliovirus vaccine should be minimized. The analytical methods above can be efficiently utilized in the development of high-integrity manufacturing processes and high-quality vaccines.
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http://dx.doi.org/10.1016/j.xphs.2020.12.012DOI Listing
December 2020

Development of syringes and vials for delivery of biologics: current challenges and innovative solutions.

Expert Opin Drug Deliv 2021 Jan 25:1-12. Epub 2021 Jan 25.

Department of Biotechnology, Graduate School of Engineering, Osaka University , Osaka, Japan.

Introduction: Several new biopharmaceutical dosage forms have developed over time, such as lyophilized vial, liquid vial, and liquid prefilled syringe formulations. This review summarizes major pharmaceutical dosage forms and their advantages, disadvantages, and countermeasures against the shortcomings of each formulation. The appropriate combination of active pharmaceutical ingredients, excipients, and containers should be selected for the safe and less burdensome administration to the patients. Finally, we note certain opinions on the future development of not only therapeutic proteins but also gene therapeutics.

Areas Covered: This review is to discuss the challenges of the development of dosage forms to improve pharmaceutical stability and how they can be overcome.

Expert Opinion: Silicone oil-free syringes are highly preferable for minimizing subvisible particles in the drug. It can be proposed that materials with less protein adsorption property are preferable for the suppression of protein aggregation. It is required to minimize adverse effects of biopharmaceuticals through proper quality control of the drug in a container, based on the understating of physicochemical stability of the protein in solution, the physicochemical properties of the container, and their combinations.
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http://dx.doi.org/10.1080/17425247.2021.1853699DOI Listing
January 2021

Critical analysis of techniques and materials used in devices, syringes, and needles used for intravitreal injections.

Prog Retin Eye Res 2021 Jan 18;80:100862. Epub 2020 Apr 18.

Department of Ophthalmology, Federal University of São Paulo, Rua Botucatu, 806, São Paulo, SP, Brazil; Department of Ophthalmology, SSM Health Saint Louis University Hospital, Saint Louis University, 1755, S. Grand Boulevard, Saint Louis, MO, USA.

Intravitreal injections have become the most commonly performed intraocular treatments worldwide. Because intravitreal injections may induce severe adverse events, such as infectious and noninfectious endophthalmitis, cataract, ocular hypertension, vitreous hemorrhage, or retinal detachment, appropriate awareness of the materials and techniques used are essential to reduce these sight-threatening complications. This review provides insights into the needles, syringes, silicone oil coating, sterilization methods, devices to assist intravitreal injections, scleral piercing techniques using needles, syringe handling, anesthesia, and safety issues related to materials and techniques. It is paramount that physicians be aware of every step involved in intravitreal injections and consider the roles and implications of all materials and techniques used. The ability to understand the theoretical and practical circumstances may definitely lead to state-of-the-art treatments delivered to patients. The most important practical recommendations are: choosing syringes with as little silicone oil as possible, or, preferably, none; avoiding agitation of syringes; awareness that most biologics (e.g., antiangiogenic proteins) are susceptible to changes in molecular properties under some conditions, such as agitation and temperature variation; understanding that improper materials and techniques may lead to complications after intravitreal injections, e.g., inflammation; and recognizing that some devices may contribute to an enhanced, safer, and faster intravitreal injection technique.
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http://dx.doi.org/10.1016/j.preteyeres.2020.100862DOI Listing
January 2021

Allosteric regulation accompanied by oligomeric state changes of Trypanosoma brucei GMP reductase through cystathionine-β-synthase domain.

Nat Commun 2020 04 15;11(1):1837. Epub 2020 Apr 15.

Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-8531, Japan.

Guanosine 5'-monophosphate reductase (GMPR) is involved in the purine salvage pathway and is conserved throughout evolution. Nonetheless, the GMPR of Trypanosoma brucei (TbGMPR) includes a unique structure known as the cystathionine-β-synthase (CBS) domain, though the role of this domain is not fully understood. Here, we show that guanine and adenine nucleotides exert positive and negative effects, respectively, on TbGMPR activity by binding allosterically to the CBS domain. The present structural analyses revealed that TbGMPR forms an octamer that shows a transition between relaxed and twisted conformations in the absence and presence of guanine nucleotides, respectively, whereas the TbGMPR octamer dissociates into two tetramers when ATP is available instead of guanine nucleotides. These findings demonstrate that the CBS domain plays a key role in the allosteric regulation of TbGMPR by facilitating the transition of its oligomeric state depending on ligand nucleotide availability.
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http://dx.doi.org/10.1038/s41467-020-15611-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160140PMC
April 2020

Effect of UVC Irradiation on the Oxidation of Histidine in Monoclonal Antibodies.

