Chinese Hamster Ovary Research Articles

Precise control of critical quality attributes, including titer and glycosylation, is essential in bioprocessing, yet conventional design‑of‑experiments methods are challenged by the high-dimensional, nonlinear design space for media and process parameters. We assemble a comprehensive glycan‑focused Chinese hamster ovary (CHO) fed‑batch dataset and develop a computational workflow (i) to train machine learning (ML) models to predict key CQAs, (ii) apply a hybrid ML+knowledge-based strategy to select potentially impactful features, and (iii) generate combinatorial media designs. The resulting models predict final titer (R2≈0.93) and major glycan metrics-mannosylation, fucosylation, galactosylation (R2≈0.79-0.95)-directly from initial media composition and process parameters without requiring spent media analysis. Feature selection shortlisted 20 features out of 76 for a second-tier validation, from which 15 were confirmed as actionable levers impacting titer and glycosylation, uncovering glycan effects independent of nucleotide sugar supplementation. Finally, we incorporated our workflow, utilizing a ML surrogate model coupled with simulated annealing, in a proof‑of‑concept active learning step, successfully proposing a media composition and process parameter combination that reduced mannosylation by 10% while increasing titer. Together, these results underscore how ML‑enhanced DOE can accelerate CHO process development and explore complex biomanufacturing spaces with greater efficiency.

A genome-scale CRISPR deletion screen in Chinese hamster ovary cells reveals essential regions of the coding and non-coding genome.

Functional expression of five refolded recombinant variants of RBD from SARS-CoV-2 in Escherichia coli.

Enhanced antibody production in Chinese hamster ovary cell cultures supplemented with barley shochu distillation by-product supernatant.

In vitro and in vivo pharmacological characterization of fentanyl analogs.

YN1548: A novel small-molecule agonist of human glucagon-like peptide-1 receptor for the treatment of type 2 diabetes mellitus and obesity.

Optimization of immobilized metal affinity chromatography for a recombinant protein expressed in CHO cells

Synthesis of 2-(3-aminopropyl)benzopyrans as potential antipsychotic agents targeting D2/D3 and 5-HT2A receptors.

Host cell protein quantitation by LC-MS. Experimental demonstration, qualification, and comparison of methods in USP 1132.1.

Mutational effects of the asparagine198 and glutamate223 residues on the human norepinephrine transporter on basal and HIV-1 Tat protein-induced inhibition of dopamine transport.

Pharmacological and electrophysiological characterization of the novel Kv7 channel inhibitor racecadotril in Parkinson's disease.

A fluorescent cholesterol analog, R-Chol, mimics the dynamics of free cholesterol in live cells.

Placental malaria (PM) causes mortality and severe morbidity in areas with stable Plasmodium falciparum transmission. The selective placental accumulation of P. falciparum-infected erythrocytes (IEs) is mediated by VAR2CSA, a PfEMP1-type parasite ligand that binds exclusively to placenta-restricted CSA. VAR2CSA-specific IgG is therefore generally restricted to women exposed to P. falciparum infection during pregnancy. However, widespread acquisition of VAR2CSA-reactive IgG outside pregnancy among Colombian and Brazilian individuals has been reported, supposedly due to cross-reactivity between VAR2CSA and the P. vivax-specific antigen PvDBP. Here, we measured levels and Fc-afucosylation of VAR2CSA-reactive IgG in plasma from pregnant Brazilian women at delivery, using full-length VAR2CSA (FV2) expressed in baculovirus-transfected insect cells (FV2BIC) or Chinese hamster ovary cells (FV2CHO) as well as the corresponding native antigen (IT4VAR04) on the IE surface. We also measured levels of IgG specific for GLURP (P. falciparum-specific) and PvDBP (P. vivax-specific). FV2CHO-specific IgG levels were lower than FV2BIC-reactive IgG levels. Furthermore, only FV2CHO-specific IgG was restricted to women exposed to P. falciparum during pregnancy. Levels of PvDBP-specific IgG were significantly higher among P. vivax-exposed pregnant women but did not correlate with FV2-specific IgG levels. Finally, FV2CHO-specific IgG was markedly Fc-afucosylated in contrast to FV2BIC-reactive IgG. Our findings caution against using levels of IgG reacting with recombinant proteins expressed in insect cells as a measure of exposure to VAR2CSA during pregnancy, at least in South America. Furthermore, our data do not support the hypothesis that exposure to PvDBP induces IgG that cross-reacts with VAR2CSA and contributes to protection against PM.

