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

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

Developing a manufacturing process to deliver a cost effective and stable liquid human rotavirus vaccine

Despite solid evidence of the success of rotavirus vaccines in saving children from fatal gastroenteritis, more than 82 million infants worldwide still lack access to a rotavirus vaccine. The main barriers to global rotavirus vaccine coverage include cost, manufacturing capacity and suboptimal efficacy in low- and lower-middle income countries. One vaccine candidate with the potential to address the latter is based on the novel, naturally attenuated RV3 strain of rotavirus, RV3-BB vaccine administered in a birth dose strategy had a vaccine efficacy against severe rotavirus gastroenteritis of 94% at 12 months of age in infants in Indonesia. To further develop this vaccine candidate, a well-documented and low-cost manufacturing process is required. A target fully loaded cost of goods (COGs) of ≤$3.50 per course of three doses was set based on predicted market requirements. COGs modelling was leveraged to develop a process using Vero cells in cell factories reaching high titers, reducing or replacing expensive reagents and shortening process time to maximise output. Stable candidate liquid formulations were developed allowing two-year storage at 2–8 °C. In addition, the formulation potentially renders needless the pretreatment of vaccinees with antacid to ensure adequate gastric acid neutralization for routine oral vaccination. As a result, the formulation allows small volume dosing and reduction of supply chain costs. A dose ranging study is currently underway in Malawi that will inform the final clinical dose required. At a clinical dose of ≤6.3 log10 FFU, the COGs target of ≤$3.50 per three dose course was met. At a clinical dose of 6.5 log10 FFU, the final manufacturing process resulted in a COGs that is substantially lower than the current average market price, 2.44 USD per dose. The manufacturing and formulation processes were transferred to BioFarma in Indonesia to enable future RV3-BB vaccine production

Human AdV-20-42-42, a promising novel adenoviral vector for gene therapy and vaccine product development

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination

TRY plant trait database – enhanced coverage and open access

Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait‐based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for ‘plant growth form’. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait–environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives

Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways

Improved poliovirus d-antigen yields by application of different Vero cell cultivation methods

Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L-1) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1 × 106 cells mL-1 during batch cultivation to 1.8, 2.7 and 5.0 × 106 cells mL-1 during semi-batch, perfusion and recirculation, respectively. The effects of these different cell culture strategies on subsequent poliovirus production were investigated. Increased cell densities allowed up to 3 times higher d-antigen levels when compared with that obtained from batch-wise Vero cell culture. However, the cell specific d-antigen production was lower when cells were infected at higher cell densities. This cell density effect is in good agreement with observations for different cell lines and virus types. From the evaluated alternative culture methods, application of a semi-batch mode of operations allowed the highest cell specific d-antigen production. The increased product yields that can easily be reached using these higher cell density cultivation methods, showed the possibility for better use of bioreactor capacity for the manufacturing of polio vaccines to ultimately reduce vaccine cost per dose. Further, the use of animal-component-free cell- and virus culture media shows opportunities for modernization of human viral vaccine manufacturing

Antigen sparing with adjuvanted inactivated polio vaccine based on Sabin strains.

Six different adjuvants, each in combination with inactivated polio vaccine (IPV) produced with attenuated Sabin strains (sIPV), were evaluated for their ability to enhance virus neutralizing antibody titres (VNTs) in the rat potency model. The increase of VNTs was on average 3-, 15-, 24-fold with adjuvants after one immunization (serotypes 1, 2, and 3, respectively). Also after a boost immunization the VNTs of adjuvanted sIPV were on average another 7-20-27 times higher than after two inoculations of sIPV without adjuvant. The results indicate that it is feasible to increase the potency of inactivated polio vaccines by using adjuvants