Sci Rep 2020 04 14;10(1):6333. Epub 2020 Apr 14.

Graduate School of Engineering, Osaka University, Osaka, Japan.

We oxidized histidine residues in monoclonal antibody drugs of immunoglobulin gamma 1 (IgG1) using ultraviolet C irradiation (UVC: 200-280 nm), which is known to be potent for sterilization or disinfection. Among the reaction products, we identified asparagine and aspartic acid by mass spectrometry. In the photo-induced oxidation of histidine in angiotensin II, O atoms from HO in the solvent were incorporated only into aspartic acid but not into asparagine. This suggests that UVC irradiation generates singlet oxygen and induces [2 + 2] cycloaddition to form a dioxetane involving the imidazole C - C bond of histidine, followed by ring-opening in the manner of further photo-induced retro [2 + 2] cycloaddition. This yields an equilibrium mixture of two keto-imines, which can be the precursors to aspartic acid and asparagine. The photo-oxidation appears to occur preferentially for histidine residues with lower pK values in IgG1. We thus conclude that the damage due to UVC photo-oxidation of histidine residues can be avoided in acidic conditions where the imidazole ring is protonated.
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http://dx.doi.org/10.1038/s41598-020-63078-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156388PMC
April 2020

Current status and issues of protein solution biophysics-Session 1SDP.

Biophys Rev 2020 Apr 4;12(2):263-264. Epub 2020 Apr 4.

Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Tokyo, Japan.

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http://dx.doi.org/10.1007/s12551-020-00671-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242577PMC
April 2020

Stirring rate affects thermodynamics and unfolding kinetics in isothermal titration calorimetry.

J Biochem 2020 Jul;168(1):53-62

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Isothermal titration calorimetry (ITC) directly provides thermodynamic parameters depicting the energetics of intermolecular interactions in solution. During ITC experiments, a titration syringe with a paddle is continuously rotating to promote a homogeneous mixing. Here, we clarified that the shape of the paddles (flat, corkscrew and small-pitched corkscrew) and the stirring rates influence on the thermodynamic parameters of protein-ligand interaction. Stirring with the flat paddle at lower and higher rate both yielded a lower exothermic heat due to different reasons. The complete reaction with no incompetent fractions was achieved only when the stirring was performed at 500 or 750 rpm using the small-pitched corkscrew paddle. The evaluation of the protein solution after 1,500 rpm stirring indicated that proteins in the soluble fraction decreased to 94% of the initial amount, among which 6% was at an unfolded state. In addition, a significant increase of micron aggregates was confirmed. Furthermore, a new approach for the determination of the unfolding kinetics based on the time dependence of the total reaction heat was developed. This study demonstrates that a proper stirring rate and paddle shape are essential for the reliable estimation of thermodynamic parameters in ITC experiments.
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http://dx.doi.org/10.1093/jb/mvaa028DOI Listing
July 2020

Dataset of microbial community structure in alcohol sprayed banana associated with ripening process.

Data Brief 2020 Apr 5;29:105216. Epub 2020 Feb 5.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Banana ripening is a complex molecular process that produces visible changes in the texture, aroma, taste and nutritional content. Ripening is controlled by genetic code, metabolic pathway and associated microbiome. We reported the microbial community structure during banana ripening with alcohol treatment to discover endophytic and epiphytic microbes. We observed the pulp and peel from the first and seventh days of Cavendish ( cv. Cavendish) from mature green fruit and treated with 70% alcohol or distilled water sum up to eight samples and applied the 16S rRNA Illumina sequencing from V3-V4 gene region. After quality check 144,368 sequences were obtained in the dataset comprising a total read length of 1,237,805 base pairs. A sum of 199 genera were successfully isolated, with genera was the most dominant genera at 56.65% and followed by more than 1% were genera , , , , , and using mothur pipelines. The highest diversity sample with 101 unique genera was belongs to distilled water treated raw bananas peel (NN1K) and the lowest diversity at 38 was belongs to distilled water treated ripe bananas pulp (NN7D). The metagenome data are available at NCBI Sequence Read Archive (SRA) database and Biosample under accession number PRJNA590572. The data contribute to discover different bacterial communities during post-harvest treatment.
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http://dx.doi.org/10.1016/j.dib.2020.105216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031138PMC
April 2020

Incorporation of Pseudo-complementary Bases 2,6-Diaminopurine and 2-Thiouracil into Serinol Nucleic Acid (SNA) to Promote SNA/RNA Hybridization.

