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This study explored the cell line-specific effects of dexamethasone (DEX) on three antibody-producing Chinese hamster ovary (CHO) clones derived from different parental host cell lines: CHO-K1, -S, and -DG44. The CHO-K1 and -DG44 cell lines displayed DEX-concentration-dependent growth inhibition but had improved viability in later culture stages, extending culture longevity. Conversely, the CHO-S cell line was less sensitive to DEX regarding cell growth but exhibited a drastic decrease in viability in the later stages of culture. The cell line-specific impact of DEX (i.e. the anti-apoptotic or pro-apoptotic effects) could have resulted from the presence or absence of the GRα-D isoform and relative level of GRα-C isoform in these cell lines. The CHO-K1 and -DG44 cell lines, which expressed GRα-D and relatively low levels of GRα-C, displayed improved cell viability in response to DEX. In contrast, the CHO-S cell line, which lacked GRα-D expression with relatively higher levels of GRα-C, exhibited pro-apoptotic effects upon exposure to DEX. DEX also influenced glucose consumption and metabolite production in all three cell lines, while its effects on antibody quality were negligible. Therefore, when supplementing glucocorticoids such as DEX in cell culture media, it is crucial to assess their ability to enhance culture performance carefully. This study could provide insights for optimizing production media to specific cell lines within the bioprocessing field. [ABSTRACT FROM AUTHOR]

BACKGROUND Honey bee venom contains complex compounds such as polypeptides, enzymes, and amines. One of the important components of bee venom is the phospholipase A2 enzyme, which is considered an important honey bee venom allergen and is also used to treat some diseases. This enzyme is found in other insects, arachnids, snakes, and mammalian cells, and its function is the hydrolysis of the second ester bond of glycerophospholipids and the release of fatty acids and lysophospholipids. Although transient transfection can produce recombinant proteins, stable cells are more suitable for high-scale production with economic efficiency. OBJECTIVES: The present study created a stable cell line to produce recombinant phospholipase A2 from honey bee (Apis mellifera) venom. METHODS: Plasmid cloning DNA vector containing phospholipase A2 gene was prepared by Macrogen Company. The recombinant plasmid was transferred to Chinese hamster ovary cells by heat shock method, and gene expression was carried out in a HamsF12 culture medium containing neomycin antibiotic. After increasing polyclonal strains containing plasmid, monoclonal clones were selected by limiting dilution. Then, monoclonal clones were propagated, the soup of the selected cells was collected and concentrated, and the protein expression was checked by sodium dodecyl-sulfate polyacrylamide gel electrophoresis test. RESULTS: The results of electrophoresis, which was performed to confirm the expression of the phospholipase A2 gene in the cell soup, showed a band with a molecular weight of 20 kilodaltons, which confirms the creation of a stable cell line for the production of recombinant phospholipase A2 honey bee venom. CONCLUSIONS: After the transient transfection of the plasmid containing this gene, several cells undergo recombination due to having repair mechanisms and putting the desired gene along with the antibiotic resistance gene in their genome. These cells can be selected and propagated by adding antibiotics to the culture medium. [ABSTRACT FROM AUTHOR]

Therapeutic monoclonal antibodies (mAbs) are biologics produced using mammalian cells and represent an important class of biotherapeutics. Aggregation in mAbs is a major challenge that can be mitigated by rigorous and reproducible upstream and downstream approaches. The impact of frequently used surfactants, like polysorbate 20, polysorbate 80, poloxamer 188, and 2‐hydroxypropyl‐beta‐cyclodextrin, on aggregation of mAbs during cell culture was investigated in this study. Their impact on cell proliferation, viability, and mAb titer was also investigated. Polysorbate 20 and polysorbate 80 at the concentration of 0.01 g/L and poloxamer 188 at the concentration of 5 g/L were found to be effective in reducing aggregate formation in cell culture medium, without affecting the cell growth or viability. Furthermore, their presence in culture media resulted in increased cell proliferation as compared to the control group. Addition of these surfactants at the specified concentrations increased monomer production while decreasing high molecular weight species in the medium. After mAbs were separated, using protein "A" chromatography, flasks with surfactant exhibited improved antibody stability, when analyzed by DLS. Thus, while producing aggregation‐prone mAbs via mammalian cell culture, these excipients may be employed as cell culture medium supplements to enhance the quality and yield of functional mAbs. [ABSTRACT FROM AUTHOR]

ABSTRACTEfficient production of bispecific antibodies (BsAbs) in single mammalian cells is essential for basic research and industrial manufacturing. However, preventing unwanted pairing of heavy chains (HCs) and light chains (LCs) is a challenging task. To address this, we created an engineering technology for preferential cognate HC/LC and HC/HC paring called FAST-Ig (Four-chain Assembly by electrostatic Steering Technology – Immunoglobulin), and applied it to NXT007, a BsAb for the treatment of hemophilia A. We introduced charged amino-acid substitutions at the HC/LC interface to facilitate the proper assembly for manufacturing a standard IgG-type BsAb. We generated CH1/CL interface-engineered antibody variants that achieved > 95% correct HC/LC pairing efficiency with favorable pharmacological properties and developability. Among these, we selected a design (C3) that allowed us to separate the mis-paired species with an unintended pharmacological profile using ion-exchange chromatography. Crystal structure analysis demonstrated that the C3 design did not affect the overall structure of both Fabs. To determine the final design for HCs-heterodimerization, we compared the stability of charge-based and knobs into hole-based Fc formats in acidic conditions and selected the more stable charge-based format. FAST-Ig was also applicable to stable CHO cell lines for industrial production and demonstrated robust chain pairing with different subclasses of parent BsAbs. Thus, it can be applied to a wide variety of BsAbs both preclinically and clinically.

