Scale-Up of Capsular Polysaccharide Production Process by Haemophilus influenzae Type b Using kLa Criterion.

Polyribosyl-ribitol-phosphate (PRP) from Haemophilus influenzae type b (Hib) is an active immunizing molecule used in the production of the vaccine against H. influenzae, and industrial production could contribute to satisfying a world demand especially in developing countries. In this sense, the aim of this study was to establish a scale-up process using the constant oxygen mass transfer coefficient (kLa) such as the criterion for production of PRP in three different sizes of bioreactor systems. Three different kLa values (24, 52 and 80 h-1) were evaluated in which the biological influence in a 1.5 L bioreactor and 52 h-1 was selected to scale-up the production process until a 75 L pilot-scale bioreactor was achieved. Finally, the fed-batch phase was started under a dissolved oxygen concentration (pO2) at 30% of the saturation in the 75 L bioreactor to avoid oxygen limitation; the performance of production presented high efficiency (9.0 g/L DCW-dry cell weight and 1.4 g/L PRP) in comparison with previous scale-up studies. The yields, productivity and kinetic behavior were similar in the three-size bioreactor systems in the batch mode indicating that kLa is possible to use for PRP production at large scales. This process operated under two stages and successfully produced DCW and PRP in the pilot scale and could be beneficial for future bioprocess operations that may lead to higher production and less operative cost.

Scale-up of capsular polysaccharide production process by Haemophilus influenzae type b using kLa criterion

Polyribosyl-ribitol-phosphate (PRP) from Haemophilus influenzae type b (Hib) is an active immunizing molecule used in the production of the vaccine against H. influenzae, and industrial production could contribute to satisfying a world demand especially in developing countries. In this sense, the aim of this study was to establish a scale-up process using the constant oxygen mass transfer coefficient (kLa) such as the criterion for production of PRP in three different sizes of bioreactor systems. Three different kLa values (24, 52 and 80 h−1) were evaluated in which the biological influence in a 1.5 L bioreactor and 52 h−1 was selected to scale-up the production process until a 75 L pilot-scale bioreactor was achieved. Finally, the fed-batch phase was started under a dissolved oxygen concentration (pO2) at 30% of the saturation in the 75 L bioreactor to avoid oxygen limitation; the performance of production presented high efficiency (9.0 g/L DCW-dry cell weight and 1.4 g/L PRP) in comparison with previous scale-up studies. The yields, productivity and kinetic behavior were similar in the three-size bioreactor systems in the batch mode indicating that kLa is possible to use for PRP production at large scales. This process operated under two stages and successfully produced DCW and PRP in the pilot scale and could be beneficial for future bioprocess operations that may lead to higher production and less operative cost.

Cell separation of Haemophilus influenzae type b through tangential microfiltration

Centrifugation techniques are frequently used to separate bacteria from the culture broth in pharmaceutical industries. Alternatively, cell separation can be performed through tangential microfiltration systems; an arguably cost-effective alternative to centrifugation. Therefore, replacement of centrifugation steps with microfiltration represents an attractive option in order to decrease production costs. An example of such use can be found in the production of the vaccine against Haemophilus influenzae type b (Hib), which relies on a centrifugation step to separate the pathogenic bacteria from the antigen-rich (exopolysaccharide) released into culture broth. The substitution of the centrifugation operation with tangential microfiltration may decrease production costs and increase vaccine availability in low-income regions. Hence, we studied the impact of diverse microfiltration systems at different production scales in the separation of Hib from its culture broth. The recovery of the exopolysaccharide - polyribosylribitol phosphate (PRP), the antigen employed in the vaccine, produced by Hib and present in the culture broth was used as a read-out for process efficiency. In sum, the use of Hydrophilic Polyvinylidene Fluoride (PVDF) membranes resulted in the highest recovery value among the tested materials; moreover, the transmembrane pressure was a paramount factor determining the recovery level. We concluded that Hib cell separation through tangential microfiltration systems represents a feasible alternative to centrifugation.

Evaluation of capsular Polysaccharide production by Haemophilus Influenzae type B in different culture media

Haemophilus influenzae b (Hib), an encapsulated Gram-negative coccobacillus, is one of the most common agents of meningitis worldwide. The Hib vaccine is included in the routine immunization schedule of several countries. The capsular polysaccharide, Poliribosyl-Ribitol-Phosphate (PRP), conjugated to a carrier protein is the antigen of the vaccine against Hib. Currently in industrial processes, Hib is cultivated in a soy peptone and yeast-extract based complex medium (MMP). This work aimed to evaluate whether a chemically defined medium could replace the MMP medium in the PRP production. The use of a defined media could offer several advantages in the whole process and would be of great interest to the industrial PRP production in vaccination programs. The PRP production ranged from 100 to 290 mg L-1 in the studied semi-defined and defined media. The formulation of several chemically defined media based on literature led to a promising composition as is the case of medium G which presented a production of 295 mg PRPL-1 with a lower biomass value of 4.8 UA (O.D.540 nm) when compared to MMP medium with 395 mg PRPL-1 and 6.45 UA respectively.

