Recombinant protein therapeutics, vaccines, and plasma products have a long record of safety. However, the use of cell culture to produce recombinant proteins is still susceptible to contamination with viruses. These contaminations cost millions of dollars to recover from, can lead to patients not receiving therapies, and are very rare, which makes learning from past events difficult. A consortium of biotech companies, together with the Massachusetts Institute of Technology, has convened to collect data on these events. This industry-wide study provides insights into the most common viral contaminants, the source of those contaminants, the cell lines affected, corrective actions, as well as the impact of such events. These results have implications for the safe and effective production of not just current products, but also emerging cell and gene therapies which have shown much therapeutic promise. The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) provides a comprehensive and forward-looking overview of industry’s experience with viral contamination of cell cultures used to produce recombinant proteins.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) The production of biologic drugs using mammalian cell production systems offers the benefits of high yield, proper protein folding, and faithful post-translational modifications. However, mammalian cell culture is vulnerable to contamination with adventitious agents, including mouse minute virus (MMV). The case study presented here demonstrates that MMV is a ubiquitous threat to CHO (Chinese hamster ovary) cell-based production of biologic drugs and that animal-free media components can be a contamination source. Compounding the risk posed by MMV, the contamination may be "silent," with no impact on cell viability and product titers. Furthermore, contamination may not be detected using in vitro virus assays, and assays based on PCR (polymerase chain reaction) are required for reliable detection. The development of effective corrective and preventative action (CAPA) was greatly aided by the identification of the source of the contamination as an animal-free recombinant media additive. The execution of a CAPA that included disposal of contaminated materials, decontamination of the facility, and replacement of the contaminated raw material allowed the resumption of MMV-free production.

Progressive lung damage in cystic fibrosis (CF) has been linked to inadequate airway mucosal hydration. We previously demonstrated that an inositol tetrakisphosphate analog, 1-O-octyl-2-O-butyryl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(propionoxymethyl)ester (INO-4995), regulates airway secretory and absorptive processes, affecting mucosal hydration by prolonged (24 h) inhibition of Na(+) and fluid absorption in CF human nasal epithelia (CFHNE). The objectives of this study were to further assess clinical potential of INO-4995 in CF through ascertaining in vivo activity in mice with CF, determining the effects of repeated administration on potency and determining cytoplasmic half-life. Uptake and metabolism of [(3)H]INO-4995 was monitored with HPLC to calculate intracellular half-life. INO-4995 was administered in vitro repeatedly over 4 to 8 days to CFHNE. Fluid absorption was assessed by blue dextran exclusion, and basal short-circuit current was measured in Ussing chambers. INO-4995 (1-100 microg/kg) was dosed intranasally either as a single dose or once per day over 4 days to gut-corrected CF mice. [(3)H]INO-4995 was rapidly taken up by epithelial cultures and converted to the active drug, which had a half-life of 40 hours. Repeated daily application of INO-4995 to CFHNE lowered the effective concentration for inhibition of fluid absorption and amiloride-sensitive short-circuit current in cultured CFHNE, and reduced nasal potential difference to nearly control levels in gut-corrected CF mice. Ca(2+)-activated Cl(-) channel activity was also boosted in cultures. Mouse nasal levels fell from abnormal levels to within 2 muA of normal with repeated exposure to 0.8 microg/kg over 4 days. These data support further development of INO-4995 for the treatment of CF.

The effects of inositol polyphosphates on ion flux in CF mucosa described here suggest a novel therapeutic approach to the treatment of cystic fibrosis. These studies indicate that transient exposure to an inositol polyphosphate analog, INO-4995, causes long lasting but ultimately reversible changes in therapeutically relevant electrophysiological properties of CF human nasal epithelia. This avoids a major problem encountered with other ion channel regulators that have been advanced as potential CF treatments, limited duration of action.