ABSTRACTThe development of virus‐like particle (VLP) production processes is often constrained by the extensive number of analytical methods required for their quantification and characterization, as well as the significant labor demands associated with these techniques. Asymmetrical flow field‐flow fractionation (AF4) coupled with in‐line detectors, such as ultraviolet (UV) and multi‐angle light scattering (MALS), presents a promising label‐free and rapid approach to simultaneously assess the quantity and quality of VLP samples. While AF4‐MALS has been widely applied for bionanoparticle characterization and quantification in final products and process development, the influence of host cell‐derived impurities on the outcome of the analysis remains underexplored.This study investigates the impact of host cell‐derived impurities, particularly host cell DNA and chromatin, on AF4‐MALS‐DLS analysis of both unpurified and purified VLP samples, using HIV‐1 gag VLPs produced in CHO cells as a model system. Our results demonstrate that DNA, chromatin, and VLPs can co‐elute due to their overlapping size distribution, which, if overlooked, may lead to imprecise determination of VLP concentrations in early process samples and inaccurate yield calculations at later stages. Nevertheless, for total particle quantification, AF4‐MALS was shown to be a suitable surrogate for nanoparticle tracking analysis, as the 90° light scattering peak area exhibited a strong linear correlation with total particle concentration. This substitution enables faster sample processing and reduces sample volume requirements.Additionally, our findings highlight the importance of particle concentration and method parameter selection, particularly the detector flow rate, when characterizing samples based on hydrodynamic radius (Rhyd). Underestimation of Rhyd due to high detector flow rates was proposed as the possible explanation for the higher‐than‐expected shape factors obtained for VLPs. These results emphasize the need for further optimization of AF4 methods to improve the separation of VLPs from host cell impurities and to ensure reliable characterization of bionanoparticles in complex mixtures.

N-terminal domains and site-specific glycosylation regulate the secretion of avian melanocortin inverse agonists, agouti signaling protein (ASIP) and agouti-related protein (AGRP).

Blinatumomab is an anti-cancer Bispecific T-cell Engager (BiTE) antibody drug approved by the Food and Drug Administration (FDA) for treating acute lymphoblastic leukemia (ALL). It targets CD3ε, which is expressed on T cells and concomitantly targets CD19, an antigen specifically expressed in B-cell leukemia. Blinatumomab-bound T cells are activated to secrete perforin and granzyme, which induce cell death in B-cell leukemia. Blinatumomab is produced in the Chinese hamster ovary (CHO) cell expression system in consideration of post-translational modification (PTM) and immunogenicity. Although the characteristics of CHO cells are similar to those of other mammalian cells, it has low yield and high production cost. In this study, we developed the yeast species Pichia pastoris-produced BiTE antibody targeting CD19 and CD3ε, herein called P. pastoris (p)-blinatumomab, which is in-house produced blinatumomab. Approximately 1.2 mg/L CD19/CD3ε-targeting BiTE antibody (p-blinatumomab) was produced from the P. pastoris by transformation with the pHIL-S1 plasmid vector encoding the sequence of blinatumomab, which was 2.4 times higher than that produced in the CHO cell expression system based on the previous study. Enzyme-linked immunosorbent assay verified that the antigen binding affinity of the purified p-blinatumomab protein for CD19 and CD3ε antigens was 1.86 and 1.43 times higher than that of the commercial blinatumomab, BLINCYTO, respectively. In addition, flow cytometry confirmed that p-blinatumomab had a similar antigen binding affinity to CD19- and CD3ε-overexpressing cells compared to the BLINCYTO. In conclusion, we suggest that P. pastoris is a useful system for expressing and producing BiTE antibody platforms for therapeutic drugs.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-21889-4.

Objective.Hyperthermia (HT) is expected to enhance the biological effects of not only conventional radiotherapy but also particle beam therapy. However, a biophysical model that comprehensively considers both radiation quality (i.e. ion type and linear energy transfer, LET) and temperature has not yet been established. This study aimed to develop a model capable of calculating the cell-killing effect of particle beam therapy combined with HT.Approach.To incorporate temperature dependence into the stochastic microdosimetric kinetic (SMK) model, we hypothesized that sublethal lesion kinetics are temperature-dependent and that the thermal enhancement effect saturates at high LET. The resulting temperature-dependent SMK (TSMK) model was validated by fitting its parameters to published survival data from five Chinese hamster ovary cell lines treated with various radiation beams and subsequent HT.Main results.The TSMK model accurately reproduced the dose-survival curves for all five cell lines under both irradiation-alone and combined HT conditions. It also successfully replicated the LET dependence of the relative biological effectiveness (RBE) and thermal enhancement ratio (TER) for both highly and minimally thermosensitive cells. The TSMK model suggests that the LET dependence of TER is ion-type specific, similar to RBE. Furthermore, for cells treated with post-irradiation HT, the RBE and TER values rise at a lower LET compared to the RBE without HT.Significance.The TSMK model is the first biophysical model capable of calculating cell survival after combined irradiation with various beams and HT while accounting for dependencies on both radiation quality and temperature. This model is expected to be instrumental in optimizing treatment conditions and developing biologically-based treatment planning systems for particle beam therapy combined with HT.

