Recombinant Protein Production in Large-Scale Agitated Bioreactors Using the Baculovirus Expression Vector System.

The production of recombinant proteins using the baculovirus expression vector system (BEVS) in large-scale agitated bioreactors is discussed in this chapter. Detailed methods of the key stages of a batch process, including host cell growth, virus stock amplification and quantification, bioreactor preparation and operation, the infection process, final harvesting, and primary separation steps for recovery of the product are presented. Furthermore, methods involved with advanced on-line monitoring and bioreactor control, which have a significant impact on the overall process success, are briefly discussed.

Production of Virus-Like Particles for Vaccination.

The ability to make a large variety of virus-like particles (VLPs) has been successfully achieved in the baculovirus expression vector system (BEVS)/insect cell system. The production and scale-up of these particles, which are mostly sought as vaccine candidates, are currently being addressed. Furthermore, these VLPs are being investigated as delivery agents for use as therapeutics. The use of host insect cells allows mass production of VLPs in a proven scalable system.

Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems.

BACKGROUND: Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. METHODS: In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. RESULTS: For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. CONCLUSIONS: This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.

Classifying bivalve larvae using shell pigments identified by Raman spectroscopy.

Because bivalve larvae are difficult to identify using morphology alone, the use of Raman spectra to distinguish species could aid classification of larvae collected from the field. Raman spectra from shells of bivalve larvae exhibit bands that correspond to polyene pigments. This study determined if the types of shell pigments observed in different species could be unique enough to differentiate larvae using chemotaxonomic methods and cluster analysis. We collected Raman spectra at three wavelengths from 25 samples of bivalve larvae representing 16 species and four taxonomic orders. Grouping spectra within general categories based on order/family relationships successfully classified larvae with cross-validation accuracies ≥92% for at least one wavelength or for all wavelengths combined. Classifications to species were more difficult, but cross-validation accuracies above 86% were observed for 7 out of 14 species when tested using species groups within orders/families at 785 nm. The accuracy of the approach likely depends on the composition of species in a sample and the species of interest. For example, high classification accuracies (85-98%) for distinguishing spectra from Crassostrea virginica larvae were achieved with a set of bivalve larvae occurring in the Choptank River in the Chesapeake Bay, USA, whereas as lower accuracies (70-92%) were found for a set of C. virginica larvae endemic to the Northeast, USA. In certain systems, use of Raman spectra appears to be a promising method for assessing the presence of certain bivalves in field samples and for validating high-throughput image analysis systems for larval bivalve studies.

Particle quantification of influenza viruses by high performance liquid chromatography.

The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5 M NaCl salt with 20 mM Tris-HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5 min. A linear response range was established between 1 × 10(9) and 1 × 10(11) virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07 × 10(8) and 4.35 × 10(9) whereas the limit of quantification was between 6.90 × 10(8) and 1.45 × 10(10)VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines.

Analytical technologies for influenza virus-like particle candidate vaccines: challenges and emerging approaches.

Influenza virus-like particle vaccines are one of the most promising ways to respond to the threat of future influenza pandemics. VLPs are composed of viral antigens but lack nucleic acids making them non-infectious which limit the risk of recombination with wild-type strains. By taking advantage of the advancements in cell culture technologies, the process from strain identification to manufacturing has the potential to be completed rapidly and easily at large scales. After closely reviewing the current research done on influenza VLPs, it is evident that the development of quantification methods has been consistently overlooked. VLP quantification at all stages of the production process has been left to rely on current influenza quantification methods (i.e. Hemagglutination assay (HA), Single Radial Immunodiffusion assay (SRID), NA enzymatic activity assays, Western blot, Electron Microscopy). These are analytical methods developed decades ago for influenza virions and final bulk influenza vaccines. Although these methods are time-consuming and cumbersome they have been sufficient for the characterization of final purified material. Nevertheless, these analytical methods are impractical for in-line process monitoring because VLP concentration in crude samples generally falls out of the range of detection for these methods. This consequently impedes the development of robust influenza-VLP production and purification processes. Thus, development of functional process analytical techniques, applicable at every stage during production, that are compatible with different production platforms is in great need to assess, optimize and exploit the full potential of novel manufacturing platforms.

