Understanding the mechanism of virus removal by Q sepharose fast flow chromatography during the purification of CHO‐cell derived biotherapeutics

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non‐electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science‐based process validation strategies to ensure viral safety of biotechnology products. Biotechnol. Bioeng. 2009; 104: 371–380 © 2009 Wiley Periodicals, Inc.     

Increased glycated calmodulin in the submandibular salivary glands of streptozotocin‐induced diabetic rats

Non‐enzymatic glycosylation, a post translational protein modification may be implicated in the diabetes complications. Calmodulin is an important calcium binding protein that complexed with Ca²⁺ may be implicated in salivary gland secretory process. Glycated calmodulin has shown to be less effective in binding calcium. The aim of this study was to determine whether the concentration of glycated‐calmodulin may be elevated in the submandibular salivary glands of streptozotocin‐induced diabetic rats. Diabetes was induced by an intraperitoneal injection of spreptozotocin, and hyperglycemia was confirmed 72 h after injection using a glucosimeter. Thirty days after the induction of diabetes, submandibular salivary glands were used for the analysis of glycated and non‐glycated calmodulin, using a glycogel B columns for separation. Glycated and non‐glycated calmodulin were assayed by an enzymatic method and by ELISA. The overall concentration of CaM (non‐glycated + glycated) in induced diabetic rats was significantly lower than in controls (p < 0.05). The concentration of non‐glycated CaM in controls was significantly higher than in experimental group (p < 0.05), while the concentration of glycated calmodulin between these groups was statistically similar (p > 0.05). Copyright © 2009 John Wiley & Sons, Ltd.     

Definitions of Night Eating in Adolescent Girls

Objective: To describe the prevalence of night eating in a community cohort of black and white girls, using different definitions of night eating as described in the literature. Research Methods and Procedures: Three‐day food diaries collected as part of the National Growth and Health Study were examined to identify episodes of night eating, which was defined in five different ways: eating >25% of daily caloric intake after the last evening meal, eating >25% of daily caloric intake after 7 pm, eating >50% of daily caloric intake after the last evening meal, eating >50% of daily caloric intake after 7 pm, or eating between 11 pm and 4:59 am. Results: Frequency of night eating varied tremendously depending on how the behavior was defined. For the least restrictive definition (>25% of total intake after last meal), 50% to 70% of girls reported one night eating event; for the most restrictive (>50% of total intake after last meal), only 1.5% of 11‐year‐old girls' diaries and 3.5% of 19‐year‐old girls' diaries contained a night eating event. The frequency of night eating decreased dramatically (typically by a factor of 10) if the inclusion criteria required multiple night eating events in a given week. Discussion: A standard definition of night eating behavior is needed to advance the field. An agreed‐on operationalized definition that includes time of day, amount of calories consumed, and a frequency criterion would enable cross‐study comparisons and encourage the examination of developmental and clinical considerations of night eating behavior.     

Ethnic Identity and Power: Cultural Contexts of Political Action in School and Society.

Ethnic Identity and Power. Cultural Contexts of Political Action in School and Society. Yali Zou and Enrique T. Trueba. eds. Albany: State University of New York Press, 1998. 452 pp.     

Nitric Oxide Prevents Alveolar Senescence and Emphysema in a Mouse Model

N^ω-nitro-L-arginine methyl ester (L-NAME) treatment induces arteriosclerosis and vascular senescence. Here, we report that the systemic inhibition of nitric oxide (NO) production by L-NAME causes pulmonary emphysema. L-NAME-treated lungs exhibited both the structural (alveolar tissue destruction) and functional (increased compliance and reduced elastance) characteristics of emphysema development. Furthermore, we found that L-NAME-induced emphysema could be attenuated through both genetic deficiency and pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1). Because PAI-1 is an important contributor to the development of senescence both in vitro and in vivo, we investigated whether L-NAME-induced senescence led to the observed emphysematous changes. We found that L-NAME treatment was associated with molecular and cellular evidence of premature senescence in mice, and that PAI-1 inhibition attenuated these increases. These findings indicate that NO serves to protect and defend lung tissue from physiological aging.     