Kinectic Models of Exopolysaccharide Production by Haemophilus Influenzae Type B

Haemophilus influenzae type b (Hib) is a pathogenic bacterium responsible for meningitis in infants and elderly. The polysaccharide present in the extracellular layer of the bacterium is an efficient antigen for vaccination when conjugated to a carrier protein. Immunization against Hib is encouraged to be extended in the developing countries, which sets a high demand on the production of the polysaccharide. The fermentation of Hib is unable to achieve high cell densities, probably due to accumulation of toxic compounds in the broth, decreasing greatly the polysaccharide production yields. In this work, a set of mathematical models were fitted to the experimental data of Hib growth in order to evaluate the kinetics of production of biomass, polysaccharide and acids as byproducts. The best model was chosen by model comparison and allowed us to conclude that polysaccharide formation is exclusively non-associated to growth but inhibited by acid, while acid formation follows a mixed associated and non-associated pattern. These results suggest the model 5.D as mathematical model to predict the fermentation kinetics process of H. influenzae.

Instability and depolymerization of the exopolysaccharide produced by Haemophilus Influenzae type B during fermentation

Haemophilus influenzae type b (Hib) is a pathogenic bacterium and the major cause of sequalae and deaths among infants due to meningitis. Hib vaccine is constituted of the exopolysaccharide from the cell capsule and conjugated to a carrier protein. The production of the polysaccharide is complicated due to low yields of production and product recovery in the downstream. In this work, the profiles of molecular mass throughout fermentation were investigated. The molecular mass decreased along fermentation time, despite the increase in concentration. The mechanisms of hydrolysis were investigated, with three possibilities considered: alkaline hydrolysis caused by the solution used to maintain pH of the fermentation; presence of hydrolytic activity from the fermentation metabolites and spontaneous hydrolysisat the temperature and pH was evaluated in the conditions of the fermentation and with purified polysaccharide. The results have suggested that there is not significant influence of the strength of the alkali solution used for pH control neither the presence of hydrolases in the supernatant; however spontaneous hydrolysis was verified in a temperature dependent manner and confirmed with purified polysaccharide.

Purification of a phosphated biopolymer by selective ethanol precipitation in presence of surfactant and sodium acetate

Bacteria have the ability to produce biopolymers with different chemical properties, for different purposes and vary according to the bacterial strains and their physiological status, and these can be used as vaccine antigens. Haemophilus influenzae type b is a microorganism pathogenic to humans, which causes several types of infections. It is classified into six serotypes, the biopolymer of serotype b (Hib) being the most virulent, known as Poly Ribosylribitolphosphate (PRP). The aim of this work was to evaluate different candidate surfactants to be used in the PRP purification step, as well as the effects of ethanol in combination with sodium acetate. From all the surfactants used, 0.5% SDS proved to be potent in eliminating protein impurities and nucleic acids and in accordance with criteria of regulatory agencies. Regarding the combination of ethanol and sodium acetate to precipitate impurities, in the first fractionation step and polysaccharide, in the second fractionation step; the best conditions were: 40% ethanol without sodium acetate in the first stage and 60% ethanol containing 7% sodium acetate in the second stage. This improved condition resulted in nearly 100% polysaccharide recovery with relative purities higher than 100 for both protein and nucleic acid. In the traditional PRP purification process the final polysaccharide recovery was around 20% at the end of the process, while the new condition will result in at least 80% and within the purity criteria established by WHO for this polysaccharide.

Study of the polyribosyl-ribitol-phosphate precipitation mechanism by salts and organic solvents.