Chem Asian J 2020 Apr 17;15(8):1266-1271. Epub 2020 Feb 17.

Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku,

Serinol nucleic acid (SNA) is a promising candidate for nucleic acid-based molecular probes and drugs due to its high affinity for RNA. Our previous work revealed that incorporation of 2,6-diaminpurine (D), which can form three hydrogen bonds with uracil, into SNA increases the melting temperature of SNA-RNA duplexes. However, D incorporation into short self-complementary regions of SNA promoted self-dimerization and hindered hybridization with RNA. Here we synthesized a SNA monomer of 2-thiouracil (sU), which was expected to inhibit base pairing with D by steric hindrance between sulfur and the amino group. To prepare the SNA containing D and sU in high yield, we customized the protecting groups on D and sU monomers that can be readily deprotected under acidic conditions. Incorporation of D and sU into SNA facilitated stable duplex formation with target RNA by suppressing the self-hybridization of SNA and increasing the stability of the heteroduplex of SNA and its complementary RNA. Our results have important implications for the development of SNA-based probes and nucleic acid drugs.
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http://dx.doi.org/10.1002/asia.201901728DOI Listing
April 2020

Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation.

Sci Rep 2020 01 30;10(1):1540. Epub 2020 Jan 30.

Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Okazaki, Aichi, 444-8787, Japan.

Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.
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http://dx.doi.org/10.1038/s41598-020-58371-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992696PMC
January 2020

Recent Achievements and Current Interests in Research on the Characterization and Quality Control of Biopharmaceuticals in Japan.

J Pharm Sci 2020 05 9;109(5):1652-1661. Epub 2020 Jan 9.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

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.
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http://dx.doi.org/10.1016/j.xphs.2020.01.001DOI Listing
May 2020

Automatic Identification of the Stress Sources of Protein Aggregates Using Flow Imaging Microscopy Images.

J Pharm Sci 2020 01 25;109(1):614-623. Epub 2019 Oct 25.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Research Department, U-Medico Inc., 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Creative Research, Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan. Electronic address:

A novel approach to identify 5 types of simulated stresses that induce protein aggregation in prefilled syringe-type biopharmaceuticals was developed. Principal components analyses of texture metrics extracted from flow imaging microscopy images were used to define subgroups of particles. Supervised machine learning methods, including convolutional neural networks, were used to train classifiers to identify subgroup membership of constituent particles to generate distribution profiles. The applicability of the stress-specific signatures for distinguishing stress source types was verified. The high classification efficiencies (100%) precipitated the collection of data from more than 20 independent experiments to train support vector machines, k-nearest neighbors, and ensemble classifiers. The performances of the trained classifiers were validated. High classification efficiencies for friability (80%-100%) and heating at 90°C (85%-100%) are indicative of high reliability of these methods for stress-stability assays while extreme variations in freeze-thawing (2%-100%) and heating at 60°C (2.25%-98.25%) indicate the unpredictability of particle composition profiles for these forced degradation conditions. We also developed subvisible particle classifiers using convolutional neural network to automatically identify silicone oil droplets, air bubbles, and protein aggregates. The developed classifiers will contribute to mitigating aggregation in biopharmaceuticals via the identification of stress sources.
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http://dx.doi.org/10.1016/j.xphs.2019.10.034DOI Listing
January 2020

Relation of Colloidal and Conformational Stabilities to Aggregate Formation in a Monoclonal Antibody.

