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Uncertainties and shortcomings associated with the current influenza vaccine production processes demand attention and exploration of new vaccine manufacture technologies. Based on a newly developed mammalian cell culture-based production process we investigated selected process parameters and describe three factors that are shown to impact productivity, process robustness and development time. They are time of infection, harvest time and virus input, or multiplicity of infection (MOI). By defining the time of infection as 4-5 days post cell seeding and harvest time as 2-3 days post-infection and comparing their effect on virus production, MOI is subsequently identified as the most impactful process parameter for live attenuated influenza vaccine (LAIV) manufacture. Infection at very low MOI (between 10(-4) and 10(-6) FFU/cell) resulted in high titer virus production (up to 30-fold productivity improvement) compared to higher MOI infections (10(-3) to 10(-2) FFU/cell). Application of these findings has allowed us to develop a platform process that can reduce the development time to approximately three weeks for an influenza vaccine manufacture process for new strains., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We demonstrate an optical technique, called laser-gain scanning microscopy (LGSM), to map dopant concentration profiles in engineered laser gain-media. The performance and application range of this technique are exampled on a Nd(3+) concentration profile embedded in a YAG transparent ceramic sample. Concentration profiles measured by both LGSM and SIMS techniques are compared and agree to within 5% over three-orders of magnitude in Nd(3+) doping level, from 0.001 at.% to 0.9 at.%. One of the unique advantages of LGSM over common physical methods such as SIMS, XPS and EMPA, is the ability to correlate optical defects with the final doping profile.

The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg-based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin-Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25-30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers > or =8.6 log(10) FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn-around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production.

Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006-2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. Based on these study results we conclude that the MDCK cell produced and egg produced vaccine strains are highly comparable., (Copyright 2010 Elsevier Ltd. All rights reserved.)

An Madin-Darby canine kidney (MDCK) cell line, 9B9-1E4, was cloned by limit dilution from a heterologous cell population and chosen as a potential production cell substrate for cell culture-based influenza vaccine manufacture. Since MDCK cells are transformed cells of canine origin, extensive characterization, including evaluation of tumorigenicity and oncogenicity, was performed to ensure the safety of this cell line for vaccine production. Injection of intact MDCK cells into adult and newborn athymic nude mice did not lead to progressive tumor formation in two separate tumorigenicity studies. In addition, neither MDCK cell lysate nor cellular DNA induced tumors in newborn rodents (athymic nude mice, hamsters and rats) in six oncogenicity studies. Observations from these studies demonstrate the low tumorigenic and oncogenic potential of the MDCK cell clone 9B9-1E4. These observations coupled with other characterization study results strongly suggest a high safety assurance level can be achieved through cell cloning and selection of low tumorigenic and oncogenic cells for influenza vaccine production., ((c) 2009 Elsevier Ltd. All rights reserved.)

A 52-year-old man with an implantable cardioverter-defibrillator (ICD) was accidentally electrocuted when a flagpole he was erecting contacted an overhead electrical power line. The electrocution injury initiated ventricular fibrillation. The ICD sensed the arrhythmia and delivered therapy, converting his rhythm to sinus rhythm. The patient had no subsequent cardiac or neurological sequelae.

To develop a cell-based live attenuated influenza vaccine (LAIV) manufacturing process, several different cell lines were evaluated by comparing the titer of viruses after infection with LAIV strains. While several cell lines have been reported to support influenza virus replication, the degree of replication and the ability to support replication of LAIV strains have not been systematically examined. MDCK cells, which have been considered as potential substrates for influenza vaccine production were evaluated in addition to Vero, MRC-5, WI-38 and FRhL cells. MRC-5, WI-38 and FRhL cells produced low to moderate titers of virus with titers equal or below 5.0 log(10) TCID(50)/mL. Both Vero and MDCK cells could support a higher level of virus replication for certain strains, however, Vero cells only produced high titers when grown in the presence of serum. MDCK cells supported high levels of vaccine virus production for multiple different LAIV subtypes in both serum containing and serum-free media. These results suggest that MDCK cell-based production can be used as an alternative production platform to the currently used egg-based LAIV production system.

