Process intensification for production of Streptococcus pneumoniae whole-cell vaccine.

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.

Process intensification for production of Streptococcus pneumoniae whole-cell vaccine

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.

Polysaccharide purification from Haemophilus influenzae type b through tangential microfiltration.

Haemophilus influenzae type b (Hib) is a human pathogen that causes meningitis in infants worldwide. Capsular polysaccharide linked to a protein has been used as an efficient vaccine, and this approach has reduced the incidence of Hib disease since its inclusion in national immunisation campaigns. The traditional polysaccharide downstream process is based on several ethanol precipitations, treatment with detergents and centrifugation. The aim of this study was to introduce tangential microfiltration (TMF) in the place of centrifugation to simplify handling and to scale up the process. The purity of the polysaccharide was RPNA=1747.2 and RPPrt=196.1 for nucleic acid and protein, respectively, meeting the quality requirements for this polysaccharide. Moreover, the polysaccharide was recognised by at specific antibody, and the ribose and phosphate contents were within the expected limits. Thus, we established a process for the purification of capsular polysaccharide produced by H. influenzae type b that is effective, robust and feasible to be scaling up.

Development of a whole cell pneumococcal vaccine: BPL inactivation, cGMP production, and stability.

Pneumococcal infections impose a large burden of disease on the human population, mainly in developing countries, and the current pneumococcal vaccines offer serotype-specific protection, but do not cover all pathogenic strains, leaving populations vulnerable to disease caused by non-vaccine serotypes. The pneumococcal whole cell vaccine is a low-cost strategy based on non-capsular antigens common to all strains, inducing serotype-independent immunity. Therefore, we developed the process for the cGMP production of this cellular vaccine. Initially, three engineering runs and two cGMP runs were performed in 60-L bioreactors, demonstrating the consistency of the production process, as evaluated by the growth curves, glucose consumption and metabolite formation (lactate and acetate). Cell recovery by tangential filtration was 92 ± 13 %. We optimized the conditions for beta-propiolactone (BPL) inactivation of the bacterial suspensions, establishing a maximum cell density of OD600 between 27 and 30, with a BPL concentration of 1:4000 (v/v) at 150 rpm and 4 °C for 30 h. BPL was hydrolyzed by heating for 2h at 37 °C. The criteria and methods for quality control were defined using the engineering runs and the cGMP Lots passed all specifications. cGMP vaccine Lots displayed high potency, inducing between 80 and 90% survival in immunized mice when challenged with virulent pneumococci. Sera from mice immunized with the cGMP Lots recognized several pneumococcal proteins in the extract of encapsulated strains by Western blot. The cGMP whole cell antigen bulk and whole cell vaccine product lots were shown to be stable for up to 12 and 18 months, respectively, based upon survival assays following i.p. challenge. Our results show the consistency and stability of the cGMP whole cell pneumococcal vaccine lots and demonstrate the feasibility of production in a developing country setting.

An alternative method for purifying and detoxifying diphtheria toxin.

Infections caused by Corynebacterium diphtheriae frequently induce situations in which very small doses of antigens injected intradermally can cause strong inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a virulence factor that can be lethal to the human organism at doses below 0.1 µg/kg of body weight. The present work proposes alternative methods of DT purification using affinity chromatography and of DT detoxification through conjugating with the polymer methoxypolyethylene glycol activated (mPEG). Tests were performed to evaluate: the formation of edemas and the presence of dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with mPEG, and the immunogenic activity of the purified and modified toxin. The results indicated that purification with Blue Sepharose was an efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein nitrogen. The modification of the Purified Toxin with mPEG did not result in the formation of edema or necrosis although it was immunogenic and stimulated the formation of antibodies that could neutralize the Purified Toxin. The toxoid obtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however.