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor 2 (HER2) has been identified as a molecular target for breast cancer therapy, such as Trastuzumab (Herceptin®). This has been shown to improve patient survival substantially. The current study is aiming to locally produce an anti-HER2 monoclonal antibody (named Trastuzumab) which has an equivalent biological properties in comparison with the original version, Herceptin®). In silico design and construction of recombinant vectors, as well as the establishment of transfected cell lines with high expression of Trastuzumab were performed. Based on the protein sequences obtained from the Drugbank, the DNA sequences encoding for the light chain (Tras-Lc) and heavy chain (Tras-Hc) of Trastuzumab were optimized and integrated into pNanogen-Hygro and pNanogen-Puro vectors, respectively. The Neon Transfection System was used to co-transfect the pNanogen-Tras-Lc-Hygro and pNanogen-Tras-Hc-Puro constructs into CHO cells. Different co-transfected single-cell-colonies selected on media supplemented with hygromycin and puromycin were used for ELISA and SDS-PAGE assays to identify the CHO cell lines which highly express Trastuzumab. Based on the present results, 30μg of both constructs were suitable for DNA co-transfection. After 07 days of culture, the highest amount of Trastuzumab (561 µg/ml) was obtained from the H06LD68 cell line.

Mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome) is an orphan genetic disease caused by deficiency of the lysosomal enzyme arylsulfatase B (ASB). The need to develop a highly productive cell line for the production of recombinant ASB, is behind the concept and relevance of this study. The most promising approach seems to be the development of CHO producer cell lines coexpressing the target ASB enzyme and an auxiliary formylglycine-generating enzyme (FGE). At the same time, it is important from a practical perspective to have the possibility of cultivating producer cell lines as suspensions free of serum or other components of animal origin. The aim of the study was to develop highly productive cell lines for the production of recombinant ASB by coexpression of the auxiliary FGE. Materials and methods: a suspension CHO cell line was used in the study. CHO cells were transfected by electroporation using the MaxCyte STX system. Monoclonal cell lines were obtained with the help of the Cell Metric system. Enzyme-linked immunosorbent assay was used for determination of ASB concentration in the culture fluid. Culture fluid samples were analysed using polyacrylamide gel electrophoresis and Western blotting. The mRNA level was measured by real-time polymerase chain reaction. Results: producer cell lines coexpressing the target ASB enzyme and auxiliary FGE were obtained. An increase in the yield of the active target ASB enzyme from 2 to 100 mg/L was achieved by selecting the optimal ratio of plasmids during transfection. The highest yield of the target ASB enzyme was achieved at the 90:10 ratio (%) of plasmids encoding the ASB and FGE genes, respectively. Conclusions: the authors developed highly productive cell lines for the production of recombinant ASB, which coexpress the target and auxiliary enzymes. The coexpression of ASB and FGE improves the growth and production characteristics of the cell line, probably due to the modification of the ASB active site. The obtained results will help resolve the problem of low enzyme yield, which is typical of this class of medicines.

Abstract Background Human matrix metalloproteinase 9 (hMMP9) is a biomarker in several diseases, including cancer, and the need for developing detectors and inhibitors of hMMP9 is increasing. As an antibody against hMMP9 can be selectively bound to hMMP9, the use of anti-MMP9 antibody presents new possibilities to address hMMP9-related diseases. In this study, we aimed to establish a stable Chinese hamster ovary (CHO) cell line for the stable production of antibodies against hMMP9. Results Weconstructed recombinant anti-hMMP9 antibody fragment-expressing genes and transfected these to CHO cells. We chose a single clone, and successfully produced a full-sized antibody against hMMP9 with high purity, sensitivity, and reproducibility. Subsequently, we confirmed the antigen-binding efficiency of the antibody. Conclusions We developed a novel recombinant anti-hMMP9 antibody via a CHO cell-based mammalian expression system, which has a high potential to be used in a broad range of medical and industrial areas.

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes.

Results from studies of the cytotoxic activity of new azolo[5,1-c][1,2,4]triazine derivatives to establish the potential of using them as antitumor agents, including for breast cancer chemotherapy, are presented. The spectral characteristics confirmed that the obtained compounds were highly pure and stable on storage. The cytotoxicity was determined using the methyltetrazolium (MTT) test against MCF-7 and CHO cell lines. The compounds were used at final concentrations from 0.25 to 10.0 μM. Two imidazo[5,1-c][1, 2, 4] triazines (5a and 5d) had calculated half-maximal inhibitory concentrations (IC50) for MCF-7 several times lower than the reference drug epirubicin. The cytotoxicity of all newly synthesized derivatives was lower than that of epirubicin with respect to untransformed CHO cells. This further indicated the potential of these compounds. The results can be used as a basis for selecting compounds 5a and 5d as substances with potential antitumor activity for further studies of their genotoxic and metabolic properties on cell models and laboratory animals. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a potent infectious disease with detrimental effects on the global swine industry and no currently vaccine available. The emergence of low-virulence CD2v-deleted mutants manifested as non-hemadsorption (non-HAD) strains represents a significant challenge to the prevention and control of ASF. In this study, we aimed to establish an indirect ELISA (IELISA) method for the identification of ASFV wild-type and CD2v-deleted strains. We integrated the CD2v protein extracellular domain sequence (CD2v-Ex, 1–588 bp) of the highly pathogenic strain China/2018/AnhuiXCGQ into the genome of suspension culture-adapted Chinese hamster Ovary-S (CHO-S) cells using lentivirus vectors (LVs). By screening, we identified a monoclonal CHO-S cell line that stably expressed secretory CD2v-Ex Protein. We then used the purified CD2v-Ex Protein as the detection antigen to establish an indirect ELISA method (CD2v-IELISA) for identification of the ASFV wild-type and CD2v-Deleted (CD2v−) strains. The CD2v-IELISA method showed excellent specificity with no cross-reaction with serum samples infected with ASFV (CD2v−), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine circovirus (PCV), porcine pseudorabies virus (PRV), swine foot and mouth disease virus (FMDV) and porcine epidemic diarrhea virus (PEDV). Furthermore, this method showed high sensitivity, allowing identification of ASFV-infected clinical serum samples up to a dilution of 1:2,560. The coefficient of variation both in and between batches was