Amiloride-sensitive, epithelial Na(+) channel (ENaC)-mediated, active absorption of Na(+) is elevated in the airway epithelium of cystic fibrosis (CF) patients, resulting in excess fluid removal from the airway lumen. This excess fluid/volume absorption corresponds to CF transmembrane regulator-linked defects in ENaC regulation, resulting in the reduced mucociliary clearance found in CF airways. Herein we show that INO-4995, a synthetic analog of the intracellular signaling molecule, D-myo-inositol 3,4,5,6-tetrakisphosphate, inhibits Na(+) and fluid absorption across CF airway epithelia, thus alleviating this critical pathology. This conclusion was based on electrophysiological studies, fluid absorption, and (22)Na(+) flux measurements in CF airway epithelia, contrasted with normal epithelia, and on electrophysiological studies in Madin-Darby canine kidney cells and 3T3 cells overexpressing ENaC. The effects of INO-4995 were long-lasting, dose-dependent, and more pronounced in epithelia from CF patients vs. controls. These findings support preclinical development of INO-4995 for CF treatment and demonstrate for the first time the therapeutic potential of inositol polyphosphate derivatives.

A crucial role in the regulation of epithelial chloride secretion is played by the phosphoinositide PtdIns(3,4,5)P3 . Membrane-permeant derivatives of this and other naturally occurring phosphoinositides have been synthesized. These derivatives, which can be bioactivated, were used in investigations on nasal epithelia of patients suffering from cystic fibrosis.

Das Phosphoinositid PtdIns(3,4,5)P3 spielt eine Schlüsselrolle bei der Regulation der epithelialen Chloridsekretion. Für Untersuchungen an Nasenepithelen von Mukoviszidose-Patienten wurden membranpermeable, bioaktivierbare Derivate natürlich vorkommender Phosphoinositide synthetisiert (siehe verallgemeinertes Schema).

Journal Article Substitution of glycine 48 with glutamic acid alters the substrate specificity of the HIV protease: second site revertants demonstrate the importance of electrostatic forces Get access Mark Moody, Mark Moody 1Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599 Search for other works by this author on: Oxford Academic PubMed Google Scholar Steve C. Pettit, Steve C. Pettit 1Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599 Search for other works by this author on: Oxford Academic PubMed Google Scholar Lorraine Everitt, Lorraine Everitt 1Lineberger Comprehensive Cancer Center, Chapel Hill, NC 27599 Search for other works by this author on: Oxford Academic PubMed Google Scholar Daniel Loeb, Daniel Loeb 1Lineberger Comprehensive Cancer Center, Chapel Hill, NC 275992Department of Microbiology and Immunology, Chapel Hill, NC 27599 Search for other works by this author on: Oxford Academic PubMed Google Scholar Clyde Hutchison III, Clyde Hutchison III 2Department of Microbiology and Immunology, Chapel Hill, NC 27599 Search for other works by this author on: Oxford Academic PubMed Google Scholar Ronald Swanstrom Ronald Swanstrom 3Department of Biochemistry and Biophysics University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Search for other works by this author on: Oxford Academic PubMed Google Scholar Protein Engineering, Design and Selection, Volume 6, Issue Supplement, 1993, Page 46, https://doi.org/10.1093/protein/6.Supplement.46-b Published: 01 January 1993

Human Immunodeficiency Virus Type-1, like other retroviruses, encodes an aspartic proteinase whose activity is required for the production of infectious virions.1–6 The protease (PR) is encoded at the 5′ end of the viral pol gene and is responsible for cleavage of the viral gag and gag/pol precursor proteins to their mature forms.7,8 Viruses with inactivating mutations in their protease domain yield immature, noninfectious particles containing unprocessed gag and gag/pol polyproteins;1 for this reason the enzyme has been a prime target for structural and biochemical studies leading to the design of inhibitors of virus replication.

The production of cytokines can be controlled by the regulation of transcription, by the regulation of translation, and by post-translational mechanisms. Therefore, to understand better the control of cytokine production, it is important to measure both concentration of the free cytokine in solution and cytokine mRNA in the cytokine-expressing cells. Traditional methods of examining gene expression, such as as Northern blots, reverse transcriptase polymerase chain reaction (RT-PCR), and RPA, tend to be labor intensive and semiquantitative. To overcome these limitations, we have developed the Xplore® (Endogen, Woburn, MA) assay for the quantification of cytokine mRNA. These microtiter plate-based assays are rapid (under 6 h), quantitative over three orders of magnitude, and have no risk of falsepositive values from contamination.KeywordsReverse Transcriptase Polymerase Chain ReactionSignal ProbeCapture ProbeSplice JunctionCytokine mRNAThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.