An alternative method for purifying and detoxifying diphtheria toxin

Infections caused by Corynebacterium diphtheriae frequently induce situations in which very small doses of antigens injected intradermally can cause strong inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a virulence factor that can be lethal to the human organism at doses below 0.1 mg/kg of body weight. The present work proposes alternative methods of DT purification using affinity chromatography and of DT detoxification through conjugating with the polymer methoxypolyethylene glycol activated(mPEG). Tests were performed to evaluate: the formation of edemas and the presence of dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with mPEG, and the immunogenic activity of the purified and modified toxin. The results indicated that purification with Blue Sepharose was an efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein nitrogen. The modification of the Purified Toxin with mPEG did not result in the formation of edema or necrosis although it was immunogenic and stimulated the formation of antibodies that could neutralize the Purified Toxin. The toxoidobtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however.

GMP-grade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis.

Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid. Liter-scale and 60-l Good Manufacturing Practice (GMP) cultures were grown in bovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected into C57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH)(3) strongly increased responses, particularly if pre-treated with phosphate. Protection was found in several tested model infections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presence of CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+ T cells were not required for protection at the time of challenge, suggesting that antibody alone was sufficient to protect against death in this model. Rabbits receiving sequential intramuscular injections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinical signs. The rabbit antiserum thus produced was active in an in vitro phagocytic killing assay and passively protected mice in the type 3 aspiration-sepsis model. Approval is being sought for human trials of this vaccine.

Fed-batch production of tetanus toxin by Clostridium tetani.

This study deals with the effects of the initial nitrogen source (NZ Case TT) level and the protocol of glucose addition during the fed-batch production of tetanus toxin by Clostridium tetani. An increase in the initial concentration of NZ Case TT (NZ(0)) accelerated cell growth, increased the consumption of the nitrogen source as well as the final yield of tetanus toxin, which achieved the highest values (50-60 L(f)/mL) for NZ(0) > or = 50 g/L. The addition of glucose at fixed times (16, 56, and 88 h) ensured a toxin yield ( approximately 60 L(f)/mL) about 33% higher than those of fed-batch runs with addition at fixed concentration ( approximately 45 L(f)/mL) and about 300% higher than those obtained in reference batch runs nowadays used at industrial scale. The results of this work promise to substantially improve the present production of tetanus toxin and may be adopted for human vaccine production after detoxification and purification.

GMP-grade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis

Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid. Liter-scale and 60-l Good Manufacturing Practice (GMP) cultures were grown in bovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected into C57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH)3 strongly increased responses, particularly if pre-treated with phosphate. Protection was found in several tested model infections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presence of CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+ T cells were not required for protection at the time of challenge, suggesting that antibody alone was sufficient to protect against death in this model. Rabbits receiving sequential intramuscular injections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinical signs. The rabbit antiserum thus produced was active in an in vitro phagocytic killing assay and passively protected mice in the type 3 aspiration-sepsis model. Approval is being sought for human trials of this vaccine.

Enzymatic profiling of tetanus and botulinum neurotoxins based on vesicle-associated-membrane protein derived fluorogenic substrates.

Botulinum (BoNT) and tetanus (TeNT) neurotoxins are bacterial zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. There are seven serotypes of BoNT, while TeNT is found in one serotype. In order to characterize their enzymatic activities and to propose serotype-differentiation an enzymatic assay based on their metalloprotease activity was developed. The assays were conducted with FRET peptides derived from SNAP-25, synaptobrevin and syntaxin. The substrates were cleaved by 2 ng/mL of toxin at different rates (K(cat)/K(M) from 0.028 to 75.9 microM.s(-)) at a single bond, as confirmed by Q-TOF mass spectrometry. Inhibition of the hydrolysis was obtained with EDTA or with specific antibodies directed to each neurotoxin. Different substrate selectivities, especially by BoNT- A and E, suggest that these substrates can be used as a putative method for clostridial toxin quantification and serotype differentiation and could be easily adapted to a high-throughput protocols.