Background: Human matrix metalloproteinase 9 (hMMP9) is a biomarker in several diseases, including cancer, and the need for developing detectors and inhibitors of hMMP9 is increasing. As an antibody against hMMP9 can be selectively bound to hMMP9, the use of anti-MMP9 antibody presents new possibilities to address hMMP9-related diseases. In this study, we aimed to establish a stable Chinese hamster ovary (CHO) cell line for the stable production of antibodies against hMMP9. Results: Weconstructed recombinant anti-hMMP9 antibody fragment-expressing genes and transfected these to CHO cells. We chose a single clone, and successfully produced a full-sized antibody against hMMP9 with high purity, sensitivity, and reproducibility. Subsequently, we confirmed the antigen-binding efficiency of the antibody. Conclusions: We developed a novel recombinant anti-hMMP9 antibody via a CHO cell-based mammalian expression system, which has a high potential to be used in a broad range of medical and industrial areas. [ABSTRACT FROM AUTHOR]

The effects that sandy everlasting, purple corn, and hedge hyssop extracts have on the viability and metabolic activity of monolayer cell cultures of the Chinese hamster ovary (CHO) cell line are studied using the MTT assay. The enhancements of the cell viability (up to 86.0 ± 8.2%) and metabolic activity (up to 196.1 ± 8.8%) are observed for all of the extracts at concentrations of 50 to 250 μg/mL. For extract concentrations of 500 to 2000 μg/mL, cell metabolic activity is suppressed (to 13.4 ± 11.3%) and the number of viable cells can fall to zero. The extracts of considered plants (sandy everlasting, purple corn, and hedge hyssop) have different effects on healthy cells, depending on the extract concentration. [ABSTRACT FROM AUTHOR]

The HIV-1 derived gp145 protein is being investigated by research groups as preclinical studies have shown high promise for this protein as a vaccine against HIV. However, one of the main challenges with manufacturing this promising protein has been ascribed to the low yield obtained in mammalian cell cultures. Significant improvements in gp145 production are needed to address this issue to test the gp145 protein as a potentially effective, safe, and affordable HIV vaccine. Here we describe the application of a novel expression technology to create GMP-grade CHO cell lines expressing approximately 50 μg/ml in non-optimized fed-batch culture, which is an order of magnitude higher than that obtained in existing processes. Top producing clones show a high degree of similarity in the glycosylation patterns of the purified protein to the reference standard. Conformational integrity and functionality was demonstrated via high-affinity binding to soluble CD4, using a panel of antibodies including VRC01, F105, Hk20, PG9 and 17b. In summary, we were able to generate CHO cell lines expressing HIV gp145 with significantly higher overall expression yields than currently accessible, and high product quality that could potentially be suitable for future studies assessing the efficacy and safety of gp145-based HIV vaccines. [ABSTRACT FROM AUTHOR]

The Chinese hamster ovary (CHO) cell line is widely used for the production of recombinant proteins due to its high growing capacity and productivity, as well as other cell lines derived later than CHO. Adapting cell culture media for each specific cell line is a key to exploit these features for cost effective and fast product generation. Media supplementation is generally addressed by means of one‐factor‐at‐a‐time or classical design of experiments approaches but these techniques may not be efficient enough in preliminary screening phases. In this study, a novel strategy consisting in folding over the Plackett–Burman design was used to increase cell growth and trastuzumab production of different CHO cell lines through supplementation with nonanimal recombinant compounds. Synergies between compounds could be detected with a reduced number of experiments by using this methodology in comparison to more conventional fractional factorial designs. In the particular case reported here, the sequential use of this modified Plackett–Burman in combination with a Box‐Behnken design led to a 1.5‐fold increase in cell growth (10 × 106 cells/mL) and a two‐fold in trastuzumab titer (122 mg/L) in suspension batch culture. [ABSTRACT FROM AUTHOR]

Hemophilia A is a X-linked recessive bleeding disorder consecutive to the lack of circulating pro-coagulant factor VIII (FVIII). The most efficient strategy to treat or prevent bleeding in patients with hemophilia A relies on replacement therapy using exogenous FVIII. Commercially available recombinant FVIII are produced using an expensive perfusion technology in stainless steel fermenters. A fed-batch fermentation technology was recently developed to produce ‘Neureight’, a full-length recombinant human FVIII, in Chinese hamster ovary (CHO) cells. Here, we investigated the structural and functional integrity and lack of increased immunogenicity of Neureight, as compared to two commercially available full-length FVIII products, Helixate and Advate, produced in baby hamster kidney or CHO cells, respectively. Our results demonstrate the purity, stability and functional integrity of Neureight with a standard specific activity of 4235 ± 556 IU/mg. The glycosylation and sulfation profiles of Neureight were similar to that of Advate, with the absence of the antigenic carbohydrate epitopes α-Gal and Neu5Gc, and with sulfation of Y1680, that is critical for FVIII binding to von Willebrand factor (VWF). The endocytosis of Neureight by human immature dendritic cells was inhibited by VWF, and its half-life in FVIII-deficient mice was similar to that of Advate, confirming unaltered binding to VWF. In vitro and in vivo assays indicated a similar immunogenicity for Neureight, Advate and Helixate. In conclusion, the production of full-length FVIII in a fed-batch fermentation mode generates a product that presents similar biochemical, functional and immunogenic properties as products developed using the classical perfusion technology. [ABSTRACT FROM AUTHOR]

