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1.
Sci Rep ; 13(1): 774, 2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36641503

RESUMO

Treatment of COVID-19 with a soluble version of ACE2 that binds to SARS-CoV-2 virions before they enter host cells is a promising approach, however it needs to be optimized and adapted to emerging viral variants. The computational workflow presented here consists of molecular dynamics simulations for spike RBD-hACE2 binding affinity assessments of multiple spike RBD/hACE2 variants and a novel convolutional neural network architecture working on pairs of voxelized force-fields for efficient search-space reduction. We identified hACE2-Fc K31W and multi-mutation variants as high-affinity candidates, which we validated in vitro with virus neutralization assays. We evaluated binding affinities of these ACE2 variants with the RBDs of Omicron BA.3, Omicron BA.4/BA.5, and Omicron BA.2.75 in silico. In addition, candidates produced in Nicotiana benthamiana, an expression organism for potential large-scale production, showed a 4.6-fold reduction in half-maximal inhibitory concentration (IC50) compared with the same variant produced in CHO cells and an almost six-fold IC50 reduction compared with wild-type hACE2-Fc.


Assuntos
COVID-19 , Aprendizado Profundo , Animais , Cricetinae , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Cricetulus , Simulação de Dinâmica Molecular , Ligação Proteica
2.
Sci Rep ; 13(1): 1200, 2023 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-36681715

RESUMO

Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr®250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.


Assuntos
Técnicas de Cultura Celular por Lotes , Ácido Láctico , Cricetinae , Animais , Humanos , Cricetulus , Células CHO , Genoma , DNA
3.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674424

RESUMO

The regulation and metabolism of the endocannabinoid system has received extensive attention for their potential neuroprotective effect in neurodegenerative diseases such as Alzheimer's disease (AD), which is characterized by amyloid ß (Aß) -induced cell toxicity, inflammation, and oxidative stress. Using in vitro techniques and two cell lines, the mouse hippocampus-derived HT22 cells and Chinese hamster ovary (CHO) cells expressing human cannabinoid receptor type 1 (CB1), we investigated the ability of endocannabinoids to inhibit Aß aggregation and protect cells against Aß toxicity. The present study provides evidence that endocannabinoids N-arachidonoyl ethanol amide (AEA), noladin and O-arachidonoyl ethanolamine (OAE) inhibit Aß42 aggregation. They were able to provide protection against Aß42 induced cytotoxicity via receptor-mediated and non-receptor-mediated mechanisms in CB1-CHO and HT22 cells, respectively. The aggregation kinetic experiments demonstrate the anti-Aß aggregation activity of some endocannabinoids (AEA, noladin). These data demonstrate the potential role and application of endocannabinoids in AD pathology and treatment.


Assuntos
Doença de Alzheimer , Endocanabinoides , Camundongos , Animais , Cricetinae , Humanos , Endocanabinoides/farmacologia , Endocanabinoides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Células CHO , Cricetulus , Doença de Alzheimer/metabolismo
4.
Int J Mol Sci ; 24(2)2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36674948

RESUMO

In Alzheimer's disease (AD), the reduction in acetylcholinesterase (AChE) enzymatic activity is not paralleled with changes in its protein levels, suggesting the presence of a considerable enzymatically inactive pool in the brain. In the present study, we validated previous findings, and, since inactive forms could result from post-translational modifications, we analyzed the glycosylation of AChE by lectin binding in brain samples from sporadic and familial AD (sAD and fAD). Most of the enzymatically active AChE was bound to lectins Canavalia ensiformis (Con A) and Lens culinaris agglutinin (LCA) that recognize terminal mannoses, whereas Western blot assays showed a very low percentage of AChE protein being recognized by the lectin. This indicates that active and inactive forms of AChE vary in their glycosylation pattern, particularly in the presence of terminal mannoses in active ones. Moreover, sAD subjects showed reduced binding to terminal mannoses compared to non-demented controls, while, for fAD patients that carry mutations in the PSEN1 gene, the binding was higher. The role of presenilin-1 (PS1) in modulating AChE glycosylation was then studied in a cellular model that overexpresses PS1 (CHO-PS1). In CHO-PS1 cells, binding to LCA indicates that AChE displays more terminal mannoses in oligosaccharides with a fucosylated core. Immunocytochemical assays also demonstrated increased presence of AChE in the trans-Golgi. Moreover, AChE enzymatic activity was higher in plasmatic membrane of CHO-PS1 cells. Thus, our results indicate that PS1 modulates trafficking and maturation of AChE in Golgi regions favoring the presence of active forms in the membrane.


