RESUMEN
Three synthesis lots of linear poly(ethyleneimine) (PEI) are compared to a fully hydrolyzed linear PEI (commercially available as PEI "Max") regarding structure, polyplex formation with plasmid DNA, and transfection of suspension-adapted HEK-293E cells. PEI "Max" binds DNA more efficiently than the other PEIs, but it is the least effective in terms of transient recombinant protein yield. One PEI lot is fractionated by means of SEC. The fractions of high-M(n) PEI are the most efficient for complex formation and transfection. Nevertheless, the highest transient recombinant protein yields are achieved with unfractionated PEI. The results demonstrate that the polydispersity and charge density of linear PEI are important parameters for gene delivery to suspension-adapted HEK-293E cells.
Asunto(s)
ADN/química , Técnicas de Transferencia de Gen , Vectores Genéticos/química , Plásmidos/química , Polietileneimina/química , Fraccionamiento Químico , ADN/genética , Expresión Génica , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Microscopía Fluorescente , Peso Molecular , Plásmidos/genética , Electricidad Estática , TransfecciónRESUMEN
In this study, a recombinant monoclonal IgG antibody was produced by transient gene expression (TGE) in suspension-adapted HEK-293E cells. The objective of the study was to determine the variation in recombinant IgG yield and glycosylation in ten independent transfections. In a ten-day batch process, the variation in transient IgG yield in the ten batches was less than 30% with the specific productivity averaging 20.2 ± 2.6 pg/cell/day. We characterized the N-glycosylation profile of each batch of affinity-purified IgG by intact protein and bottom-up mass spectrometry. Four major glycans were identified at Asn(297) in the ten batches with the maximum relative deviation for a single glycoform being 2.5%. In addition, within any single transfection there was little variation in glycoforms over the ten-day culture. Our experimental data indicate that with TGE, the production of recombinant IgG with little batch-to-batch variation in volumetric yield and protein glycosylation is feasible, even in a non-instrumented cultivation system as described here.
Asunto(s)
Inmunoglobulina G/química , Polisacáridos/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Glicopéptidos/análisis , Glicosilación , Células HEK293 , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.
Asunto(s)
Anticuerpos Monoclonales/química , Espectrometría de Masa por Ionización de Electrospray , Secuencia de Aminoácidos , Animales , Línea Celular , Transporte de Electrón , Humanos , Inmunoglobulina G/química , Ratones , Datos de Secuencia Molecular , Procesamiento Proteico-PostraduccionalRESUMEN
Despite our increasing topological knowledge on regulation networks in model bacteria, it is largely unknown which of the many co-occurring regulatory events actually control metabolic function and the distribution of intracellular fluxes. Here, we unravel condition-dependent transcriptional control of Escherichia coli metabolism by large-scale (13)C-flux analysis in 91 transcriptional regulator mutants on glucose and galactose. In contrast to the canonical respiro-fermentative glucose metabolism, fully respiratory galactose metabolism depends exclusively on the phosphoenol-pyruvate (PEP)-glyoxylate cycle. While 2/3 of the regulators directly or indirectly affected absolute flux rates, the partitioning between different pathways remained largely stable with transcriptional control focusing primarily on the acetyl-CoA branch point. Flux distribution control was achieved by nine transcription factors on glucose, including ArcA, Fur, PdhR, IHF A and IHF B, but was exclusively mediated by the cAMP-dependent Crp regulation of the PEP-glyoxylate cycle flux on galactose. Five further transcription factors affected this flux only indirectly through cAMP and Crp by increasing the galactose uptake rate. Thus, E. coli actively limits its galactose catabolism at the expense of otherwise possible faster growth.
Asunto(s)
Acetilcoenzima A/metabolismo , Carbono/metabolismo , Galactosa/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucosa/metabolismo , Isótopos de Carbono/metabolismo , Ciclo del Ácido Cítrico , AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucólisis , Glioxilatos/metabolismo , Marcaje Isotópico , Oxígeno/metabolismo , Fosfoenolpiruvato/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.