In this study, in vitro and in vivo methods were used to evaluate the cytotoxicity and genotoxicity properties of silver nanoparticles (Ag-NPs) made from a crude ethanolic extract of Salacia chinensis. The test Ag-NPs had no cytotoxicity on the fibroblast cell line at a concentration of 100 μg/mL, according to the MTT assay results. The Chinese hamster ovary (CHO) cell line treated with varied concentrations of test Ag-NPs, with a maximum concentration of 200 μg/mL, did not exhibit any appreciable genotoxic activity, either by comparing the results with positive controls of genotoxicity caused by Methyl methane sulfonate and Benzo (a) pyrene at the concentration of 20 μg/mL, the lack of genotoxicity was established. An in vivo study in Swiss albino mice using various concentrations (250, 500, and 1000 mg/kg) of test Ag-NPs, which were compared with positive controls, further confirmed this in vitro result pattern. Contrary to the genotoxicity caused by the positive control, mouse bone marrow micronucleus testing findings revealed the absence of genotoxicity. These findings imply that at the measured doses, the Ag-NPs produced from the crude ethanolic extract of Salacia chinensis do not exhibit any cytotoxicity or genotoxicity. • The AgNPs not causing cytotoxicity on fibroblast cellline at100 μg/mL concentration. • No chromosomal aberration found in CHO cellline at 200 μg mL−1 dosage of AgNPs. • The AgNPs not inducing any genotoxicity on both in-vitro and in-vivo studies. • The animal study proved that absence of genotoxicity on bone marrow micronucleus test. • S. chinensis synthesized AgNPs not causing cytotoxicity and genotoxicity. [ABSTRACT FROM AUTHOR]

An intensively developing trend in modern biotechnology is the production of recombinant antibodies and proteins. Recombinant antibodies have immunogenic properties and can stay in the blood for a long time; neutralize pathogens, toxins, and viruses; and destroy tumor cells. Recombinant proteins are widely used to cure, prevent, and diagnose diseases. A topical trend in modern medical biotechnology is the creation of prolonged-action drugs based on proteins obtained by genetic engineering methods. The issue of improving the specificity and selectivity of preparations based on enzymes and antibodies awaits solution. All this can be implemented using the approaches of modern combinatorial chemistry and biology, as well as quantum-mechanical calculations. [ABSTRACT FROM AUTHOR]

Chitosan nanoparticles (CS NPs) were prepared as a carrier for Human papillomavirus type 16 HPV-16) E7 gene and their gene transfection ability were evaluatedin vitro. The plasmid expressing green fluorescent protein (pEGFP) was used as a reporter gene. Gel electrophoresis demonstrated full binding of CS NPs with the pDNA. The transfection of CS-pEGFP NPs was efficient in CHO cells and the expression of green fluorescent proteins was well observed. The expression of E7 proteins was confirmed under SDS-PAGE and western blot analysis. As a conclusion CS NPs may serve as an effective nonviral carrier for delivery of nucleotides into eukaryotic cells. [ABSTRACT FROM PUBLISHER]

Efficient production of bispecific antibodies (BsAbs) in single mammalian cells is essential for basic research and industrial manufacturing. However, preventing unwanted pairing of heavy chains (HCs) and light chains (LCs) is a challenging task. To address this, we created an engineering technology for preferential cognate HC/LC and HC/HC paring called FAST-Ig (Four-chain Assembly by electrostatic Steering Technology - Immunoglobulin), and applied it to NXT007, a BsAb for the treatment of hemophilia A. We introduced charged amino-acid substitutions at the HC/LC interface to facilitate the proper assembly for manufacturing a standard IgG-type BsAb. We generated CH1/CL interface-engineered antibody variants that achieved > 95% correct HC/LC pairing efficiency with favorable pharmacological properties and developability. Among these, we selected a design (C3) that allowed us to separate the mis-paired species with an unintended pharmacological profile using ion-exchange chromatography. Crystal structure analysis demonstrated that the C3 design did not affect the overall structure of both Fabs. To determine the final design for HCs-heterodimerization, we compared the stability of charge-based and knobs into hole-based Fc formats in acidic conditions and selected the more stable charge-based format. FAST-Ig was also applicable to stable CHO cell lines for industrial production and demonstrated robust chain pairing with different subclasses of parent BsAbs. Thus, it can be applied to a wide variety of BsAbs both preclinically and clinically.

Background: Chinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of recombinant genes in particular clones early in the development stage. Results: The transcriptome profiles of CHO clones with stable and unstable recombinant protein production were investigated over 10-weeks of cultivation, using a DNA microarray. We identified 14 genes that were differentially expressed between the stable and unstable clones already at 2 weeks from the beginning of the cultivation. Their expression was validated by reverse-transcription quantitative real-time PCR (RT-qPCR). Furthermore, the k-nearest neighbour algorithm approach shows that the combination of the gene expression patterns of only five of these 14 genes is sufficient to predict stable recombinant protein production in clones in the early phases of cell-line development. Conclusions: The exact molecular mechanisms that cause unstable recombinant protein production are not fully understood. However, the expression profiles of some genes in clones with stable and unstable recombinant protein production allow prediction of such instability early in the cell-line development stage. We have thus developed a proof-of-concept for a novel approach to eliminate unstable clones in the CHO-Dhfr expression system, which saves time and labour-intensive work in cell-line development. [ABSTRACT FROM AUTHOR]

Developing stable Chinese hamster ovary (CHO) cell lines for biotherapeutics is an irreversible process and therefore, key quality attributes, such as sequence variants, must be closely monitored during cell line development (CLD) to avoid delay in the developmental timeline, and more importantly, to assure product safety and efficacy. Sequence variants, defined as unintended amino acid substitution in recombinant protein primary structure, result from alteration at either the DNA or the protein level. Here, for the first time, we report the application of transcriptome sequencing (RNAseq) in an IgG1 monoclonal antibody (mAb) CLD campaign to detect, identify, and eliminate cell lines containing low-level point mutations in recombinant coding sequence. Among the top eleven mAb producers chosen from transfectant, clone or subclone stages, three of the cell lines contained either missense or nonsense point mutations at a low level of less than 2%. Subsequent LC/MS/MS characterization detected ∼3% sequence variants with an amino acid change from Ser to Leu at residue 117 in the heavy chain of transfectants 11 and 27. This substitution is consistent with the RNAseq finding of a C/T mutation located at 407 base pair (TCA→TTA) in the heavy chain coding sequence. Here we demonstrate that RNAseq is a rapid and highly sensitive method to identify low-level genetic mutation de novo corresponding to the amino acid substitution that elicits sequence variant(s). Its implementation in CLD constitutes an early and effective step in identifying desired CHO expression cell lines. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1077-1085, 2015 [ABSTRACT FROM AUTHOR]

