Accelerating bioprocess development by analysis of all available data: A USP case study

datasets (e.g. time series, quality measurements). By analyzing all available data, bioprocess development can be accelerated. This can only be achieved by having a clearly defined data logging and analysis strategy. Here, we present a case study using available data from the development and optimization of the upstream process (USP) of Sabin inactivated polio vaccine (IPV) using animal component free medium. IPV production using attenuated Sabin strains instead of wild type polio viruses is an initiative supported by the World Health Organization. This change is favorable to reduce the risk of outbreaks during IPV production. Optimizing this process using only animal free components reduces operational costs and lowers the risk of adverse effects related to animal derived compounds. During the process development, 40 bioreactors at scales ranging from 2.3 to 16 L were run. For optimization and robustness studies, design of experiments (DoE) was performed and several USP operational parameters were varied. These included operational mode (batch vs semi-batch), multiplicity of infection (MOI) and time of infection (TOI). This data was routinely analyzed using factors based on DoE methodology. With the new strategy, it became possible to scrutinize all data from the 40 USP development runs in a single data study. The total data package that was analyzed; this included the DoE response parameters, all offline data (e.g. cell, substrate and product concentrations), all data generated by the bioreactor control systems (T, pH, DO, DOCO), and derived calculations (specific rates like µ and qglu). This analysis showed which parameters were most important regarding the bioreactor performance. This USP case study showed that with the new strategy a more detailed, reliable and exact view on the most important parameters regarding bioreactor performance could be obtained. In order to do this, a feature based approach supported by the inCyght® software was utilized. It consisted of logging all data into a database, which was used to determine data integrity for all variables and batches. Exact phase information (cell growth, virus production phase) and other meta information are transferred into the database for each batch. This allowed outliers to be visually determined and certain variables to be excluded from the analysis (i.e. those that did not fluctuate). Univariate outlier detection technique was used to further determine outliers. Principal component analysis (PCA) was used to gain a multivariate process understanding and partial least squares (PLS) regression was performed to identify correlations. This result determined the best subset of variables to be fitted by using multiple linear regression (MLR). Future experiments will focus on the relevant parameters highlighted by this approach. This strategy was applied for the analysis of previously produced data. Further development will use this data analysis methodology for continuous accelerated process development, intensified DoE and integrated process modelling

A platform approach for the production of Hand, Foot, Mouth Disease vaccines

Hand, Foot and Mouth Disease (HFMD) is an endemic childhood disease in Southeast Asia, with substantial disease burden affecting millions of children each year. Occasionally the central nervous system is involved causing serious and sometimes fatal neurological complications. HFMD outbreaks are also observed outside the Asia-Pacific countries. HFMD can be caused by multiple enteroviruses of which the best known virus is EVA71. However, also other enteroviruses such as CVA6, CVA10 and CVA16 can cause the disease. Inactivated EVA71 vaccines are registered in China, but in order to prevent all HFMD cases, multivalent vaccines are warranted. Intravacc is developing an HFMD combination vaccine. Here we used our rescue platform to generate the starting materials required for vaccine production. Infectious clones from EVA71_B4, EVA71_C4, CVA6, CVA10 and CVA16 were constructed and the corresponding enteroviruses were rescued. Virus seeds were produced on Vero cells in animal component free medium. Rescued enteroviruses could efficiently replicate, resulting in seed lots with high viral titers. This rescue platform has the major advantage that clinical isolates are not required to obtain the starting material to produce a vaccine, thus mitigating the risk that other, unwanted, viruses are also present. Next to that, the virus source is pre-designed, controlled and well documented. Please click Download on the upper right corner to see the full abstract

