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Root cause analysis (RCA) is one of the most prominent tools used to comprehensively evaluate a biopharmaceutical production process. Despite of its widespread use in industry, the Food and Drug Administration has observed a lot of unsuitable approaches for RCAs within the last years. The reasons for those unsuitable approaches are the use of incorrect variables during the analysis and the lack in process understanding, which impede correct model interpretation. Two major approaches to perform RCAs are currently dominating the chemical and pharmaceutical industry: raw data analysis and feature-based approach. Both techniques are shown to be able to identify the significant variables causing the variance of the response. Although they are different in data unfolding, the same tools as principal component analysis and partial least square regression are used in both concepts. Within this article we demonstrate the strength and weaknesses of both approaches. We proved that a fusion of both results in a comprehensive and effective workflow, which not only increases better process understanding. We demonstrate this workflow along with an example. Hence, the presented workflow allows to save analysis time and to reduce the effort of data mining by easy detection of the most important variables within the given dataset. Subsequently, the final obtained process knowledge can be translated into new hypotheses, which can be tested experimentally and thereby lead to effectively improving process robustness.

Sabin-IPV (or sIPV, inactivated polio vaccine based on attenuated Sabin strains) is anticipated to replace the oral polio vaccine for the endgame in polio eradication. Optimization of sIPV production will lead to a better economically feasible vaccine. To assist process optimization, we studied Sabin type 1 poliovirus (PV) infection kinetics on Vero cells in controlled bioreactor vessels. The aim of our study was to develop a descriptive mathematical model able to capture the dynamics of adherent Vero cell growth and PV infection kinetics in animal component free medium. The model predicts the cell density, metabolites profiles, and viral yields in time. We found that the multiplicity of infection (MOI) and the time of infection (TOI) within the investigated range did not affect maximal PV yields, but they did affect the process time. The latter may be reduced by selecting a low TOI and a high MOI. Additionally, we present a correlation between viral titers and D-antigen, a measure for immunogenicity, of Sabin type 1 PV. The developed model is adequate for further studies of the cell metabolism and infection kinetics and may be used to identify control strategies to increase viral productivity. Increased viral yields reduce costs of polio vaccines with large implications on public health.

Industrial-scale inactivated polio vaccine (IPV) production dates back to the 1960s when at the Rijks Instituut voor de Volksgezondheid (RIV) in Bilthoven a process was developed based on micro-carrier technology and primary monkey kidney cells. This technology was freely shared with several pharmaceutical companies and institutes worldwide. In this contribution, the history of one of the first cell-culture based large-scale biological production processes is summarized. Also, recent developments and the anticipated upcoming shift from regular IPV to Sabin-IPV are presented. Responding to a call by the World Health Organization (WHO) for new polio vaccines, the development of Sabin-IPV was continued, after demonstrating proof of principle in the 1990s, at the Netherlands Vaccine Institute (NVI). Development of Sabin-IPV plays an important role in the WHO polio eradication strategy as biocontainment will be critical in the post-OPV cessation period. The use of attenuated Sabin strains 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 to clinical trial material production, process development, optimization and formulation research is being carried out to further optimize the process and reduce cost per dose. Also, results will be shown from large-scale (to prepare for future technology transfer) generation of Master- and Working virus seedlots, and clinical trial material (for phase I studies) production. Finally, the planned technology transfer to vaccine manufacturers in low and middle-income countries is discussed.

Historical manufacturing data can potentially harbor a wealth of information for process optimization and enhancement of efficiency and robustness. To extract useful data multivariate data analysis (MVDA) using projection methods is often applied. In this contribution, the results obtained from applying MVDA on data from inactivated polio vaccine (IPV) production runs are described. Data from over 50 batches at two different production scales (700-L and 1,500-L) were available. The explorative analysis performed on single unit operations indicated consistent manufacturing. Known outliers (e.g., rejected batches) were identified using principal component analysis (PCA). The source of operational variation was pinpointed to variation of input such as media. Other relevant process parameters were in control and, using this manufacturing data, could not be correlated to product quality attributes. The gained knowledge of the IPV production process, not only from the MVDA, but also from digitalizing the available historical data, has proven to be useful for troubleshooting, understanding limitations of available data and seeing the opportunity for improvements.

