Martin Rhiel

Simultaneous Measurements of Glucose, Glutamine, Ammonia, Lactate, and Glutamate in Aqueous Solutions by Near-Infrared Spectroscopy

Calibration models are generated and evaluated for the measurement of five different components in synthetic mixtures prepared in aqueous solutions. Mixtures of glucose, glutamine, ammonia, lactate, and glutamate were prepared to simulate concentration levels expected during routine bioreactor fermentation processes. Near-IR spectra were collected from these solutions over the spectral range from 5000 to 4000 cm−1. This spectral information was used to build individual multivariate calibration models for each analyte. Models were constructed on the basis of partial least-squares regression of raw and Fourier filtered absorbance spectra. Each analyte could be detected selectively with mean percent errors of prediction ranging from 4 to 8%.

Comparison of Trichoplusia ni BTI-Tn-5B1-4 (High Five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures

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.

On-line determination of animal cell concentration

A new approach for the indirect determination of cell concentration in the case of nonconstant metabolic rates has been developed. The specific glucose-uptake rate was shown to be nonconstant in batch cultures of free suspended and immobilized CHO SSF3 cells. Time-independent models correlating the specific rate to the limiting substrate concentration were established, thus providing a continuous determination of the specific rate through on-line measurement of the limiting substrate. The method could be applied to determine on-line cell concentration in both free suspended and immobilized cell cultures. Results were verified off-line by crystal violet nuclei counting. The predicted cell concentration was in very good agreement with the off-line reference during the whole exponential-growth phase, until the specific glucose-uptake rate tended to zero.

Simultaneous measurement of glucose and glutamine in aqueous solutions by near infrared spectroscopy.

A method is described for measuring the concentrations of both glucose and glutamine in binary mixtures from near infrared (NIR) absorption spectra. Spectra are collected over the range from 5000–4000/cm (2.0–2.5μm) with a 1-mm optical path length. Glucose absorbance features at 4710, 4400, and 4300/cm and glutamine features at 4700, 4580, and 4390/cm provide the analytical information required for the measurement. Multivariate calibration models are generated by using partial least squares (PLS) regression alone and PLS regression combined with a preprocessing digital Fourier filtering step. The ideal number of PLS factors and spectral range are identified separately for each analyte. In addition, the optimum Fourier filter parameters are established for both compounds. The best overall analytical performance is obtained by combining Fourier filtering and PLS regression. Glucose measurements are established over the concentration range from 1.66–59.91 mM, with a standard error of prediction (SEP) of 0.32 mM and a mean percent error of 1.84%. Glutamine can be measured over the concentration range from 1.10–30.65 mM with a SEP of 0.75 mM and a mean percent error of 6.67%. These results demonstrate the analytical utility of NIR spectroscopy for monitoring glucose and glutamine levels in mammalian and insect cell cultures.

Ammonia measurements in mammalian cell culture media with a diffuse reflectance-based fiberoptic ammonia sensor

A solid-state, diffuse reflectance-based fiberoptic sensor is described for quantifying ammonia. This sensor is constructed by immobilizing chlorophenol red, a weak acid chromophoric indicator dye, in a microporous polypropylene membrane. A flow-injection analysis system is used to carry a 10-μL aliquot of the sample across the treated membrane. Ammonia in the sample diffuses through the air-filled pores within the membrane structure before reacting with the indicator dye. A reversible acid-base reaction between ammonia and chlorophenol red results in a measurable change in the reflectance at 560 nm. Response characteristics include a peak response within 20 s, a limit of detection of 0.2 ± 0.1 mM ammonia, and a dynamic range of up to 60 mM ammonia. The analytical utility of this sensor is demonstrated by measuring ammonia levels in 48 individual samples collected during the growth of PC3 human prostate cancer cells in a typical serum-containing growth medium. Accuracy of the proposed sensor is verified by a comparison of results from this sensor to those from a conventional enzyme assay.

INDIRECT BIOMASS DETERMINATION IN CASE OF NON-CONSTANT METABOLIC RATES

A novel approach for indirect biomass determination in case of non-constant specific metabolic rates is reported. Specific glucose consumption rate was determined during a first calibration culture for both free suspended and immobilized cells. It was expressed as a linear function of the limiting substrate, i. e. glucose for suspension and oxygen for immobilized cell cultures. For validation, these time-independent models were applied on a secondculture presenting a different inoculum cell density. On-line monitoring of the limiting substrate enabled a continuous determination of the specific metabolic rate and thus on-line cell number prediction, which was verified with an off-line cell counting technique.