Cytotoxic T-lymphocyte-associated protein 4-Ig (CTLA4-Ig) produced using Chinese hamster ovary (CHO) cell lines is a fusion protein of CTLA4 and the Fc region of antibody. In the present study, we identified and overexpressed genes capable of increasing sialic acid levels in CTLA4-Ig to develop cell lines using glycoengineering technology. CTLA4-Ig was produced using CHO cells overexpressing N -acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST). The conditions were wild type (WT), overexpression (GnT-IV, GnT-V, and α2,6-ST), and co-overexpression (GnT-IV and α2,6-ST, and GnT-V and α2,6-ST). GnT-IV and GnT-V were transfected into CHO cells to determine tri-antennary structure formation in CTLA4-Ig. CHOGnT-IV (cells overexpressing GnT-IV) showed the highest tri-antennary structures of glycans. Compared to CHOWT, neutral and mono-sialylated glycans decreased (−10.9% and −18.6%, respectively), while bi- and tri-sialylated N -glycans increased (4.1% and 85.7%, respectively) in CHOGnT-IV∙ST (cells co-overexpressing GnT-IV and α2,6-ST). The sum of the relative quantities of neutral N -glycans decreased from 32.0% to 28.5%, while that of sialylated N -glycans increased from 68.0% to 71.5% in CHOGnT-IV∙ST. These results are the first to demonstrate the co-overexpression of especially GnT-IV and α2,6-ST, which is an effective strategy to increase sialic acid levels and the tri-antennary structure of CTLA4-Ig produced using CHO cell lines. • Protein-specific glycoengineered CHO cells were constructed. • Overexpression of GnT-IV played a major role in antennary structure formation of CTLA4-Ig. • Co-overexpression of GnT-IV and α2,6-ST made highly sialylated CTLA4-Ig. [ABSTRACT FROM AUTHOR]

VEGF and its receptors, especially VEGFR2 (KDR), are known to play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies. This study was aimed at developing a fully human IgG1 antibody (mAb-04) constructed from a phage-derived scFv, targeting the VEGF/VEGFR2 pathway. Firstly, an innovative transfection system, containing two recombinant expression vectors (pMH3 and pCApuro), were introduced into CHO-s cells and clones with higher yield selected accordingly. After an optimal fermentation condition was determined, fed-batch fermentation was performed in 5-L bioreactor with a final yield up to 60 mg/L. Further, cell proliferation, wound healing, transwell invasion, tube formation and chick embryo chorioallantoic membrane assays showed significant anti-angiogenic activity of mAb-04 in vitro and in vivo. In addition, the results of Western blotting indicated the ability of mAb-04 to inhibit VEGF-induced VEGFR2 signaling pathway. Finally, ADCC assay demonstrated that mAb-04 is capable of mediating tumor cell killing in presence of effector cells. This study has therefore proved that the full-length antibody targeting human VEGFR2 has potential clinical applications in the treatment of cancer and other diseases where pathological angiogenesis is involved. [ABSTRACT FROM AUTHOR]

In this study, the light chain ( κ) and heavy chain ( γ) sequences of the monoclonal antibody against vascular endothelial growth factor (VEGF) were sub-cloned into the eukaryotic pcDNA3.1 (+) (Hygro) and the pcDNA3.1 (+) (Neo) expression vectors using the traditional and homologous recombination methods. To express the antibody, the recombinant plasmids were transfected into the Chinese hamster ovary (CHO) and the K562 cell lines. The recombinant antibody was then purified using the protein A affinity chromatography. Furthermore, in order to demonstrate the inhibition of VEGF-induced mitogenesis of the recombinant antibody, the bovine aorta endothelial like cells were employed. The results showed specialization and conjunction of the recombinant antibody to the VEGF. It was also indicated that the antibody expression in the K562 cell lines was higher than the CHO cell lines. Furthermore, the in vitro VEGF inhabitation of the recombinant antibodies which were produced from the K562 cell line, and the CHO cell line, were similar. This proved that the K562 cell line is a good substitute for the CHO cell line in the production of the recombinant antibodies. [ABSTRACT FROM AUTHOR]

Most of the recombinant biotherapeutics employed today to combat severe illnesses, for example, various types of cancer or autoimmune diseases, are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization toward rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels. Especially proteomics workflows reached new levels in proteome coverage and data quality because of advances in high-resolution mass spectrometry instrumentation. One type of workflow concentrates on spatial proteomics by usage of subcellular fractionation of organelles with subsequent shotgun mass spectrometry proteomics and machine learning algorithms to determine the subcellular localization of large portions of the cellular proteome at a certain time point. Here, we present the first subcellular spatial proteome of a CHO-K1 cell line producing high titers of recombinant antibody in comparison to the spatial proteome of an antibody-producing plasma cell-derived myeloma cell line. Both cell lines show colocalization of immunoglobulin G chains with chaperones and proteins associated in protein glycosylation within the endoplasmic reticulum compartment. However, we report differences in the localization of proteins associated to vesicle-mediated transport, transcription, and translation, which may affect antibody production in both cell lines. Furthermore, pairing subcellular localization data with protein expression data revealed elevated protein masses for organelles in the secretory pathway in plasma cell-derived MPC-11 (Merwin plasma cell tumor-11) cells. Our study highlights the potential of subcellular spatial proteomics combined with protein expression as potent workflow to identify characteristics of highly efficient recombinant protein-expressing cell lines. Data are available via ProteomeXchange with identifier PXD029115., Competing Interests: Conflict of interest S. F. is an employee of Boehringer Ingelheim Pharma GmbH & Co KG, which develops and sells pharmaceuticals. All other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