Assuntos
Acetilcolinesterase , Doença de Alzheimer , Cricetinae , Animais , Humanos , Acetilcolinesterase/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Doença de Alzheimer/metabolismo , Lectinas/metabolismo , Encéfalo/metabolismo , Cricetulus , Presenilina-2/genética , Mutação
5.
Molecules ; 28(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36615612

RESUMO

Pain is a common clinical symptom among patients. Although various opioid analgesics have been developed, their side effects hinder their application. This study aimed to develop a novel opioid analgesic, HAGD (H-Tyr-D-AIa-GIy-Phe-NH2), with limited side effects. In vivo studies on mouse models as well as in vitro studies on Chinese hamster ovary (CHO) cells expressing human mu, delta, or kappa opioid receptors (CHOhMOP, CHOhDOP, and CHOhKOP, respectively) and human sperm were conducted. Compared with subcutaneous morphine (10 mg/kg), subcutaneous HAGD (10 mg/kg) produced equipotent or even greater antinociception with a prolonged duration by activating mu/delta opioid receptors in preclinical mouse pain models. The analgesic tolerance, rewarding effects (i.e., conditioned place preference and acute hyperlocomotion), and gastrointestinal transit inhibition of HAGD were significantly reduced compared with those of morphine. Both HAGD and morphine exhibited a withdrawal response and had no impacts on motor coordination. In CHOhMOP and CHOhDOP, HAGD showed specific and efficient intracellular Ca2+ stimulation. HAGD had minimal impact on human sperm motility in vitro, whereas 1 × 10-7 and 1 × 10-8 mol/L of morphine significantly declined sperm motility at 3.5 h. Overall, HAGD may serve as a promising antinociceptive compound.


Assuntos
Analgésicos Opioides , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Cricetinae , Humanos , Masculino , Camundongos , Animais , Analgésicos Opioides/efeitos adversos , Receptores Opioides delta , Células CHO , Motilidade Espermática , Cricetulus , Sêmen , Morfina/efeitos adversos , Dor/induzido quimicamente , Dor/tratamento farmacológico , Receptores Opioides mu/agonistas
6.
Sci Rep ; 13(1): 1482, 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36707606

RESUMO

Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.


Assuntos
Glucose , Cricetinae , Animais , Cricetulus , Células CHO , Proteínas Recombinantes/metabolismo , Proliferação de Células
7.
Bioorg Med Chem Lett ; 80: 129120, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36587872

RESUMO

GPR88 is an orphan G protein-coupled receptor which has been implicated in a number of striatal-associated disorders. Herein we describe the synthesis and pharmacological characterization of the first GPR88 radioligand, [3H]RTI-33, derived from a synthetic agonist RTI-13951-33. [3H]RTI-33 has a specific activity of 83.4 Ci/mmol and showed one-site, saturable binding (KD of 85 nM) in membranes prepared from stable PPLS-HA-hGPR88-CHO cells. A competition binding assay was developed to determine binding affinities of several known GPR88 agonists. This radioligand represents a powerful tool for future mechanistic and cell-based ligand-receptor interaction studies of GPR88.


Assuntos
Proteínas de Transporte , Receptores Acoplados a Proteínas G , Cricetinae , Animais , Cricetulus , Receptores Acoplados a Proteínas G/agonistas , Ensaio Radioligante
8.
Int J Mol Med ; 51(2)2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36660940