Avaliação da produção de toxina tetânica por Clostridium tetani cultivado por processos fermentativos descontínuo e descontínuo alimentado / Evaluation of the toxin production for Clostridium tetani cultivated by batch fermentative and fed-batch process

A toxina tetânica é uma proteína sintetizada pelo bacilio Clostridium tetani que após destoxificação através da ação do formol, continua apresentando propriedades antigênicas e imunogênicas, obtendo a denominação toxóide tetânico. A síntese dessa proteína ocorre quando esse bacilo encontra-se na sua forma vegetativa e em meio de cultura específico relativamente complexo contendo glicose e peptonas. O efeito simultâneo de diferentes níveis de glicose (Go) e N-Z Case TT® (NZo) como fontes de carbono e nitrogênio, respectivamente, na produção de toxina tetânica foi investigada nesta parte do trabalho em cultivo estático por meio de planejamento fatorial em estrela com cinco níveis e avaliado por metodologia de superfície de resposta, com a finalidade de otimização do processo. O valor mais alto de toxina tetânica encontrado, correspondente a Go=9,7 g/L e NZo = 43,5 g/L, foi 79% maior que aqueles obtidos em condições padrões de cultivo (Go = 8,0 g/L e NZo = 25,0 g/L.Também foram realizados cultivos de C. tetani utilizando o processo descontínuo alimentado com diferentes protocolos para a correção da concentração de glicose no meio de cultivo ao longo do tempo em diferentes concentrações iniciais de N-Z Case TT®. Dois grupos de ensaios foram executados: a) experimentos realizados com a correção da concentração de glicose para 3,0 g/L nos instantes 16, 56 e 88 h e b) experimentos com correção inicial da concentração de glicose para 3,0 g/L e após esta cair para 1-1,5 g/L. O primeiro protocolo de correção da concentração de glicose e NZo = 50,0 g/L foram as melhores condições para obtenção de toxina tetânica Nestas condições, o título de toxina tetânica foi 300% maior que aqueles obtidos em cultivos padrão...

Effect of medium composition on the production of tetanus toxin by Clostridium tetani.

The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G0) and N-Z Case TT (NZ0) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five-level star-shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin (72 Lf/mL), achieved at G0= 9.7 g/L and NZ0= 43.5 g/L, was 80% higher than that obtained with standard cultivations (G0= 8.0 g/L and NZ0= 25.0 g/L).

Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification.

Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two-chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge. The low-molecular-mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin. With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73-82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx. The rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti-recombinant fragment C antibody, and the cleavage was in a single bond (Gln-Phe) with purified and crude TTx. Besides, ELISA applied to the anti-TTx serum produced at our Institute showed cross-reaction with every fraction of the crude TTx extract. Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10. The results obtained suggest that the use of the new fluorescent substrate, Abz-synaptobrevin(73-82)-EDDnp, enables easy and quick determination of TTx. It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose).

Tetanus toxoid: 1. purification in 50 kD molecular weight cut-off membrane

The tetanus anatoxin (M.W.=150,000 dALTONS) is the fundamental component for Tetanus Toxoid (TT) combined vaccines production and must follow the parameters established by World Health Organization (WHO). Among other qualifications the purity degree must be equal or greater than 1,000 Lf/mg PN and the residual formaldehyde must be at maximum level of 0,02 per cent. The Tangential Flow Filtration technique with 50,000 Nominal Molecular Weight Limit (NMWL) membranes and gel filtration in Sephadex G-50 were associated to purify 18 consecutive lots of tetanus anatoxin

Otimizaçäo do processo de purificaçäo industrial de toxóide tetânico por gel filtraçäo / Improvement in the industrial purification of tetanus toxoid by gel filtration

A metodologia industrial para obtençäo de Toxóide Tetânico(T.T.) atual, consiste na pré-precipitaçäo pelo (NH4)2SO4 seguida de gel filtraçäo em Sephadex G-50, com o qual obtém-se grau médio de pureza de 2.300 Lf/mgNP, sendo que o mínimo exigido pela O.M.S. é 1.000Lf/mgNP. Visando basicamente a diminuiçäo do tempo empregado no processo de purificaçäo do T.T., utilizamos resinas cromatográficas de 2§ e 3§ geraçäo, o Sephacryl S-100HR e o Superdex 75 prepgrade, respectivamente, que possibilitaram a eliminaçäo da etapa de pré-precipitaçäo pelo (NH4)2SO4 obtendo-se resultados com grau de pureza ao redor de 3.000Lf/mgNP. A atividade imunogênica destes produtos quando absorvidos pelo Al (OH)3 em concentraçäo de 15Lf/ml foi de 4,5UI/ml em cobaios. A O.M.S. recomenda que o T.T. induza a formaçäo de no mínimo 2UI/ml nestes animais. O rendimento do processo de purificaçäo foi de 88,46//, que é similar ao obtido no processo atual. A metodologia introduzida permitiu a diminuiçäo do tempo empregado neste processamento assim como a possibilidade de trabalhar-se com purificaçäo em sistema fechado (coluna cromatográfica)