MedImmune Vaccines has engineered a live, attenuated chimeric virus that could prevent infections caused by parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV), causative agents of acute respiratory diseases in infants and young children. The work here details the development of a serum-free Vero cell culture production platform for this virus vaccine candidate. Efforts to identify critical process parameters and optimize culture conditions increased infectious virus titers by approximately 2 log(10) TCID(50)/ml over the original serum-free process. In particular, the addition of a chemically defined lipid concentrate to the pre-infection medium along with the shift to a lower post-infection cultivation temperature increased virus titers by almost 100-fold. This improved serum-free process achieved comparable virus titers to the serum-supplemented process, and demonstrated consistent results upon scale-up: Vero cultures in roller bottles, spinner flasks and bioreactors reproducibly generated maximum infectious virus titers of 8 log(10) TCID(50)/ml.

The detection of small threats, such as explosives, drugs, and chemical weapons, concealed or encased in surrounding material, is a major concern in areas from security checkpoints to UneXploded Ordnance (UXO) clearance. Techniques such as X-ray and trace detection are often ineffectual in these applications. Thermal neutron analysis (TNA) provides an effective method for detecting concealed threats. This paper shows the effectiveness of Ancore's SPEDS, based on TNA, in detecting concealed liquid threats and differentiating live from inert mortar shells.

SeaPODDS (Sea Portable Drug Detection System) is a non-intrusive tool for detecting concealed threats in hidden compartments of maritime vessels. This system consists of an electronic neutron generator, a gamma-ray detector, a data acquisition computer, and a laptop computer user-interface. Although initially developed to detect narcotics, recent algorithm developments have shown that the system is capable of correctly classifying a threat into one of four distinct categories: narcotic, explosive, chemical weapon, or radiological dispersion device (RDD). Detection of narcotics, explosives, and chemical weapons is based on gamma-ray signatures unique to the chemical elements. Elements are identified by their characteristic prompt gamma-rays induced by fast and thermal neutrons. Detection of RDD is accomplished by detecting gamma-rays emitted by common radioisotopes and nuclear reactor fission products. The algorithm phenomenology for classifying threats into the proper categories is presented here.

We examined, in young adult women, the association between current low dose oral contraceptive (OC) use and plasma levels of C-reactive protein (CRP), an acute phase reactant predictive of cardiovascular disease risk. We conducted a cross-sectional analysis of 30 healthy, non-smoking, non-obese women (18 OC users and 12 nonusers) who were subjects in a randomized diet-controlled trial of the effects of soy intake on sex hormone metabolism. The study was sited at a university outpatient general clinical research center. Fasting plasma CRP levels were measured 4 times during 2 menstrual cycles (2 mid-follicular phase and 2 mid-luteal phase) using a high-sensitivity CRP assay. Differences between OC users and nonusers were examined by 3-way analysis of variance. Multiple regression was used to examine the relationship between OC use and CRP. There were no significant differences in baseline demographic characteristics between OC users and nonusers. Plasma CRP levels (mean +/- SE) were 2 times higher among OC users than among non-users (2.0 +/- 0.2 versus 0.9 +/- 0.3 mg/l, p<0.0001) independent of diet assignment, diet treatment order, and phase of the menstrual cycle. In a multivariate model, OC use predicted 32 percent of the variance in CRP levels (p<0.0001). As all CRP levels were within a previously established normal range, further study is indicated to establish the clinical significance of the observed elevated CRP levels in OC users.

Attractive economics and short development timelines have often been cited as reasons for using bacteria to express eukaryotic proteins on a commercial scale. Nevertheless, routine techniques for bacterial expression of heterologous proteins are beset by a variety of technical and legal difficulties. In particular, the use of plasmids to express foreign proteins, popular promoter systems, protein fusion partners, and histidine tags and the recovery of proteins from inclusion bodies are affected by a host of issued patents. Chromosomally encoded leaderless fusions (CELF) offer a variety of technical and legal advantages over existing bacterial expression systems. In this study, we show that CELF can be used to produce a wide assortment of eukaryotic proteins at 10-liter fermentation scale., (Copyright 1998 Academic Press.)

Removable dies used in fixed prosthodontics typically exhibit movement. A solid working cast permits the technician to perfect the interproximal contacts of fixed prostheses. This saves the clinician time at the insertion appointment. This article describes a quick and easy procedure for making a solid working cast with easily visualized margins.