RESUMO

Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, which is highly expressed on tumor cells. As EpCAM plays a crucial role in cell adhesion, survival, proliferation, stemness, and tumorigenesis, it has been considered as a promising target for tumor diagnosis and therapy. Anti­EpCAM monoclonal antibodies (mAbs) have been developed and have previously demonstrated promising outcomes in several clinical trials. An anti­EpCAM mAb, EpMab­37 (mouse IgG1, kappa) was previously developed by the authors, using the cell­based immunization and screening method. In the present study, a defucosylated version of anti­EpCAM mAb (EpMab­37­mG2a­f) was generated to evaluate the antitumor activity against EpCAM­positive cells. EpMab­37­mG2a­f recognized EpCAM­overexpressing CHO­K1 (CHO/EpCAM) cells with a moderate binding­affinity [dissociation constant (KD)=2.2x10­8 M] using flow cytometry. EpMab­37­mG2a­f exhibited potent antibody­dependent cellular cytotoxicity (ADCC) and complement­dependent cytotoxicity (CDC) for CHO/EpCAM cells by murine splenocytes and complements, respectively. Furthermore, the administration of EpMab­37­mG2a­f significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab­37­mG2a­f also exhibited a moderate binding­affinity (KD=1.5x10­8 M) and high ADCC and CDC activities for a colorectal cancer cell line (Caco­2 cells). The administration of EpMab­37­mG2a­f to Caco­2 tumor­bearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab­37­mG2a­f never suppressed the xenograft tumor growth of Caco­2 cells in which EpCAM was knocked out. On the whole, these results indicate that EpMab­37­mG2a­f may exert antitumor activities against EpCAM­positive cancers and may thus be a promising therapeutic regimen for colorectal cancer.


Assuntos
Anticorpos Monoclonais , Neoplasias Colorretais , Cricetinae , Humanos , Animais , Camundongos , Anticorpos Monoclonais/uso terapêutico , Células CACO-2 , Xenoenxertos , Molécula de Adesão da Célula Epitelial , Cricetulus , Neoplasias Colorretais/tratamento farmacológico , Imunoglobulina G , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral
9.
J Biol Chem ; 299(1): 102803, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36529291

RESUMO

Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, that is, induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF-7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.


Assuntos
Adenocarcinoma , Fator de Crescimento do Tecido Conjuntivo , Animais , Cricetinae , Humanos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Cricetulus , Proteínas de Sinalização Intercelular CCN/genética , Proteínas de Sinalização Intercelular CCN/metabolismo , Peptídeos , Proteínas Recombinantes
10.
J Chromatogr A ; 1687: 463701, 2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36502645

RESUMO

Engineered multi-specific monoclonal antibodies (msAbs) and antibody fragments offer valuable therapeutic options against metabolic disorders, aggressive cancers, and viral infections. The advancement in molecular design and recombinant expression of these next-generation drugs, however, is not equaled by the progress in downstream bioprocess technology. The purification of msAbs and fragments requires affinity adsorbents with orthogonal biorecognition of different portions of the antibody structure, namely its Fc (fragment crystallizable) and Fab (fragment antigen-binding) regions or the CH1-3 and CL chains. Current adsorbents rely on protein ligands that, while featuring high binding capacity and selectivity, need harsh elution conditions and suffer from high cost, limited biochemical stability, and potential release of immunogenic fragments. Responding to these challenges, we undertook the de novo discovery of peptide ligands that target different regions of human Fab and enable product release under mild conditions. The ligands were discovered by screening a focused library of 12-mer peptides against a feedstock comprising human Fab and Chinese hamster ovary host cell proteins (CHO HCPs). The identified ligands were evaluated via binding studies as well as molecular docking simulations, returning excellent values of binding capacity (Qmax ∼ 20 mg of Fab per mL of resin) and dissociation constant (KD = 2.16·10-6 M). Selected ligand FRWNFHRNTFFP and commercial Protein L ligands were further characterized by measuring the dynamic binding capacity (DBC10%) at different residence times (RT) and performing the purification of polyclonal and monoclonal Fabs from CHO-K1 cell culture fluids. The peptide ligand featured DBC10% ∼ 6-16 mg/mL (RT of 2 min) and afforded values of yield (93-96%) and purity (89-96%) comparable to those provided by Protein L resins.