J Pharm Sci 2020 01 24;109(1):308-315. Epub 2019 Oct 24.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki 444-8787, Japan. Electronic address:

Aggregation of therapeutic monoclonal antibodies has a potential risk of immunogenicity, requiring minimization of aggregate formation. We have developed a fitting formula for antibody aggregation at 40°C based on physicochemical parameters, including colloidal and conformational stabilities. An IgG1 monoclonal antibody, MAb-T, was formulated in 24 combinations of different buffer types and pH with or without sodium chloride. The fitting formula for monomer loss was successfully established by nonlinear regression analysis of the results from accelerated stability testing. Calculated monomer fraction values by the fitting formula were strongly correlated with experimental values (R = 0.92). The model includes secondary virial coefficient, B, as the representative parameter of colloidal stability, and aggregation temperature, T, representing conformational stability. Then, we examined charge state, conformational flexibility, and thermal unfolding profile of MAb-T to clarify the molecular basis for the different aggregation propensities in sodium acetate buffer and in sodium citrate buffer at the same pH and buffer concentration. We concluded that the accumulation of citrate anions on the surface of MAb-T is the primary source of the less colloidal and conformational stabilities, resulting in the higher aggregation propensity in sodium citrate buffer.
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http://dx.doi.org/10.1016/j.xphs.2019.10.038DOI Listing
January 2020

Assessment of the Injection Performance of a Tapered Needle for Use in Prefilled Biopharmaceutical Products.

J Pharm Sci 2020 01 22;109(1):515-523. Epub 2019 Oct 22.

Research Department, U-Medico Inc., 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Creative Research, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan. Electronic address:

The design of injection devices, including prefilled syringes (PFSs) and autoinjectors, requires an understanding of the optimization of injection conditions. The injection of highly concentrated biopharmaceuticals can lead to exceptionally high injection forces, due to their high viscosity. To overcome this challenge, a tapered needle has been recently developed by Terumo Corporation. In the present study, we measured the injection forces in PFSs equipped with 24G-29G tapered needle (29G TNN), 27G thin-wall needle (27G TW), and 29G TW using several model and pharmaceutical protein solutions. The injection forces measured in the 29G TNN PFSs were lower than those in 29G TW for all solutions, similar to those in 27G TW PFSs for Newtonian solutions, and were lower than those in the 27G TW PFSs for non-Newtonian solutions which demonstrated shear-thinning behavior. No significant changes in aggregates or micron-size particle concentrations were observed upon injection, regardless of the needle type. Mathematical modeling supported the experimental findings that under similar flow rate conditions injection pressure in a tapered needle is lower than that in a cylindrical needle. Our results indicate that there are advantages of using tapered needles for the injection of biopharmaceutical formulations particularly those showing shear-thinning behavior.
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http://dx.doi.org/10.1016/j.xphs.2019.10.033DOI Listing
January 2020

Structural characterization of HypX responsible for CO biosynthesis in the maturation of NiFe-hydrogenase.

Commun Biol 2019 18;2:385. Epub 2019 Oct 18.

1Department of Creative Research, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki 444-8787 Japan.

Several accessory proteins are required for the assembly of the metal centers in hydrogenases. In NiFe-hydrogenases, CO and CN are coordinated to the Fe in the NiFe dinuclear cluster of the active center. Though these diatomic ligands are biosynthesized enzymatically, detail mechanisms of their biosynthesis remain unclear. Here, we report the structural characterization of HypX responsible for CO biosynthesis to assemble the active site of NiFe hydrogenase. CoA is constitutionally bound in HypX. Structural characterization of HypX suggests that the formyl-group transfer will take place from N-formyl-THF to CoA to form formyl-CoA in the N-terminal domain of HypX, followed by decarbonylation of formyl-CoA to produce CO in the C-terminal domain though the direct experimental results are not available yet. The conformation of CoA accommodated in the continuous cavity connecting the N- and C-terminal domains will interconvert between the extended and the folded conformations for HypX catalysis.
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http://dx.doi.org/10.1038/s42003-019-0631-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802093PMC
April 2020

Cooperative Binding of KaiB to the KaiC Hexamer Ensures Accurate Circadian Clock Oscillation in Cyanobacteria.

Int J Mol Sci 2019 Sep 13;20(18). Epub 2019 Sep 13.

Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.

