Local temperatures inside a 75-microns-i.d. capillary under electrophoresis conditions are measured noninvasively with a Raman microprobe. The method is based on the temperature dependence of the water O-H stretch equilibrium between weakly bent and strongly bent hydrogen bonded species. With calibration against a known temperature standard, this technique is shown to be capable of obtaining spatially resolved intracapillary temperature measurements with an accuracy of +/- 1.0 degree C and a precision of +/- 0.1 degree C. Intracapillary temperatures ranging from 25 to 70 degrees C are observed over the range of buffer compositions and electric fields used. Differences between local temperatures and average capillary temperatures are observed at all operating conditions. The difficulty of an accurate theoretical description of heat dissipation under CE conditions is discussed.
As more protein biopharmaceuticals are produced using mammalian cell culture techniques, it becomes increasingly important for the biopharmaceutical industry to have tools to characterize the cell culture media and evaluate its impact on the cell culture performance. Exposure of the cell culture media to light, temperature stress, or adventitious introduction of low-level organisms during preparation can lead to the generation of chemical degradants or metabolites of the media components, which are potentially detrimental to the cell culture process. In this work, we applied a liquid chromatography-mass spectrometry based metabolomics methodology for the investigation of a media lot used for a mammalian cell culture process that had resulted in low growth rate and failure to meet required viable cell density (VCD). The study led to the observation of increased levels of tryptophan oxidation products and a riboflavin degradant, lumichrome, in the malfunctioning media lot, relative to working media lots. A compound found 7-fold higher in the working media lots appeared to be tetrahydropentoxyline, a condensation product of glucose and tryptophan. A second compound found at an over 50-fold higher level in the malfunctioning media lot with a proposed molecular formula of C(21)H(17)N(3)O(3) from high-resolution mass spectrometry (HRMS) analysis remains unknown, although it is confirmed to be a degradant of tryptophan in the media. A study of the cell culture media performed under stress conditions using fluorescent light and heat showed that the media powder was highly resistant to light-induced degradation, while solution media could be easily degraded after brief light exposure. It is therefore suspected that inadvertent exposure of the media to light during preparation and storage has resulted in the poor performance of the media causing the low growth and VCD in the cell culture process.
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