RESUMO
Baculoviruses have enormous potential for use as biopesticides to control insect pest populations without the adverse environmental effects posed by the widespread use of chemical pesticides. However, continuous baculovirus production is susceptible to DNA mutation and the subsequent production of defective interfering particles (DIPs). The amount of DIPs produced and their genome length distribution are of great interest not only for baculoviruses but for many other DNA and RNA viruses. In this study, we elucidate this aspect of virus replication using baculovirus as an example system and both experimental and modeling studies. The existing mathematical models for the virus replication process consider DIPs as a lumped quantity and do not consider the genome length distribution of the DIPs. In this study, a detailed population balance model for the cell-virus culture is presented, which predicts the genome length distribution of the DIP population along with their relative proportion. The model is simulated using the kinetic Monte Carlo algorithm, and the results agree well with the experimental results. Using this model, a practical strategy to maintain the DIP fraction to near to its maximum and minimum limits has been demonstrated.
Assuntos
Genoma Viral , Nucleopoliedrovírus/fisiologia , Spodoptera/virologia , Replicação Viral , Animais , Linhagem Celular , Método de Monte CarloRESUMO
Accumulation of baculovirus defective interfering particle (DIP) and few polyhedra (FP) mutants is a major limitation to continuous large-scale baculovirus production in insect-cell culture. Although overcoming these mutations would result in a cheaper platform for producing baculovirus biopesticides, little is known regarding the mechanism of FP and DIP formation. This issue was addressed by comparing DIP production of wild-type (WT) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with that of a recombinant AcMNPV (denoted Ac-FPm) containing a modified fp25k gene with altered transposon insertion sites that prevented transposon-mediated production of the FP phenotype. In addition to a reduction in the incidence of the FP phenotype, DIP formation was delayed on passaging of Ac-FPm compared with WT AcMNPV. Specifically, the yield of DIP DNA in Ac-FPm was significantly lower than in WT AcMNPV up to passage 16, thereby demonstrating that modifying the transposon insertion sites increases the genomic stability of AcMNPV. A critical component of this investigation was the optimization of a systematic method based on the use of pulsed-field gel electrophoresis (PFGE) to characterize extracellular virus DNA. Specifically, PFGE was used to detect defective genomes, determine defective genome sizes and quantify the amount of defective genome within a heterogeneous genome population of passaged virus.
Assuntos
Elementos de DNA Transponíveis , Vírus Defeituosos/genética , Nucleopoliedrovírus/crescimento & desenvolvimento , Nucleopoliedrovírus/genética , Animais , Vírus Defeituosos/isolamento & purificação , Lepidópteros/virologia , Nucleopoliedrovírus/ultraestrutura , Deleção de Sequência , Inoculações Seriadas , VirulênciaRESUMO
Low-cost, large-scale production of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) using continuous insect cell culture is seriously hindered by the accumulation of AcMNPV mutants. Specifically, few-polyhedra (FP) mutants, with a reduced yield of occluded virus (polyhedra) and decreased infectivity, usually accumulate upon passaging in cell culture. FP mutations result from transposon insertions in the baculovirus fp25k gene, leading to significantly reduced levels of FP25K protein synthesis. This study evaluated the effects of removing the transposon insertion sites from the wild-type baculovirus fp25k gene; the mutated virus was denoted Ac-FPm. Specifically, this study involved a detailed comparison of wild-type (WT) AcMNPV and Ac-FPm with regard to the proportion of cells having polyhedra, number of polyhedra per cell, the fraction of empty polyhedra, number of occlusion-derived viruses per polyhedron, number of nucleocapsids in the nuclei, FP25K protein synthesis and genetic analysis of the fp25k gene. Removal of TTAA transposon insertion sites from the fp25k gene stabilized FP25K protein synthesis and delayed the appearance of the FP phenotype from passage 5 to passage 10. Electron micrographs revealed that more virus particles were found inside the nuclei of cells infected with Ac-FPm than in the nuclei of cells infected with WT AcMNPV (at passage 10). Abnormalities, however, were observed in envelopment of nucleocapsids and virus particle occlusion within Ac-FPm polyhedra. Thus, the FP phenotype appeared in spite of continued FP25K protein synthesis, suggesting that mechanisms other than fp25k gene disruption can lead to the FP phenotype.