African swine fever (ASF) is a potent infectious disease with detrimental effects on the global swine industry and no currently vaccine available. The emergence of low-virulence CD2v-deleted mutants manifested as non-hemadsorption (non-HAD) strains represents a significant challenge to the prevention and control of ASF. In this study, we aimed to establish an indirect ELISA (IELISA) method for the identification of ASFV wild-type and CD2v-deleted strains. We integrated the CD2v protein extracellular domain sequence (CD2v-Ex, 1-588 bp) of the highly pathogenic strain China/2018/AnhuiXCGQ into the genome of suspension culture-adapted Chinese hamster Ovary-S (CHO-S) cells using lentivirus vectors (LVs). By screening, we identified a monoclonal CHO-S cell line that stably expressed secretory CD2v-Ex Protein. We then used the purified CD2v-Ex Protein as the detection antigen to establish an indirect ELISA method (CD2v-IELISA) for identification of the ASFV wild-type and CD2v-Deleted (CD2v - ) strains. The CD2v-IELISA method showed excellent specificity with no cross-reaction with serum samples infected with ASFV (CD2v - ), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine circovirus (PCV), porcine pseudorabies virus (PRV), swine foot and mouth disease virus (FMDV) and porcine epidemic diarrhea virus (PEDV). Furthermore, this method showed high sensitivity, allowing identification of ASFV-infected clinical serum samples up to a dilution of 1:2,560. The coefficient of variation both in and between batches was <10% with good reproducibility and a high compliance rate of 99.4%. This CD2v-IELISA method developed here is of great significance for the prevention, control and purification of ASFV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jiang, Jiang, Li, Wan, Wang, Wang, Shi, Zhao, Song, Zhu, Ji and Zhang.)

Inverse agonists are useful active ingredient of drugs clinically used to treat diseases mainly involving receptors endowed with nonendogenous agonist induced activity (constitutive or basal activity). SP-1e and SP-1g are the first two potent and highly selective β3-adrenoceptor inverse agonists [EC50=181 nM (IA=- 64%) and 136 nM (IA=-73%), respectively], which their peculiar activity seems due to the absolute configurations of the two stereogenic centres present in each molecule. Rat proximal colon motility measurements allowed their further pharmacological characterization and pA2 values determination by Schild analysis (7.89 and 8.16, respectively). The purpose of our work is a further characterization of our novel β3-adrenoceptor agonists (SP-1a-d, SP-1f,1h) and inverse agonists (SP-1e and SP1g) on rat proximal colon motility and a confirmation of their inverse agonist nature in a more complex system like the functional test on rat proximal colon. Male Wistar rats segment of the proximal colon were placed in organ baths containing Krebs solution. Muscle tension was recorded isotonically. Cumulative β3-AR agonists doses experiments were performed for each test compound: isoprenaline, BRL37344, SP-1a-d, SP-1f and SP-1h were dissolved in Krebs. The EC50 values of each agonists and pA2 of inverse agonists were determined. SP- 1a-d, SP-1f and SP-1h in rat colon have a muscle relaxing effect thus confirming their partial agonist activity found in CHO-K1 cell line. SP-1e and SP-1g behaved as antagonists with pA2 values of 7.89 and 8.16, respectively. In conclusion, experiments carried out by using isolated rat proximal colon allowed us to determine the pA2 values of the two β3-AR inverse agonists and add knowledge on the behavior of a novel set of compounds and their possible value as agents useful whenever is necessary to also control the colon motility. [ABSTRACT FROM AUTHOR]

Clonally derived recombinant cell lines are highly desired to achieve consistent production of recombinant biotherapeutics. Despite repeated rounds of cloning by limiting dilution or single cell cloning, the resulting cell lines have often been observed to diverge, becoming a heterogeneous population and losing productivity over long-term sub-culturing. To understand the underlying molecular mechanisms, we developed quantitative polymerase chain reaction (qPCR) assays for the analysis of transgene copy number distribution in single recombinant cells isolated from Chinese hamster ovary (CHO) cell lines. Single cells were obtained by fluorescence activated cell sorting (FACS) technology and lysed directly in 96-well plates. qPCR assays were then applied to analyze the quantity and distribution of transgenes in those single cells. Results revealed multiple types of transgene copy number distribution profiles from those clonally derived CHO cell lines. The cell lines that maintained productivity over time displayed relatively constant and homogeneous transgene copy number distributions; while most of those cell lines exhibiting a loss of productivity over time showed varying degrees of transgene copy number heterogeneity and distribution drift with passaging. Some cell lines showed the existence of a significant portion of cells lacking the transgenes (referred to as negative cells in this study) and the percentage of those negative cells increased with subsequent generations. Criteria based on transgene copy number distribution profiles were developed to assess cell line suitability for clinical applications, which include (i) percentage of negative cells; (ii) standard deviation of qPCR threshold cycle ( Ct) value, a measure of population heterogeneity; (iii) mean Ct changes during aging, a measure of population drift. By implementing these criteria, undesirable cell lines were eliminated for further clinical and commercial applications. Biotechnol. Bioeng. 2012; 109:1713-1722. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background. Gene electrotransfer is a non-viral gene delivery method that requires successful electroporation for DNA delivery into the cells. Changing the direction of the electric field during the pulse application improves the efficacy of gene delivery. In our study, we tested a pipette tip with integrated electrodes that enables changing the direction of the electric field for electroporation of cell suspension for gene electrotransfer. Materials and methods. A new pipette tip consists of four cylindrical rod electrodes that allow the application of electric pulses in different electric field directions. The experiments were performed on cell suspension of CHO cells in phosphate buffer. Plasmid DNA encoding for green fluorescent protein (GFP) was used and the efficiency of gene electrotransfer was determined by counting cells expressing GFP 24 h after the experiment. Results. Experimental results showed that the percentage of cells expressing GFP increased when the electric field orientation was changed during the application. The GFP expression was almost two times higher when the pulses were applied in orthogonal directions in comparison with single direction, while cell viability was not significantly affected. Conclusions. We can conclude that results obtained with the described pipette tip are comparable to previously published results on gene electrotransfer using similar electrode geometry and electric pulse parameters. The tested pipette tip, however, allows work with small volumes/samples and requires less cell manipulation. [ABSTRACT FROM AUTHOR]

