Ionic interfaces and diphtheria toxoid interactions.

We present here a systematic study on the purification of the diphtheria toxoid (Dtxd) produced at the Instituto Butantan, by adding only one step on the entire process of its production. Aliquots of 1.0 ml of Dtxd were added to an equal amount of Q-Sepharose previously equilibrated with 500 mM Tris, pH 5.0-9.0 (increments of 0.5 pH units). The best condition for the Dtxd monomer adsorption was achieved at pH 9.0. The best condition for desorption was obtained with 300 mM NaCl. After studying the gel binding capacity for Dtxd, a column (C20/20) equilibrated with 500 mM Tris, pH 9.0, was prepared. The purification factor for Dtxd was 1.5. The final recovery of Dtxd was 68.75%, with 90.31% purity. The process methodology presented here is a very realistic sequence of separation steps, which is perfectly compatible with the production requirements. Vaccination with "toxoid highly purified toxin" is known to confer a strong immunity on people in the absence of undesirable reactions, which led experts of European Pharmacopoeia to recommend its use both for children and adult vaccination.

Calibration of replacement international standard and European Pharmacopoeia Biological Reference Preparation for Diphtheria Toxoid, Adsorbed.

We report here the characterisation of a preparation of diphtheria toxoid, adsorbed, and its calibration by twenty laboratories in fourteen countries in terms of the Second International Standard (I.S.) for Diphtheria Toxoid, Adsorbed, coded sample A (DIXA) using the established World Health Organisation (WHO)/European Pharmacopoeia (Ph Eur) challenge methods. The replacement standard preparation was found to have a unitage of 160 IU/ampoule on the basis of its calibration by in vivo bioassay. Stability was assessed within the collaborative study, and as part of candidate characterisation. Results suggest that the replacement standard will have satisfactory stability. This study also provided an opportunity to investigate serology as alternative to in vivo bioassay for potency testing of diphtheria vaccines. Six laboratories participated by performing serology according to in-house protocol. The calibration of the replacement standard in a mouse Vero cell assay gave a significantly higher results than in the established WHO/Ph Eur methods. Based on the results of this study and with the agreement of participants, the candidate standard was established as the Third International Standard for Diphtheria Toxoid, Adsorbed (coded 98/560) by the WHO Expert Committee of Biological Standardization in October 1999. The same preparation was also established as the second Ph Eur Biological Reference Preparation (Ph Eur BRP, batch no. 3) by the Steering Committee of the Biological Standardisation Programme of the European Directorate for the Quality of Medicines and approved by the European Pharmacopoeia Commission.

Physicochemical analysis of purified diphtheria toxoids: is toxoided then purified the same as purified then toxoided?

Diphtheria toxin can be converted into a highly immunogenic and irreversibly detoxified vaccine either using the conventional process in which the crude toxin is formalinised and subsequently purified (DTxd(conv)) or by detoxification of the highly purified toxin (DTxd(new)). In this study, both DTxd(new) and DTxd(conv) were evaluated by use of physico-chemical methods. For some methods, results were also compared to those obtained for cross-reacting material (CRM197), which is a non-toxic mutant of diphtheria toxin. DTxd(new) was assayed to have a specific purity of at least 2300 LF/mg protein N, thereby exceeding Pharm. Eur. requirements by up to 35%. Superior purity of DTxd(new) could also be demonstrated by size exclusion HPLC experiments and by amino acid composition studies. Far-UV circular dichroism spectroscopy revealed that the secondary structure of DTxd(new) almost resembled that of CRM197, suggesting only minor molecular changes during detoxification. This study worked out differences between purified diphtheria toxoids. Physico-chemical means revealed the advantages of DTxd(new) being the purer and more defined product, thus making it highly efficient for its use as a vaccine carrier as well as a component of vaccine combinations.

Capillary electrophoretic analysis of meningococcal polysaccharide-diphtheria toxoid conjugate vaccines.