Assuntos
Elementos de DNA Transponíveis , DNA Viral/genética , Lepidópteros/virologia , Nucleopoliedrovírus/fisiologia , Proteínas Virais/genética , Replicação Viral , Animais , Mutagênese Insercional , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/crescimento & desenvolvimento , FenótipoRESUMO
The insect cell culture/baculovirus system has three primary applications: (1) recombinant protein synthesis, (2) biopesticide synthesis, and (3) as a model system (e.g., for studying apoptosis). The fundamental techniques involved in these applications are described throughout this book. In this chapter, the most widely techniques are summarized and the reader is directed to detailed information found elsewhere in this book. Furthermore, many useful tips and the author's personal preferences that are rarely published are discussed in this chapter along with quantitative methods to characterize cell growth, baculovirus infection, and metabolism.
Assuntos
Baculoviridae/genética , Insetos/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Contagem de Células , Linhagem Celular , Proliferação de Células , Insetos/citologia , Insetos/genética , Proteínas Recombinantes/genéticaRESUMO
The insect cell culture/baculovirus system has three primary applications: (1) recombinant protein synthesis, (2) biopesticide synthesis, and (3) as a model system (e.g., for studying apoptosis). The fundamental techniques involved in these applications are described throughout this book. In this chapter the most widely used techniques are summarized and the reader is directed to detailed information found elsewhere in this book. Furthermore, many useful tips and my personal preferences that are rarely published are discussed in this chapter along with quantitative methods to characterize cell growth, baculovirus infection, and metabolism.
Assuntos
Células/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/fisiologia , Células/citologia , Células/virologia , DNA Recombinante/genética , Insetos/citologia , Insetos/virologia , Oxigênio/metabolismoRESUMO
A problem in the mass production of recombinant proteins and biopesticides using insect cell culture is CO2 accumulation. This research investigated the effect of elevated CO2 concentration on insect cell growth and metabolism. Spodoptera frugiperda Sf-9 insect cells were grown at 20% air saturation, 27(°) C, and a pH of 6.2. The cells were exposed to a constant CO2 concentration by purging the medium with CO2 and the headspace with air. The population doubling time (PDT) of Sf-9 cells increased with increasing CO2 concentration. Specifically, the PDT for 0-37, 73, 147, 183, and 220 mm Hg CO2 concentrations were 23.2 ± 6.7, 32.4 ± 7.2, 38.1 ± 13.3, 42.9 ± 5.4, and 69.3 ± 35.9 h (n = 3 or 4, 95% confidence level), respectively. The viability of cells in all experiments was above 90%, i.e., while increased CO2 concentrations inhibited cell growth, it did not affect cell viability. The osmolality for all bioreactor experiments was observed to be 300-360 mOsm/kg, a range that is known to have a negligible effect on insect cell culture. Elevated CO2 concentration did not significantly alter the cell specific glucose consumption rate (2.5-3.2 × 10(-17) mol/cell s), but slightly increased the specific lactate production rate from -3.0 × 10(-19) to 10.2 × 10(-19) mol/cell s. Oxidative stress did not contribute to CO2 inhibition in uninfected Sf-9 cells as no significant increase in the levels of lipid hydroperoxide and protein carbonyl concentrations was discovered at elevated CO2 concentration. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:465-469, 2016.
Assuntos
Dióxido de Carbono/farmacologia , Células/efeitos dos fármacos , Células/metabolismo , Spodoptera/citologia , Animais , Reatores Biológicos , Dióxido de Carbono/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células CultivadasRESUMO
Glycosylation, an important post-translation modification, could alter biological activity or influence the clearance rates of glycoproteins. We report here the first example of a heterozygous protein deficiency leading to metabolic alteration of N-glycan structures in residual secreted protein. Analysis of C1 esterase inhibitor (C1INH) glycans from normal individuals and patients with hereditary deficiency of C1INH demonstrated identical O-glycan structures but the N-glycans of patients with a heterozygous genetic deficiency were small, highly charged and lacked sialidase releasable N-acetylneuraminic acid. Structural studies indicate that the charge character of these aberrant N-glycan structures may result from the presence of mannose-6-phosphate residues. These residues might facilitate secretion of C1INH through an alternate lysosomal pathway, possibly serving as a compensatory mechanism to enhance plasma levels of C1INH in these deficient patients.
