Biosensing-based quality control monitoring of the higher-order structures of therapeutic antibody domains.

Advanced biopharmaceutical manufacturing requires novel process analytical technologies for the rapid and sensitive assessment of the higher-order structures of therapeutic proteins. However, conventional physicochemical analyses of denatured proteins have limitations in terms of sensitivity, throughput, analytical resolution, and real-time monitoring capacity. Although probe-based sensing can overcome these limitations, typical non-specific probes lack analytical resolution and provide little to no information regarding which parts of the protein structure have been collapsed. To meet these analytical demands, we generated biosensing probes derived from artificial proteins that could specifically recognize the higher-order structural changes in antibodies at the protein domain level. Biopanning of phage-displayed protein libraries generated artificial proteins that bound to a denatured antibody domain, but not its natively folded structure, with nanomolar affinity. The protein probes not only recognized the higher-order structural changes in intact IgGs but also distinguished between the denatured antibody domains. These domain-specific probes were used to generate response contour plots to visualize the antibody denaturation caused by various process parameters, such as pH, temperature, and holding time for acid elution and virus inactivation. These protein probes can be combined with established analytical techniques, such as surface plasmon resonance for real-time monitoring or plate-based assays for high-throughput analysis, to aid in the development of new analytical technologies for the process optimization and monitoring of antibody manufacturing.

Alternative autophagy dampens UVB-induced NLRP3 inflammasome activation in human keratinocytes.

Sunlight exposure results in an inflammatory reaction of the skin commonly known as sunburn, which increases skin cancer risk. In particular, the ultraviolet B (UVB) component of sunlight induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses. Here, we explore the intracellular machinery that maintains skin homeostasis by suppressing UVB-induced inflammasome activation in human keratinocytes. We found that pharmacological inhibition of autophagy promoted UVB-induced NLRP3 inflammasome activation. Unexpectedly, however, gene silencing of Atg5 or Atg7, which are critical for conventional autophagy, had no effect, whereas gene silencing of Beclin1, which is essential not only for conventional autophagy but also for Atg5/Atg7-independent alternative autophagy, promoted UVB-induced inflammasome activation, indicating an involvement of alternative autophagy. We found that damaged mitochondria were highly accumulated in UVB-irradiated keratinocytes when alternative autophagy was inhibited, and they appear to be recognized by NLRP3. Overall, our findings indicate that alternative autophagy, rather than conventional autophagy, suppresses UVB-induced NLRP3 inflammasome activation through the clearance of damaged mitochondria in human keratinocytes and illustrate a previously unknown involvement of alternative autophagy in inflammation. Alternative autophagy may be a new therapeutic target for sunburn and associated cutaneous disorders.

Subcellular localization and ER-mediated cytotoxic function of α1A and α1ACT in spinocerebellar ataxia type 6.

Spinocerebellar ataxia type 6 (SCA6) is a polyglutamine (polyQ) disease, which is caused by the elongation of CAG repeats encoding polyQ in the CACNA1A gene. The CACNA1A gene encodes two proteins, namely, α1A (a subunit of the plasma membrane calcium channel), which is translated in its entire length, and α1ACT, which is translated from the second cistron, and both proteins have a polyQ tract. The α1A-polyQ and α1ACT-polyQ proteins with an elongated polyQ stretch have been reported to form aggregates in cells and induce neuronal cell death, but the subcellular localization of these proteins and their cytotoxic properties remain unclear. In this study, we first analyzed SCA6 model mice and found that α1A-polyQlong localized mainly to the Golgi apparatus, whereas a portion of α1ACT-polyQlong localized to the nucleus. Analysis using Neuro2a cells also showed similar subcellular localizations of these proteins, and a proportion of both proteins localized to the endoplasmic reticulum (ER). Cytotoxic studies demonstrated that both proteins induce both the ER stress response and apoptosis, indicating that they are able to induce ER stress-induced apoptosis.

An Overview of Golgi Membrane-Associated Degradation (GOMED) and Its Detection Methods.