Protein-polysaccharide conjugate vaccines are large, complex molecules that present challenges in terms of characterization. Free solution capillary electrophoresis using alkaline (pH 9-10) borate or glycine/NaOH buffers permitted electrophoresis of diphtheria toxoid (Dt) or meningococcal polysaccharide-Dt conjugates as well-formed, relatively broad peaks. Resolution was observed between the conjugate and the Dt peaks for some serotypes; however, overlap between Dt and one or more conjugate peaks was observed with both buffers. Inclusion of SDS in the separation buffer at a level above the critical micelle concentration allowed separation of Dt and Dt-conjugates of meningococcal serotypes A, C, Y and W135. Using borate/SDS, a linear relationship between peak area and Dt concentration was observed between approximately 2 mg/ml and 20 microg/ml. A linear relationship between Dt peak area and injection times from 10 to 50 seconds was also observed. Capillary electrophoresis may therefore be a useful method for quantifying free protein level in meningococcal-Dt 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.

Evaluación del rendimento en la purificación de toxoide diftérico con sulfato de amonio y filtración en gel / Evaluation of yeld of diphteric toxoid purified by amnonium sulfate and gel filtration

Con objeto de eliminar impurezas que pueden dar lugar a reacciones secundarias se concentraron lotes de toxoide diftérico con sulfato de amonio mediante la técnica utilizada en el Instituto de Biológicas del Departamento de Salud Pública de Boston, Massachussetts, E.U.A., el cual consiste en precipitaciones fraccionadas a diferentes concentraciones de sulfato de amonio. Posteriormente los toxoides concentrados se separaron haciéndolos pasar por una columna de sephadex G-100. A las fracciones obtenidas se les determinó la presencia de proteínas, midiendo la densidad óptica de la muestra a 280 nm. Los rendimientos obtenidos fueron para toxoides concentrados de 55 a 94% dando como promedio 81.23% y los resultados con toxoides purificados de 63 a 92% y el promedio de 79.5%. Este estudio permitió integrar los métodos antes mencionados a la producción del Instituto con buenos rendimientos

[Improved culture of Corynebacterium diphtheriae for obtaining toxin-anatoxin]. / Sovershenstvovanie kul'tivirovaniia difteriinykh korinebakterii dlia polucheniia toksina-anatoksina.

The work deals with the optimization of the cultivation of C. diphtheriae with a view to obtaining diphtheria toxin--toxoid on the basis of the study of the kinetics of microbial growth and toxin formation. The combined cultivation process consisting of 3 cycles has been experimentally developed and realized under industrial conditions. The use of this cultivation method has made it possible to obtain more balanced cultures and to standardize the biological properties of the biomass and toxin thus obtained. The characteristic features of the toxoid preparations obtained on the basis of this method are a high degree of purification, good immunogenic properties and constant fractional composition; their degree of homogeneity approximates the molecular homogeneous state.

Results with a new DTP vaccine in Japan.

Adverse reactions to DTP became a social problem in Japan in 1975 which brought a fall of immunization rates and an epidemic of pertussis which had its peak in 1979. Development of a new DTP vaccine with less adverse reactions was urgently needed and an acellular pertussis vaccine was developed which has replaced whole cell pertussis vaccines. More than 15 million doses of the new DTP vaccine has been administered since 1981 in Japan. Immunization acceptance reached 80% in 1982 and the incidence of pertussis has shown a definite decline. Temperature elevation to 38 degrees C or above following immunization with the new DTP vaccine occurred in approximately 1% of all vaccinees. Local reactions were less frequent after the first dose but observed in 40-50% of vaccinees after the second or later doses. However, local reactions were transient and subsided within a few days. Three cases who developed CNS symptoms have been reported for an incidence of 0.19 per one million doses. Attack rates in home contacts below 5 years of age were 84.2% in non-vaccinees and 5.5% in children who had received two or more doses of DTP. The estimated efficacy rate was 93.5%. Anti-LPF and anti-FHA titers after two or three doses of DTP were similar to those in the convalescent stage of natural pertussis infections. Antibody titers of children immunized with the new DTP were similar to those of children immunized with whole cell pertussis vaccines.

