RESUMEN
Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements.
Asunto(s)
Cricetulus , Proteínas Recombinantes , Células CHO , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/genética , Cricetinae , Humanos , Transposasas/genética , Transposasas/metabolismoRESUMEN
PURPOSE: Chronic exposure to ionizing radiation (IR) at low doses (<100 mGy) has been insufficiently studied to understand fully the risk to health. Relatively little knowledge exists regarding how species and healthy tissues respond at the protein level to chronic exposure to low doses of IR, and mass spectrometric-based profiling of protein expression is a powerful tool for studying changes in protein abundance. MATERIALS AND METHODS: SDS gel electrophoresis, LC-MS/MS mass spectrometry-based approaches and bioinformatic data analytics were used to detect proteomic changes following chronic exposure to moderate/low doses of radiation in adults and normally developed Medaka fish (Oryzias latipes). RESULTS: Significant variations in the abundance of proteins involved in thyroid hormone signaling and lipid metabolism were detected, which could be related to the gonadal regression phenotype observed after 21.04 mGy and 204.3 mGy/day exposure. The global proteomic change was towards overexpression of proteins in muscle and skin, while the opposite effect was observed in internal organs. CONCLUSION: The present study provides information on the impacts of biologically relevant low doses of IR, which will be useful in future research for the identification of potential biomarkers of IR exposure and allow for a better assessment of radiation biosafety regulations.
Asunto(s)
Oryzias , Animales , Cromatografía Liquida , Biología Computacional , Proteómica , Radiación Ionizante , Espectrometría de Masas en TándemRESUMEN
PURPOSE: Ionizing radiation is found naturally in the environment. Low doses of IR may have beneficial applications, yet there is also potential for detrimental long-term health effects. Impacts following exposure to low levels of IR have been refractory to identification and quantification. Glycoprotein glycosylation is vital to cell-cell communication and organismal function, and sensitive to changes in an organism's macro- and cellular environment. We investigated whether accumulated low doses of IR (LoDIR) affect the N-linked glycoprotein glycans using Medaka fish (Oryzias latipes). MATERIALS AND METHODS: State-of-the-art methods in radiation exposure and glycan analysis were applied to study N-glycan changes after 190 day exposure at three different rates of gamma irradiation (2.25, 21.01, and 204.3 mGy/day) in wild-type adult Medaka. Tissue N-glycans were analyzed following enzymatic release from extracted proteins. RESULTS: N-linked glycan profiles are dominated by complex type N-glycans modified with terminal sialic acid and core fucose. Fucosylation and sialylation of N-linked glycoprotein glycans are affected by LoDIR and a subset of N-glycans are involved in the organismal radio-response. CONCLUSION: This is the first indication that the glycome can be interrogated for biomarkers that report the impact of chronic exposure to environmental stressors, such as low-level IR.