Assuntos
Angioedema/genética , Polissacarídeos/química , Polissacarídeos/metabolismo , Deficiência de Proteína/genética , Serpinas/metabolismo , Angioedema/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Proteínas Inativadoras do Complemento 1 , Proteína Inibidora do Complemento C1 , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Glicosilação , Heterozigoto , Humanos , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/genética , Valores de Referência , Serpinas/química , Serpinas/deficiência , Serpinas/genéticaRESUMO
LNCaP prostate cancer cells were grown under four unique cultivation conditions. Two types of bioreactor systems were used to observe the influence of low-shear culture conditions allowing for three-dimensional growth: a) a perfusion rotating wall vessel (RWV) bioreactor; and b) a high aspect ratio vessel (HARV) RWV bioreactor, with periodic medium exchanges (fed-batch). In addition, two growth methods utilized tissue culture flasks (TCFs): a) unaltered or conventional TCFs; and b) poly(2-hydroxyethyl methacrylate) [poly(HEMA)] coated TCFs, to inhibit cell attachment. Comparisons were drawn based on qualitative observation of cell morphology and quantitative metabolic data. Similar cellular metabolism was demonstrated for cells grown under each condition. The degree of aggregation, however, varies considerably. Spherical shaped aggregates with diameters of 1 to 3 mm were produced when cells were grown within the perfusion-RWV bioreactor. All other growth conditions produced irregular shaped aggregates of various sizes. Quantitative results demonstrated the expected glucose utilization concomitant with lactate accumulation. Immunohistochemical evaluations were unremarkable for all four cultivation conditions. Results demonstrate that use of the perfusion-RWV bioreactor is advantageous in obtaining spherical aggregates, while grown in a controlled environment.
Assuntos
Técnicas de Cultura/métodos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Reatores Biológicos , Caderinas/metabolismo , Adesão Celular , Agregação Celular , Glucose/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Ácido Láctico/metabolismo , Masculino , Poli-Hidroxietil Metacrilato/química , Antígeno Prostático Específico/metabolismo , Células Tumorais CultivadasRESUMO
A probe-type oxygen sensor was developed utilizing a radioluminescent (RL)-based light source and a ruthenium-based sensing chemistry for monitoring the dissolved oxygen (DO) concentration in a modified version of the NASA-designed high aspect ratio vessel (HARV), a batch rotating wall vessel. This sensor provided the means to monitor the DO concentration in the HARV without influencing the flow pattern, thereby retaining the low shear HARV environment conducive to the formation of 3-dimensional cell aggregates. This sensor lost significant signal as a result of exposure to the first three autoclave cycles, but only minimal change in signal was observed following exposure to subsequent autoclave cycles. A new calibration model requiring only one fitted parameter was developed that accurately fit data over the entire range from 0% to 100% oxygen saturation. The ability for DO concentration control within the vessel was demonstrated by using this sensor to monitor the DO concentration inside the HARV.
Assuntos
Reatores Biológicos/microbiologia , Técnicas de Cultura de Células/instrumentação , Consumo de Oxigênio/fisiologia , Oxigênio/análise , Oxigênio/metabolismo , Espectrometria de Fluorescência/instrumentação , Spodoptera/metabolismo , Transdutores , Animais , Calibragem , Técnicas de Cultura de Células/métodos , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Retroalimentação/fisiologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodos , Spodoptera/crescimento & desenvolvimentoRESUMO
Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/mL produced by Sf-9. Kinetic analysis showed that Vmax and Km of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the Vmax and Km of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man(2-8)GlcNAc2 and FucMan(3 or 4)GlcNAc2) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The proportions of these oligosaccharide structures were different in the two cell lines. Man4GlcNAc2 and FucMan4GlcNAc2 were the major rSEAP N-glycans produced in Sf-9 cells, while Man2GlcNAc2 was the major rSEAP N-glycan produced in Tn-5B1-4 cells.
Assuntos
Fosfatase Alcalina/metabolismo , Vetores Genéticos , Nucleopoliedrovírus/genética , Polissacarídeos/química , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/isolamento & purificação , Animais , Sequência de Carboidratos , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Humanos , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
It has previously been demonstrated that baculovirus infection of the Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines leads to oxidative stress as measured by protein and membrane lipid oxidation and that this oxidative damage contributes to cell death. As a result of these findings, it was hypothesized that baculovirus infection stimulates superoxide radical (O(2)(.-)) synthesis in the mitochondria and that the resulting O(2)(.-) accumulation overwhelms the cells' antioxidant defenses. We investigated the ability of manganese superoxide dismutase (MnSOD) expression (which reduces O(2)(.-) to H(2)O(2) to overcome the oxidative damage caused by baculovirus infection. It was found that MnSOD expression significantly reduced oxidative damage in baculovirus-infected Tn-5B1-4 cells but had no significant effect on oxidative damage in baculovirus-infected Sf-9 cells. The results are consistent with the hypothesis that O(2)(.-) accumulation in the mitochondria is at least partially responsible for the oxidative damage resulting from the baculovirus infection of insect cells.