Inactivated poliovirus vaccine: current production methods and new developments.

The biotechnologic developments during the last decade have led to the production of inactivated poliovirus vaccine (IPV) on an industrial scale and at economically acceptable costs. Replacement of primary monkey kidney cells by subcultured monkey kidney, Vero, or human diploid cells as substrate for virus multiplication as well as the introduction of the microcarrier culture technique have made cell and virus cultivation in large fermentors of 100-1,000 liters feasible. Procedures for processing virus harvests into highly concentrated purified vaccines were developed; also, the safety and potency control tests were improved and simplified. It has been demonstrated that these more potent poliovirus vaccines, either alone or in combination with diphtheria-tetanus-pertussis vaccine, induce a high immunity with reduced vaccination schedules. The overall costs of vaccination will be reduced considerably in this way. In addition, the results of biochemical and immunologic studies indicate that neutralizing antibodies can be induced by the viral proteins alone. These findings open up promising perspectives for production of subunit poliovirus vaccines with use of recombinant DNA and synthetic antigen, a method that has already proved feasible for producing vaccine against foot and mouth disease. These new techniques may lead to a further reduction of production costs and will improve the safety of the vaccine.

Comparison of gel filtration and ammonium sulphate precipitation in the purification of diphtheria toxin and toxoid.

Crude diphtheria toxin and toxoid were subjected to purification by gel filtration and stepwise ammonium sulphate precipitation. The various fractions obtained by the purification procedures were studied by immunological methods. A high molecular weight fraction of glycoprotein nature was present in all of the crude preparations studied. The fraction was antigenically non-identical with the real toxin or toxoid and did not have its origin in the culture medium. It showed a long flocculation time when tested against equine diphtheria toxoid antiserum. The fraction could be removed from the crude preparations by gel filtration or by precipitation with 21% (w/v) ammonium sulphate. When comparing toxoids purified by each of these methods, the method of gel filtration resulted in a somewhat higher degree of purity, suggesting that this method would be more suitable than the AS precipitation method for the purification of diphtheria toxoid.

A versatile method for purification of diphtheria toxoid for immunization purposes.

Diphtheriae growth supernatant. The toxoid was quantitatively recovered from the immunosorbent in two fractions: the first in 0.005 M hydochloric acid and the second in 6' M urea containing 0.1 M HCL. Both fractions were soluble in aqueous solvents, and both had a similar specific activity: between 200 and 250 Lf/mg protein, and about 1.5 protective unit (ED50) per l Lf unit, as assayed in guinea pigs. Using the same procedure, the specific activity of a commerical toxoid preparation, purified and concentrated, was increrased two fold, from 130 Lf/mg to 260 Lf/mg protein.

New method for the large-scale preparation of diphtheria toxoid: purification of toxin.

Diphtheria toxin of high purity was prepared in batch cultures of 1 to 40 liters by procedures capable of processing 200-liter batches without modification. The procedure incorporates preliminary purification of the growth medium and, after deep fermentation or sulfate culture of Corynebacterium diphtheriae, both concentration and partial purification of the toxin by membrane ultrafiltration. Final purification is achieved by Sephadex G-100 gel filtration. Purities of 2,000 to 2,500 flocculation units per mg of protein nitrogen (260 to 410 flocculation units per unit of absorbance at 280 nm) were routinely obtained with only a 10% loss of toxin. The toxin appeared pure on immunoelectrophoresis and ultracentrifugation, and only minor amounts of lower-molecular-weight impurities were revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Significant advantages of the procedure are its rapidity and reproducibility and the fact that all stages are performed at 4 C in neutral isotonic buffer.