Catechins prevent monoclonal antibody fragmentation during production via fed-batch culture of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used for the industrial production of therapeutic monoclonal antibodies (mAbs). To meet the increasing market demands, high productivity, and quality are required in cell culture. One of the critical attributes of mAbs, from a safety perspective, is mAb fragmentation. However, methods for preventing mAbs fragmentation in CHO cell culture are limited. In this study, we observed that the antibody fragment content increased with increasing titers in fed-batch cultures for all three cell lines expressing recombinant antibodies. Adding copper sulfate to the culture medium further increased the fragment content, suggesting the involvement of reactive oxygen species (ROS) in the fragmentation process. Though antioxidants may be helpful to scavenge ROS, several antioxidants are reported to decrease the productivity of CHO cells. Among the antioxidants examined, we observed that the addition of catechin or (-)-epigallocatechin gallate to the culture medium prevented fragmentation content by about 20% and increased viable cell density and titer by 30% and 10%, respectively. Thus, the addition of catechins or compounds of equivalent function would be beneficial for manufacturing therapeutic mAbs with a balance between high titers and good quality.

Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.

New high-throughput screening method for Chinese hamster ovary cell lines expressing low reduced monoclonal antibody levels: application of a system controlling the gas phase over cell lysates in miniature bioreactors and facilitating multiple sample setup.

Interchain disulfide bonds in monoclonal antibodies may be reduced during large-scale mAb production using Chinese hamster ovary (CHO) cells. This reaction lowers the mAb product yield and purity; however, it may be prevented by screening cell lines that are unsusceptible to reduction and using them in mAb production. Antibody reduction susceptibility may be cell line-dependent. To the best of our knowledge, however, an efficient method of screening reduction-unsusceptible CHO cell lines has not been previously reported. Here, we report a novel screening method that can simultaneously detect and identify mAb reduction susceptibility in lysates containing ≤ 48 CHO cell lines. This evaluation system was equally effective and generated similar results at all culture scales, including 250 mL, 3 L, and 1000 L. Furthermore, we discovered that reduction-susceptible cell lines contained higher total intracellular nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ concentrations than reduction-unsusceptible cell lines, regardless of whether they expressed immunoglobulin (Ig)G4 or IgG1. NADPH or NADP+ supplementation in the lysate of reduction-unsusceptible cells resulted in mAb reduction. Application of the innovative CHO cell line screening approach could mitigate or prevent reductions in large-scale mAb generation from CHO cells.

Clip Formation in the Complementarity Determining Region of Bevacizumab Lowers Monomer Stability and Affinity for Both FcRn and FcγR: A Comprehensive Characterization of the Clipped Variant Including its Higher Order Structure.

The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, have been reported. However, the properties of CDR-clipped variants are not fully understood because of difficulties in separating them from intact mAbs under non-denaturing conditions due to similarities in size. We have established a method for separating CDR-clipped variants under non-denaturing conditions using an appropriate size exclusion chromatography column.1 In this report, we provide a comprehensive characterization of a CDR-clipped variant from bevacizumab. The variant exhibited a lower pI, a higher tendency to form dimers, and a lower affinity for both neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR). The effects of clip formation in CDR H3 on the higher order structure were analyzed by hydrogen/deuterium exchange mass spectrometry, and the observed changes in the structures of the VH, CH2, and VL domains were in agreement with the lowered affinity for antigen, FcRn, and FcγR. These findings suggest that clip formation in the CDR may affect the efficacy, safety, and pharmacokinetics of pharmaceutical mAbs.

The N93D mutation of the human T-cell leukemia virus type 1 envelope glycoprotein found in symptomatic patients enhances neuropilin-1 b1 domain binding.