The central oscillator generating cyanobacterial circadian rhythms comprises KaiA, KaiB, and KaiC proteins. Their interactions cause KaiC phosphorylation and dephosphorylation cycles over approximately 24 h. KaiB interacts with phosphorylated KaiC in competition with SasA, an output protein harboring a KaiB-homologous domain. Structural data have identified KaiB-KaiC interaction sites; however, KaiB mutations distal from the binding surfaces can impair KaiB-KaiC interaction and the circadian rhythm. Reportedly, KaiB and KaiC exclusively form a complex in a 6:6 stoichiometry, indicating that KaiB-KaiC hexamer binding shows strong positive cooperativity. Here, mutational analysis was used to investigate the functional significance of this cooperative interaction. Results demonstrate that electrostatic complementarity between KaiB protomers promotes their cooperative assembly, which is indispensable for accurate rhythm generation. SasA does not exhibit such electrostatic complementarity and noncooperatively binds to KaiC. Thus, the findings explain KaiB distal mutation effects, providing mechanistic insights into clock protein interplay.
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http://dx.doi.org/10.3390/ijms20184550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769508PMC
September 2019

The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III.

Sci Rep 2019 08 16;9(1):11957. Epub 2019 Aug 16.

Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, 444-8787, Japan.

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering.
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http://dx.doi.org/10.1038/s41598-019-48323-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697678PMC
August 2019

A head-to-toe dimerization has physiological relevance for ligand-induced inactivation of protein tyrosine receptor type Z.

J Biol Chem 2019 10 15;294(41):14953-14965. Epub 2019 Aug 15.

Division of Molecular Neurobiology, National Institute for Basic Biology (NIBB), 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan

Protein-tyrosine phosphatase (PTPase) receptor type Z (PTPRZ) has two receptor isoforms, PTPRZ-A and -B, containing tandem intracellular PTP-D1 and -D2 domains, with only D1 being active. Pleiotrophin (PTN) binding to the extracellular PTPRZ region leads to inactivation of its PTPase activity, thereby facilitating oligodendrocyte precursor cell (OPC) differentiation and myelination in the central nervous system. However, the mechanisms responsible for PTN-induced PTPRZ inactivation remain unclear. We herein report that the crystal structure of the intracellular region of PTPRZ (PTPRZ-ICR) shows a "head-to-toe"-type dimer conformation, with D2 masking the catalytic site of D1. MS analyses revealed that PTPRZ-ICR proteins remain in monomer-dimer equilibrium in aqueous solution and that a substrate-derived inhibitory peptide or competitive inhibitor (SCB4380) specifically bind to the monomer form in a 1:1 ratio. A D2 deletion (ΔD2) or dimer interface mutation (DDKK) disrupted dimer formation, but SCB4380 binding was maintained. Similar to WT PTPRZ-B, monomer-biased PTPRZ-B-ΔD2 and PTPRZ-B-DDKK variants efficiently dephosphorylated p190RhoGAP at Tyr-1105 when co-expressed in BHK-21 cells. The catalytic activities of these variants were not suppressed by PTN treatment, but were inhibited by the cell-permeable PTPase inhibitor NAZ2329. Of note, the PTN treatment did not enhance OPC differentiation in primary cultured glial cells from ΔD2 or PTPase-inactive PTPRZ-B (CS) mutant knock-in mice. Our results thus indicate that PTN-induced PTPRZ inactivation results from dimer formation of the intracellular tandem PTP domains in a head-to-toe configuration, which is physiologically relevant to the control of OPC differentiation .
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http://dx.doi.org/10.1074/jbc.RA119.007878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791311PMC
October 2019

Publisher Correction: Dynamic structural states of ClpB involved in its disaggregation function.

Nat Commun 2019 Jul 12;10(1):3079. Epub 2019 Jul 12.

Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, 920-1192, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-019-11204-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626010PMC
July 2019

ATP hydrolysis by KaiC promotes its KaiA binding in the cyanobacterial circadian clock system.

Life Sci Alliance 2019 06 3;2(3). Epub 2019 Jun 3.

Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan

The cyanobacterial clock is controlled via the interplay among KaiA, KaiB, and KaiC, which generate a periodic oscillation of KaiC phosphorylation in the presence of ATP. KaiC forms a homohexamer harboring 12 ATP-binding sites and exerts ATPase activities associated with its autophosphorylation and dephosphorylation. The KaiC nucleotide state is a determining factor of the KaiB-KaiC interaction; however, its relationship with the KaiA-KaiC interaction has not yet been elucidated. With the attempt to address this, our native mass spectrometric analyses indicated that ATP hydrolysis in the KaiC hexamer promotes its interaction with KaiA. Furthermore, our nuclear magnetic resonance spectral data revealed that ATP hydrolysis is coupled with conformational changes in the flexible C-terminal segments of KaiC, which carry KaiA-binding sites. From these data, we conclude that ATP hydrolysis in KaiC is coupled with the exposure of its C-terminal KaiA-binding sites, resulting in its high affinity for KaiA. These findings provide mechanistic insights into the ATP-mediated circadian periodicity.
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http://dx.doi.org/10.26508/lsa.201900368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549140PMC
June 2019

Mutational and Combinatorial Control of Self-Assembling and Disassembling of Human Proteasome α Subunits.