To optimize Chinese Hamster Ovary (CHO) cell culture to recombinant protein therapeutic production, we stably overexpressed survivin and cyclin D1 in three CHO DG44-derived cell lines. The modifications conferred increases of 56-94% in S-phase fractions and decreases of 33-43% in early-stage apoptosis fractions. Clone 6.3, which expressed the highest levels of survivin and cyclin D1, reached significantly greater cell densities in suspension (2.7 × 10 cells/ml) following serum deprivation. Nude mice inoculated with the modified cells showed no tumorigenesis suggesting that the CHO DG44-derived cell lines are viable candidates for biopharmaceutical production. [ABSTRACT FROM AUTHOR]

In an attempt to develop high producing mammalian cell lines expressing glucagon-like-peptide-1-antibody fusion proteins (GLP-1), we have noted that the N-terminal GLP-1 portion of the fusion protein was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. The majority of the N-terminal clipped product appeared to be due to the removal of the entire biologically active peptide (30 amino acids) from the intact molecule. A number of parameters that influenced the degradative process were investigated. Additionally, protease inhibitors specific for each class of protease were tested. Results suggested that one or more serine-threonine class of protease(s) were involved in this process and inhibitors that are specific for this class of protease, including benzamidine hydrochloride could significantly inhibit the proteolytic degradation of the fusion proteins. Identification of the specific proteases involved in this process by shotgun proteomics methodology will pave the way for engineering the CHOK1SV cell line which will serve as a superior host for the production of future fusion protein products. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 [ABSTRACT FROM AUTHOR]

Abstract: Exposure of humans to benzene present in environment may lead to adverse chronic effects—even at the genetic level. However, the mechanism of its genotoxicity is not well understood. In the present study, in vitro genotoxicity of benzene (BZ) and its major metabolites [p-benzoquinone (BQ), hydroquinone (HQ), catechol (CT), 1,2,4-benzenetriol (BT) and trans–trans muconic acid (MA)] at concentrations 0.5–50μM, was assessed in Chinese hamster ovary (CHO) cells employing the alkaline Comet assay, cytokinesis blocked micronucleus (CBMN) assay, flow cytometric analysis of micronucleus (flow MN) and chromosome aberration (CA) test. The data revealed significant (P <0.05) concentration-dependent response in all end points. HQ was found to be the most potent DNA damaging metabolite in the Comet assay followed by BQ>BT>CT>BZ>MA. Both CBMN and flow MN assays revealed a good correlation in their results, where BQ and MA exhibited maximum and minimum micronucleus induction respectively. Significant chromosomal aberrations were induced mainly by BQ, BT and HQ, with moderate response shown by CT and BZ and least by MA. The results demonstrated the utility of sensitive techniques like Comet assay and flow cytometry for determination of MN, to quantify in vitro genotoxicity at low levels and also suggested that partly non-repaired DNA damage could cause adverse health effects in human population exposed to benzene. In silico studies using different endpoints of genotoxicity and molecular docking studies with human topoisomerase-II alpha, a major DNA repair enzyme were also conducted. These corroborated the results obtained from the in vitro data, pointing to a direct relationship of the observed genotoxicity with the structural properties and various interactions of metabolites with the enzyme. This comprehensive study demonstrated that genotoxicity of benzene in mammalian cells is mainly due to the inhibition of topoisomerase by the metabolites. [Copyright &y& Elsevier]

Milky sap isolated from Chelidonium majus L. (Greater Celandine) serves as a rich source of various biologically active substances such as alkaloids, flavonoids and phenolic acids. Previous research showed that the activity of Ch. majus milky sap may depend also on the presence of biologically active proteins. The goal of this study was to evaluate the biological effect of two nucleases isolated from Ch. majus milky sap, CMN1 of 20 kDa and CMN2 of 36 kDa, on HeLa and CHO tumour cell lines. Both studied nucleases together with other proteins in the sap of the plant are involved in stress and defence reactions against different pathogens. After 48 h incubation of CMN1 and CMN2 only with HeLa cells, the depend- ence between the number of apoptotic lesions and the concentration of applied nuclease was observed. The highest proapoptotic activity was induced by 13.3 ng/ml concentration of CMN2 collected in May (62 ± 3% HeLa cells were apoptotic). Moreover, the proportion of necrotic cells in all concentrations of the nucleases and both cell lines was relatively low (1-8 ± 0.5%). In summary, results of this study show that purified nucleases CMN1 and CMN2 isolated from Ch. majus milky sap exhibit apoptotic activity in HeLa tumour cell line, but not in CHO cells, without inflamanatory reaction. [ABSTRACT FROM AUTHOR]

An interaction between adenosine A2A and dopamine D2 receptors has been demonstrated previously. It is generally found that agonist treatment internalizes receptors, including A2A and D2, whereas less is known of the long-term effects involved in the agonist-mediated trafficking of A2A and D2 receptors. Furthermore, the possible influence of the antagonists on receptor trafficking is still undefined. The present studies focus on the long-term effects of A2A and D2 agonist and D2 antagonist treatments on both A2A and D2 receptor trafficking studied at three different time intervals—3, 15, and 24 h. In addition, with the fluorescence resonance energy transfer technique, formation of heteromeric A2A and D2 receptor complexes was shown in the cotransfected CHO cell line. Confocal microscopy analysis showed that a 3-h treatment with the D2 agonist induced coaggregation of A2A/D2 receptors. These A2A/D2 receptor coaggregates internalized after 15 h with a recruitment of the receptors back to the cell membrane after 24 h. In contrast to the effects of the agonist treatment, a 3-h treatment with the D2-like antagonist raclopride increased both A2A and D2 immunoreactivity, indicating that the D2 antagonist stabilizes the D2 receptor and thereby reduces the internalization of both of the A2A and D2 receptors. Taken together, an activation of either A2A and D2 receptor or blockade of D2 receptors will cause long-lasting changes in A2A and D2 receptor trafficking. [ABSTRACT FROM AUTHOR]

