A novel procedure is proposed as a method to characterize the chemical basis of selectivity for m... more 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.
Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and ... more Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Five cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Five cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Five cell cultures, but not in Sf-9 cell cultures. In addition, High Five cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Five cell cultures. It was also found that the medium had a significant effect on High Five cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM.
The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell line has received considerable attention ... more The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell line has received considerable attention as a host for the baculovirus expression vector system. In the present study, suspension cultures were used to compare Tn-5B1-4 cell aggregates and cells selected to grow predominantly as individual cells. No significant difference was found between cell aggregates and cells growing predominantly individually in regard to cell growth rate, glucose consumption and lactate accumulation, and specific recombinant protein synthesis levels. In addition, the levels of recombinant protein synthesis were considerably higher than those produced by the commonly used Spodoptera frugiperda Sf-9 insect cell line.
Near infrared spectroscopy is demonstrated as a suitable method for monitoring real time cell den... more 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.
... References. BH UTTER WQRTH EINEMANN Variation in recombinant protein expression levels among ... more ... References. BH UTTER WQRTH EINEMANN Variation in recombinant protein expression levels among clones of lepidopteran cell populations Murali K. Pasumarthy and ... at 37C until sufficient color was developed, and then the reaction was stopped using 10 (wt vol) citric acid. ...
A novel procedure is proposed as a method to characterize the chemical basis of selectivity for m... more 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.
Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and ... more Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Five cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Five cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Five cell cultures, but not in Sf-9 cell cultures. In addition, High Five cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Five cell cultures. It was also found that the medium had a significant effect on High Five cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM.
The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell line has received considerable attention ... more The Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell line has received considerable attention as a host for the baculovirus expression vector system. In the present study, suspension cultures were used to compare Tn-5B1-4 cell aggregates and cells selected to grow predominantly as individual cells. No significant difference was found between cell aggregates and cells growing predominantly individually in regard to cell growth rate, glucose consumption and lactate accumulation, and specific recombinant protein synthesis levels. In addition, the levels of recombinant protein synthesis were considerably higher than those produced by the commonly used Spodoptera frugiperda Sf-9 insect cell line.
Near infrared spectroscopy is demonstrated as a suitable method for monitoring real time cell den... more 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.
... References. BH UTTER WQRTH EINEMANN Variation in recombinant protein expression levels among ... more ... References. BH UTTER WQRTH EINEMANN Variation in recombinant protein expression levels among clones of lepidopteran cell populations Murali K. Pasumarthy and ... at 37C until sufficient color was developed, and then the reaction was stopped using 10 (wt vol) citric acid. ...
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