Int J Mol Sci 2019 May 9;20(9). Epub 2019 May 9.

School of Physical Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8787, Japan.

Eukaryotic proteasomes harbor heteroheptameric α-rings, each composed of seven different but homologous subunits α1-α7, which are correctly assembled via interactions with assembly chaperones. The human proteasome α7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric α6. We comprehensively characterized the oligomeric state of human proteasome α subunits and demonstrated that only the α7 subunit exhibits this unique, self-assembling property and that not only α6 but also α4 can disrupt the α7 double ring. We also demonstrated that mutationally monomerized α7 subunits can interact with the intrinsically monomeric α4 and α6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.
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http://dx.doi.org/10.3390/ijms20092308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539845PMC
May 2019

Temperature-controlled repeatable scrambling and induced-sorting of building blocks between cubic assemblies.

Nat Commun 2019 03 29;10(1):1440. Epub 2019 Mar 29.

Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, 153-8902, Japan.

Separation of a homogeneous mixture of different components to reach an ordered out-of-equilibrium state in solution has attracted continuous attention. While this can be achieved using external chemical fuels or photo energy, an alternative energy source is heat. Here we realize a temperature-controlled cycle of transitions between ordered and disordered states based on a mixture of two kinds of building blocks that self-assemble into cubic structures (nanocubes). An almost statistical mixture of nanocubes (disordered state) is thermodynamically most stable at lower temperature (25 °C), while homoleptic assemblies composed of single components are preferentially produced at higher temperature (100 °C) followed by rapid cooling. The scrambling of the building blocks between the nanocubes takes place through the exchange of free building blocks dissociated from the nanocubes. Based on this mechanism, it is possible to accelerate, retard, and perfectly block the scrambling by the guest molecules encapsulated in the nanocubes.
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http://dx.doi.org/10.1038/s41467-019-09495-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441092PMC
March 2019

Ionic liquids and protein folding-old tricks for new solvents.

Biophys Rev 2019 Apr 19;11(2):209-225. Epub 2019 Mar 19.

Institute for Protein Research, Osaka University, 3-1-Yamada-oka, Suita, Osaka, 565-0871, Japan.

One important aspect of the green chemistry revolution has been the use of ionic liquids as the solvent in liquid-phase enzymatic catalysis. An essential requirement for protein enzyme function is the correct folding of the polypeptide chain into its functional "native" state. Quantitative assessment of protein structure may be carried out either empirically, or by using model-based characterization procedures, in which the parameters are defined in terms of a standard reference state. In this short note, we briefly outline the nature of the parameters associated with different empirical and model-based characterization procedures and point out factors which affect their interpretation when using a base solvent different from water. This review principally describes arguments developed by Wakayama et al., Protein Solubility and Amorphous Aggregation: From Academic Research to Applications in Drug Discovery and Bioindustry, 2019, edited by Y. Kuroda and F. Arisaka; CMC Publishing House. Sections of that work are translated from the original Japanese and republished here with the full permission of CMC Publishing Corporation.
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http://dx.doi.org/10.1007/s12551-019-00509-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441443PMC
April 2019

Identification of IgG1 Aggregation Initiation Region by Hydrogen Deuterium Mass Spectrometry.

J Pharm Sci 2019 07 6;108(7):2323-2333. Epub 2019 Mar 6.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki 444-8787, Japan. Electronic address:

Antibody aggregates are a potential risk for immunogenicity; therefore, rational approaches to improve associated aggregation properties need to be developed. Here, we report the amino acid region responsible for aggregation initiation. Two types of therapeutic IgG1 antibody monomer samples were prepared: IgG1 mAb40-3M stored at 40°C for 3 months, which existed in monodisperse state, and the monomer mAb65-5m, which was dissociated from small soluble aggregates by heating at 65°C for 5 min. Hydrogen deuterium exchange mass spectrometry of mAb40-3M identified 2 sites in the Fc region (site 1, F239-M256; site 2, S428-G450) with increased exchange rates. Site 1 includes a region reported as being susceptible to structural change induced by stress. Exposure of site 1 was undetected after 2 months of storage at 40°C but was subsequently detectable after 3 months. As site 2 is spatially close to site 1, the structural change of site 1 could propagate site 2. Besides these 2 regions, hydrogen deuterium exchange mass spectrometry of mAb65-5m identified an exposure of I257-W281 in Fc (site 3), within which a peptide sequence with high aggregation tendency was discovered. We thus concluded that exposure of site 3 is a trigger for the association of a partially denatured antibody.
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http://dx.doi.org/10.1016/j.xphs.2019.02.023DOI Listing
July 2019

An Assessment of the Ability of Submicron- and Micron-Size Silicone Oil Droplets in Dropped Prefillable Syringes to Invoke Early- and Late-Stage Immune Responses.

J Pharm Sci 2019 07 18;108(7):2278-2287. Epub 2019 Feb 18.

U-Medico Inc, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, Japan, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan; Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.
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http://dx.doi.org/10.1016/j.xphs.2019.02.002DOI Listing
July 2019

SDS-induced oligomerization of Lys49-phospholipase A from snake venom.

Sci Rep 2019 02 20;9(1):2330. Epub 2019 Feb 20.

Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi, 980-8577, Japan.

Phospholipase A (PLA) is one of the representative toxic components of snake venom. PLAs are categorized into several subgroups according to the amino acid at position 49, which comprises either Asp49, Lys49, Arg49 or Ser49. Previous studies suggested that the Lys49-PLA assembles into an extremely stable dimer. Although the behavior on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions suggested the presence of intermolecular disulfide bonds, these bonds were not observed in the crystal structure of Lys49-PLA. The reason for this discrepancy between the crystal structure and SDS-PAGE of Lys49-PLA remains unknown. In this study, we analyzed a Lys49-PLA homologue from Protobothrops flavoviridis (PflLys49-PLA BPII), by biophysical analyses including X-ray crystallography, SDS-PAGE, native-mass spectrometry, and analytical ultracentrifugation. The results demonstrated that PflLys49-PLA BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.
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http://dx.doi.org/10.1038/s41598-019-38861-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382788PMC
February 2019

Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4.

Sci Rep 2019 02 6;9(1):1503. Epub 2019 Feb 6.

Department of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, 599-8531, Japan.

Several dog allergens cause allergic reactions in humans worldwide. Seven distinct dog allergens, designated Canis familiaris allergen 1 to 7 (Can f 1-Can f 7), have been identified thus far. Can f 6 shows high sequence similarity and cross-reactivity with Fel d 4 and Equ c 1, major cat and horse allergens, respectively. This study was conducted on the allergenic epitopes of Can f 6 based on its structural characterization. We demonstrated that sera from 18 out of 38 (47%) dog-sensitized patients reacted to recombinant Can f 6 protein (rCan f 6). We then determined the crystal structure of rCan f 6 by X-ray crystallography, which exhibited a conserved tertiary structural architecture found in lipocalin family proteins. Based on the tertiary structure and sequence similarities with Fel d 4 and Equ c 1, we predicted three IgE-recognizing sites that are possibly involved in cross-reactivity. Substituting three successive amino acids in these sites to triple alanine decreased IgE reactivity to the allergen. However, the degree of reduction in IgE reactivity largely depended on the site mutated and the serum used, suggesting that Can f 6 is a polyvalent allergen containing multiple epitopes and Can f 6-reactive sera contain varied amounts of IgE recognising individual Can f 6 epitopes including those predicted in this study. We also demonstrated that the predicted epitopes are partly involved in IgE cross-reactivity to Fel d 4. Interestingly, the effect of the mutation depended on whether the protein was structured or denatured, indicating that the bona fide tertiary structure of Can f 6 is essential in determining its IgE epitopes.
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http://dx.doi.org/10.1038/s41598-018-38134-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365566PMC
February 2019

[JIRA's Activity toward the Global Convergence of Medical Device Regulation].

Authors:
Susumu Uchiyama

Nihon Hoshasen Gijutsu Gakkai Zasshi 2019;75(1):125-127

Japan Medical Imaging and Radiological Systems Industries Association, International Department.

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http://dx.doi.org/10.6009/jjrt.2019_JSRT_75.1.125DOI Listing
July 2019