Assuntos
Anticorpos Monoclonais , Peptídeos , Cricetinae , Animais , Humanos , Cricetulus , Ligantes , Células CHO , Simulação de Acoplamento Molecular , Ligação Proteica , Peptídeos/química , Cromatografia de Afinidade
11.
Talanta ; 254: 124187, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36549134

RESUMO

The biopharmaceutical industry extensively employs Chinese hamster ovary (CHO) cell culture for monoclonal antibody production. Amino acids represent an essential source of nutrients in all CHO cell culture media, and their concentration is known to significantly impact cell viability, titre, and monoclonal antibody critical quality attributes. In this study, a robust Fourier transform near-infrared spectroscopy (FT-NIR) based quantification method has been developed for of all 20 amino acids (0-24 mM), as well as concentrations of glucose (0-6.7 mg mL-1), lactate (0-2.7 mg mL-1), and trastuzumab (0-2.5 mg mL-1) in the CHO cell culture. Near infra-red absorbance spectrum in the range of 4000-11,000 cm-1 were acquired, and spectra pre-processing through smoothening and derivatives were employed to enhance key characteristic signals. High-performance liquid chromatography with pre-column derivatization was used as the orthogonal analytical tool for quantification. Principal component analysis and partial least squares regression were employed for region selection and calibration model development, respectively. The results demonstrate that a good calibration statistic with the acceptable coefficient of determinations for both calibration (Rc2 = 0.94-0.99) and prediction (Rp2 = 0.83-0.98) could be achieved, along with high RPD values (>3) for all components except alanine (2.4). The external validation study also exhibited a satisfactory outcome (REV2 = 0.89-0.99, RMSE = 0.04-1.04), validating the model's ability to predict the concentrations of the respective species. The calibration models were successfully applied for at-line monitoring of two perfusion runs on a 10 L scale. To our knowledge, this is the first application where NIR spectroscopy-based measurement of all 20 amino acids in mammalian cell culture samples has been demonstrated. The proposed tool can play a critical role as biopharma manufacturers implement continuous processing as well as for facilitating process analytical technology-based control of mammalian cell culture processes.


Assuntos
Aminoácidos , Espectroscopia de Luz Próxima ao Infravermelho , Cricetinae , Animais , Células CHO , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Cricetulus , Técnicas de Cultura de Células/métodos , Análise dos Mínimos Quadrados , Anticorpos Monoclonais , Calibragem
12.
Methods Mol Biol ; 2557: 755-764, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512249

RESUMO

Sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate the uptake and synthesis of cholesterol and fatty acids in mammalian cells. SREBP cleavage-activating protein (SCAP) is an endoplasmic reticulum (ER) protein that binds newly synthesized SREBP, retaining it in the ER where SREBP is inactive. SCAP binds cholesterol and functions as the cholesterol sensor in this regulatory system. Under low cholesterol conditions, SCAP escorts SREBP from the ER to the Golgi apparatus where two proteases sequentially cleave and activate SREBP. Given their central importance in maintaining cellular lipid homeostasis, other mechanisms exist to regulate SREBP activity, such as control of protein synthesis and degradation. Here, we describe methods to assay ER-to-Golgi transport of SCAP in vitro using immunofluorescence microscopy and two different cell systems, Chinese hamster ovary (CHO) cells stably expressing hamster GFP-SCAP and human HeLa cells transiently expressing human GFP-SCAP. These methods will permit investigators to determine if cellular perturbations act by affecting the ER-to-Golgi transport of SCAP.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Esteróis , Cricetinae , Animais , Humanos , Esteróis/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Células CHO , Células HeLa , Cricetulus , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Golgi/metabolismo , Retículo Endoplasmático/metabolismo , Colesterol/metabolismo , Microscopia de Fluorescência , Transporte Proteico
13.
Water Res ; 230: 119512, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36580801

