RESUMEN
OBJECTIVES: To evaluate the characteristics of a novel human cell line, F2N78, including growth performance, physicochemical properties, and biological activity via direct comparison with CHO cells. RESULTS: The culture performance and physicochemical properties of antibodies produced from F2N78 and CHO cells were compared. For charge variants, antibodies produced from F2N78 cells contained a greater acidic charge variants than CHO cells. Regarding main glycoforms, degree of galactosylation was 52% in CT-A produced from F2N78 cells compared to CHO cells (37%). For sialic acid forms, α-2,6-linked sialic acid and N-acetylneuraminic acid (NANA) residues were observed in antibodies produced from F2N78 cells. In contrast, only α-2,3 linked sialic acid forms were detected in antibodies produced from CHO cells, and NANA and N-glycolylneuraminic acid were detected. Hybrid structure and bisecting structure were only observed in F2N78 cells. CONCLUSIONS: F2N78 cells stably produced antibodies with human specific N-glycan. The novel expression system based on human cells may facilitate the development of an alternative host cell for production of recombinant proteins.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Línea Celular/citología , Glicosilación , Animales , Anticuerpos Monoclonales/química , Células CHO , Cromatografía Líquida de Alta Presión , Cricetulus , Humanos , Células Híbridas/citología , Ácidos Neuramínicos/química , Polisacáridos/química , Ácidos Siálicos/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The human cell line rF2N78 produces an antibody with a high galactosylation ratio which resembles human IgG. However, it has been observed that the aglycosylated antibody starts to appear when glucose is depleted. To determine whether glucose depletion is a main cause for aglycosylation of the antibody, fed-batch cultures of rF2N78 cells were performed using different feeding cocktails (glucose only, nutrient feeding cocktail without glucose, and nutrient feeding cocktail with glucose). In the fed-batch culture with nutrient feeding cocktail without glucose, aglycosylated antibody was produced in a later phase of culture, when glucose was depleted. Approximately 44 % of antibodies produced were aglycosylated at the end of culture. In contrast, aglycosylated antibody was not produced in cultures with glucose feeding. The expression levels of oligosaccharyl transferases determined by Western blot analysis were similar among the cultures, suggesting that aglycosylation of the antibody was not due to altered expression of oligosaccharyl transferases under glucose-deficient conditions. Thus, it is likely that glucose deficiency led to insufficiency of the precursor for glycosylation and induced aglycosylation of the antibody. Taken together, glucose feeding in fed-batch cultures successfully prevented occurrence of aglycosylated antibody during the cultures, confirming that glucose depletion is a main cause for aglycosylation of antibody.
Asunto(s)
Anticuerpos/metabolismo , Medios de Cultivo/química , Glucosa/metabolismo , Anticuerpos/genética , Técnicas de Cultivo Celular por Lotes , Línea Celular , Glicosilación , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0-7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.
Asunto(s)
Anticuerpos Antivirales/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Medios de Cultivo/química , Metabolismo/efectos de los fármacos , Metabolismo/efectos de la radiación , Anticuerpos Antivirales/genética , Técnicas de Cultivo de Célula , Línea Celular , Glicosilación/efectos de los fármacos , Glicosilación/efectos de la radiación , Humanos , Concentración de Iones de Hidrógeno , Virus de la Rabia/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Suspensiones , Tecnología Farmacéutica/métodos , TemperaturaRESUMEN
The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID-19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self-dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc-mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID-19 in several countries, can effectively trap SARS-CoV-2 virus-like particles in fresh human airway mucus. IN-006, a reformulation of regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN-006 resulted in 100-fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN-006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS-CoV-2 and other respiratory pathologies.
RESUMEN
BACKGROUND AND AIMS: CT-P13 is the first biosimilar monoclonal antibody to infliximab, and was recently approved in the European Union, Japan, Korea, and USA for all six indications of infliximab. However, studies directly assessing the biologic activity of CT-P13 versus inflximab in the context of inflammatory bowel disease [IBD] are still scanty. In the present study, we aimed to compare the biological activities of CT-P13 and infliximab with specific focus on intestinal cells so as to gain insight into the potential biosimilarity of these two agents for treatment of IBD. METHODS: CT-P13 and infliximab were investigated and compared by in vitro experiments for their neutralisation ability of soluble tumour necrosis factor alpha [sTNFα] and membrane-bound tumour necrosis factor alpha [mTNFα], suppression of cytokine release by reverse signalling, induction of regulatory macrophages and wound healing, and antibody-dependent cell cytotoxicity [ADCC]. RESULTS: CT-P13 showed similar biological activities to infliximab as gauged by neutralisation of soluble TNFα, as well as blockade of apoptosis and suppression of pro-inflammatory cytokines in intestinal Caco-2 cells. Infliximab and CT-P13 equally induced apoptosis and outside-to-inside signals through transmembrane TNFα [tmTNFα]. Moreover, regulatory macrophage induction and ensuing wound healing were similarly exerted by CT-P13 and infliximab. However, neither CT-P13 nor infliximab exerted any significant ADCC of ex vivo-stimulated peripheral blood monocytes or lamina propria mononuclear cells from IBD patients. CONCLUSIONS: These findings indicate that CT-P13 and infliximab exert highly similar biological activities in intestinal cells, and further support a mechanistic comparability of these two drugs in the treatment of IBD.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Biosimilares Farmacéuticos/farmacología , Fármacos Gastrointestinales/farmacología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Infliximab/farmacología , Intestinos/efectos de los fármacos , Anticuerpos Monoclonales/uso terapéutico , Biosimilares Farmacéuticos/uso terapéutico , Células CACO-2/efectos de los fármacos , Citocinas/metabolismo , Fármacos Gastrointestinales/uso terapéutico , Humanos , Técnicas In Vitro , Infliximab/uso terapéutico , Intestinos/citología , Intestinos/inmunología , Macrófagos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
A human hybrid cell line, F2N78, was developed by somatic fusion of HEK293 and Namalwa cells for the production recombinant biopharmaceutical proteins. In this study, we performed perfusion culture to verify its potential in culture process used for human cell expression platform. Cell viability could be maintained over 90% and high viable cell density was obtained at higher than 1.0 × 10(7) cells/mL by bleeding process in perfusion culture. The cells were adapted well in both culture modes, but there were apparent differences in protein quality. Compared to fed-batch culture, degalactosylated forms such as G0F and G0 as well as Man5 showed no significant increases in perfusion culture. In terms of charge variants, acidic peaks increased, whereas main peaks constantly decreased according to the length of culture period in both methods.