Toxina tetânica: produçäo e purificaçäo em escala industrial / Tetanus toxin: production and purification in large scale

A obtençäo de toxina tetânica purificada em grandes volumes é um passo fundamental para a produçäo de Toxóide Tetânico. A utilizaçäo de vapor para a esterilizaçäo do meio de cultura para a produçäo desta toxina apresentava títulos baixos de toxina tetânica. O emprego da filtraçäo convencional teve como consequência índices insatisfatórios de pureza (Lf/mgNP) e de rendimento (porcentagem) do processo. Com o objetivo de encontrar soluçöes para estas etapas do processo produtivo, foi utilizada a esterilizaçäo por cartuchos para o meio de cultura e a filtraçäo tangencial através de um sistema Prostak, associado à ultrafiltraçäo molecular em 30Kd para a toxina tetânica. A nova metodologia trouxe aumentos significativos em pureza (Lf/mgNP) e em rendimento (porcentagem). A tecnologia empregada e a comparaçäo dos resultados obtidos nos diferentes processos säo apresentadas neste trabalho

Estabelecimento da concentracao antimicrobiana eficaz de tiomersal em toxoide tetanico / Determination of effective antimicrobial concentration of thimerosal in tetanus toxoid

Amostras de taxoide tetanico, contendo quatro concentracoes de tiomersal (0,005; 0,010; 0,015 e 0,020%) dentro dos limites recomendados oficialmente,foram submetidos ao teste de eficacia antimicrobiana do conservante. Com relacao as exigencias da Farmacopeia Americana XXII, apenas Staphylococcus aureus se mostrou resistente as quatro concentracoes, enquanto que para Pseudomonas aeruginosa, Candida albicans e Aspergillus niger o sistema conservador atendeu as exigencias oficiais, mesmo na menor concentracao. O teor de tiomersal em toxoide tetanico podera ser de 0,005%

Purificaçäo industrial do toxóide tetânico por gel filtraçäo / Industrial purification of tetanus toxoids by gel filtration

Com o fracionamento pelo sulfato de amônio ou com a precipitaçäo alcoólica, säo obtidos toxóides tetânicos com pureza ao redor de 500 Lf/mg N.P. incompatíveis com as exigências da OMS, de 1000 a 1200 Lf/mg N.P. Foram descritas técnicas eficazes com a utilizaçäo da gel filtraçäo em Sephadex G200, G100 e G75 que, no entanto, pela longa duraçäo do processo, dificultam a produçäo em escala industrial. Introduzimos , assim, uma metodologia baseada na combinaçäo da pré-precipitaçäo pelo sulfato de amônio com a gel filtraçäo em Sephadex G50 com a qual obtivemos toxóides tetânicos com grau médio reprodutivo de pureza ao redor de 2.312,75 Lf/mg N.P. e resposta imunogênica satisfatória

Preparaçäo e teste da toxina botulínica tipo "E" e do seu anti-soro específico / Preparation and test of botulinum toxins type \"E\" and its specific immune sera

Os autores descrevem o método utilizado na preparaçäo do soro antibotulínico tipo E, que näo apresenta reaçäo cruzada com a toxina heteróloga, pela hiperimunizaçäo de equídeos, através do antígeno absorvido pelo alúmen de potássio (sulfato duplo de alumínio e potássio). Empregando o esquema de imunizaçäo anteriormente proposto, conseguiram obter mistura de plasma hiperimunes o qual, após purificado pelo método de Pope, modificado, concentrou até níveis na ordem de 750 a 1.000 UI/ml. Este produto foi diluído convenientemente para que contivesse 500 UI/ml de antitoxina