RESUMO

Disinfecting reclaimed water for safe reuse can produce toxic disinfection by-products such as adsorbable organic bromine (AOBr). Irradiating stored reclaimed water with sunlight is a "green" and free method for eliminating some toxic disinfection by-products, but the effects of irradiation with sunlight on ozonated reclaimed water containing bromide are not well understood. In this study, AOBr was found at concentrations of 171-180 (µg Br)/L in ozonated reclaimed water containing bromide at a concentration of 2 (mg Br)/L and dissolved organic carbon at a concentration of ∼5 (mg C)/L. Irradiation with sunlight degraded 53-74% of the AOBr in two reclaimed water samples in 8 h, and the pseudo-first-order rate constants (k) were 0.09-0.17 h-1. The concentration of tribromomethane, a typical Br-containing disinfection by-product, was decreased by >96% by irradiation for 8 h (k = 0.42-0.47 h-1). Irradiation with sunlight decreased the toxicity of ozonated reclaimed water to Chinese hamster ovary cells. Irradiation with sunlight decreased the degree of intracellular oxidative stress and oxidative damage caused by ozonated reclaimed water. Irradiation with sunlight for 8 h decreased cytotoxicity of the ozonated reclaimed water samples by 79% and 65%. The change in AOBr concentration correlated with the change in toxicity (R2=0.69, p<0.05). The relationships between sunlight wavelength and decreases in the AOBr concentration and toxicity were assessed, and it was found that UV in sunlight was predominantly responsible for decreasing the AOBr concentration and toxicity by reclaimed water. During irradiation for 8 h, UV was responsible for 65%-66% of the decrease in the AOBr concentration and 65-79% of the decrease in reclaimed water induced cytotoxicity. Irradiation with sunlight is a promising method for degrading AOBr and detoxifying ozonated reclaimed water during storage to allow the water to be reused.


Assuntos
Poluentes Químicos da Água , Purificação da Água , Cricetinae , Animais , Bromo , Água , Luz Solar , Brometos , Células CHO , Cricetulus , Poluentes Químicos da Água/análise , Desinfecção , Purificação da Água/métodos
14.
Appl Microbiol Biotechnol ; 107(2-3): 769-783, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36536089

RESUMO

Recombinant Chinese hamster ovary (CHO) cell line development for complex biotherapeutic production is conventionally based on the random integration (RI) approach. Due to the lack of control over the integration site and copy number, RI-generated cell pools are always coupled with rigorous screening to find clones that satisfy requirements for production titers, quality, and stability. Targeted integration into a well-defined genomic site has been suggested as a possible strategy to mitigate the drawbacks associated with RI. In this work, we employed the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system in combination with the Bxb1 recombinase-mediated cassette exchange (RMCE) system to generate an isogenic transgene-expressing cell line. We successfully utilized the CRIS-PITCh system to target a 2.6 kb Bxb1 landing pad with homology arms as short as 30 bp into the upstream region of the S100A gene cluster, achieving a targeting efficiency of 10.4%. The platform cell line (PCL) with a single copy of the landing pad was then employed for the Bxb1-mediated landing pad exchange with an EGFP encoding cassette to prove its functionality. Finally, to accomplish the main goal of our cell line development method, the PCL was applied for the expression of a secretory glycoprotein, human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2). Taken together, on-target, single-copy, and stable expression of the transgene over long-term cultivation demonstrated our CRIS-PITCh/RMCE hybrid approach might possibly improve the cell line development process in terms of timeline, specificity, and stability. KEY POINTS: • CRIS-PITCh system is an efficient method for single copy targeted integration of the landing pad and generation of platform cell line • Upstream region of the S100A gene cluster of CHO-K1 is retargetable by recombinase-mediated cassette exchange (RMCE) approach and provides a stable expression of the transgene • CRIS-PITCh/Bxb1 RMCE hybrid system has the potential to overcome some limitations of the random integration approach and accelerate the cell line development timeline.


Assuntos
Genoma , Recombinases , Cricetinae , Animais , Humanos , Células CHO , Cricetulus , Recombinases/genética , Transgenes
15.
Clin Drug Investig ; 43(1): 37-44, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36478528

