Quality by design approach for viral clearance by protein a chromatography

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.     

Glycine mineralization in situ closely correlates with soil carbon availability across six North American forest ecosystems

Free amino acids (FAA) constitute a significant fraction of dissolved organic nitrogen (N) in forest soils and play an important role in the N cycle of these ecosystems. However, comparatively little attention has been given to their role as labile carbon (C) substrates that might influence the metabolic status of resident microbial populations. We hypothesized that the residence time of simple C substrates, such as FAA, are mechanistically linked to the turnover of endogenous soil C pools. We tested this hypothesis across a latitudinal gradient of forested ecosystems that differ sharply with regard to climate, overstory taxon, and edaphic properties. Using a combined laboratory and field approach, we compared the turnover of isotopically labeled glycine in situ to the turnover of mineralizable soil C (C_min) at each site. The turnover of glycine was rapid (residence times <2 h) regardless of soil type. However, across all ecosystems glycine turnover rates were strongly correlated with indices of soil organic matter quality. For example, C:N ratios for the upper soil horizons explained ~80% of the variability observed in glycine turnover, and there was a strong positive correlation between in situ glycine-C turnover and C_min measured in the laboratory. The turnover of glycine in situ was better explained by changes in soil C availability than cross-ecosystem variation in soil temperature or concentrations of dissolved inorganic N and FAA-N. This suggests the consumption of these low-molecular-weight substrates by soil microorganisms may be governed as much by the overall decomposability of soil C as by N limitation to microbial growth.     

Increase in Candida parapsilosis Fungemia in Critical Care Units: A 6-Years Study

Objectives

We aimed to asses possible clinically significant differences between C. parapsilosis and other candida species candidemia receiving care in the intensive care unit (ICU) setting.

Methods

The study included 118 adult patients diagnosed as candidemia after admission to the ICU of a university hospital between January 2004 and December 2009. Data about demographic characteristics, underlying diseases, and risk factors for ICU-related candidemia were collected.

Results

During the study period, 118 patients with candidemia were identified among 2,853 patients admitted into the ICU. Candidemia was seen in 41.4 cases per 1,000 ICU admissions. The overall incidence of candidemia in ICU patients during the study period was 2.09 per 1,000 hospital admissions. Of the isolates, 18.6% were C. albicans and 81.4% were C. non-albicans. The species most frequently isolated was C. parapsilosis (66.1%, 78/118). The distribution of other Candida spp. was as follows: 15 had C. tropicalis (12.7%) and 3 had C. glabrata (2.5%). By Statistical analysis, when patients with candidemia who had C. parapsilosis were compared with other Candida spp., the following factors were found to be significantly associated with C. parapsilosis fungemia; intravascular catheters (p = 0.008), malignity (p = 0.049) and age (p = 0.039). Relationship was found between C. tropicalis and hematologic malignancies (p = 0.001).

Conclusions

When infections with a high mortality such as candidemia is suspected in critically ill patients, it is important to know local risk factors and epidemiological distributions of causative agents in selection of empirical and effective antifungal treatment.     

The Ca2+-binding protein calretinin is selectively enriched in a subpopulation of the epithelial rests of Malassez

During tooth development, the inner and outer enamel epithelia fuse by mitotic activity to produce a bilayered epithelial sheath termed Hertwig’s epithelial root sheath (HERS). The epithelial rests of Malassez (ERM) are the developmental residues of HERS and remain in the adult periodontal ligament (PDL). Although the cellular regulation of the Ca²⁺-binding proteins parvalbumin, calbindin-D28k, and calretinin has been reported in the inner and outer enamel epithelia during tooth development, an involvement of Ca²⁺-binding proteins in the ERM has not so far been characterized. Among the three Ca²⁺-binding proteins tested (calbindin D28k, parvalbumin, calretinin), we have only been able to detect calretinin in a subpopulation of adult rat molar ERM, by using quantitative immunohistochemical and confocal immunofluorescence techniques. TrkA (a marker for ERM) is present in numerous epithelial cell clusters, whereas calretinin has been localized in the cytosol and perinuclear region of a subpopulation of TrkA-positive cells. We conclude that, in inner and outer enamel epithelial cells, Ca²⁺ is regulated by calbindin, parvalbumin, and calretinin during tooth development, whereas in the ERM of adult PDL, Ca²⁺ is regulated only by calretinin. The expression of Ca²⁺-binding proteins is restricted in a developmental manner in the ERM.     

