An effective, simple and low-cost pretreatment for culture clarification in tetanus toxoid production.

Chemically inactivated tetanus toxin (tetanus toxoid, TT), purified from cultures of a virulent Clostridium tetani strain, is the active pharmaceutical ingredient of anti-tetanus vaccines. Culture clarification for TT production and is usually performed by filtration-based techniques. Final clarification of the culture supernatant is achieved by passage through 0.2 µm pore size filtering membranes. Large particles removal (primary clarification) before final filtration (secondary clarification) reduces costs of the overall clarification process. With this aim, chitosan-induced particle aggregation was assessed as an alternative for primary clarification. Three chitosan variants were tested with similar results. Optimal clarification of culture supernatant was achieved by the addition of 8 mg chitosan per l of culture. Extrapolation analysis of filter sizing results indicate that 100 l of chitosan-treated supernatant can be finally filtered with a 0.6 m2 normal filtration cartridge of 0.45 + 0.2 µm pore size. The clarified material is compatible with current standard downstream processing techniques for TT purification. Thus, chitosan-induced particle aggregation is a suitable operation for primary clarification.

Binding and cleavage (BINACLE) assay for the functional in vitro detection of tetanus toxin: applicability as alternative method for the safety testing of tetanus toxoids during vaccine production.

Tetanus toxoids (i.e. chemically inactivated preparations of tetanus neurotoxin) are used for the production of tetanus vaccines. In order to exclude the risk of residual toxicity or of a "reversion to toxicity", each batch of tetanus toxoid is subject to strict safety testing. Up to now, these prescribed safety tests have to be performed as in vivo toxicity tests in guinea pigs. However, as animal tests are generally slow, costly and ethically disputable, a replacement by an in vitro method would be desirable. A suitable alternative method would have to be able to sensitively detect already low concentrations of active tetanus neurotoxin in matrices containing large amounts of inactivated toxoid molecules. We have developed a method which detects active tetanus neurotoxin molecules based on their specific receptor-binding capacity as well as their proteolytic activity. By taking into account two relevant functional characteristics, this combined "BINding And CLEavage" (BINACLE) assay more reliably discriminates between toxic and detoxified molecules than other in vitro assays which solely rely on one single toxin function (e.g. endopeptidase assays). Data from an in-house validation show that the BINACLE assay is able to detect active tetanus neurotoxin with a detection limit comparable to the in vivo test. The sensitive detection of active toxin which has been spiked into toxoid samples from different manufacturers could also be demonstrated. Specificity and precision of the method have been shown to be satisfactory. The presented data indicate that for toxoid batches from some of the most relevant European vaccine manufacturers, the BINACLE assay may represent a potential alternative to the prescribed animal safety tests. In addition, this novel method may also provide a convenient tool for monitoring batch-to-batch consistency during toxoid production.

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.

Production and purification of ovine anti-tetanus antibody.

We used the ovine as bioreactor for the production and optimization of anti-tetanus toxin antibody. Four female sheep were immunized with human tetanus vaccine (TT-alum) every two weeks for 16 weeks, after which serum was collected and its titer was estimated by ELISA. The highest titer obtained was 39,000 IU ml-1. To optimize a purification protocol for ovine anti-tetanus toxin, we used four procedures; weak anion (DEAE-Sephadex), weak cation (CM-Sephadex), ammonium sulfate precipitation alone or in combination with caprylic acid. Fifty percent saturation with ammonium sulfate combined with caprylic acid gave us the highest yield of protein with specific activity and the purest Fab product.

Use of sephacryl high resolution (HR) in tetanus anatoxin purification.

The tetanus purified anatoxin is used in the preparation of the tetanus toxoid and multiple vaccines (dT, DT and DTP), all of them strictly following specifications established by the WHO with a minimum antigenic purity equal to 1,000 Lf/mgPN. Aiming to establish more sensitive and accurate methods for purification, samples from four different lots of tetanus anatoxin were submitted to gel filtration in twenty independent trials using the Sephacryl S-100 HR and S-200 HR resins. The Authors were careful to optimize their parameters of performance as to sample volume, elution and selectivity flow for tetanus anatoxin purification, allowing their use in industrial scale. The Sephacryl S-100 HR resin presented the best selectivity, that is, the best separation, allowing a greater linear-flow and, consequently, the best purity index. Satisfactory results were also achieved with the Sephacryl S-200 HR resin after optimization of chromatographic parameters for elution flow and volume of the sample applied. The good results of purification obtained, as well as the high chemical stability, have pointed out both the Sephacryl S-100 HR and S-200 HR resins as equally efficient for industrial production.

Rapid and complete adsorption of unconjugated protein from protein-polysaccharide conjugate vaccines.