RESUMO

BACKGROUND: Follitropin delta, a novel recombinant follicle-stimulating hormone (rFSH) preparation derived from a human cell line, has different pharmacokinetic and pharmacodynamic properties compared with existing rFSH preparations expressed by Chinese hamster ovary cells (CHO). OBJECTIVES: The objective of this study was to assess the pharmacokinetic characteristics, dose proportionality, and safety of follitropin delta in healthy Chinese women. METHODS: This was a phase I, randomized, open-label study. Twenty-four healthy Chinese women were randomized (1:1:1) to receive a single subcutaneous administration of follitropin delta 12, 18, or 24 µg. The pharmacokinetic parameters (maximum observed serum concentration [Cmax], time to reach Cmax [tmax], area under the serum concentration-time curve from dosing to infinity [AUC∞], and elimination phase half-life [t½]) of follitropin delta were derived using noncompartmental analysis. RESULTS: Following a single subcutaneous administration of follitropin delta 12, 18, or 24 µg, mean Cmax (0.388, 0.677, and 0.825 ng/mL, respectively) and AUC∞ (41.3, 62.9, and 83.1 h·ng/mL, respectively) increased in a dose-proportional manner. The median tmax was 24 h, and the mean t½ was in the range of 50.5-60.9 h. All treatment-related adverse events were categorized as mild, except for one case of urticaria from the follitropin delta 18-µg dose group which was considered moderate. Only one woman presented with elevation of alanine transaminase and aspartate aminotransferase at the follow-up visit, which was reported as a treatment-emergent adverse event. There were no injection-site reactions and none of the participants showed any confirmed presence of treatment-induced anti-FSH antibodies. CONCLUSIONS: The administration of single doses of follitropin delta to healthy Chinese women demonstrated dose-proportional pharmacokinetics over the dose range of 12-24 µg, and these doses were well tolerated. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov registration no. NCT04150861.


Assuntos
Hormônio Foliculoestimulante Humano , Hormônio Foliculoestimulante , Cricetinae , Animais , Humanos , Feminino , Células CHO , Cricetulus , Hormônio Foliculoestimulante Humano/efeitos adversos , Hormônio Foliculoestimulante Humano/farmacocinética
16.
Biotechnol Adv ; 62: 108080, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36526238

RESUMO

Bone morphogenetic proteins (BMPs) are a group of growth factors with the clinical potential to regulate cartilage and bone formation. Functionally active mature recombinant human BMPs (rhBMPs), produced primarily in Chinese hamster ovary (CHO) cells for clinical applications, are considered difficult to express because they undergo maturation processes, signaling pathways, or endocytosis. Although BMPs are a family of proteins with similar mature domain sequence identities, their individual properties are diverse. Thus, understanding the properties of individual rhBMPs is essential to improve rhBMP production in CHO cells. In this review, we discuss various approaches to improve rhBMP production in CHO cells by understanding the overall maturation process, signaling pathways and endocytosis of individual rhBMPs.


Assuntos
Proteínas Morfogenéticas Ósseas , Endocitose , Cricetinae , Animais , Humanos , Cricetulus , Células CHO , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
Bioelectrochemistry ; 149: 108319, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36375440

RESUMO

The reversal of the electric field direction inhibits various biological effects of nanosecond electric pulses (nsEP). This feature, known as "bipolar cancellation," enables interference targeting of nsEP bioeffects remotely from stimulating electrodes, for prospective applications such as precise cancer ablation and non-invasive deep brain stimulation. This study was undertaken to achieve the maximum cancellation of electroporation, by quantifying the impact of the pulse shape, duration, number, and repetition rate across a broad range of electric field strengths. Monolayers of endothelial cells (BPAE) were electroporated in a non-uniform electric field. Cell membrane permeabilization was quantified by YO-PRO-1 (YP) dye uptake and correlated to local electric field strength. For most conditions tested, adding an opposite polarity phase reduced YP uptake by 50-80 %. The strongest cancellation, which reduced YP uptake by 95-97 %, was accomplished by adding a 50 % second phase to 600-ns pulses delivered at a high repetition rate of 833 kHz. Strobe photography of nanosecond kinetics of membrane potential in single CHO cells revealed the temporal summation of polarization by individual unipolar nsEP applied at sub-MHz rate, leading to enhanced electroporation. In contrast, there was no summation for bipolar pulses, and increasing their repetition rate suppressed electroporation. These new findings are discussed in the context of bipolar cancellation mechanisms and remote focusing applications.