A live attenuated RSV vaccine, process development studies

Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory tract disease in infants and young children. A vaccine to prevent the high burden of disease caused by RSV is urgently needed, but not available. A live attenuated respiratory syncytial virus (RSV) vaccine for intranasal delivery is currently under development at Intravacc. The vaccine concept comprises a live Glycoprotein-complemented RSVΔG virus. This G-RSVΔG virus is generated by proliferation of an RSVΔG on G-expressing Vero cells. The vaccine thus contains virus particles that have the G-protein on their surface but not in their RNA genomes. This recombinant virus is highly attenuated compared to wild type RSV and therefore presents a live attenuated vaccine candidate for RSV infection. A vaccine production process has been setup for the production of Phase I clinical lots. In short, the production process steps are: cell and virus culture, clarification, continuous flow density gradient ultracentrifugation, ultra/diafiltration, filling and lyophilization. An example of process development is the design of the cell and virus culture method. Using the statistical design of experiment approach the virus culture has been optimized to both virus yield and harvest quality. As RSV is a filamentous virus, the optimization of harvest quality with respect to purification opportunities is pivotal. This DoE was done at lab-scale bioreactors (2-L) and the chosen conditions were successfully scaled-up to 50-L single use bioreactors. Preparation of preclinical and clinical lots is done at this scale. The pre-clinical studies were successful. In the cotton rat model, the G-RSVΔG vaccine is safe, immunogenic and protects against challenge with wild type RSV. The following step, a clinical Phase I study, is planned

Third generation vaccine for world eradication of poliomyelitis

Great efforts have been undertaken by the World Health Organization to achieve eradication of poliomyelitis, a paralytic disease. At present, two different vaccines are available: inactivated polio vaccine (IPV) developed by Salk based on chemical inactivation of the virus and oral polio vaccine (OPV) developed by Sabin based on live attenuated virus strains. The risks associated with IPV concern the safety of the production process as it is based on highly virulent wild type strains, and in contrast, the OPV risks are associated with the reversibility of the attenuated viruses to a transmissible paralytic form. There is therefore a need for a new generation polio vaccines capable to overcome outbreaks and manufacturing risks. With the evolution of molecular virology of Sabin vaccine strains, it is now possible to design extremely genetically stable and hyperattenuated strains without the associated reversion risks. Sabin poliovirus strains were therefore genetically modified giving rise to the third generation of polio vaccine strains [1, 2]. In the present work we have explored the possibility of using the already well-established IPV production process, developed at our site [3] and integrated worldwide [4] for the production and manufacturing of third generation of IPV strains. Specifically, we have produced third generation vaccines in animal component free medium and at 50-L pilot scale. The product obtained did show acceptable yields and was immunogenic in rats. Together, our results indicate that the third generation vaccine strains produced under the flexible platform process are potential candidates which provide increased biosafety during manufacturing which is necessary after polio eradication. In addition, the flexibility and scalability of the process constitute a platform for the production of a large range of vaccines worldwide. 1. Knowlson, S., et al., New Strains Intended for the Production of Inactivated Polio Vaccine at Low-Containment After Eradication. PLoS Pathog, 2015. 11(12): p. e1005316. 2. Macadam, A.J., et al., Rational design of genetically stable, live-attenuated poliovirus vaccines of all three serotypes: relevance to poliomyelitis eradication. J Virol, 2006. 80(17): p. 8653-63. 3. Thomassen, Y.E., et al., Scale-down of the inactivated polio vaccine production process. Biotechnol Bioeng, 2013. 110(5): p. 1354-65. 4. Wezel, v., Monolayer growth systems: Homogeneous unit processes. Spier, R. E. and Griffiths, J. B., eds., 1985: p. 266-281

DEVELOPMENT OF INACTIVATED POLIO VACCINE FROM ATTENUATED SABIN STRAINS FOR CLINICAL STUDIES AND TECHNOLOGY-TRANSFER PURPOSES

Recently, responding to WHO’s call for new polio vaccines, the development of Sabin-IPV (injectable, formalin-Inactivated Polio Vaccine, based on attenuated ‘Sabin’ polio virus strains) was initated at NVI. This activity plays an important role in the WHO polio eradication strategy. The use of Sabin instead of wild-type Salk polio strains will provide additional safety during vaccine production. Initially, the Sabin-IPV production process will be based on the scale-down model of the current, and well-established, Salk-IPV process. In parallel, process development, optimization and formulation research is being carried out to further modernize the process and reduce cost per dose. The lab-scale accelerated process development, product characterization, clinical lot production, and preparations for technology transfer will be discussed. Multivariate data analysis (MVDA) was applied on data from current IPV production (more than 60 Vero cell culture based runs) to extract relevant information, like operating ranges. Subsequently, based on the MVDA analysis, a 3-L scale-down model of the current twin 750-L bioreactors has been setup. Currently, in this lab-scale process, cell and virus culture approximate the large-scale and process improvement studies are in progress. This includes the application of increased cell densities, animal component free media, and DOE optimization in multiple parallel bioreactors. Also, results will be shown from large-scale (to prepare for future technology transfer) generation and testing of Master- and Working virus seedlots, and clinical lot (for phase I studies) production under cGMP conditions. The obtained product was used for immunogenicity studies in rats. It was shown that Sabin-IPV induces a good immune response, and a comparison will be made to regular Salk-IPV. Finally, technology transfer to vaccine manufacturers in low and middle–income countries will take place. For that, an international Sabin-IPV manufacturing course, including practical training at pilot-scale, is being setup

Identification of six new susceptibility loci for invasive epithelial ovarian cancer.

Genome-wide association studies (GWAS) have identified 12 epithelial ovarian cancer (EOC) susceptibility alleles. The pattern of association at these loci is consistent in BRCA1 and BRCA2 mutation carriers who are at high risk of EOC. After imputation to 1000 Genomes Project data, we assessed associations of 11 million genetic variants with EOC risk from 15,437 cases unselected for family history and 30,845 controls and from 15,252 BRCA1 mutation carriers and 8,211 BRCA2 mutation carriers (3,096 with ovarian cancer), and we combined the results in a meta-analysis. This new study design yielded increased statistical power, leading to the discovery of six new EOC susceptibility loci. Variants at 1p36 (nearest gene, WNT4), 4q26 (SYNPO2), 9q34.2 (ABO) and 17q11.2 (ATAD5) were associated with EOC risk, and at 1p34.3 (RSPO1) and 6p22.1 (GPX6) variants were specifically associated with the serous EOC subtype, all with P < 5 × 10(-8). Incorporating these variants into risk assessment tools will improve clinical risk predictions for BRCA1 and BRCA2 mutation carriers.COGS project is funded through a European Commission's Seventh Framework Programme grant (agreement number 223175 ] HEALTH ]F2 ]2009 ]223175). The CIMBA data management and data analysis were supported by Cancer Research.UK grants 12292/A11174 and C1287/A10118. The Ovarian Cancer Association Consortium is supported by a grant from the Ovarian Cancer Research Fund thanks to donations by the family and friends of Kathryn Sladek Smith (PPD/RPCI.07). The scientific development and funding for this project were in part supported by the US National Cancer Institute GAME ]ON Post ]GWAS Initiative (U19 ]CA148112). This study made use of data generated by the Wellcome Trust Case Control consortium. Funding for the project was provided by the Wellcome Trust under award 076113. The results published here are in part based upon data generated by The Cancer Genome Atlas Pilot Project established by the National Cancer Institute and National Human Genome Research Institute (dbGap accession number phs000178.v8.p7). The cBio portal is developed and maintained by the Computational Biology Center at Memorial Sloan ] Kettering Cancer Center. SH is supported by an NHMRC Program Grant to GCT. Details of the funding of individual investigators and studies are provided in the Supplementary Note. This study made use of data generated by the Wellcome Trust Case Control consortium, funding for which was provided by the Wellcome Trust under award 076113. The results published here are, in part, based upon data generated by The Cancer Genome Atlas Pilot Project established by the National Cancerhttp://dx.doi.org/10.1038/ng.3185This is the Author Accepted Manuscript of 'Identification of six new susceptibility loci for invasive epithelial ovarian cancer' which was published in Nature Genetics 47, 164–171 (2015) © Nature Publishing Group - content may only be used for academic research

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good

Search for stop and higgsino production using diphoton Higgs boson decays

Results are presented of a search for a "natural" supersymmetry scenario with gauge mediated symmetry breaking. It is assumed that only the supersymmetric partners of the top-quark (stop) and the Higgs boson (higgsino) are accessible. Events are examined in which there are two photons forming a Higgs boson candidate, and at least two b-quark jets. In 19.7 inverse femtobarns of proton-proton collision data at sqrt(s) = 8 TeV, recorded in the CMS experiment, no evidence of a signal is found and lower limits at the 95% confidence level are set, excluding the stop mass below 360 to 410 GeV, depending on the higgsino mass