Assuntos
Baculoviridae/fisiologia , Lepidópteros/enzimologia , Lepidópteros/virologia , Estresse Oxidativo , Superóxido Dismutase/biossíntese , Transfecção/métodos , Viroses/enzimologia , Viroses/virologia , Animais , Baculoviridae/genética , Linhagem Celular , Peroxidação de Lipídeos , Proteínas Recombinantes/metabolismo , Spodoptera/enzimologia , Spodoptera/virologia , Superóxido Dismutase/genéticaRESUMO
Near infrared spectroscopy is demonstrated as a suitable method for monitoring real time cell density and concentrations of glucose and lactate during insect cell cultivation. The utility of this approach is illustrated during the cultivation of Trichoplusia ni BTI-Tn-5B1-4 insect cells in a stirred-tank bioreactor. On-line near infrared measurements are made by passing unaltered culture medium through an autoclavable near infrared flow-through sample cell during the cultivation process. Single-beam near infrared spectra were collected over the combination spectral range (5000-4000cm(-1)) through a 1.5mm path length sample. Cell density calibration model was established by uni-variable linear regressions with measured mean absorbance values of on-line spectra collected during a cultivation run. Calibration models are generated for glucose and lactate by regression analysis of both off line and on line spectra collected during a series of pre-measurement cultivation runs. Analyte-specific calibration models are generated by using a combination of spectra from both natural, unaltered samples and samples spiked with known levels of glucose and lactate. Spiked samples are used to destroy concentration correlations between solutes, thereby enhancing the selectivity of the calibration models. Absorbance spectra are used to build partial least squares calibration models for glucose and lactate. The calibration model for cell density corresponds to a univariate linear regression calibration model based on the mean absorbance between 4750 and 4250cm(-1). The standard errors of prediction are 1.54mM, 0.83mM, and 0.38×10(6)cells/mL for the glucose, lactate, and cell density models, respectively.
Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Glucose/metabolismo , Ácido Láctico/metabolismo , Animais , Reatores Biológicos , Calibragem , Contagem de Células , Linhagem Celular , Insetos , Análise de Regressão , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
The effects of dissolved oxygen (DO) concentration on virally infected insect cells were investigated in 3-L bioreactor culture. Specifically, cultures of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) were infected with Autographa californica multiple nucleopolyhedrovirus expressing secreted alkaline phosphatase (SEAP). Following infection at a DO concentration of 50% air saturation, the DO concentration was adjusted to a final value of either 190%, 50%, or 10% air saturation. Recombinant SEAP production, cell viability, protein carbonyl content, and thiobarbituric acid reactive substances (TBARS) content were monitored. The increases in protein carbonyl and TBARS contents are taken to be indicators of protein oxidation and lipid oxidation, respectively. DO concentration was found to have no noticeable effect on SEAP production or cell viability decline in the Sf-9 cell line. In the Tn-5B1-4 cell line, cells displayed an increased peak SEAP production rate for 190% air saturation and displayed an increased rate of viability decline at increased DO concentration. Protein carbonyl content showed no significant increase in the Sf-9 cell line by 72 h postinfection (pi) at any DO concentration but showed a twofold increase at 10% and 50% DO concentration and a threefold increase at 190% DO concentration by 72 h pi in Tn-5B1-4 cells. TBARS content was found to increase by approximately 50% in Sf-9 cells and by approximately twofold in Tn-5B1-4 cells by 72 h pi with no clear relationship to DO concentration. It is hypothesized that oxygen uptake changes due to the viral infection process may bear a relation to the observed increases in protein and lipid oxidation and that lipid oxidation may play an important role in the death of virally infected insect cells.
Assuntos
Estresse Oxidativo , Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Spodoptera/metabolismo , Animais , Spodoptera/citologia , Spodoptera/virologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
An adaptive calibration procedure is used to build selective multivariate calibration models for the measurement of glucose, lactate, glutamine, and ammonia in undiluted serum-based cell culture media. This adaptive procedure removes metabolism-induced covariance between these analytes in a series of calibration samples collected during the cultivation of PC-3 human prostate cancer cells. Partial least-squares calibration models are generated from single-beam near-infrared (NIR) spectra collected over the 4800- to 4200-cm(-1) combination spectral range. Calibration models were generated with both the full spectral range and optimized spectral ranges. In both cases, the number of model factors was optimized and model validity was determined by comparing analyte concentrations predicted from a series of independent and unaltered samples that were obtained during a subsequent cultivation of the PC-3 cells. Similar analytical performance was achieved with fewer model factors when the optimized spectral range was used. The lowest standard errors of prediction were 0.82, 0.94, 0.55, and 0.76 mM for glucose, lactate, glutamine, and ammonia, respectively. Different spectral ranges were optimal for each analyte and the optimized spectral range coincided with the distinguishing spectral features of the analyte. The results of this study demonstrate that NIR spectroscopy can be used effectively in the off-line measurement of important nutrients (glucose and glutamine) and byproducts (lactate and ammonia) in a serum-based animal cell culture medium.