Blockade of multiple but not single cytokines abrogates downregulation of angiotensin II type-I receptors and anticipates septic shock

In this prospective, randomized animal study, the role of proinflammatory cytokines in the pathogenesis sepsis-induced circulatory failure with downregulation of angiotensin-II-type-I-(AT₁)-receptors was investigated. Sepsis in wild-type mice and in mice with deficiencies for TNF-α, IL-1β, IFN-γ or IL-6 was induced by cecal ligation and puncture (CLP) and wild-type mice were injected with cytokines. Animals were treated with glucocorticoids or small interfering RNA (siRNA) targeting single or multiple cytokines or NF-κB. Vascular smooth muscle cells (VSMCs) were incubated with cytokines. CLP resulted in circulatory failure and a significant downregulation of AT₁-receptors. Injection of single proinflammatory cytokines also strongly downregulated AT₁-receptors paralleled by a markedly endogenous liberation of further cytokines, whereas, simultaneous blockade of these endogenously activated cytokines by dexamethasone prevented downregulation of AT₁-receptors. Furthermore, inhibition of multiple but not single cytokines by treatment with siRNA against multiple cytokines or NF-κB significantly attenuated CLP-induced AT₁-receptor downregulation and prevented septic circulatory failure. Our data demonstrate that downregulation of AT₁-receptors during sepsis is due to multiple but not single cytokines and define a relevant role for NF-κB in the pathogenesis of septic shock.     

Isometric back muscle endurance: An EMG study on the criterion validity of the Ito test

The validity of the Sorensen test as a measure for back muscle endurance is controversial due to a possible impact of hip extensor muscles. The aim of this study was to investigate the criterion validity of an alternative test (Ito test) compared to the Sorensen test. Both procedures were performed by 29 healthy subjects (11 women) for 5 s and until exhaustion (randomized order). EMG activity was measured from 3 lumbar back and 3 hip extensor muscles. Muscular involvement in test positions was calculated as percentage of maximal voluntary contraction (MVC). Muscle fatigue was determined by the normalized regression coefficient of the median frequencies of the EMG power spectrum (NMF_slope). Prediction of holding time by NMF_slope values was investigated using regression analysis. In the test positions, the hamstring muscles were activated to a higher MVC percentage in the Sorensen than in the Ito test, while the iliocostalis muscle was less activated. Similarly, the iliocostalis (p = 0.006) and the multifidi muscles (p = 0.03) significantly contributed to predict holding time in the Ito test, whereas the multifidi muscles (p = 0.001) and the semitendinosus muscle (p = 0.046) did so in the Sorensen test. The results of this study indicate that the Ito test might present a valuable alternative for testing back muscle endurance in LBP patients.     

The effects of acute changes in temperature and oxygen availability on cardiac performance in winter flounder (Pseudopleuronectes americanus)

Studies on how flatfish cardiovascular function responds to environmental challenges are limited, and have largely relied upon indirect methodologies (i.e. Fick principle). Thus, we measured dorsal aortic blood pressure (P_DA) and cardiac function in 8 and 15 °C-acclimated flounder exposed to graded hypoxia, and in 8 °C-acclimated fish exposed to an acute temperature increase to their critical thermal maximum (CTM). The extent of bradycardia in 8 °C-acclimated fish (decrease in heart rate of 41%) was consistent with that observed for other teleosts, as was this species' CTM (25.8 ± 0.5 °C) and its cardiac response to increasing temperature. However, this study provides further examples of how cardiovascular function is controlled differently in the flounder as compared with other fishes. First, the onset of bradycardia in 8 °C-acclimated fish occurred earlier than expected for this inactive and hypoxia-tolerant species (60% water air saturation). Second, resting cardiac output was similar in flounder acclimated to 8 and 15 °C (～ 15 mL min^? 1 kg^? 1), and hypoxic bradycardia was surprisingly absent at 15 °C. Finally, systemic vascular resistance decreased when flounder were exposed to elevated temperature, and this resulted in a 26% fall in P_DA. These are novel findings, however, the extent to which the flounder's behaviour influenced some of the results is unclear.     

Sustained lung activity of a novel chimeric protein, SOD2/3, after intratracheal administration

Delivery of recombinant superoxide dismutase to the lung is limited by its short half-life and poor tissue penetration. We hypothesized that a chimeric protein, SOD2/3, containing the enzymatic domain of manganese superoxide dismutase (SOD2) and the heparan-binding domain of extracellular superoxide dismutase (SOD3), would allow for the delivery of more sustained lung and pulmonary vascular antioxidant activity compared to SOD2. We administered SOD2/3 to rats by intratracheal (i.t.), intraperitoneal (i.p.), or intravenous (i.v.) routes and evaluated the presence, localization, and activity of lung SOD2/3 1 day later using Western blot, immunohistochemistry, and SOD activity gels. The effect of i.t. SOD2/3 on the pulmonary and systemic circulation was studied in vivo in chronically catheterized rats exposed to acute hypoxia. Active SOD2/3 was detected in lung 1 day after i.t. administration but not detected after i.p. or i.v. SOD2/3 administration or i.t. SOD2. The physiologic response to acute hypoxia, vasoconstriction in the pulmonary circulation and vasodilation in the systemic circulation, was enhanced in rats treated 1 day earlier with i.t. SOD2/3. These findings indicate that i.t. administration of SOD2/3 effectively delivers sustained enzyme activity to the lung as well as pulmonary circulation and has a longer tissue half-life compared to native SOD2. Further testing in models of chronic lung or pulmonary vascular diseases mediated by excess superoxide should consider the longer tissue half-life of SOD2/3 as well as its potential systemic vascular effects.