Human T-cell leukemia virus type 1 (HTLV-1) infection of host cells is mainly mediated by interactions with the viral envelope glycoprotein surface unit (SU) and three host receptors: heparan sulfate proteoglycan, neuropilin-1 (Nrp1), and glucose transporter type 1. Residues 90-94 of SU are considered as a Nrp1 binding site, and our previous results show that an SU peptide consisting of residues 85-94 can bind directly to the Nrp1 b1 domain with a binding affinity of 7.4 µM. Therefore, the SU peptide is expected to be a good model to investigate the SU-Nrp1 interaction. Recently, the N93D mutation in the Nrp1 b1 binding region of the SU was identified in symptomatic patients with HTLV-1 infections in the Brazilian Amazon. However, it remains unclear how the SU-N93D mutation affects Nrp1 b1 binding. To elucidate the impact of the substituted Asp93 of SU on Nrp1 b1 binding, we analyzed the interaction between the SU-N93D peptide and Nrp1 b1 using isothermal titration calorimetry and nuclear magnetic resonance. The SU-N93D peptide binds directly to Nrp1 b1 with a binding affinity of 3.5 µM, which is approximately two-fold stronger than wild-type. This stronger binding is likely a result of the interaction between the substituted residue Asp93 of the N93D peptide and the four residues Trp301, Lys347, Glu348, and Thr349 of Nrp1 b1. Our results suggest that the interaction of SU Asp93 with the four residues of Nrp1 b1 renders the high affinity of the N93D mutant for Nrp1 b1 binding during HTLV-1 entry.

Identification and Characterization of a Monoclonal Antibody Variant Species with a Clipping in the Complementarity Determining Region Isolated by Size Exclusion Chromatography Under Native Conditions.

The content of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Peptide bonds in the hinge region of mAbs are susceptible to hydrolysis, generating Fc-Fab fragments, which are associated with lower efficacy than the intact antibody. Fc-Fab fragments can be separated from intact antibody molecules under native conditions by size exclusion chromatography (SEC). Although several fragments generated by a clip in the complementarity determining region (CDR) have been reported, their efficacies have not been analyzed. This is because these fragments could not be separated from intact antibodies under native conditions owing to their similar molecular sizes. Here, we report that bevacizumab variant with clipping in the CDR, with the resulting fragments remaining intact in the variant, can be isolated under native conditions by selecting an adequate SEC column.

The prevention of an anomalous chromatographic behavior and the resulting successful removal of viruses from monoclonal antibody with an asymmetric charge distribution by using a membrane adsorber in highly efficient, anion-exchange chromatography in flow-through mode.

Anion exchange (AEX) chromatography in the flow-through mode is a widely employed purification process for removal of process/product-related impurities and exogenous/endogenous viruses from monoclonal antibodies (mAbs). The pH of the mobile phase for AEX chromatography is typically set at half a unit below the isoelectric point (pI) of each mAb (i.e., pI - 0.5) or lower and, in combination with a low ionic strength, these conditions are usually satisfactory for both the recovery of the mAb and removal of impurities. However, we have recently encountered a tight binding of mAb1 to AEX resins under these standard chromatographic conditions. This anomalous adsorption behavior appears to be an effect of the asymmetric charge distribution on the surface of the mAb1. We found that mAb1 did not bind to the AEX resins if the mobile phase has a much lower pH and higher ionic strength, but those conditions would not allow adequate virus removal. We predicted that the use of membrane adsorbers might provide effective mAb1 purification, since the supporting matrix has a network structure that would be less susceptible to interactions with the asymmetric charge distribution on the protein surface. We tested the Natriflo HD-Q AEX membrane adsorber under standard chromatographic conditions and found that mAb1 flowed through the membrane adsorber, resulting in successful separation from murine leukemia virus. This AEX membrane adsorber is expected to be useful for process development because mAbs can be purified under similar standard chromatographic conditions regardless of their charge distributions.

Changes of urine metabolite profiles are induced by inactivated influenza vaccine inoculations in mice.

The safety evaluation of vaccines is critical to avoid the development of side effects in humans. To increase the sensitivity of detection for toxicity tests, it is important to capture not only pathological changes but also physiological changes. 1H nuclear magnetic resonance (NMR) spectroscopy analysis of biofluids produces profiles that show characteristic responses to changes in physiological status. In this study, mouse urine metabolomics analysis with 1H NMR was performed using different influenza vaccines of varying toxicity to assess the usefulness of 1H NMR in evaluating vaccine toxicity. Two types of influenza vaccines were used as model vaccines: a toxicity reference vaccine (RE) and a hemagglutinin split vaccine. According to the blood biochemical analyses, the plasma alanine transaminase levels were increased in RE-treated mice. Changes in metabolite levels between mice administered different types of influenza vaccines were observed in the 1H NMR spectra of urine, and a tendency toward dosage-dependent responses for some spectra was observed. Hierarchical clustering analyses and principal component analyses showed that the changes in various urine metabolite levels allowed for the classification of different types of vaccines. Among them, two liver-derived metabolites were shown to largely contribute to the formation of the cluster. These results demonstrate the possibility that urine metabolomics analysis could provide information about vaccine-induced toxicity and physiological changes.

NMR characterization of the interaction between Bcl-xL and the BH3-like motif of hepatitis B virus X protein.

Hepatitis B virus X protein (HBx) possesses a BH3-like motif that directly interacts with the anti-apoptotic proteins, Bcl-2 and Bcl-xL. Here we report the interaction between the HBx BH3-like motif and Bcl-xL, as revealed by nuclear magnetic resonance spectroscopy. Our results showed that this motif binds to the common BH3-binding hydrophobic groove on the surface of Bcl-xL, with a binding affinity of 89 µM. Furthermore, we examined the role of the tryptophan residue (Trp120) in this motif in Bcl-xL binding using three mutants. The W120A mutant showed weaker binding affinity (294 µM) to Bcl-xL, whereas the W120L and W120F mutants exhibited almost equivalent binding affinity to the wild-type. These results indicate that the bulky hydrophobic residues are important for Bcl-xL binding. The findings will be helpful in understanding the apoptosis networks between viral proteins and host factors.

Loss of Glutamate Decarboxylase 67 in Somatostatin-Expressing Neurons Leads to Anxiety-Like Behavior and Alteration in the Akt/GSK3ß Signaling Pathway.

Major depressive disorder (MDD) is a highly prevalent psychiatric disorder worldwide. Several lines of evidence suggest that the dysfunction of somatostatin (SOM) neurons is associated with the pathophysiology of MDD. Importantly, most SOM neurons are γ-aminobutyric acid (GABA) interneurons. However, whether the dysfunction of GABAergic neurotransmission from SOM neurons contributes to the pathophysiology of MDD remains elusive. To address this issue, we investigated the emotional behaviors and relevant molecular mechanism in mice lacking glutamate decarboxylase 67 (GAD67), an isoform of GABA-synthesizing enzyme, specifically in SOM neurons (SOM-GAD67 mice). The SOM-GAD67 mice exhibited anxiety-like behavior in the open-field test without an effect on locomotor activity. The SOM-GAD67 mice showed depression-like behavior in neither the forced swimming test nor the sucrose preference test. In addition, the ability to form contextual fear memory was normal in the SOM-GAD67 mice. Furthermore, the plasma corticosterone level was normal in the SOM-GAD67 mice both under baseline and stress conditions. The expression ratios of p-AktSer473/Akt and p-GSK3ßSer9/GSK3ß were decreased in the frontal cortex of SOM-GAD67 mice. Taken together, these data suggest that the loss of GAD67 from SOM neurons may lead to the development of anxiety-like but not depression-like states mediated by modification of Akt/GSK3ß activities.

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies.

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.

Observation of Protein and Lipid Membrane Structures in a Model Mimicking the Molecular-Crowding Environment of Cells Using Neutron Scattering and Cell Debris.

The interior of living cells is a molecular-crowding environment, where large quantities of various molecules coexist. Investigations into the nature of this environment are essential for an understanding of both the elaborate biological reactions and the maintenance of homeostasis occurring therein. The equilibrium states of biological macromolecular systems are affected by molecular-crowding environments unmatched by in vitro diluted environments; knowledge about crowding effects is still insufficient due to lack of relevant experimental studies. Recent developments in the techniques of in-cell NMR and large-scale molecular dynamics simulation have provided new insights into the structure and dynamics of biological molecules inside the cells. This study focused on a new experimental technique to directly observe the structure of a specific protein or membrane in condensed crowder solutions using neutron scattering. Deuterated whole-cell debris was used to reproduce an environment that more closely mimics the interior of living cells than models used previously. By the reduction of the background scattering from large amounts of cell debris, we successfully extracted structure information for both small globular protein and small unilamellar vesicle (SUV) from the concentrated cell-debris solution up to a weight ratio of 1:60 for protein/crowder and 1:40 for SUV/crowder.

In-frame Val216-Ser217 deletion of KIT in mild piebaldism causes aberrant secretion and SCF response.

BACKGROUND: Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. OBJECTIVE: The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. METHODS: We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. RESULTS: A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val216 and Ser217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF); however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. CONCLUSION: These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism.

A novel neuropilin-1-binding sequence in the human T-cell lymphotropic virus type 1 envelope glycoprotein.

Entry of human T-cell lymphotropic virus type 1 (HTLV-1) into host cells is mainly mediated by interactions between the viral envelope glycoprotein surface unit (SU) and three host receptors: glucose transporter type 1, heparin/heparan sulfate proteoglycan, and neuropilin-1 (Nrp1). Here, we analyzed the interaction between HTLV-1 SU and Nrp1 using nuclear magnetic resonance and isothermal titration calorimetry. We found that two SU peptides, residues 85-94 and residues 304-312, bound directly to the Nrp1 b1 domain with affinities of 7.4 and 17.7 µM, respectively. The binding modes of both peptides were almost identical to those observed for Tuftsin and vascular endothelial growth factor A binding to the Nrp1 b1 domain. These results suggest that the C-terminal region of HTLV-1 SU contains a novel site for direct binding of virus to the Nrp1 b1 domain. Our biophysical characterization of the SU peptides may help in developing inhibitors of HTLV-1 entry.

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies.

Hepatitis B virus X protein (HBx) is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level.

Identification of Candidate Genes for Generalized Tonic-Clonic Seizures in Noda Epileptic Rat.

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

Structural basis for Ccd1 auto-inhibition in the Wnt pathway through homomerization of the DIX domain.

Wnt signaling plays an important role in governing cell fate decisions. Coiled-coil-DIX1 (Ccd1), Dishevelled (Dvl), and Axin are signaling proteins that regulate the canonical pathway by controlling the stability of a key signal transducer ß-catenin. These proteins contain the DIX domain with a ubiquitin-like fold, which mediates their interaction in the ß-catenin destruction complex through dynamic head-to-tail polymerization. Despite high sequence similarities, mammalian Ccd1 shows weaker stimulation of ß-catenin transcriptional activity compared with zebrafish (z) Ccd1 in cultured cells. Here, we show that the mouse (m) Ccd1 DIX domain displays weaker ability for homopolymerization than that of zCcd1. Furthermore, X-ray crystallographic analysis of mCcd1 and zCcd1 DIX domains revealed that mCcd1 was assembled into a double-helical filament by the insertion of the ß1-ß2 loop into the head-to-tail interface, whereas zCcd1 formed a typical single-helical polymer similar to Dvl1 and Axin. The mutation in the contact interface of mCcd1 double-helical polymer changed the hydrodynamic properties of mCcd1 so that it acquired the ability to induce Wnt-specific transcriptional activity similar to zCcd1. These findings suggest a novel regulatory mechanism by which mCcd1 modulates Wnt signaling through auto-inhibition of dynamic head-to-tail homopolymerization.

Solution structure of the first RNA recognition motif domain of human spliceosomal protein SF3b49 and its mode of interaction with a SF3b145 fragment.

The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598-631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of ß1-α1-ß2-ß3-α2-ß4. Furthermore, a docking model based on NOESY measurements suggests that residues 607-616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix-helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull-down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix-helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145.