Precipitation has been widely applied to purification and fractionation of biological macromolecules. Several physical-chemical factors contribute to the destabilization of those solutions, such as the nature of solvent employed, presence of salts, temperature, and concentration of the macromolecule. In the case of charged biopolymers, electrostatic forces are the major contributors to their stability in solution. However, the role of each variable and the exact mechanism of precipitation are not completely understood yet. The aim of this work was to study the precipitation of polyribosyl-ribitol-phosphate (PRP, a linear homogeneous anionic biopolymer) in presence of salts and non-solvents, in order to contribute to the elucidation of its precipitation mechanism. The solvents tested (acetone, ethanol, and isopropanol) presented distinct dielectric constants. The salts used (NH4Cl, NaCl, KCl, MgCl2, and CaCl2) differ by their cations. For each salt concentration, the solvent fraction that induces precipitation was identified and the dielectric constant of the bulk solution was calculated. Precipitation always occurred at well-defined combinations of solvents and salts. At low concentration of monovalent salts, there was a linear correlation between the logarithm of the salt concentration and the inverse of the medium dielectric constant at a defined precipitation point. This is a strong indication that the stability of the solution depends almost exclusively on the balance of electrostatic forces. This behavior is compatible with the DLVO modeling of colloidal systems. When divalent salts were used, low concentrations of the counterion were sufficient to induce precipitation, due to a phenomenon called ionic condensation. Apparently, PRP precipitates when around 90% of its charges are neutralized, value that is similar to charge neutralization for DNA precipitation.

Study of the polyribosyl-ribitol-phosphate precipitation mechanism by salts and organic solvents

Precipitation has been widely applied to purification and fractionation of biological macromolecules. Several physical-chemical factors contribute to the destabilization of those solutions, such as the nature of solvent employed, presence of salts, temperature, and concentration of the macromolecule. In the case of charged biopolymers, electrostatic forces are the major contributors to their stability in solution. However, the role of each variable and the exact mechanism of precipitation are not completely understood yet. The aim of this work was to study the precipitation of polyribosyl-ribitol-phosphate (PRP, a linear homogeneous anionic biopolymer) in presence of salts and non-solvents, in order to contribute to the elucidation of its precipitation mechanism. The solvents tested (acetone, ethanol, and isopropanol) presented distinct dielectric constants. The salts used (NH4Cl, NaCl, KCl, MgCl2, and CaCl2) differ by their cations. For each salt concentration, the solvent fraction that induces precipitation was identified and the dielectric constant of the bulk solution was calculated. Precipitation always occurred at well-defined combinations of solvents and salts. At low concentration of monovalent salts, there was a linear correlation between the logarithm of the salt concentration and the inverse of the medium dielectric constant at a defined precipitation point. This is a strong indication that the stability of the solution depends almost exclusively on the balance of electrostatic forces. This behavior is compatible with the DLVO modeling of colloidal systems. When divalent salts were used, low concentrations of the counterion were sufficient to induce precipitation, due to a phenomenon called ionic condensation. Apparently, PRP precipitates when around 90% of its charges are neutralized, value that is similar to charge neutralization for DNA precipitation.

Study of the polyribosyl-ribitol-phosphate precipitation mechanism by salts and organic solvents

Precipitation has been widely applied to purification and fractionation of biological macromolecules. Several physical-chemical factors contribute to the destabilization of those solutions, such as the nature of solvent employed, presence of salts, temperature, and concentration of the macromolecule. In the case of charged biopolymers, electrostatic forces are the major contributors to their stability in solution. However, the role of each variable and the exact mechanism of precipitation are not completely understood yet. The aim of this work was to study the precipitation of polyribosyl-ribitol-phosphate (PRP, a linear homogeneous anionic biopolymer) in presence of salts and non-solvents, in order to contribute to the elucidation of its precipitation mechanism. The solvents tested (acetone, ethanol, and isopropanol) presented distinct dielectric constants. The salts used (NH4Cl, NaCl, KCl, MgCl2, and CaCl2) differ by their cations. For each salt concentration, the solvent fraction that induces precipitation was identified and the dielectric constant of the bulk solution was calculated. Precipitation always occurred at well-defined combinations of solvents and salts. At low concentration of monovalent salts, there was a linear correlation between the logarithm of the salt concentration and the inverse of the medium dielectric constant at a defined precipitation point. This is a strong indication that the stability of the solution depends almost exclusively on the balance of electrostatic forces. This behavior is compatible with the DLVO modeling of colloidal systems. When divalent salts were used, low concentrations of the counterion were sufficient to induce precipitation, due to a phenomenon called ionic condensation. Apparently, PRP precipitates when around 90% of its charges are neutralized, value that is similar to charge neutralization for DNA precipitation.