We have developed a rapid and inexpensive approach to remove unconjugated protein from protein-polysaccharide conjugate vaccines, without using gel filtration or ultrafiltration. We employ porous particles that adsorb the protein, whether bound or free, but with a pore size that allows only the unconjugated protein to enter the particle. Using limited amounts of media there is preferential binding of the unconjugated protein over the high molecular weight protein-polysaccharide conjugate. Adsorption of the unconjugated protein is rapid, with greater than 90% recovery of the conjugate. The approach is applicable to both neutral and charged polysaccharides and is not dependent on the chemistry used to make the conjugate vaccine. We have used this method to prepare tetanus toxoid-polysaccharide conjugates and found their immunogenicity in mice comparable to conjugates prepared using gel filtration. The method described can be used to reduce the cost and increase the yields of protein-polysaccharide conjugate vaccines.

Rational approaches to reduce adverse reactions in man to vaccines containing tetanus and diphtheria toxoids.

Adverse reactions to routine vaccines are obstacles to the mass vaccination campaigns. Though the absolute safety of any injectable vaccine cannot be guaranteed, the adverse side effects to vaccines can be minimized by practicing existing scientific knowledge. Adverse side effects to tetanus and diphtheria toxoids have been known for many years and there have been ways to minimize these reactions. These procedures did not get wide acceptance, because the current partially purified tetanus and diphtheria vaccines meet the regulatory requirements and the manufacturers are reluctant to change the established procedures of production due to the amount of work involved in the regulatory issues under the current Good Manufacturing Practices (GMP). Due to the recent epidemic of diphtheria in the independent states of the former Soviet Union, and its potential for spread to other European Countries, vaccination campaigns with tetanus and diphtheria vaccines received a new boost with several international agencies. In this report, we review the causes for adverse reactions to tetanus and diphtheria vaccines and offer practical suggestions for minimizing these reactions. The major issues in minimizing adverse reactions to these vaccines include: (1) purifying the toxins before detoxification as the reactogenic accessory antigens get covalently bound to the toxins during detoxification; (2) either using well-tolerated adjuvants which do not elicit the production of antigenic specific IgE antibodies responsible for adverse reactions or by using non-adjuvanted highly immunogenic polymerized antigens; (3) checking the status of immunity by recently developed rapid serological methods or by the Schick skin-test for diphtheria to avoid allergic or Arthus-type reactions. These approaches are applicable to industrial scales and would result in a pure, less reactogenic and better characterized toxoids antigens which would be more suitable for combined vaccines comprising highly purified acellular pertussis components, polysaccharide-protein conjugates and other antigens.

Global DTP manufacturing capacity and capability. Status report: January 1995.

A recently completed survey of 63 manufacturers of diphtheria-tetanus-pertussis (DTP) vaccine and its components in 42 countries shows that there is potentially a large excess installed capacity for DTP production. However, many manufacturers are not producing to capacity, and demand and supply for this vaccine are not matched in individual countries. About half of all countries producing DTP vaccine and its components do not have fully functional national control systems, and some countries are performing none of the critical functions for an effective control of quality. Thus, potential for export of excess capacity is limited. The data collected indicate much homogeneity in the preparation of diphtheria and tetanus toxoids. Nearly all manufacturers use the same seeds and similar purification methods, but there is variability in whether purification is done before or after conversion of toxin to toxoid. About 10% of all manufacturers do not meet WHO-defined standards of purity for these toxoids. There is much more heterogeneity in the pertussis seed strains and the methods of purification used. The formulation of DTP vaccine differs considerably among producers. Potency testing is not being done by the WHO-recommended method by about 50% of manufacturers on lots of diphtheria and tetanus toxoids for release. Testing of irreversibility of conversion of toxin to toxoid, a WHO-specified safety test, is also not being done on each lot of diphtheria toxoid by 15% of manufacturers surveyed nor on each lot of tetanus toxoid vaccine by 30% of manufacturers surveyed. Access to technology to develop new DTP-based combination vaccines will be delayed if these manufacturers cannot ensure consistent high quality vaccine for their target populations. The results and conclusions suggest areas for future activities to strengthen the supply and quality of DTP and DTP-based combination vaccines.

Beef allergy and the Persian Gulf syndrome.

It is suggested that the Persian Gulf Syndrome (PGS) is caused by beef allergy. In the first symptomless phase, as a result of an energetic US Army immunizing program, using sera with adjuvants to produce detectable antibody levels, the subjects not only developed immunity to the targeted substances, but also became sensitized to one or more of the other substances in the immunizing sera, and specifically to beef protein. The subjects remained healthy while in the war zone on a restricted diet essentially free from beef, but developed PGS after they came home, and were again able to obtain steaks and hamburgers.

Neutralizing antibodies and immunoprotection against pertussis and tetanus obtained by use of a recombinant pertussis toxin-tetanus toxin fusion protein.

