IPV v2.0 : upgrading the established inactivated polio vaccine production process

Y.E. Thomassen

Research output: Thesisinternal PhD, WU


The first vaccine against poliovirus (PV), the causative agent of poliomyelitis, was developed in the 1950s by Jonas Salk. The vaccine (IPV) consists of an injected dose of purified and inactivated wild-type PVs (all three serotypes). Soon after this discovery, at the Rijks Instituut voor de Volksgezondheid (RIV) in Bilthoven, an industrial-scale production process for IPV was developed based on micro-carrier technology and primary monkey kidney cells. In the 1970s, the manufacturing of IPV was scaled up to 300-L by the development of well-controlled bioreactors for cell culture, the so-called “Bilthoven Units” (originally used for bacterial fermentations). In 2004, the Vero cell line was introduced to replace the then used tertiary monkey kidney cells followed by a scale-up from 700 to 1,500-L (from two 350-L to two 750-L bioreactors). IPV manufacturing has been part of the regular vaccine manufacturing activities in Bilthoven ever since the establishment of the IPV production process.


With polio eradication on our doorstep, the World Health Organization (WHO) is pursuing a new IPV based on non-wild-type strains to increase the biosafety of vaccine manufacturing. In addition, and due to the pending cessation of oral polio vaccines (OPV), the global demand for affordable IPV is increasing. To accommodate these questions two research programs were started at the Netherlands Vaccine Institute (now Institute for Translational Vaccinology). One concerned optimization of the current conventional IPV production process, the other the manufacturing of an affordable sIPV, an IPV based on the attenuated Sabin PV strains normally used for OPV production. The technology of the sIPV production process, developed in the latter project, is also aimed to be transferred to developing countries manufacturers (Chapter 2).


From the substantial history in polio vaccine production in Bilthoven, a valuable dataset has been generated. Data from over 50 batches at two different production scales (700-L and 1,500-L) were analyzed using multivariate data analysis (MVDA). This statistical method is stimulated by the ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) to improve scientific understanding of production processes for troubleshooting and improved process control. The initial 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 cell- and virus culture 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 (Chapter 3).


One of the gaps in the data was located in the product quantification during processing. The available assay used for determining the D-antigen concentration in in-process samples had high variability. A so-called fast ELISA was developed and qualified for analysis of polio D-antigen. The original 20h-protocol was optimized by minimizing the total incubation time to 1h, 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. This fast D-antigen ELISA was suitable for measurement of D-antigen concentrations in the different matrixes present during the different unit-operations in the production process (Chapter 4).


To accommodate research to improve and optimize the current cIPV production process an up-to-date lab-scale version encompassing the legacy inactivated polio vaccine production process was set-up based on the knowledge obtained during the MVDA of historical data. This lab-scale version was designed to be representative of the large scale, meaning a scale-down model, to allow experiments for process optimization that can be readily applied to manufacturing scale. 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. The developed 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 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 has been 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 (Chapter 5).


The upcoming of disposables in GMP manufacturing triggered the study on alternatives for the clarification unit (Chapter 5) and alternative Wave-type, bioreactors (Chapter 6). This type of bioreactors makes use of sterilized disposable bags, which could be beneficial in a GMP environment, for example to reduce the cleaning validation burden, or to facilitate change-over to another product using the same equipment. Wave-type bioreactors make use of vertical (standard rocking motion-type) or both vertical and horizontal displacement (CELL-tainer®(CELLution Biotech BV)) for mixing instead of an impeller, which is used in stirred tank reactors. Using the design of experiments (DoE) approach, models for the mixing times in both the CELL-tainer®and the BIOSTAT®CultiBag RM (Sartorius Stedim Biotech) bioreactor (standard rocking motion-type) were developed. The conditions for cultivation of Vero cells in the CELL-tainer®bioreactor were chosen based on comparable mixing times. Vero cells growing adherent to Cytodex 1 microcarriers were cultivated in the CELL-tainer®and in the BIOSTAT®CultiBag RM. Vero cell growth in both bioreactors was comparable with respect to the growth characteristics and main metabolite production and consumption rates. Additionally, poliovirus production in both bioreactors was shown to be similar.


In view of WHOs pursuit towards an IPV manufacturing process with increased biosafety, the development of sIPV was taken up. Prior to large scale production of clinical lots, an initial proof of principle study was done (Chapter 2). Starting from the conventional IPV (cIPV) production process, minimal adaptations, such as lower virus cultivation temperature, were implemented. Also, the selected disposable filter unit (Chapter 5) was implemented. To quickly prepare sIPV clinical lots and show proof-of-principle of sIPV in human, no further process optimization and/or modernization was done. sIPV was produced at industrial scale followed by formulation of both plain and aluminium adjuvanted sIPV (Chapter 7). The final products met the quality criteria, were immunogenic in rats, showed no toxicity in rabbits and could be released for testing in the clinic. While an immunogenic product, both in animals as in humans was prepared, the product yield was extremely low and further process development will be needed to obtain an affordable sIPV.


Especially the yield of Sabin PV type 2 after ion exchange chromatography was low. To determine if this effect could be due to a difference in the isoelectric point (pI) of the poliovirus a method for pImeasurement of live virus was developed (Chapter 8). A method for analyzing biological hazardous components (biological safety level 2) was set up for the capillary isoelectric focusing-whole column imaging detection (CIEF-WCID) analyzer. This method is based on closed circuits. Subsequently, the pI’s of complete intact polioviruses were determined. The polioviruses that were analyzed are the commonly used viruses for the production of IPV - Mahoney (type 1), MEF-1 (type 2), and Saukett (type 3) - as well as for OPV - Sabin types 1, 2, and 3. The determined pI's were 6.2 for Mahoney, 6.7 for MEF-1, and 5.8 for Saukett. The pI's of Sabin types 1, 2, and 3 viruses were 7.4, 7.2, and 6.3, respectively. With a pIof 7.2, Sabin PV type 2 is prone to self-aggregation at the pH used during chromatography (pH 7.0). Self-aggregation was thus suggested to be the main cause of low product yield and prevention of this self-aggregation was suggested to be the main focus point for process optimization.


Besides optimization of the downstream processing, optimization of the upstream processing, i.e. increased virus yields after cell and virus culture was studied (Chapter 9). 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 × 106cells mL-1during batch cultivation to 1.8, 2.7 and 5.0 × 106cells mL-1during 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.


To assess the affordability of sIPV the manufacturing costs were determined (Chapter 10). The sIPV manufacturing costs, when produced as described in Chapter 7, indicate the requirement for process optimizations. However, the manufacturing costs can be reduced at least a factor 2 when implementing the upstream processing optimization, ACF media and a semi-batch process, as described in Chapter 9. Assuming improvements in downstream processing will result in a process with yields comparable to the cIPV process costs can be further lowered. In addition, a scale-up from two 350-L bioreactors to two 1,000-L bioreactors would nearly halve the manufacturing costs resulting in a more than cost competitive sIPV. These costs analysis showed that an affordable sIPV is feasible.  

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Wijffels, Rene, Promotor
  • Bakker, W.A.M., Co-promotor, External person
  • van der Pol, L.A., Co-promotor, External person
Award date15 Apr 2014
Place of PublicationWageningen
Print ISBNs9789461738561
Publication statusPublished - 2014


  • poliomyelitis
  • inactivated vaccines
  • vaccine development
  • bioreactors

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