Autophagy is a cellular mechanism that utilizes lysosomes to degrade its own components and is performed using Atg5 and other molecules originating from the endoplasmic reticulum membrane. On the other hand, we identified an alternative type of autophagy, namely, Golgi membrane-associated degradation (GOMED), which also utilizes lysosomes to degrade its own components, but does not use Atg5 originating from the Golgi membranes. The GOMED pathway involves Ulk1, Wipi3, Rab9, and other molecules, and plays crucial roles in a wide range of biological phenomena, such as the regulation of insulin secretion and neuronal maintenance. We here describe the overview of GOMED, methods to detect autophagy and GOMED, and to distinguish GOMED from autophagy.

Optimization of Flow Imaging Microscopy Setting Using Spherical Beads with Optical Properties Similar to Those of Biopharmaceuticals.

Flow imaging microscopy (FIM) is widely used to characterize biopharmaceutical subvisible particles (SVPs). The segmentation threshold, which defines the boundary between the particle and the background based on pixel intensity, should be properly set for accurate SVP quantification. However, segmentation thresholds are often subjectively and empirically set, potentially leading to variations in measurements across instruments and operators. In the present study, we developed an objective method to optimize the FIM segmentation threshold using poly(methyl methacrylate) (PMMA) beads with a refractive index similar to that of biomolecules. Among several candidate particles that were evaluated, 2.5-µm PMMA beads were the most reliable in size and number, suggesting that the PMMA bead size analyzed by FIM could objectively be used to determine the segmentation threshold for SVP measurements. The PMMA bead concentrations measured by FIM were highly consistent with the indicative concentrations, whereas the PMMA bead size analyzed by FIM decreased with increasing segmentation threshold. The optimal segmentation threshold where the analyzed size was closest to the indicative size differed between an instrument with a black-and-white camera and that with a color camera. Inter-instrument differences in SVP concentrations in acid-stressed recombinant adeno-associated virus (AAV) and protein aggregates were successfully minimized by setting an optimized segmentation threshold specific to the instrument. These results reveal that PMMA beads can aid in determining a more appropriate segmentation threshold to evaluate biopharmaceutical SVPs using FIM.

Determination of the optimal connector length to enhance stability of backbone-circularized granulocyte colony-stimulating factor.

Improving protein stability is important for industrial applications, and one promising method for achieving this is backbone circularization. As connector length affects stability, predicting and elucidating a more stable connector length is necessary for development of the backbone circularization method. However, the relationship between connector length and protein stability has not been completely elucidated. Here, we determined the most stable connector length for granulocyte colony-stimulating factor by changing one residue at a time to produce connector length variants and then measuring their thermal stability. Analysis of the local structures obtained from the predicted structures of the circularized variants revealed that an approach using helix length, dihedral backbone angle, and number of unbonded hydrogen bond donors and acceptors is suitable for identifying connector lengths with higher stability.

Involvement of casein kinase 1 epsilon/delta (Csnk1e/d) in the pathogenesis of familial Parkinson's disease caused by CHCHD2.

Parkinson's disease (PD) is a common neurodegenerative disorder that results from the loss of dopaminergic neurons. Mutations in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) gene cause a familial form of PD with α-Synuclein aggregation, and we here identified the pathogenesis of the T61I mutation, the most common disease-causing mutation of CHCHD2. In Neuro2a cells, CHCHD2 is in mitochondria, whereas the T61I mutant (CHCHD2T61I ) is mislocalized in the cytosol. CHCHD2T61l then recruits casein kinase 1 epsilon/delta (Csnk1e/d), which phosphorylates neurofilament and α-Synuclein, forming cytosolic aggresomes. In vivo, both Chchd2T61I knock-in and transgenic mice display neurodegenerative phenotypes and aggresomes containing Chchd2T61I , Csnk1e/d, phospho-α-Synuclein, and phospho-neurofilament in their dopaminergic neurons. Similar aggresomes were observed in a postmortem PD patient brain and dopaminergic neurons generated from patient-derived iPS cells. Importantly, a Csnk1e/d inhibitor substantially suppressed the phosphorylation of neurofilament and α-Synuclein. The Csnk1e/d inhibitor also suppressed the cellular damage in CHCHD2T61I -expressing Neuro2a cells and dopaminergic neurons generated from patient-derived iPS cells and improved the neurodegenerative phenotypes of Chchd2T61I mutant mice. These results indicate that Csnk1e/d is involved in the pathogenesis of PD caused by the CHCHD2T61I mutation.

