Feasibility of spectral pH measurement during the low-pH virus inactivation step of continuous therapeutic antibody production.

Acidic virus inactivation is commonly used during production of biotherapeutic products to provide virus safety in case of undetected virus contamination. Accurate pH measurement is required to ensure the product pH reaches a virus-inactivating level (typically 3.5-3.7), and a level post-inactivation that is appropriate for later purification steps (typically 5.5-7.5). During batch low-pH inactivation in discrete tanks, potentiometric glass probes are appropriate for measuring pH. During continuous inactivation for 2-3 weeks in an enclosed product stream, probe calibration drift and lag may lead to poor accuracy, and operational difficulties when compensating for drift. Monitoring the spectral response of compounds (indicators) in the product stream whose spectra are pH-sensitive offers a possible alternative way to measure pH without these drawbacks. Such indicators can already exist in the stream (intrinsic) or can be added (extrinsic). Herein are reported studies evaluating the feasibility of both.Promising ultraviolet screening results with the two extrinsics studied, thiamine and ascorbic acid, led to the addition of both to product stream samples titrated to different potentiometric pH values in the 3.3-4.5 range (a representative range encountered during continuous inactivation), and attempts to model pH using sample ultraviolet spectra. One model, based on variability in six spectral attributes, was able to predict pH of an independent sample set within ±0.07 units at the 95% confidence level. Since a typical inactivating pH tolerance is ±0.1 units, the results show that extrinsic indicators potentially can measure inactivation pH with sufficient accuracy. Suggested future steps and an alternative approach are presented.

A novel approach for using dielectric spectroscopy to predict viable cell volume (VCV) in early process development.

Online monitoring of viable cell volume (VCV) is essential to the development, monitoring, and control of bioprocesses. The commercial availability of steam-sterilizable dielectricspectroscopy probes has enabled successful adoption of this technology as a key noninvasive method to measure VCV for cell-culture processes. Technological challenges still exist, however. For some cell lines, the technique's accuracy in predicting the VCV from probepermittivity measurements declines as the viability of the cell culture decreases. To investigate the cause of this decrease in accuracy, divergences in predicted vs. actual VCV measurements were directly related to the shape of dielectric frequency scans collected during a cell culture. The changes in the shape of the beta dispersion, which are associated with changes in cell state, are quantified by applying a novel "area ratio" (AR) metric to frequency-scanning data from the dielectric-spectroscopy probes. The AR metric is then used to relate the shape of the beta dispersion to single-frequency permittivity measurements to accurately predict the offline VCV throughout an entire fed-batch run, regardless of cell state. This work demonstrates the possible feasibility of quantifying the shape of the beta dispersion, determined from frequency-scanning data, for enhanced measurement of VCV in mammalian cell cultures by applying a novel shape-characterization technique. In addition, this work demonstrates the utility of using changes in the shape of the beta dispersion to quantify cell health.

Nuclear protein-induced bending and flexing of the hypoxic response element of the rat vascular endothelial growth factor promoter.

Bending and flexing of DNA may contribute to transcriptional regulation. Because hypoxia and other physiological signals induce formation of an abasic site at a key base within the hypoxic response element (HRE) of the vascular endothelial growth factor (VEGF) gene (FASEB J. 19, 387-394, 2005) and because abasic sites can introduce flexibility in model DNA sequences, in the present study we used a fluorescence resonance energy transfer-based reporter system to assess topological changes in a wild-type (WT) sequence of the HRE of the rat VEGF gene and in a sequence harboring a single abasic site mimicking the effect of hypoxia. Binding of the hypoxia-inducible transcriptional complex present in hypoxic pulmonary artery endothelial cell nuclear extract to the WT sequence failed to alter sequence topology whereas nuclear protein binding to the modified HRE engendered considerable sequence flexibility. Topological effects of nuclear proteins on the modified VEGF HRE were dependent on the transcription factor hypoxia-inducible factor-1 and on formation of a single-strand break at the abasic site mediated by the coactivator, Ref-1/Ape1. These observations suggest that oxidative base modifications in the VEGF HRE evoked by physiological signals could be a precursor to single-strand break formation that has an impact on gene expression by modulating sequence flexibility.

Oxidants in signal transduction: impact on DNA integrity and gene expression.

Physiological stimuli using reactive oxygen species (ROS) as second messengers caused nucleotide-specific base modifications in the hypoxic response element of the VEGF gene in lung vascular cells, with the 3' guanine of the HIF-1 DNA recognition sequence uniformly targeted. Modeling this effect by replacing the targeted guanine with an abasic site increased incorporation of HIF-1 and the bi-functional DNA repair enzyme and transcriptional coactivator, Ref-1/Ape1, into the transcriptional complex and engendered more robust reporter gene expression. Oxidants generated in the context of physiological signaling thus affect nuclear DNA integrity and may facilitate gene expression by optimizing DNA-protein interactions.