Assuntos
Eletroporação , Células Endoteliais , Cricetinae , Animais , Cricetulus , Permeabilidade da Membrana Celular , Células CHO
18.
Biotechnol Prog ; 38(1): e3211, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34549552

RESUMO

Chinese hamster ovary (CHO) cells are by far the most important mammalian cell lines used for producing antibodies and other therapeutic proteins. It is critical to fully understand their physiological conditions during a bioprocess in order to achieve the highest productivity and the desired product quality. Flow cytometry technology possesses unique advantages for measuring multiple cellular attributes for a given cell and examining changes in cell culture heterogeneity over time that can be used as metrics for enhanced process understanding and control strategy. Flow cytometry-based assays were utilized to examine the progression of cell cycle and apoptosis in three case studies using different antibody-producing CHO cell lines in both fed-batch and perfusion bioprocesses. In our case studies, we found that G0/G1 phase distribution and early apoptosis accumulation responded to subtle changes in culture conditions, such as pH shifting or momentary glucose depletion. In a perfusion process, flow cytometry provided an insightful understanding of the cell physiological status under a hypothermic condition. More importantly, these changes in cell cycle and apoptosis were not detected by a routine trypan blue exclusion-based cell counting and viability measurement. In summary, integration of flow cytometry into bioprocesses as a process analytical technology tool can be beneficial for establishing optimum process conditions and process control.


Assuntos
Apoptose , Técnicas de Cultura de Células , Animais , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Células CHO , Ciclo Celular , Cricetinae , Cricetulus , Citometria de Fluxo
19.
Biotechnol Prog ; 38(6): e3290, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36537257

RESUMO

Antigen binding fragments (Fab) are a promising class of therapeutics as they maintain high potency while having significantly smaller size relative to full-length antibodies. Because Fab molecules are aglycosylated, many expression platforms, including prokaryotic, yeast, and mammalian cells, have been developed for their expression, with Escherichia coli being the most commonly used Fab expression system. In this study, we have examined production of a difficult to express Fab molecule in a targeted integration (TI) Chinese Hamster Ovary (CHO) host. Without a need for extensive host or process optimization, as is usually required for E. coli, by simply using different vector configurations, clones with very high Fab expression titers were obtained. In this case, by increasing heavy chain (HC) gene copy numbers, clones with titers of up to 7.4 g/L in the standard fed-batch production culture were obtained. Our findings suggest that having a predetermined transgene integration site, as well as the option to optimize gene copy number/dosage, makes CHO TI hosts an effective system for expression of Fab molecules, allowing Fab expression using platform process and without significant process development efforts.


Assuntos
Fragmentos Fab das Imunoglobulinas , Proteínas Recombinantes , Animais , Cricetinae , Células CHO , Cricetulus , Dosagem de Genes , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transgenes
20.
Int J Mol Sci ; 23(24)2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36555637

RESUMO

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts effective neuroprotective activity through its specific receptor, PAC1-R. We accidentally discovered that as a positive allosteric modulator (PAM) of PAC1-R, the small-molecule PAM (SPAM1) has a hydrazide-like structure, but different binding characteristics, from hydrazide for the N-terminal extracellular domain of PAC1-R (PAC1-R-EC1). SPAM1 had a significant neuroprotective effect against oxidative stress, both in a cell model treated with hydrogen peroxide (H2O2) and an aging mouse model induced by D-galactose (D-gal). SPAM1 was found to block the decrease in PACAP levels in brain tissues induced by D-gal and significantly induced the nuclear translocation of PAC1-R in PAC1R-CHO cells and mouse retinal ganglion cells. Nuclear PAC1-R was subjected to fragmentation and the nuclear 35 kDa, but not the 15 kDa fragments, of PAC1-R interacted with SP1 to upregulate the expression of Huntingtin (Htt), which then exerted a neuroprotective effect by attenuating the binding availability of the neuron-restrictive silencer factor (NRSF) to the neuron-restrictive silencer element (NRSE). This resulted in an upregulation of the expression of NRSF-related neuropeptides, including PACAP, the brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH), and synapsin-1 (SYN1). The novel mechanism reported in this study indicates that SPAM1 has potential use as a drug, as it exerts a neuroprotective effect by regulating NRSF.


Assuntos
Fármacos Neuroprotetores , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Cricetinae , Camundongos , Animais , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Fármacos Neuroprotetores/farmacologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Cricetulus , Peróxido de Hidrogênio
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