We report the large-scale production of VHH antibody fragments, raised against the azo-dye RR-6, by the yeast Saccharomyces cerevisiae. The secretion efficiency of the VHH fragments was correlated with their overall hydrophobicity. With a 15 m3 fed-batch fermentation, 1.3 kg VHHs were produced. Purification was done by removal of biomass and product concentration. During induction, the specific production rate qp was 0.213 g.[kg dryweight]−1.h−1.

Mass transfer, mixing times and power consumption were measured in rigid disposable stirred tank bioreactors and compared to those of a traditional glass bioreactor. The volumetric mass transfer coefficient and mixing times are usually determined at high agitation speeds in combination with sparged aeration as used for single cell suspension and most bacterial cultures. In contrast, here low agitation speeds combined with headspace aeration were applied. These settings are generally used for cultivation of mammalian cells growing adherent to microcarriers. The rigid disposable vessels showed similar engineering characteristics compared to a traditional glass bioreactor. On the basis of the presented results appropriate settings for adherent cell culture, normally operated at a maximum power input level of 5 W m−3, can be selected. Depending on the disposable bioreactor used, a stirrer speed ranging from 38 to 147 rpm will result in such a power input of 5 W m−3. This power input will mix the fluid to a degree of 95% in 22 ± 1 s and produce a volumetric mass transfer coefficient of 0.46 ± 0.07 h−1. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1269–1276, 2014

The anticipated increase in the demand for inactivated polio vaccines resulting from the success in the polio eradication program requires an increase in production capacity and cost price reduction of the current inactivated polio vaccine production processes. Improvement of existing production processes is necessary as the initial process development has been done decades ago. An up-to-date lab-scale version encompassing the legacy inactivated polio vaccine production process was set-up. This lab-scale version should be representative of the large scale, meaning a scale-down model, to allow experiments for process optimization that can be readily applied. Initially the separate unit operations were scaled-down at setpoint. Subsequently, the unit operations were applied successively in a comparative manner to large-scale manufacturing. This allows the assessment of the effects of changes in one unit operation to the consecutive units at small-scale. Challenges in translating large-scale operations to lab-scale are discussed, and the concessions that needed to be made are described. The current scale-down model for cell and virus culture (2.3-L) presents a feasible model with its production scale counterpart (750-L) when operated at setpoint. Also, the current scale-down models for the DSP unit operations clarification, concentration, size exclusion chromatography, ion exchange chromatography, and inactivation are in agreement with the manufacturing scale. The small-scale units can be used separately, as well as sequentially, to study variations and critical product quality attributes in the production process. Finally, it is shown that the scale-down unit operations can be used consecutively to prepare trivalent vaccine at lab-scale with comparable characteristics to the product produced at manufacturing scale.

A fast ELISA was developed and qualified for analysis of polio D-antigen. The original 20 h-protocol was optimized by minimizing the total incubation time to 1 h, and by replacing the signal reagent 3,3',5,5'-tetramethylbenzidine by a chemiluminogenic signal reagent with a theoretical low intrinsic background and high dynamic range.

Saccharomyces cerevisiae carrying a multicopy integrated expression vector containing the gene encoding a Llama antibody fragment, has been cultivated in continuous cultures both under carbon and nitrogen limiting conditions with galactose as the sole carbon source. VHH-R2 expression was under control of the inducible GAL7 promoter. Induction however, was independent of the galactose consumption rate and maximal at all growth rates. VHH-R2 was secreted with 70% efficiency at all growth rates and under both limitations. The specific production rate increased linear with increasing growth rate in a growth-associated manner. However, when grown under nitrogen limitation at growth rates above 0.09 h(-1), the extracellular VHH-R2 was less active or part of the VHH-R2 was in an inactive form. From our results we conclude that to obtain a maximal amount of VHH per kilogram biomass per hour, VHH production should be done in carbon limited continuous cultures at high specific growth rates.