X-CHIP: an integrated platform for high-throughput protein crystallization and on-the-chip X-ray diffraction data collection.

The X-CHIP (X-ray Crystallization High-throughput Integrated Platform) is a novel microchip that has been developed to combine multiple steps of the crystallographic pipeline from crystallization to diffraction data collection on a single device to streamline the entire process. The system has been designed for crystallization condition screening, visual crystal inspection, initial X-ray screening and data collection in a high-throughput fashion. X-ray diffraction data acquisition can be performed directly on-the-chip at room temperature using an in situ approach. The capabilities of the chip eliminate the necessity for manual crystal handling and cryoprotection of crystal samples, while allowing data collection from multiple crystals in the same drop. This technology would be especially beneficial for projects with large volumes of data, such as protein-complex studies and fragment-based screening. The platform employs hydrophilic and hydrophobic concentric ring surfaces on a miniature plate transparent to visible light and X-rays to create a well defined and stable microbatch crystallization environment. The results of crystallization and data-collection experiments demonstrate that high-quality well diffracting crystals can be grown and high-resolution diffraction data sets can be collected using this technology. Furthermore, the quality of a single-wavelength anomalous dispersion data set collected with the X-CHIP at room temperature was sufficient to generate interpretable electron-density maps. This technology is highly resource-efficient owing to the use of nanolitre-scale drop volumes. It does not require any modification for most in-house and synchrotron beamline systems and offers a promising opportunity for full automation of the X-ray structure-determination process.

A comparison of accurate mass techniques for the structural elucidation of fluconazole.

Mass spectrometry plays a major role in the structural elucidation and characterisation of drug candidates and related substances. Accurate mass data allow the mathematical prediction of molecular formula of both precursor and fragment ions. In this paper, a comparison of the accurate mass data obtained for the fragmentation of fluconazole, an antifungal drug, by three different methods is made: electron ionisation (EI) using a magnetic sector instrument; electrospray ionisation (ES) using a Fourier transform ion cyclotron mass spectrometer (FTICRMS); and ES using a quadrupole-time-of-flight mass spectrometer (Q-ToF). It is clear from the data obtained that mass accuracy is not simply a function of instrument resolution. The subtle differences observed between collisionally activated dissociation (CAD) and sustained off-resonance collisionally activated dissociation (SORI-CAD) spectra are explained as a consequence of the excitation process. The advantages and disadvantages of the three techniques are discussed within the context of structural elucidation.

Medical and midwifery students: how do they view their respective roles on the labour ward?

BACKGROUND: It has been suggested that much of the medical and midwifery student curricula on normal pregnancy and birth could be taught as a co-operative effort between obstetric and midwifery staff. One important element of a successful combined teaching strategy would involve a determination of the extent to which the students themselves identify common learning objectives. AIM: The aim of the present study was to survey medical and midwifery students about how they perceived their respective learning roles on the delivery suite. METHODS: A descriptive cross-sectional survey study was undertaken. The study venue was an Australian teaching and tertiary referral hospital in obstetrics and gynaecology Survey participants were medical students who had just completed a 10 week clinical attachment in obstetrics and gynaecology during the 5th year of a six year undergraduate medical curriculum and midwifery students undertaking a one year full-time (or two year part-time) postgraduate diploma in midwifery. RESULTS: Of 130 and 52 questionnaires distributed to medical and midwifery students, response rates of 72% and 52% were achieved respectively The key finding was that students reported a lesser role for their professional colleagues than they identified for themselves. Some medical students lacked an understanding of the role of midwives as 8%, 10%, and 23% did not feel that student midwives should observe or perform a normal birth or neonatal assessment respectively. Of equal concern, 7%, 22%, 26% and 85% of student midwives did not identify a role for medical students to observe or perform a normal birth, neonatal assessment or provide advice on breastfeeding respectively. SUMMARY: Medical and midwifery students are placed in a competitive framework and some students may not understand the complementary role of their future colleagues. Interdisciplinary teaching may facilitate co-operation between the professions and improve working relationships.