Cue to Acid-Induced Long-Range Conformational Changes in an Antibody Preceding Aggregation: The Structural Origins of the Subpeaks in Kratky Plots of Small-Angle X-ray Scattering.

Antibody aggregation, followed by acid denaturation and neutralization of pH, is one of the reasons why the production of therapeutic monoclonal antibodies (mAbs) is expensive. Determining the structural details of acid-denatured antibodies is important for understanding their aggregation mechanism and for antibody engineering. Recent research has shown that monoclonal antibodies of human/humanized immunoglobulin G1 (IgG1) become smaller globules at pH 2 compared to their native structure at pH 7. This acid-denatured species is unstable at pH 7 and prone to aggregation by neutralization of pH. Small-angle X-ray scattering (SAXS) data have revealed an acid-induced reduction in the subpeaks in Kratky plot, indicating conformational changes that can lead to aggregation. The subpeaks are well resolved at pH > 3 but less pronounced at pH ≤ 2. One of the weakened subpeaks indicates loosely organized inter-region (Fab-Fab and Fab-Fc) correlations due to acid denaturation. However, the structural origin of the other subpeak (called q3 peak in this study) has not been established because its q region could represent the various inter-region, inter-domain, and intra-domain correlations in IgG1. In this study, we aimed to untangle the effects of domain-domain correlations on Kratky's q3 peak based on the computed SAXS of the crystal structure of IgG1. The q3 peak appeared in the static structure and was more prominent in the Fc region than in the Fab or isolated domains. Further brute-force analysis indicated that longer domain-domain correlations, including the inter-region, also positively contribute to Kratky's q3 peak. Thus, the distortion of the Fc region and a longer inter-region correlation initiate acid denaturation and aggregation.

Getting Smaller by Denaturation: Acid-Induced Compaction of Antibodies.

Protein denaturation is a ubiquitous process that occurs both in vitro and in vivo. While our molecular understanding of the denatured structures of proteins is limited, it is commonly accepted that the loss of unique intramolecular contacts makes proteins larger. Herein, we report compaction of the immunoglobulin G1 (IgG1) protein upon acid denaturation. Small-angle X-ray scattering coupled with size exclusion chromatography revealed that IgG1 radii of gyration at pH 2 were ∼75% of those at a neutral pH. Scattering profiles showed a compact globular shape, supported by analytical ultracentrifugation. The acid denaturation of proteins with a decrease in size is energetically costly, and acid-induced compaction requires an attractive force for domain reorientation. Such intramolecular aggregation may be widespread in immunoglobulin proteins as noncanonical structures. Herein, we discuss the potential biological significance of these noncanonical structures of antibodies.

Absence of ULK1 decreases AMPK activity in the kidney, leading to chronic kidney disease progression.

AMP-activated protein kinase (AMPK) inactivation in chronic kidney disease (CKD) leads to energy status deterioration in the kidney, constituting the vicious cycle of CKD exacerbation. Unc-51-like kinase 1 (ULK1) is considered a downstream molecule of AMPK; however, it was recently reported that the activity of AMPK could be regulated by ULK1 conversely. We demonstrated that AMPK and ULK1 activities were decreased in the kidneys of CKD mice. However, whether and how ULK1 is involved in the underlying mechanism of CKD exacerbation remains unknown. In this study, we investigated the ULK1 involvement in CKD, using ULK1 knockout mice. The CKD model of Ulk1-/- mice exhibited significantly exacerbated renal function and worsening renal fibrosis. In the kidneys of the CKD model of Ulk1-/- mice, reduced AMPK and its downstream ß-oxidation could be observed, leading to an energy deficit of increased AMP/ATP ratio. In addition, AMPK signaling in the kidney was reduced in control Ulk1-/- mice with normal renal function compared to control wild-type mice, suggesting that ULK1 deficiency suppressed AMPK activity in the kidney. This study is the first to present ULK1 as a novel therapeutic target for CKD treatment, which regulates AMPK activity in the kidney.

Characterization and Value Assignment of a Monoclonal Antibody Reference Material, NMIJ RM 6208a, AIST-MAB.