Low Abdominal NIRS Values and Elevated Plasma Intestinal Fatty Acid-Binding Protein in a Premature Piglet Model of Necrotizing Enterocolitis

To identify early markers of necrotizing enterocolitis (NEC), we hypothesized that continuous abdominal near-infrared spectroscopy (A-NIRS) measurement of splanchnic tissue oxygen saturation and intermittent plasma intestinal fatty-acid binding protein (pI-FABP) measured every 6 hours can detect NEC prior to onset of clinical symptoms. Premature piglets received parenteral nutrition for 48-hours after delivery, followed by enteral feeds every three hours until death or euthanasia at 96-hours. Continuous A-NIRS, systemic oxygen saturation (SpO₂), and heart rate were measured while monitoring for clinical signs of NEC. Blood samples obtained at 6-hour intervals were used to determine pI-FABP levels by ELISA. Piglets were classified as fulminant-NEC (f-NEC), non-fulminant-NEC (nf-NEC) and No-NEC according to severity of clinical and histologic features. Of 38 piglets, 37% (n=14) developed nf-NEC, 18% (n=7) developed f-NEC and 45% (n=17) had No-NEC. There were significant differences in baseline heart rate (p=0.008), SpO₂ (p<0.001) and A-NIRS (p<0.001) among the three groups. A-NIRS values of NEC piglets remained lower throughout the study with mean for f-NEC of 69±3.8%, 71.9±4.04% for nf-NEC, and 78.4±1.8% for No-NEC piglets (p<0.001). A-NIRS <75% predicted NEC with 97% sensitivity and 97% specificity. NEC piglets demonstrated greater variability from baseline in A-NIRS than healthy piglets (10.1% vs. 6.3%; p=0.04). Mean pI-FABP levels were higher in animals that developed NEC compared to No-NEC piglets (0.66 vs. 0.09 ng/mL;p<0.001). In f-NEC piglets, pI-FABP increased precipitously after feeds (0.04 to 1.87 ng/mL;p<0.001). pI-FABP levels increased in parallel with disease progression and a value >0.25ng/mL identified animals with NEC (68% sensitivity and 90% specificity). NIRS is a real-time, non-invasive tool that can serve as a diagnostic modality for NEC. In premature piglets, low A-NIRS in the early neonatal period and increased variability during initial feeds are highly predictive of NEC, which is then confirmed by rising plasma I-FABP levels. These modalities may help identify neonates with NEC prior to clinical manifestations of disease.     

The Vaginal Microbiota over an 8- to 10-Year Period in a Cohort of HIV-Infected and HIV-Uninfected Women

BackgroundWe identified predominant vaginal microbiota communities, changes over time, and how this varied by HIV status and other factors in a cohort of 64 women.MethodsBacterial DNA was extracted from reposited cervicovaginal lavage samples collected annually over an 8–10 year period from Chicago Women’s Interagency HIV Study participants: 22 HIV-negative, 22 HIV-positive with stable infection, 20 HIV-positive with progressive infection. The vaginal microbiota was defined by pyrosequencing of the V1/V2 region of the 16S rRNA gene. Scheduled visits included Bacterial vaginsosis (BV) screening; clinically detected cases were referred for treatment. Hierarchical clustering identified bacterial community state types (CST). Multinomial mixed effects modeling determined trends over time in CST, by HIV status and other factors.ResultsThe median follow-up time was 8.1 years (range 5.5–15.3). Six CSTs were identified. The mean relative abundance (RA) of Lactobacillus spp. by CST (with median number of bacterial taxa) was: CST-1–25.7% (10), CST-2–27.1% (11), CST-3–34.6% (9), CST-4–46.8% (9), CST-5–57.9% (4), CST-6–69.4% (2). The two CSTs representing the highest RA of Lactobacillus and lowest diversity increased with each additional year of follow-up (CST-5, adjusted odds ratio (aOR) = 1.62 [95% CI: 1.34–1.94]; CST-6, aOR = 1.57 [95 CI: 1.31–1.89]), while the two CSTs representing lowest RA of Lactobacillus and higher diversity decreased with each additional year (CST-1, aOR = 0.89 [95% CI: 0.80–1.00]; CST-2, aOR = 0.86 [95% CI: 0.75–0.99]). There was no association between HIV status and CST at baseline or over time. CSTs representing lower RA of Lactobacillus were associated with current cigarette smoking.ConclusionsThe vaginal microbial community significantly improved over time in this cohort of women with HIV and at high risk for HIV who had regular detection and treatment referral for BV.