Assuntos
Técnicas de Cultura de Células/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Algoritmos , Animais , Calibragem , Bovinos , Meios de Cultura/química , Meios de Cultura/metabolismo , Humanos , Análise dos Mínimos Quadrados , Masculino , Modelos Químicos , Modelos Estatísticos , Análise Multivariada , Soroalbumina Bovina/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/normas , Espectroscopia de Luz Próxima ao Infravermelho/estatística & dados numéricos , Células Tumorais CultivadasRESUMO
PC-3 human prostate cancer cells have been cultivated in a rotating wall vessel in which glucose, lactate, and glutamine profiles were monitored noninvasively and in real time by near-infrared (NIR) spectroscopy. The calibration models were based on off-line spectra from tissue culture experiments described previously (Rhiel et al., Biotechnol Bioeng 77:73-82). Monitoring performance was improved by Fourier filtering of the spectra and initial off-set adjustment. The resulting standard errors of predictions were 0.95, 0.74, and 0.39 mM for glucose, lactate, and glutamine, respectively. The concentration of ammonia could not be accurately measured from the same spectra. In addition, metabolite uptake and production rates were determined for PC-3 prostate cancer cells during exponential growth in batch-mode cultivation. Cells grew with a doubling time of 21 h and consumed glucose and glutamine at rates of 6.8 and 1.8 x 10(-17) mol/cell.s, respectively. This resulted in lactate and ammonia production rates of 11.9 and 1.3 x 10(-17) mol/cell.s, respectively. Compared with other monitoring technologies, this technology has many advantages for spaceflights and stand-alone units; for instance, calibration can be performed at one time and then applied in a reagentless, low-maintenance way at a later time. The resulting concentration information can be incorporated into closed-loop control schemes, thereby leading to better in vitro models of in vivo behavior.
Assuntos
Reatores Biológicos , Neoplasias da Próstata/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho , Amônia/análise , Amônia/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Glucose/análise , Glucose/metabolismo , Glutamina/análise , Glutamina/metabolismo , Humanos , Ácido Láctico/análise , Ácido Láctico/metabolismo , Masculino , PerfusãoRESUMO
A novel procedure is proposed as a method to characterize the chemical basis of selectivity for multivariate calibration models. This procedure involves submitting pure component spectra of both the target analyte and suspected interferences to the calibration model in question. The resulting model output is analyzed and interpreted in terms of the relative contribution of each component to the predicted analyte concentration. The utility of this method is illustrated by an analysis of calibration models for glucose, sucrose, and maltose. Near-infrared spectra are collected over the 5000-4000-cm(-)(1) spectral range for a set of ternary mixtures of these sugars. Partial least-squares (PLS) calibration models are generated for each component, and these models provide selective responses for the targeted analytes with standard errors of prediction ranging from 0.2 to 0.7 mM over the concentration range of 0.5-50 mM. The concept of the proposed pure component selectivity analysis is illustrated with these models. Results indicate that the net analyte signal is solely responsible for the selectivity of each individual model. Despite strong spectral overlap for these simple carbohydrates, calibration models based on the PLS algorithm provide sufficient selectivity to distinguish these commonly used sugars. The proposed procedure demonstrates conclusively that no component of the sucrose or maltose spectrum contributes to the selective measurement of glucose. Analogous conclusions are possible for the sucrose and maltose calibration models.
Assuntos
Carboidratos/análise , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Calibragem , Carboidratos/química , Glucose/análise , Glucose/química , Análise dos Mínimos Quadrados , Modelos Lineares , Maltose/análise , Maltose/química , Modelos Estatísticos , Análise Multivariada , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho/estatística & dados numéricos , Sacarose/análise , Sacarose/químicaRESUMO
The effect of dissolved oxygen concentration on human secreted alkaline phosphatase (SEAP) glycosylation by the insect cell-baculovirus expression system was investigated in a well-controlled bioreactor. Oligomannose-type N-linked glycans (i.e., Man2 to Man6 and Man3F) were present in SEAP produced by Spodoptera frusiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines. The relative amounts of the most highly processed glycans (i.e., Man3F and Man2 in the SEAP from Sf-9 and Tn-5B1-4 cells, respectively) were significantly higher at 50% of air saturation than at either 10% or 190% of air saturation. That is, glycan processing was inhibited at both low and high dissolved oxygen concentrations.