Monoclonal antibodies have been established as the largest product class of biopharmaceuticals. Since extensive characterization is required for development and quality control of monoclonal antibody, a widely available reference material (RM) is needed. Herein, a humanized IgG1κ monoclonal antibody reference material, RM 6208-a, AIST-MAB, was established by the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). The monoclonal antibody solution was produced as a pharmaceutical grade using a Chinese hamster ovary-derived cell line. The assigned indicative value represents the concentration of the antibody with a heterotetrameric structure including oligomeric forms, determined by an amino acid analysis using isotope dilution mass spectrometry, and their homogeneity and stability were assessed. In addition to antibody concentration, various physicochemical properties, including peptide mapping data, charge variants, and aggregates, were examined. This RM is intended for use in validation of analytical procedures and instruments such as a system suitability test for quantification of antibody. It is also intended for comparing and evaluating the results of antibody analyses across analytical methods and analytical laboratories such as inter-laboratory comparison. Both the material and the set of data from our study provide a tool for an accurate and reliable characterization of product quality attributes of monoclonal antibodies in biopharmaceutical and metrology communities.

Live-cell imaging to analyze intracellular aggregation of recombinant IgG in CHO cells.

Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality.

Investigations into the Potential of Using Open Source CFD to Analyze the Differences in Hemodynamic Parameters for Aortic Dissections (Healthy versus Stanford Type A and B).

BACKGROUND: The objective of this study was to develop a method to evaluate the effects of an aortic dissection on hemodynamic parameters by conducting a comparison with that of a healthy (nondissected) aorta. Open-source software will be implemented, no proprietary software/application will be used to ensure accessorily and repeatability, in all the data analysis and processing. Computed tomography (CT) images of aortic dissection are used for the model geometry segmentation. Boundary conditions from literature are implemented to computational fluid dynamics (CFD) to analyze the hemodynamic parameters. METHODS: A numerical simulation model was created by obtaining accurate 3-dimensional geometries of aortae from CT images. In this study, CT images of 8 cases of aortic dissection (Stanford type-A and type-B) and 3 cases of healthy aortae are used for the actual aorta model geometry segmentation. These models were exported into an open-source CFD software, OpenFOAM, where a simplified pulsating flow was simulated by controlling the flow pressure. Ten cycles of the pulsatile flow (0.50 sec/cycle) conditions, totaling 5 sec, were calculated. RESULTS: The pressure distribution, wall shear stress (WSS) and flow velocity streamlines within the aorta and the false lumen were calculated and visualized. It was found that the flow velocity and WSS had a high correlation in high WSS areas of the intermittent layer between the true and false lumen. Most of the Stanford type-A dissections in the study showed high WSS, over 38 Pa, at the systole phase. This indicates that the arterial walls in type-A dissections are more likely to be damaged with pulsatile flow. CONCLUSIONS: Using CFD to estimate localized high WSS areas may help in deciding to treat a type-A or B dissection with a stent graft to prevent a potential rupture.

Nano-Microscopy of Therapeutic Antibody Aggregates in Solution.

Scanning electron-assisted dielectric microscopy (SE-ADM) is a new microscope technology developed to observe the fine structure of biological samples in aqueous solution. One main advantage of SE-ADM is that it does not require sample pretreatment, including dehydration, drying, and staining, which is indispensable in conventional scanning electron microscopy (SEM) and can cause sample deformation. In addition, the sample is not directly irradiated with an electron beam in SE-ADM, further avoiding damage. The resolution of SE-ADM is higher than that of an optical microscope, which is typically used for observing biological samples in a solution, allowing for the observation of the detailed structure of samples. Considering these advantages, we applied SE-ADM to observe aggregates of therapeutic immunoglobulin G (IgG) of various sizes and shapes in an aqueous solution. In this chapter, we outline the step-by-step procedure for observing aggregates of monoclonal antibodies using SE-ADM and the subsequent analysis of the particle distribution and calculation of the fractal dimension using SE-ADM image data. The proposed method for particle analysis is highly reliable with respect to size measurement and can determine the diameter of a sample with an accuracy of ±20%, a precision of ±10%, and a lower limit of quantification of ≤50 nm. Further, by calculating the fractal dimension of the image, it is possible to classify the shape of the aggregates and determine the mechanism of aggregation.

