Proceedings of the 2023 Viral Clearance Symposium, Session 8: Cell Banks, Advanced Technologies (ATMPs, NGS).

The Cell Banks, Advanced Technologies (ATMPs, NGS) session at the 2023 Viral Clearance Symposium (VCS) focused on the assurance of high virus safety profiles of advanced technology medicinal products (ATMPs) by implementation of advanced virus detection methods using rapid and sensitive technologies, such as next-generation sequencing (NGS). All presentations in this session made the need to replace in vivo testing for viruses by new technologies that have been demonstrated to be incomparably broad in their detection capabilities and can even detect unknown viruses. An evaluation of historical data collected by the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) from their members' in vivo and in vitro adventitious virus test experience as well as on using NGS was presented. The data convincingly supported the necessity to replace in vivo testing with faster, broader, more sensitive, more accurate, and more specific virus detection methods. Additionally, a collaborative study-initiated by the CAACB-with the goal to revisit traditional adventitious agent testing by using targeted NGS to replace in vivo and in vitro tests for well-known and broadly used Chinese hamster ovary (CHO) cells was presented, including the planned risk-assessment approach using prior knowledge and historical data. Overall, this session demonstrated that the use of new virus detection methods, such as NGS, represents a great opportunity to provide sufficient viral safety margins, specifically, for ATMPs, where downstream virus clearance is not possible. This path forward is also supported by the final ICH Q5A(R2) guideline.

Hybridized quantum dot, silica, and gold nanoparticles for targeted chemo-radiotherapy in colorectal cancer theranostics.

Multimodal nanoparticles, utilizing quantum dots (QDs), mesoporous silica nanoparticles (MSNs), and gold nanoparticles (Au NPs), offer substantial potential as a smart and targeted drug delivery system for simultaneous cancer therapy and imaging. This method entails coating magnetic GZCIS/ZnS QDs with mesoporous silica, loading epirubicin into the pores, capping with Au NPs, PEGylation, and conjugating with epithelial cell adhesion molecule (EpCAM) aptamers to actively target colorectal cancer (CRC) cells. This study showcases the hybrid QD@MSN-EPI-Au-PEG-Apt nanocarriers (size ~65 nm) with comprehensive characterizations post-synthesis. In vitro studies demonstrate the selective cytotoxicity of these targeted nanocarriers towards HT-29 cells compared to CHO cells, leading to a significant reduction in HT-29 cell survival when combined with irradiation. Targeted delivery of nanocarriers in vivo is validated by enhanced anti-tumor effects with reduced side effects following chemo-radiotherapy, along with imaging in a CRC mouse model. This approach holds promise for improved CRC theranostics.

Cx1Mab-1: A Novel Anti-mouse CXCR1 Monoclonal Antibody for Flow Cytometry.

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 have been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb, Cx1Mab-1 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Cx1Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx1Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6 × 10-9 M and 2.1 × 10-8 M, respectively. Furthermore, Cx1Mab-1 could detect mCXCR1 by Western blot analysis. These results indicated that Cx1Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.

PMab-314: An Anti-Giant Panda Podoplanin Monoclonal Antibody.

The giant panda (Ailuropoda melanoleuca) is one of the important species in worldwide animal conservation. Because it is essential to understand the disease of giant panda for conservation, histopathological analyses of tissues are important to understand the pathogenesis. However, monoclonal antibodies (mAbs) against giant panda-derived proteins are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. PDPN is also overexpressed in various human tumors, which are associated with poor prognosis. Here, an anti-giant panda PDPN (gpPDPN) mAb, PMab-314 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening method. PMab-314 recognized N-terminal PA16-tagged gpPDPN-overexpressed Chinese hamster ovary-K1 cells (CHO/PA16-gpPDPN) in flow cytometry. The KD value of PMab-314 for CHO/PA16-gpPDPN was determined as 1.3 × 10-8 M. Furthermore, PMab-314 is useful for detecting gpPDPN in western blot analysis. These findings indicate that PMab-314 is a useful tool for the analyses of gpPDPN-expressed cells.

Enhancing the productivity and proliferation of CHO-K1 cells by oncoprotein YAP (Yes-associated protein).

CHO cells are extensively employed in biological drug industry to manufacture therapeutic proteins. Nevertheless, production of biopharmaceuticals faces obstacles such as limited growth and inadequate productivity. Employing host cell engineering techniques for CHO cells serves as a valuable approach to address the constraints encountered in biologics manufacturing. Despite advancements, most techniques focus on specific genes to address individual cellular challenges. The significance of YAP, transcriptional co-activator, cannot be overstated due to its involvement in regulating organ size and tumor formation. YAP's influence extends to various cellular processes and is regulated by kinase cascade in the Hippo pathway, which phosphorylates serine residues in specific LATS recognition motifs. Activation of YAP has been observed to impact both the size and quantity of cells. This research investigates the effects of YAP5SA on proliferation, apoptosis, and productivity in CHO-K1 cells. YAP5SA, with mutations in all five LATS-target sites, is selected for its heightened activity and resistance to repression through the Hippo-LATS1/2 kinase signaling pathway. Plasmid harboring YAP5SA was transfected into EPO-CHO and the influence of YAP5SA overexpression was investigated. According to our findings, transfection of EPO-CHO cells with YAP5SA exhibited a substantial enhancement in CHO cell productivity, resulting in a 3-fold increase in total protein and EPO, as well as a 1.5-fold increase in specific productivity. Additionally, it significantly contributes in augmenting viability, size, and proliferation. Overall, the findings of this study exemplify the potential of utilizing YAP5SA to impact particular cellular mechanisms, thereby presenting an avenue for customizing cells to fulfill production demands. KEY POINTS: • YAP5SA in CHO cells boosts growth, reduces apoptosis, and significantly improves productivity. • YAP5SA regulates genes involved in proliferation, survival, and mTOR activation. • YAP5SA increases productivity by improving cell cycle, c-MYC expression, and mTOR pathway.

Characterization of cellular responses and cell lysis to elevated hydrodynamic stress from benchtop perfusion bioreactors.

The effective design of perfusion cell culture is currently challenging regarding balancing the operating parameters associated with the hydrodynamic conditions due to increased system complexity. To address this issue, cellular responses of an industrial CHO cell line to different types of hydrodynamic stress in benchtop perfusion bioreactors originating from agitation, sparging, and hollow fibers (HF) in the cell retention devices were systematically investigated here with the analysis of cell lysis. It was found that cell lysis was very common and most associated with the sparging stress, followed by the HF and lastly the agitation, consequently heavily impacting the estimation of process descriptors related to biomass. The results indicated that the agitation stress led to a reduced cell growth with a shift toward a more productive phenotype, suggesting an energy redirection from biomass formation to product synthesis, whereas the sparging stress had a small impact on the intracellular metabolic flux distribution but increased the cell death rate drastically. For HF stress, a similar cell maintenance profile was found as the sparging while the activity of glycolysis and the TCA cycle was significantly impeded, potentially leading to the lack of energy and thus a substantial decrease in cell-specific productivity. Moreover, a novel concept of volume average shear stress was developed to further understand the relations of different types of stress and the observed responses for an improved insight for the perfusion cell culture.

Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways.

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.

Cannabidiol exhibits anxiolytic-like effects and antipsychotic-like effects in mice models.

Cannabidiol (CBD), a non-psychoactive compound derived from the cannabis plant, has been confirmed to induce anxiolytic-like and antipsychotic-like effects. However, the exact mechanisms remain unclear. This study substantiated CBD's interaction with the 5-HT1A receptor (5-HT1AR) in vitro (CHO cells expressing human 5-HT1AR) and in vivo (rat lower lip retraction test, LLR test). We then assessed the impact of CBD in mice using the stress-induced hyperthermia (SIH) model and the phencyclidine (PCP)-induced negative symptoms of schizophrenia model, respectively. Concurrently, we investigated whether WAY-100635, a typical 5-HT1AR antagonist, could attenuate these effects. Furthermore, the neurotransmitter changes through high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were studied. Results revealed that CBD exhibits selective 5-HT1AR agonists-mediated effects in the rat lower lip retraction test, aligning with the robust agonistic (EC50 = 1.75 µM) profile observed in CHO cells. CBD at 3 mg/kg significantly reduced SIH (ΔT), a response that WAY-100635 abolished. Chronic administration of CBD at 100 mg/kg mitigated the increase in PCP-induced immobility time in the forced swim test (FST) and tail suspension test (TST). Moreover, it induced significant alterations in gamma-aminobutyric acid (GABA) and norepinephrine (NE) levels within the hippocampus (HPC). Thus, we concluded that the 5-HT1AR mediates CBD's anxiolytic-like effects. Additionally, CBD's effects on the negative symptoms of schizophrenia may be linked to changes in GABA and NE levels in the hippocampus. These findings offer novel insights for advancing the exploration of CBD's anxiolytic-like and antipsychotic-like effects.

Novel purification platform based on multimodal preparative scale separation of mAb fragments and aggregates.

Monoclonal antibodies (mAbs) continue to dominate the biopharmaceutical industry. Certain mAbs are prone to fragmentation and clipping and in these cases, adequate removal of these species is critical during manufacturing. Fragments can be generated during fermentation, purification, storage, formulation, and administration. Their addition to the acidic charge-variant of the purified mAb has been reported to decrease stability and potency of the final product. However, contrary to mAb aggregation, manufacturers have not given much attention to removal of fragments and clipped species and as a result most conventional mAb platforms offer at best limited capabilities for their removal. In this study, we propose a novel purification platform that uses multimodal chromatography and achieves complete removal of a range of mAb fragments and clipped products (25-120 kDa). The utility of the platform has been successfully demonstrated for 2 IgG1s and 2 IgG4s. Further, adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has been achieved. Finally, the platform was able to deliver adequate removal of high molecular weight impurities (<1 %) and a 30 % clearance of the acidic charge variant. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform.

Host cell protein networks as a novel co-elution mechanism during protein A chromatography.

Host cell proteins (HCPs) are process-related impurities of therapeutic proteins produced in for example, Chinese hamster ovary (CHO) cells. Protein A affinity chromatography is the initial capture step to purify monoclonal antibodies or Fc-based proteins and is most effective for HCP removal. Previously proposed mechanisms that contribute to co-purification of HCPs with the therapeutic protein are either HCP-drug association or leaching from chromatin heteroaggregates. In this study, we analyzed protein A eluates of 23 Fc-based proteins by LC-MS/MS to determine their HCP content. The analysis revealed a high degree of heterogeneity in the number of HCPs identified in the different protein A eluates. Among all identified HCPs, the majority co-eluted with less than three Fc-based proteins indicating a drug-specific co-purification for most HCPs. Only ten HCPs co-purified with over 50% of the 23 Fc-based proteins. A correlation analysis of HCPs identified across multiple protein A eluates revealed their co-elution as HCP groups. Functional annotation and protein interaction analysis confirmed that some HCP groups are associated with protein-protein interaction networks. Here, we propose an additional mechanism for HCP co-elution involving protein-protein interactions within functional networks. Our findings may help to guide cell line development and to refine downstream purification strategies.

Goat mammary epithelial cells provide a better expression system for production of recombinant human bone morphogenetic protein 2 compared to Chinese hamster ovarian cells.

Bone Morphogenetic Protein 2 (BMP2), a member of the Transforming Growth Factor-ß (TGF-ß) super family of proteins and is instrumental in the repair of fractures. The synthesis of BMP2 involves extensive post-translational processing and several studies have demonstrated the abysmally low production of rhBMP2 in eukaryotic systems, which may be due to the short half-life of the bioactive protein. Consequently, production costs of rhBMP2 are quite high, limiting its availability to the general populace. Therefore, there is an urgent need to identify better in-vitro systems for large scale production of rhBMP2. In the present study, we have carried out a comparative analysis of rhBMP2 production by the conventionally used Chinese Hamster ovarian cells (CHO) and goat mammary epithelial cells (GMEC), upon transfection with appropriate construct. Udder gland cells are highly secretory, and we reasoned that such cells may serve as a better in-vitro model for large scale production of rhBMP2. Our results indicated that the synthesis and secretion of bioactive rhBMP2 by goat mammary epithelial cells was significantly higher as compared to that by CHO-K1 cells. Our results provide strong evidence that GMECs may serve as a better alternative to other mammalian cells used for therapeutic protein production.

Effects of process intensification on homogeneity of an IgG1:κ monoclonal antibody during perfusion culture.

The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products' efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs). KEY POINTS: • Molecular integrity may suffer with increasing process intensity. • Galactosylated and sialylated N-glycans may decrease. • Perfusion culture appears to maintain protein charge structure.

Safety evaluation of vitamin K2 (menaquinone-7) via toxicological tests.

This study aims to evaluate the safety of MK-7 produced by fermentation process using a Bacillus subtilis var. natto strain for human ingestion via acute oral toxicity, repeated dose 90-day oral toxicity, 28-day recovery test, and genotoxicity tests. The acute oral toxicity test results indicated that all subjects survived at the dose of 5000 mg/kg with no toxic effects. For the repeated dose 90-day oral toxicity test, MK-7 was administered to rats at 500, 1500, and 4500 mg/kg for 90 d. No abnormal findings were detected in clinical observations or in clinical pathological and histopathological examinations. The no-observed-adverse-effect level(NOAEL) was determined to be 4500 mg/kg/d, the maximum dose tested. For the evaluation of genotoxicity, reverse mutation, chromosomal aberration, and micronucleus tests were performed. In the reversion mutation test, vitamin K2 did not induce reversion in bacterial strains, and no chromosomal abnormality was observed in the chromosomal abnormality test using Chinese hamster lung cells. In the micronucleus test, micronuclei were not induced using ICR mouse bone marrow cells. All the toxicity test results suggest that vitamin K2 produced by fermentation processes using Bacillus subtilis var. natto induced no toxicological changes under the experimental conditions.

Pharmacologic Characterization of Substituted Nitazenes at µ, κ, and Δ Opioid Receptors Suggests High Potential for Toxicity.

The benzimidazole opioids (substituted nitazenes) are highly potent µ opiod receptor (MOR) agonists with heroin- or fentanyl-like effects. These compounds have caused hospitalizations and fatal overdoses. We characterized the in vitro pharmacology and structure-activity relationships of 19 nitazenes with substitutions at three positions of the benzimidazole core. Affinities were assessed using agonist radioligand binding assays at human µ, κ, and Δ opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in CHO cells. Notably, for MOR binding, nine substituted nitazenes had significantly higher affinities than fentanyl including N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, and N-desethyl isotonitazene; 13 had subnanomolar affinities. Only metodesnitazene and flunitazene had significantly lower affinities than fentanyl. Affinities for the substituted nitazenes at KOR and DOR relative to MOR were 46- to 2580-fold and 180- to 1280-fold lower, respectively. Functional activities were assessed using [35S]GTPγS binding assays. Four nitazenes had subnanomolar potencies at MOR: N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, N-pyrrilidino protonitazene and N-desethyl isotonitazene. Ten substituted nitazenes had significantly higher potencies than fentanyl. All tested nitazenes were full MOR agonists. Potencies at KOR and DOR relative to MOR were 7.3- to 7920-fold and 24- to 9400-fold lower, respectively. Thus, many of these compounds are high affinity/high potency MOR agonists with elevated potential to elicit toxicity and overdose at low doses. SIGNIFICANCE STATEMENT: Substituted nitazenes are a growing public health threat. Although the 19 nitazenes tested vary in their opioid receptor pharmacology, a number are very high affinity, high potency, and high efficacy compounds- higher than fentanyl. Their pharmacology suggests high potential for harm.

Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing.

Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to various coronaviruses, including the lingering SARS-CoV-2, as this coronavirus must bind to ACE2 to allow for its entry into host cells. ACE2Fc, an investigational new drug developed by Henlius (Shanghai China), has passed the Phase I clinical trial, but its huge amount of charge isoforms and complicated charge heterogeneity posed a challenge to charge variant investigation in pharmaceutical development. We employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed characterization of enriched ACE2Fc fractions, to unveil the structural origins of charge heterogeneity in ACE2Fc expressed by recombinant CHO cells. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, the highest allowable voltage to maximize the FF-IEF separation window and a mild Protein A elution method for preservation of protein structural integrity. Through peptide mapping and other characterizations, we revealed that the intricate profiles of ACE2Fc charge heterogeneity are mainly caused by highly sialylated multi-antenna N-glycosylation. In addition, based on fraction characterization and in silico glycoprotein model analysis, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards Spike (S) protein of SARS-CoV-2. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a quantitative sialylation-activity relationship in ACE2Fc.

The ß2-adrenergic receptor associates with CXCR4 multimers in human cancer cells.

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and ß2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that ß2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

Characterization of Sodium Channel Peptides Obtained from the Venom of the Scorpion Centruroides bonito.

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na+ channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (ß-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide ß-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

Establishment of a Novel Anti-Mouse CCR1 Monoclonal Antibody C1Mab-6.

C-C motif chemokine receptor 1 (CCR1/CD191) is a member of G-protein-coupled receptors and is expressed on myeloid cells, such as neutrophils and macrophages. Because the CCR1 signaling promotes tumor expansion in the tumor microenvironment (TME), the modification of TME is an effective strategy for cancer therapy. Although CCR1 is an attractive target for solid tumors and hematological malignancies, therapeutic agents for CCR1 have not been approved. Here, we established a novel anti-mouse CCR1 (mCCR1) monoclonal antibody (mAb), C1Mab-6 (rat IgG2b, kappa), using the Cell-Based Immunization and Screening method. Flow cytometry and Western blot analyses showed that C1Mab-6 recognizes mCCR1 specifically. The dissociation constant of C1Mab-6 for mCCR1-overexpressed Chinese hamster ovary-K1 was determined as 3.9 × 10-9 M, indicating that C1Mab-6 possesses a high affinity to mCCR1. These results suggest that C1Mab-6 could be a useful tool for targeting mCCR1 in preclinical mouse models.

CHO cells for virus-like particle and subunit vaccine manufacturing.

Chinese Hamster Ovary (CHO) cells, employed primarily for manufacturing monoclonal antibodies and other recombinant protein (r-protein) therapeutics, are emerging as a promising host for vaccine antigen production. This is exemplified by the recently approved CHO cell-derived subunit vaccines (SUV) against respiratory syncytial virus (RSV) and varicella-zoster virus (VZV), as well as the enveloped virus-like particle (eVLP) vaccine against hepatitis B virus (HBV). Here, we summarize the design, production, and immunogenicity features of these vaccine and review the most recent progress of other CHO-derived vaccines in pre-clinical and clinical development. We also discuss the challenges associated with vaccine production in CHO cells, with a focus on ensuring viral clearance for eVLP products.

A simple and sensitive differential digestion method to analyze adeno-associated virus residual host cell proteins by LC-MS.

Many methods using liquid chromatography-mass spectrometry (LC-MS) have been established for identifying residual host cell proteins (HCPs) to aid in the process development and quality control of therapeutic proteins. However, the use of MS-based techniques for adeno-associated virus (AAV) is still in its infancy, with few methods reported and minimal information available on potentially problematic HCPs. In this study, we developed a highly sensitive and effective differential digestion method to profile residual HCPs in AAV. Unlike direct digestion, which completely digests both AAV and HCPs, our differential digestion method takes advantage of AAV's unique characteristics to maintain the integrity of AAV while preferentially digesting HCPs under denaturing and reducing conditions. This differential digestion method requires only several micrograms of sample and significantly enhances the identification of HCPs. Furthermore, this method can be applied to all five different AAV serotypes for comprehensive HCP profiling. Our work fills a gap in AAV HCP analysis by providing a sensitive and robust strategy for detecting, monitoring, and measuring HCPs.