Serum-free suspension culture technology for animal cells involves the division and proliferation of cells in serum-free medium as single cells or cell clusters within shaking flasks or bioreactors. This approach enables large-scale cell culture, enhances the yield and quality of biopharmaceuticals, reduces costs, and broadens the applications of animal cells. Serum-free suspension culture of adherent cells (e.g., Madin–Darby canine kidney (MDCK), Chinese hamster ovary (CHO), Vero, baby hamster kidney (BHK-21), and human embryonic kidney (HEK293) cells) has been successfully achieved through direct or indirect adaptation, medium optimization, and genetic engineering. Additionally, novel suspension cell lines, such as duck embryonic stem (EB66) and human retinoblastoma (PER.C6) cells, have been developed as potential new substrates for biopharmaceutical production. This review examines animal cell suspension culture technology and its applications in viral vaccines, recombinant proteins, and monoclonal antibodies, providing insights into the development and utilization of this important technology.

Canine B-cell lymphoma is a malignant hematologic neoplastic disease, with diffuse large B-cell lymphoma being the predominant subtype. This subtype highly expresses canine CD20 on its surface. Most clinical treatments are chemotherapeutic, with the standard treatment being the CHOP regimen. In immunotherapy, rituximab is effective for treating human B-cell lymphoma. However, its inability to bind natural canine CD20 renders it ineffective in treating canine B-cell lymphoma. The main objective of this study was to establish camel-canine chimeric antibodies and validate their in vitro biological activity. A Bactrian camel was immunized with the CD20 extracellular region peptide. The VHH fragment was amplified using nested PCR, ligated to the phage vector pMECS, and used to construct a phage antibody library with a capacity of 3.4 × 1010. After three rounds of enrichment and screening, 92 clones were selected for phage ELISA. Clones 4, 5, 8, 30, 43 and 46 exhibited high binding affinities for canine CD20. These strains were induced and purified using a prokaryotic expression system to obtain anti-canine CD20 single-domain antibodies. ELISA, Western blotting and flow cytometry analysis showed that the anti-canine CD20 single-domain antibody specifically binds to canine CD20. Cellular immunofluorescence validation revealed that the anti-canine CD20 single-domain antibody specifically binds to CD20 on the surface of Raji cells. The anti-canine CD20 single-domain antibody was subsequently ligated to the canine Fc fragment to construct a camel-canine chimeric antibody. The camel-canine chimeric antibody was expressed in CHO-S cells, and ELISA revealed that the chimeric antibody can specifically bind to the canine CD20 extracellular region polypeptide and the cell surface CD20 in Raji cells. In vitro killing assays revealed that the camel-canine chimeric antibody effectively mediated the targeting of canine peripheral blood mononuclear cells to Raji cells. These results indicate that the camel-canine chimeric antibody has strong anti-canine B-cell lymphoma activity and provide a basis for further development and clinical application of anti-canine B-cell lymphoma drugs.

Chinese hamster ovary (CHO) cells, widely utilised in biopharmaceutical production, experience various stressors during cell culture that can affect protein expression and folding, particularly within the endoplasmic reticulum (ER). Mild hypothermia is widely employed in CHO cell bioproduction to improve recombinant protein yield and quality; however, its impact on ER-associated pathways, particularly those governing protein folding and stress responses, remains insufficiently characterised. Mass spectrometry-based proteomics allows for the identification and relative quantification of proteins, enabling detailed insights into protein expression, modifications, and functional networks. This study investigates the impact of mild hypothermic conditions (31°C) on the whole cell proteome and ubiquitinated proteome of CHO cells, with a specific focus on ER proteins and ER stress. Using high-resolution mass spectrometry, we conducted a comprehensive proteomic and ubiquitinated proteomic analysis to quantify changes in protein abundance and ubiquitinated peptides under mild hypothermia. The downregulation of several proteins involved in the glycosylation of nascent polypeptides at 31°C, including DDOST, P4HB, PRKSCH and LMAN1, in all cell lines studied suggests that mild hypothermic shock disrupts the cell's normal ability to fold new proteins, leading to ER stress as the misfolded proteins build up. When this is coupled with the maintained cell viability and increased productivity at 31°C, it indicates the ER stress response can mitigate the build-up of misfolded proteins. The differential regulation of the transcription factor eIF2α, downregulated in non-producer cells but upregulated in producer cells at 31°C, suggests that recombinant protein-producing CHO cells possess a more adaptive ER stress response, enabling more efficient function under hypothermic culture conditions. Enhanced ubiquitination of misfolded protein substrates highlights an increased reliance on ER-associated degradation (ERAD) pathways to alleviate proteotoxic stress, as well as the wide range of biological processes that are regulated by ubiquitination as part of the hypothermic stress response. These findings provide new insights into the cellular adaptation mechanisms of CHO cells to mild hypothermia, with implications for optimising bioproduction strategies to improve yield and quality of therapeutic proteins. Our study highlights the importance of understanding the more complex aspects of the proteome and how this additional layer of detail can open new avenues for CHO cell engineering.