Desenvolvimento de um processo de extração da L-asparaginase II, do Periplasma, por choque osmótico

A Leucemia Linfoide Aguda (LLA) é uma das leucemias mais importantes e, segundo o INCA, atingiu nos últimos anos a margem de 80% entre crianças menores de cinco anos. Sendo causa multifatorial, a prevenção não é tão simples. Assim, a L-Asparaginase II vem recebendo destaque mundial como uma estratégia no tratamento desta patologia. A enzima possui afinidade pela asparagina, hidrolisando-a e indisponibilizando para as células neoplásicas que necessitam como fonte de nutriente; e sem este, as células entram em remissão e morrem. Contudo, o Brasil ainda não possui produção nacional deste biofármaco, o que resulta em maior custo e risco de insuficiência na distribuição. Existem estudos em andamento, no entanto a produção desta enzima é complexa, envolvendo processos desde clonagem e cultivo do microrganismo, onde será produzida a enzima, até a extração e posterior purificação. Há três métodos mais utilizados de extração: sonicação e pérola de vidro, que causam a lise total da célula, gerando mais impurezas, e o choque osmótico que por uma perturbação na osmolaridade celular libera a enzima do periplasma, tornando o processo mais vantajoso, facilitando a purificação. O objetivo deste estudo foi estabelecer e padronizar condições ideais da extração propondo uma estratégia de escalonamento com robustez. Foi realizado ensaios para definir concentração de soluções I e II; avaliar a concentração celular; avaliar a relação de volume de solução/concentração celular e uma estratégia de escalonamento. A melhor condição das soluções foi: Tris-HCl 200 mM; Sacarose 20%; EDTA 5 mM (Sol I) e Água (Sol II). Verificou-se que a atividade enzimática aumenta em função da DO600, até certo ponto, com volume das soluções constante. Para melhor rendimento, foi necessário encontrar uma proporção entre concentração celular e o volume de solução. O planejamento fatorial realizado, com este propósito, apresentou inconsistências, estatisticamente, devido ao método de Nessler ainda estar em definição. No ensaio de escalonamento verificou-se escape a enzima no sobrenadante I, resultando em baixo rendimento. Conclui-se que é necessário um estudo mais detalhado para definir a proporção de soluções X DO600; bem como uma investigação cautelosa do escape enzimático precoce.

Selection of a mathematical model for the kinetics of Haemophilus influenzae type b using akaike's in information criterion

Haemophilus influenzae type b (Hib) vaccine is made up from its capsular polysaccharide (PRP). Low productivity of the polysaccharide during cell growth increases the final cost of this vaccine. Hib achieves low levels of cellular concentration in vitro due to the inhibition caused by acetate. The Akaike Information Criterion (AIC) was used in this work for selecting models of microbial growth. The application to the case of the multivariate models is outlined and the procedure is carried out using data from Hib cultures. From 4 models of biomass and 15 of acetate and PRP, one could be selected with great evidence for support. The use of AIC has shown to be robust and free of subjectivity, and it was able to define a kinetic model that is adequate for the cell growth and the production of its PRP over a wide range of culture conditions. The exponential inhibition factor was found to be the best for modelling inhibition of cell growth by acetate, while the hyperbolic factor was the best for inhibition of PRP formation. The acetate formation was found to have both growth associated and non associated types. PRP formation was found to be only growth associated.

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.

Study of the chemical stability of the capsular polysaccharide produced by Haemophilus influenzae type b.

Haemophilus influenzae type b (Hib) is a human pathogen that causes severe infections such as pneumonia, sepsis and meningitis. Vaccines for Hib infections are based on its capsular polysaccharide conjugated to a protein. This conjugated Hib antigen is included as one of the components of polyvalent vaccines and accounts for more than 50% of the total cost of the formulations. The instability of the polysaccharide is responsible for the high cost of the vaccine. In this study, the factors affecting the spontaneous degradation of the polysaccharide from Hib were evaluated based on the decrease in its molecular mass, as measured by size-exclusion chromatography. Temperature and pH were found to be the most significant variables, and the results showed that the conditions of bacterial cell growth (37 °C and pH 7.5) are favourable for depolymerization. An increase in the concentration of sodium ions up to 200 mM intensified the effect of pH, allowing higher rates of depolymerization at lower pH values, whereas the presence of magnesium ions showed no effects.

Improvement in the purification process of the capsular polysaccharide from Haemophilus influenzae type b by using tangential ultrafiltration and diafiltration.

Capsular polysaccharide produced by Haemophilus influenzae b (Hib) is the main virulent agent and used as the antigen in the vaccine formulation. In this study, an improved process of polysaccharide purification was established based on tangential flow ultrafiltration using detergents (cocamidopropyl betaine and sodium deoxycholate), two selective ethanol precipitations steps, and extensive enzymatic hydrolysis as strategy. The relative purity (RP) related to protein and nucleic acids were 122~263 and 294~480, respectively, and compatible with the specifications established by the World Health Organization for Hib vaccine, RP≥100. These results make this process simple, cheaper, efficient, environmentally friendly, and prone to be scaled up.