Effect of backbone circularization on colloidal stability: Compaction of unfolded structures improves aggregation resistance of granulocyte colony-stimulating factor.

Aggregation of protein therapeutics can lead to immunogenicity and loss of function in vivo. Its effective prevention requires an understanding of the conformational and colloidal stability of protein and the improvement of both. Granulocyte colony-stimulating factor (G-CSF), which is one of the most widely used protein therapeutics, was previously shown to be conformationally stabilized by connecting its N- and C-termini with amide bonds (backbone circularization). In this study, we investigated whether circularization affects the colloidal stability of proteins. Colloidal stability was indirectly assessed by analyzing the aggregation behavior of G-CSF variants using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). Consequently, we found that the unfolded structure of circularized G-CSF was more compact than non-circularized G-CSF, and that backbone circularization improved its aggregation resistance against chemical denaturation by guanidine hydrochloride (GdnHCl). The improved aggregation resistance suggests that the expansion tolerance of circularized G-CSF in the unfolded state increased its colloidal stability. Thus, backbone circularization is an excellent method for enhancing the colloidal and the conformational stability of protein with minimal sequence changes. It is therefore expected to be effective in extending the storage stability of protein therapeutics, enhancing their biological stability.

Local disorder of the C-terminal segment of the heavy chain as a common sign of stressed antibodies evidenced with a peptide affinity probe specific to non-native IgG.

Therapeutic antibodies have many biopharmaceutical applications; however, characterization of their higher-order structures is a major concern in quality control. We have developed AF.2A1, an artificial protein, that specifically recognizes non-native, structured IgGs. We performed binding assays using various types of IgGs and fragments to investigate the mechanisms by which AF.2A1 interacts with the non-native IgG. AF.2A1 recognized the acid-stressed IgGs from human, mouse, and rat, but not rabbit. Binding assays using the human IgG1 fragments revealed that an interface emerged by deleting five C-terminal residues. We conclude that AF.2A1 recognizes an exposed hydrophobic core centered on the Trp417. Our results concur with those of the previous studies showing that C-terminal structural changes occur early during antibody denaturation and aggregation. Our findings explain the molecular rationale for using AF.2A1 in quality control of biopharmaceutical IgGs.

Polyploid engineering by increasing mutant gene dosage in yeasts.

The yeast Saccharomyces cerevisiae, widely used for ethanol production, is one of the best-understood biological systems. Diploid strains of S. cerevisiae are preferred for industrial use due to the better fermentation efficiency, in terms of vitality and endurance as compared to those of haploid strains. Whole-genome duplications is known to promote adaptive mutations in microorganisms, and allelic variations considerably contribute to the product composition in ethanol fermentation. Although fermentation can be regulated using various strains of yeast, it is quite difficult to make fine adjustment of each component in final products. In this study, we demonstrate the use of polyploids with varying gene dosage (the number of copies of a particular gene present in a genome) in the regulation of ethanol fermentation. Ethyl caproate is one of the major flavouring agents in a Japanese alcoholic beverage called sake. A point mutation in FAS2 encoding the α subunit of fatty acid synthetase induces an increase in the amount of caproic acid, a precursor of ethyl caproate. Using the FAS2 as a model, we generated and evaluated yeast strains with varying mutant gene dosage. We demonstrated the possibility to increase mutant gene dosage via loss of heterozygosity in diploid and tetraploid strains. Productivity of ethyl caproate gradually increased with mutant gene dosage among tetraploid strains. This approach can potentially be applied to a variety of yeast strain development via growth-based screening.

Wipi3 is essential for alternative autophagy and its loss causes neurodegeneration.

Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.

Autophagy involvement in oncogenesis.

Various clinical and experimental findings have revealed the causal relationship between autophagy failure and oncogenesis, and several mechanisms have been suggested to explain this relationship. We recently proposed two additional mechanisms: centrosome number dysregulation and the failure of autophagic cell death. Here, we detail the mechanical relationship between autophagy failure and oncogenesis.

Identification of a phosphorylation site on Ulk1 required for genotoxic stress-induced alternative autophagy.

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy.