Advanced process monitoring: the ultimate alternative.

Parametric release has the possibility to be the ultimate alternative for the use of animal tests. It involves the release of a product using evaluation of tightly controlled physical parameters, without the performance of a test on the final product. This practice is strengthened if there is a lack of validity for the final test to indicate quality or consistency. That suggests that in-process monitoring should replace the finished product controls when among others, batch-to-batch consistency has been formally validated. Currently, parametric release is a common practice for terminally sterilised and aseptically filled biologicals. An example is the production of inactivated cells of Bordetella pertussis which shows that new techniques are able to monitor the process in detail. Furthermore, it is shown that the production of Bordetella pertussis is well defined and highly predictable. In parallel with the tests for terminally sterilised biologicals, mouse potency tests are statistically hardly capable of indicating batch-to-batch variation. Monitoring on-line process parameters provides a better assurance of consistency and quality, thus giving the opportunity for batch release and regulatory approval without the use of animal tests.

Dual-substrate utilization by Bordetella pertussis.

To improve the cultivation of Bordetella pertussis and take advantage of the newest techniques in monitoring and control, a quantitative description of substrate utilisation is necessary. Growth of the organism is limited by two main substrates. However neither interactive nor non-interactive modelling seem appropriate. A model that combines essential and enhanced kinetics was developed based on experimental observation. Instead of fitting all model parameters at once, a step-wise experimentation procedure was used. Finally two cultivations showed the accuracy of the model.

Rational medium design for Bordetella pertussis: basic metabolism.

In current Bordetella pertussis media ammonium accumulates because of an imbalance in the nitrogen:carbon ratio of the substrates used, which is one of the factors limiting cell density in fed-batch cultures. The aim of this study was to map B. pertussis catabolic and anabolic capabilities, in order to design a medium that avoids ammonium accumulation, while substrates are metabolised completely. Besides the known dysfunctional glycolysis, B. pertussis also possessed a partially dysfunctional citric-acid cycle. Although ammonium accumulation was avoided by adding various carbon sources to medium with glutamate, nuclear magnetic resonance (NMR) showed excretion of acetate, acetoacetate and beta-hydroxy-butyrate, thereby reducing the biomass yield. Acetoacetate and beta-hydroxy-butyrate were also formed in Verwey, B2 and modified Stainer-Scholte medium. Electron microscopy in combination with NMR showed that cells early on in these cultures contained poly-hydroxy-butyrate (PHB) globules, which disappeared later during the culture, coinciding with the appearance of beta-hydroxy-butyrate and/or acetoacetate. No globules nor metabolite excretion was detected when lactate in combination with glutamate were used as substrates. Thus, metabolite excretion and ammonium accumulation were avoided, while the yield of 8.8 g C-mol-1 compared favourably with literature values, averaging 6.5 g C-mol-1. Optimisation of this medium for pertussis toxin production will be reported in a separate article.

Antigenic and immunogenic properties of inactivated polio vaccine made from Sabin strains.

Inactivated polio vaccine (IPV) was prepared with the Sabin strains, normally used for the attenuated live vaccine. The vaccine was characterized with respect to its antigenicity as determined by ELISA and biosensor analysis and its immunogenicity in rats. Compared with the vaccine prepared with virulent strains (Mahoney, MEF and Saukett) some distinct differences were found with regard to the interaction with glass vials (types 1 and 2) and antigenicity (type 3). The most profound difference however, was the immunogenicity of types 1 and 2. Standardized on the amount of virus, type 1 Sabin-IPV was about 3 times more immunogenic as compared to Mahoney-IPV. The immunogenicity of type 2 Sabin-IPV on the other hand was reduced approximately 10-fold, compared to MEF-IPV. Type 3 Sabin and Saukett IPV were comparable in immunogenicity but differences in antigenicity were evident. The lack of correlation between antigenicity and immunogenicity demonstrate that current IPV standards are not suitable to quantify IPV made from Sabin strains. The results indicate that future Sabin-IPV vaccines may have to contain virus amounts that are very different as compared to current IPV vaccines.

Quantification of cell-associated and free antigens in Bordetella pertussis suspensions by antigen binding ELISA.

In order to achieve batch-to-batch consistency of whole-cell pertussis vaccines, properties relevant for protection and safety should be characterised. Therefore, ELISAs to quantify pertussis toxin (PT), filamentous haemagglutinin (FHA), 92 kD outer membrane protein (92 kD-OMP) and pertactin (PRN) in Bordetella pertussis (B. pertussis) suspensions were developed. In this paper the influence of the bacterial growth stage on antigen production and antigen release into the supernatant was studied for pertussis strains 134, 509 and CS. The levels of cell-associated and free antigens during growth were strongly strain and antigen dependent. Because of this, the proportion of cell-associated antigens changed during cultivation for all three strains. Substantial amounts of PT and PRN were released into the supernatant, while little free FHA and 92 kD-OMP were found. The amount of cell-associated FHA declined rapidly during growth, whereas cell-associated 92 kD-OMP contents increased. These findings demonstrate that, although antigen exposure and release differ from strain to strain, the main factor that determines the antigen production and release is the growth phase.

Comparison of enhanced potency inactivated poliovirus vaccine (EIPV) versus standard oral poliovirus vaccine (OPV) in Thai infants.

Enhanced potency inactivated poliovirus vaccine (EIPV), combined with diphtheria-tetanus-pertussis (DTP) vaccine, was compared with oral poliovirus vaccine (OPV) regarding immunogenicity in Thai infants, vaccinated at 2, 4 and 6 months of age. EIPV induced significantly higher seroconversion rates than OPV to all 3 poliovirus types after the second and third immunization. After 3 doses of each vaccine, at 7 months of age, all infants receiving EIPV proved seropositive for poliovirus type 1, type 2 and type 3 neutralizing antibodies, whereas of those receiving OPV, 9% remained seronegative (titre < 1:4) for type 1 (p = 0.0042) and 11% for type 3 (p = 0.0013). All participating children were given an additional dose of OPV at the age of 9 months and tested again at 12 months of age. At that point, virtually all infants had poliovirus neutralizing antibodies, but the geometric mean titres to each poliovirus type were significantly higher in the vaccinees who had received EIPV. It is concluded that the greater immunogenicity of EIPV vis-à-vis 3 doses of OPV may be biologically significant for protection against poliovirus types 1 and 3 in countries where cases of poliomyelitis occur in young children. These findings warrant considering EIPV, alone or in combination with OPV, for an immunization programme in Thailand and similar countries in the future.