Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.
Cricetinae ; Animals ; Humans ; Cricetulus ; CHO Cells ; Epigenesis, Genetic/genetics* ; DNA Methylation ; Gene Expression Regulation ; Recombinant Proteins/genetics*

In response to the market demand for therapeutic antibodies, the upstream cell culture scale and expression titer of antibodies have been significantly improved, while the production efficiency of downstream purification process is relatively fall behind, and the downstream processing capacity has become a bottleneck limiting antibody production throughput. Using monoclonal antibody mab-X as experimental material, we optimized the caprylic acid (CA) precipitation process conditions of cell culture fluid and low pH virus inactivation pool, and studied two applications of using CA treatment to remove aggregates and to inactivate virus. Based on the lab scale study, we carried out a 500 L scale-up study, where CA was added to the low pH virus inactivation pool for precipitation, and the product quality and yield before and after precipitation were detected and compared. We found that CA precipitation significantly reduced HCP residuals and aggregates both before and after protein A affinity chromatography. In the aggregate spike study, CA precipitation removed about 15% of the aggregates. A virus reduction study showed complete clearance of a model retrovirus during CA precipitation of protein A purified antibody. In the scale-up study, the depth filtration harvesting, affinity chromatography, low pH virus inactivation, CA precipitation and depth filtration, and cation exchange chromatography successively carried out. The mixing time and stirring speed in the CA precipitation process significantly affected the CA precipitation effect. After CA precipitation, the HCP residue in the low pH virus inactivation solution decreased 895 times. After precipitation, the product purity and HCP residual meet the quality criteria of monoclonal antibodies. CA precipitation can reduce the chromatography step in the conventional purification process. In conclusion, CA precipitation in the downstream process can simplify the conventional purification process, fully meet the purification quality criterion of mab-X, and improve production efficiency and reduce production costs. The results of this study may promote the application of CA precipitation in the purification of monoclonal antibodies, and provide a reference for solving the bottleneck of the current purification process.
Cricetinae ; Animals ; Antibodies, Monoclonal/metabolism* ; Caprylates/chemistry* ; Cell Culture Techniques ; Chromatography, Affinity ; CHO Cells ; Cricetulus ; Chemical Precipitation

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.
Cricetinae ; Humans ; Animals ; Mice ; Immunoglobulin M/genetics* ; Antibodies, Viral ; CHO Cells ; Cricetulus ; Hybridomas ; Recombinant Fusion Proteins

Anti-reflective nanocoatings that mimic the eyes of fruit flies are biodegradable materials with great market potential for a variety of optical devices that require anti-reflective properties. Microbial expression of retinin provides a new idea for the preparation of nanocoatings under mild conditions compared to physicochemical methods. However, the current expression level of retinin, the key to anti-reflective coating, is low and difficult to meet mass production. In this study, we analyzed and screened the best expression hosts for Drosophila-derived retinin protein, and optimized its expression. Chinese hamster ovary (CHO) cells were identified as the efficient expression host of retinin, and purified retinin protein was obtained. At the same time, the preparation method of lanolin nanoemulsion was explored, and the best anti-reflective ability of the nano-coating was determined when the ratio of specific concentration of retinin protein and wax emulsion was 16:4, the pH of the nano-coating formation system was 7.0, and the temperature was 30 ℃. The enhanced antireflective ability and reduced production cost of artificial antireflective nanocoatings by determining the composition of nanocoatings and optimizing the concentration, pH and temperature of system components may facilitate future application of artificial green degradable antireflective coatings.
Animals ; Cricetinae ; CHO Cells ; Emulsions ; Cricetulus ; Drosophila ; Eye Proteins ; Drosophila Proteins

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×10⁷ U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.
Swine ; Animals ; Cricetinae ; Interferon-gamma/pharmacology* ; Cricetulus ; CHO Cells ; Sincalide ; Recombinant Proteins/pharmacology* ; Antiviral Agents/pharmacology*

The aim of this study was to produce E^rns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified E^rns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-E^rns was constructed based on the gene sequence of BVDV-1 NADL strain. The E^rns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-E^rns into CHO cells. The expression and purification of the E^rns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the E^rns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified E^rns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the E^rns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the E^rns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log₁₀ was induced in rabbits. In this study, purified BVDV E^rns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.
Rabbits ; Animals ; Cricetinae ; Cricetulus ; CHO Cells ; Antibodies, Viral ; Diarrhea Viruses, Bovine Viral/genetics* ; Antibodies, Monoclonal/genetics* ; Diarrhea ; Viral Vaccines/genetics*