Functional interactions between adenosine A2A and dopamine D2 receptors have been demonstrated both at the D2 agonist-binding and second messenger levels. The present studies use a [3H]dopamine-binding assay as a sensitive measure of A2A receptor-mediated modulation of D2 receptors. Co-incubation with an A2A receptor agonist increased the K d value of high-affinity [3H]dopamine-binding sites of the D2 receptor without changing their B max values in a cotransfected cell line. This interaction was shown to be subtype specific, as the A2A receptor agonist did not modulate the affinity of the D1 receptor for [3H]dopamine. The domains of the D2 receptor important for the A2A/D2 receptor interaction were studied with chimeric dopamine D2/D1 receptors. The results showed that the A2A receptor agonist still strongly reduced the affinity of a D2/D1 chimera with the sixth trans-membrane (TM) domain and third extracellular loop from the D1 receptor. However, the A2A receptor agonist was not able to modulate a D2/D1 chimeric receptor containing the fifth and sixth TM domains and the third intracellular and extracellular loops from the D1 receptor, indicating that the fifth TM domain and/or the third intracellular loop may be involved in the interaction between A2A and D2 receptors. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that Chinese hamster ovary (CHO) cells transfected with the angiotensin II AT1 receptor gene containing only the coding region, presented tachyphylaxis to the total inositol phosphate (InsPs) and Ca2+ responses mediated by angiotensin II and [2-lysine]angiotensin II ([Lys2]angiotensin II). Now we have evaluated the possible role of the 3′-untranslated region of the angiotensin AT1 receptor mRNA in modulating the angiotensin AT1 receptor-mediated cellular responses. The binding parameters, as well as the Ca2+ and InsPs responses induced by angiotensin II and [Lys2]angiotensin II were similar in cells transfected with the angiotensin AT1 receptor with or without the 3′-untranslated region sequence. In cells transfected with the receptor containing the 3′-untranslated region sequence, angiotensin II-induced Ca2+ and InsPs responses were desensitized by repeated stimulations, whereas [Lys2]angiotensin II caused desensitization of InsPs production but not of Ca2+ uptake in these cells. Our results suggest that the 3′-untranslated region plays a role in modulating cell signalling involved in the tachyphylaxis of angiotensin AT1 receptor-mediated Ca2+ responses. [Copyright &y& Elsevier]

The manifestation of tachyphylaxis to angiotensin II in Chinese hamster ovary (CHO) cells expressing the rat angiotensin II AT1 receptor was investigated. The cells were transfected with a cDNA fragment containing the complete coding region of the angiotensin II AT1A receptor gene, as well as 56 bp of its 3′- and 52 bp of its 5′-untranslated regions. These cells (CHO-AT1) responded to angiotensin II by increases in intracellular Ca2+ concentration and inositol phosphate turnover, which were inhibited upon repeated administrations, characterizing the tachyphylaxis phenomenon. In contrast to smooth muscle cells, which are rendered tachyphylactic to angiotensin II but not to [2-lysine]angiotensin II ([Lys2]angiotensin II), this analogue induced responses in CHO-AT1 cells that were also inhibited upon repeated administrations. A smooth muscle cell line, which showed tachyphylaxis only to angiotensin II, became tachyphylactic also to [Lys2]angiotensin II after transfection with the angiotensin II AT1 receptor gene. Our findings suggest that posttranscriptional control directed by the 3′- or the 5′-untranslated regions in the angiotensin II AT1 receptor gene may play a role in modulating the signal transduction pathways involved in the mechanism of angiotensin II tachyphylaxis. [Copyright &y& Elsevier]

The human brain voltage-gated Na+ channel type IIA α subunit was cloned and stably expressed in Chinese hamster ovary cells and its biophysical and pharmacological properties were studied using whole-cell voltage-clamp. Fast, transient inward currents of up to –8000 pA were elicited by membrane depolarization of the recombinant cells. Channels activated at –50 mV and reached maximal activation at –10 mV to 0 mV. The reversal potential was 62±2 mV which is close to the Na+ equilibrium potential. The half-maximal activation and inactivation voltages were –24±2 mV and –63±1 mV, respectively. Currents were reversibly blocked by tetrodotoxin with a half-maximal inhibition of 13 nM. The effects of four commonly used anti-convulsant drugs were examined for the first time on the cloned human type IIA channel. Lamotrigine and phenytoin produced concentration- and voltage-dependent inhibition of the type IIA currents, whereas, sodium valproate and gabapentin (up to 1 mM) had no effect. These results indicate that recombinant human type IIA Na+ channels conduct tetrodotoxin-sensitive Na+ currents with similar properties to those observed in recombinant rat brain type IIA and native rat brain Na+ channels. This stable cell line should provide a useful tool for more detailed characterization of therapeutic modulators of human Na+ channels. [ABSTRACT FROM AUTHOR]

The proprotein processing enzyme furin is the mammalian prototype of a novel family of subtilisin-like serine endoproteases which possess cleavage specificity for sites involving multiple basic amino acid residues and are involved in the processing of precursor proteins of a variety of regulatory peptides and proteins. One of the limiting steps in the engineering of mammalian cells designed for the overproduction of secreted proteins is the endoproteolytic cleavage of the precursor molecule to its mature biologically active form. The extremely low level of endogenous furin is likely the reason why cells are not able to fully mature overexpressed precursor proteins to their mature form. Here, we report a CHO-derived cell line genetically engineered for the production of high levels of recombinant proteins that need such endoproteolytic maturation. First, the human furin cDNA under the control of the cytomegalovirus early promoter and enhancer was introduced and overexpressed in a DHFR-deficient CHO cell line. A permanent cell line CHO-D3-FUR was established that expressed biologically active furin. Subsequently, to demonstrate the capacity of CHO-D3-FUR cells to produce recombinant proteins in a fully matured form, two derivative cell lines were established that overexpressed the von Willebrand factor (vWF) and transforming growth factor β1 (TGFβ1); CHO-D3-vWF and CHO-D3-TGFβ1, respectively. Both derivative cell lines were able to produce relatively high levels of recombinant protein in a fully matured and biologically active form. Our results illustrate the potential of the CHO-D3-FUR cell line in the production of recombinant secretory proteins that need endoproteolytic activation at the consensus furin cleavage sequence Arg-X-Lys/Arg-Arg. [ABSTRACT FROM AUTHOR]