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BACKGROUND In mammalian cell culture processes, viable cell density ( VCD) is one of the most crucial parameters for monitoring of the current status of cell growth and protein production. Among the various real-time monitoring tools available for VCD in Chinese hamster ovary ( CHO) cell culture, dielectric spectroscopy is one of the more reliable due to its simplicity, robustness and accuracy. These attributes enable efficient monitoring and control of the corresponding processes. However, one of the major drawbacks of dielectric spectroscopy is frequent deterioration of accuracy during the late-growth phase of CHO cell culture. This can be caused by abrupt changes in the cell physiology, as well as environmental conditions. RESULTS In this study, various kinds of chemometric tools were employed to understand the spectral dynamics of dielectric spectroscopy during extended CHO cell culture, including different cell growth phases. Then, the performance of different multivariate regression models were compared with conventional Cole-Cole equation-based approaches. The models were compared in their ability to estimate VCD in CHO cell culture from dielectric spectra from several batches of bioreactor operations. Multivariate analysis of dielectric spectral datasets clearly revealed the time-varying evolution of spectral dynamics during the transition between cell growth phases. The best prediction performance from the dielectric spectroscopy was realized using a regression model based on locally-weighted partial least squares ( LWPLS). This estimation model best compensated for spectral dynamics observed during the different cell growth phases. CONCLUSION Dielectric spectroscopy combined with LWPLS allows achievement of optimal and stable estimation of VCD during CHO cell culture, regardless of inherent differences in cell growth phases. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

ABSTRACT Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)-type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. Biotechnol. Bioeng. 2014;111: 95-103. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Separation of viruses and other contaminants from protein therapeutics using anion exchange membrane adsorbers is a successful new approach to viral clearance; however, the fundamental phenomena that control performance are not well understood. For example, the kinetics of adsorption to the anion exchange surface may limit clearance, but has yet to be characterized experimentally and mathematically. In the present study, surface plasmon resonance was used to determine the adsorption kinetics for five large biological molecules: phage PP7, phage ΦX174, phage PR772, thyroglobulin, and DNA. Rate constants were incorporated into a kinetic model of chromatography to illustrate the impact on performance. [ABSTRACT FROM AUTHOR]

The major histocompatibility complex (MHC) of the BALB/c mouse contains three genes encoding classical class I molecules, as well as at least 32 nonclassical class I genes. Although much is known about the genes encoding the classical class I molecules, the majority of the nonclassical genes have not been characterized. This report describes a newly identified nonclassical class I gene, Thy19.4, which contains an open reading frame and resembles in several regards the genes encoding classical class I molecules. The similarities include shared amino acid sequence motifs which suggest that the putative Thy19.4 molecule may assume a tertiary structure similar to that of the classical class I molecules, as well as widespread transcription in a variety of tissues. However, unlike the classical class I genes, the Thy19.4 gene maps approximately 1 cm distal to the Tla region of the MHC, in the same region as the gene encoding the Hmt element of the maternally transmitted antigen. [ABSTRACT FROM AUTHOR]

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2, D3, and D4, in addition to H-2Dand H-2L. We have determined the DNA sequence of the D2gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2gene is similar to other class I genes. It also contains similar 5′ regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2 molecule, we generated an exon-shuffled construct containing the promoter and exons 1-3, encoding the signal peptide, α1, and α2 external domains of the D2gene linked to exons 4-8, encoding the α3, transmembrane and cytoplasmic domains, of the H-2Dgene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the α3 domain of H-2D, as well as by other class I-specific mAbs. Although D2 is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene. [ABSTRACT FROM AUTHOR]

The viral and transmissible spongiform encephalopathy (TSE) safety of therapeutics of biological origin (biologicals) is greatly influenced by the nature and degree of variability of the source material and by the mode of purification. Plasma-derived and recombinant DNA products currently have good viral safety records, but challenges remain. In general, large enveloped viruses are easier to remove from biologicals than small ‘naked’ viruses. Monoclonal antibodies and recombinant DNA biopharmaceuticals are derived from relatively homogeneous source materials and purified by multistep schemes that are robust and amenable to scientific analysis and engineering improvement. Viral clearance is more challenging for blood and cell products, as they are complex and labile. Source selection (e.g. country of origin, deferral for CJD risk factors) currently occupies the front line for ensuring that biologicals are free of TSE agents, but robust methods for their clearance from products are under development. [Copyright &y& Elsevier]