Objective: To explore the immunogenicity and influencing factors of hepatitis B vaccination based on different vaccination schedules among chronic kidney disease (CKD) patients. Methods: CKD patients who participated in randomized controlled trials in four hospitals in Shanxi province and completed three doses of 20 µg vaccination (at months 0, 1 and 6) and four doses of 20 µg or 60 µg vaccination (at months 0, 1, 2, and 6) were surveyed from May 2019 to July 2020.According to the ratio of 1∶1∶1, 273 CKD patients were divided into 3 groups randomly. Quantification of the anti-hepatitis B surface antigen-antibody (anti-HBs) in serum samples was performed using chemiluminescent microparticle immunoassay at months 1 and 6 after the entire course of the vaccinations. The positive rate, high-level positive rate, geometric mean concentration (GMC) of anti-HBs, and the influencing factors were analyzed by χ² tests, analysis of variance, unconditional logistic regression analysis. Results: A total of 273 CKD patitents were participants.The positive rates in the CKD patients with four doses of 20 µg vaccination (92.96%,66/71) or 60 µg vaccination (93.15%, 68/73) were higher than that in the CKD patients with three doses of 20 µg vaccination (81.69%, 58/71) at month one after the full course of the vaccinations (P<0.05). The GMCs of anti-HBs showed similar results (2 091.11 mIU/ml and 2 441.50 mIU/ml vs. 1 675.21 mIU/ml) (P<0.05). The positive rate was higher in the CKD patients with four doses of 60 µg vaccination (94.83%,55/58) than in those with three doses of 20 µg vaccination (78.79%,52/66) (P<0.05) at month six after the full course of the vaccinations. And the GMC of anti-HBs in the patients with four doses of 60 µg vaccination (824.28 mIU/ml) was significantly higher than those in the patients with 3 or 4 doses of 20 µg vaccination (639.74 mIU/ml and 755.53 mIU/ml) (P<0.05). After controlling the confounding factors, the positive rate in the CKD patients with four doses of 60 µg vaccination were 3.19 (95%CI: 1.02-9.96) and 5.32 (95%CI: 1.27-22.19) times higher than those in the patients with three doses of 20 µg vaccination at months 1 and 6 after the full course of the vaccinations, respectively. The positive rate in CKD patients without immune suppression or hormone therapy was 3.33 (95%CI: 1.26-8.80) and 4.78 (95%CI: 1.47-15.57) times higher than those in the patients with such therapy, respectively. Conclusions: Four doses of 20 µg or 60 µg hepatitis B vaccination could improve the immunogenicity in patients with CKD. And four doses of 60 µg vaccination might play a positive role in maintaining anti-HBs in this population. The immunogenicity in the CKD patients with immune suppression or hormone therapy was poor.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Follow-Up Studies ; Hepatitis B/prevention & control* ; Hepatitis B Antibodies ; Hepatitis B Surface Antigens ; Hepatitis B Vaccines ; Humans ; Immunization, Secondary ; Renal Insufficiency, Chronic ; Vaccination

Chinese hamster ovary (CHO) cells are the preferred host cells for the production of complex recombinant therapeutic proteins. Adenine phosphoribosyltransferase (APRT) is a key enzyme in the purine biosynthesis step that catalyzes the condensation of adenine with phosphoribosylate to form adenosine phosphate AMP. In this study, the gene editing technique was used to knock out the aprt gene in CHO cells. Subsequently, the biological properties of APRT-KO CHO cell lines were investigated. A control vector expressed an enhanced green fluorescent protein (EGFP) and an attenuation vector (containing an aprt-attenuated expression cassette and EGFP) were constructed and transfected into APRT-deficient and wild-type CHO cells, respectively. The stable transfected cell pools were subcultured for 60 generations and the mean fluorescence intensity of EGFP in the recombinant CHO cells was detected by flow cytometry to analyze the EGFP expression stability. PCR amplification and sequencing showed that the aprt gene in CHO cell was successfully knocked out. The obtained APRT-deficient CHO cell line had no significant difference from the wild-type CHO cells in terms of cell morphology, growth, proliferation, and doubling time. The transient expression results indicated that compared with the wild-type CHO cells, the expression of EGFP in the APRT-deficient CHO cells transfected with the control vector and the attenuation vector increased by 42%±6% and 56%±9%, respectively. Especially, the EGFP expression levels in APRT-deficient cells transfected with the attenuation vector were significantly higher than those in wild-type CHO cells (P < 0.05). The findings suggest that the APRT-deficient CHO cell line can significantly improve the long-term expression stability of recombinant proteins. This may provide an effective cell engineering strategy for establishing an efficient and stable CHO cell expression system.
Adenine/metabolism* ; Adenine Nucleotides ; Adenine Phosphoribosyltransferase/genetics* ; Adenosine Monophosphate ; Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Recombinant Proteins/genetics*

To enhance recombinant protein production by CHO cells, We compared the impact of overexpression of metabolic enzymes, namely pyruvate carboxylase 2 (PYC2), malate dehydrogenase Ⅱ (MDH2), alanine aminotransferase Ⅰ (ALT1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase Ⅰ (CPSⅠ), and metabolism related proteins, namely taurine transporter (TAUT) and Vitreoscilla hemoglobin (VHb), on transient expression of anti-hLAG3 by ExpiCHO-S. Overexpression of these 7 proteins could differentially enhance antibody production. OTC, CPSI, MDH2, and PYC2 overexpression could improve antibody titer by 29.2%, 27.6%, 24.1%, and 20.3%, respectively. Specifically, OTC and MDH2 could obviously improve early-stage antibody production rate and the culture period was shortened by 4 days compared with that of the control. In addition, OTC and MDH2 had little impact on the affinity of anti-hLAG3. In most cases, overexpression of these proteins had little impact on the cell growth of ExpiCHO-S. MDH2 and ALT1 overexpression in H293T cells could also improve antibody production. Overall, overexpression of enzymes involved in cellular metabolism is an effective tool to improve antibody production in transient expression system.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Enzymes/metabolism* ; Recombinant Proteins/genetics*

Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.
Animals ; Batch Cell Culture Techniques ; Bioreactors ; standards ; CHO Cells ; Cell Count ; Computer Simulation ; Cricetinae ; Cricetulus ; Industrial Microbiology ; instrumentation ; methods