The currently available diphtheria-tetanus-whole-cell pertussis (DTP) vaccines are associated with a variety of problems, including undesirable side effects and inconsistent efficacy. These problems are probably related to the poor definition of such vaccines, especially with respect to the whole-cell component against pertussis. Ideal vaccines should include only immunoprotective antigens with no toxin activity. As an initial step towards obtaining a well-defined and simplified DTP vaccine, a pertussis toxin-tetanus toxin chimeric protein was constructed. A soluble form of the pertussis toxin S1 subunit was fused to the protective fragment C of tetanus toxin, and the recombinant hybrid protein was produced in Escherichia coli. The 75-kDa fusion protein (p75) was overexpressed as a soluble molecule and purified to near homogeneity by two consecutive chromatographic steps. Purified p75 retained its ability to bind to ganglioside GT1b, the receptor for tetanus toxin, and to be recognized by protective and neutralizing anti-pertussis toxin antibodies specific for conformational epitopes. When administered to mice, the hybrid protein was found to be nontoxic but immunogenic. In addition, it was capable of inducing strong protection against tetanus and some protection against pertussis, as well as eliciting a pertussis toxin-neutralizing antibody response. Although the levels of anti-pertussis toxin antibodies were rather low, neutralizing titers of the immunized mice correlated well with anti-pertussis toxin titers, indicating that protective epitopes are conserved in the recombinant protein.

[An evaluation of the quality of tetanus anatoxins by gel electrophoresis]. / Otsenka kachestva stolbniachnykh anatoksinov metodom gel'-fil'tratsii.

The study of tetanus toxoids obtained from different manufacturers in the USSR has shown that these preparations exhibit molecular heterogeneity. The method of gel filtration has made it possible to find out that tetanus toxoids from different manufacturers differ in the degree of their purification. The preparations produced by the manufacturing enterprises in Perm and Ufa have been found to contain considerably less ballast substances than the preparations produced in Moscow.

Antibody response of pregnant women to two different absorbed tetanus toxoids.

The antibody response in pregnant women vaccinated with either of two different adsorbed tetanus toxoids has been studied. One vaccine (A), prepared by toxoiding purified tetanus toxin followed by its adsorption onto calcium phosphate, exhibited a low titre expressed as international immunizing units, 69 IIU/0.5 ml. The other vaccine (B), prepared by purifying formalinized crude tetanus toxin and adsorbing it onto aluminium phosphate showed a high titre, 212 IIU/0.5 ml. No significant differences between titres of circulating antibodies were obtained after the first injection of either vaccine, but titres after the second injection were much higher for vaccine A as compared with those obtained using vaccine B. The results showed that the immune response in human beings is not correlated to titres expressed in IIU. These results confirm that other methods should be adopted for evaluating the potency of vaccines. A simplified technique based on the comparison of circulating antitoxin levels after vaccination of mice has recently been proposed.

[The immunogenic capacity of soluble "vaccinia" antigen (fractionated smallpox vaccine) administered simultaneously with inactivated typhoid vaccine and with purified and concentrated tetanus anatoxin]. / La capacité immunogène de l'antigène soluble "vaccinia" (vaccin fractionné antivariolique) administré simultanément avec le vaccin typhoïdique inactivé et avec l'anatoxine tétanique purifiée et concentrée.

In this study we have investigated the immune humoral response in the associated vaccination with smallpox, tetanus and typhoid fractionated vaccine (trivaccine) administered in two series at 1 month interval, by dermojet, in a group of young people of 18-20 years old. The results were comparatively estimated with those obtained in two groups of young people of the same age (control group), separately immunized with two components of the tri-vaccine: fractionated smallpox vaccine and tetanus toxoid, following-up the humoral response to the two vaccine components. It was find that, at the end of the surveillance period, similar results were obtained for the testing group and control group, the antibody titers (in geometric mean) presenting very close values: 1/1,140,463 for the testing group, and 1/1,053,583 for the control group against vaccinia component, and 1/1,86,880,586 for the testing group and 1/79,900,431 for the control group, against the tetanus component. The results obtained entitle us to propose this vaccination scheme for the vaccination practice.

Serological survey of immune response to tetanus toxoid using antitoxin enzyme immunoassay.

A method for testing serum tetanus immunoglobulin (TIG) was set up using enzyme immunoassay kit (EIA kit) and prepared by competitive principle in this laboratory. Forty-eight human sera tested by both EIA and toxin neutralization test (NT) in mice were in good agreement. Determination coefficient (r2) of the EIA and NT was 0.922. The lowest detectable dose was 0.1 IU/mL ELISA value. Many authors believe this ELISA value to be a safe protection TIG level. For serological survey of immune response to tetanus toxoid, sera from two groups of subjects were examined. Among 83 subjects, each over 40 years of age, only 47.0% were positive to TIG EIA level. Forty-six subjects with undetectable EIA titres to tetanus toxoid (TT) were immunized against two doses of TT at an interval of four weeks; 44 (95.6%) demonstrated TIG concentration of greater than or equal to 0.2 IU/mL within two weeks to one month following the last dose vaccination. The TIG positive rates of 725 school students' sera, selected by multistage sampling from 2395 specimens, were reported. Those were collected by simple random sampling from geographical area in the North, South, Central, Central Mountain, and East areas of Taiwan according to age groups. They were 80.8%, 65.1%, 56.0%, and 42.0% to the age groups of 0-4, 5-9, 10-14, and 15-17 years respectively. The probabilities of each near by two groups were p less than 0.1; p less than 0.05; p less than 0.05. The differences of positive rates in the last three groups were significant.