RESUMEN
In recent years, many biological-based products have been developed, representing a significant fraction of income in the pharmaceutical market. Ion exchange chromatography is an important downstream step for the purification of target recombinant proteins present in clarified cell extracts, together with many other unknown impurities. This work develops a robust approach to model and simulate the purification of untagged heterologous proteins, so that the improved conditions to carry out an ion exchange chromatography are identified in a rational basis prior to the real purification run itself. Purification of the pneumococcal surface protein A (PspA4Pro) was used as a case study. This protein is produced by recombinant Escherichia coli and is a candidate for the manufacture of improved pneumococcal vaccines. The developed method combined experimental and computational procedures. Different anion exchange operating conditions were mapped in order to gather a broad range of representative experimental data. The equilibrium dispersive and the steric mass action equations were used to model and simulate the process. A training strategy to fit the model and separately describe the elution profiles of PspA4Pro and other proteins of the cell extract was applied. Based on the simulation results, a reduced ionic strength was applied for PspA4Pro elution, leading to increases of 14.9% and 11.5% for PspA4Pro recovery and purity, respectively, compared to the original elution profile. These results showed the potential of this method, which could be further applied to improve the performance of ion exchange chromatography in the purification of other target proteins under real process conditions.
RESUMEN
The use of conjugate vaccines remains an effective intervention to prevent pneumococcal diseases. In order to expand vaccine coverage, the inclusion of pneumococcal proteins as carriers is a propitious alternative that has been explored over the past few years. In this study, pneumococcal surface protein A (PspA) clade 1, family 1 (PspA1) and clade 3, family 2 (PspA3) were used as carrier proteins for pneumococcal capsular polysaccharide serotype 6B (Ps6B). Employing an improved reductive amination chemistry, 50% of Ps6B was incorporated to each protein, PspA1 and PspA3. The effect of chemical modifications in Ps6B and PspA was assessed by an antigenicity assay and circular dichroism, respectively. Fragmentation and oxidation decreased the antigenicity of Ps6B while conjugation improved antigenicity. In the same manner, introduction of adipic acid dihydrazide (ADH) reduced PspA secondary structure content, which was partially restored after conjugation. Immunization of Ps6B-PspA1 and Ps6B-PspA3 conjugates in mice induced specific IgG antibodies against the Ps6B and the protein; and anti-PspA antibodies had functional activity against two pneumococcal strains with different serotypes. These results suggest that chemical coupling between Ps6B and PspA did not affect antigenic epitopes and support the further development of PspA as a carrier protein in pneumococcal conjugate vaccines to provide broader protection.
RESUMEN
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.
RESUMEN
Ion exchange chromatography is extensively used in the purification of biological compounds. Reliable mathematical models describing this chromatographic technique are available and can be used to improve the performance of this separation step. However, the use of synthetic mixtures for model development hampers the application of this approach with real cell extracts processed in downstream operations. This work presents an original approach for handling non-synthetic genuine mixtures of proteins, which was applied in the purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro). First, evaluation was made of the efficiency of steric mass action (SMA) and modified Langmuir isotherms, which were separately used together with the equilibrium dispersive model (EDM). The data used for parameter estimation and model validation were obtained from anion exchange chromatography runs (employing Q-Sepharose FF), applied to real cell extracts produced by different cultivation strategies. Simulations showed that the models were able to describe the complex mixtures of unknown proteins. Next, the EDM and SMA approaches were used to separately describe the profile of PspA4Pro and the pool of protein impurities eluted together. The simulations showed that PspA4Pro tended to elute at the beginning of the peak, enabling the establishment of an alternative elution schedule that provided a 34% increase in the purity achieved using the anion exchange chromatography.
RESUMEN
Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 °C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 ± 2.5% of PspA4Pro with 97.8 ± 0.36% purity and reduced endotoxin concentration by >99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/genética , Extracción Líquido-Líquido/métodos , Streptococcus pneumoniae/química , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Clonación Molecular , Detergentes/química , Endotoxinas/aislamiento & purificación , Escherichia coli/química , Escherichia coli/metabolismo , Fermentación , Expresión Génica , Glicerol/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Lactoferrina/química , Lactosa/metabolismo , Presión , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Streptococcus pneumoniae/metabolismoRESUMEN
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.
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Haemophilus influenzae tipo b , Polisacáridos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/análisis , Reactores Biológicos , Precipitación Química , Filtración , Haemophilus influenzae tipo b/metabolismo , Ácidos Nucleicos/análisis , Fósforo/análisis , Polisacáridos Bacterianos/metabolismoRESUMEN
Despite the substantial beneficial effects of incorporating the 7-valent pneumococcal conjugate vaccine (PCV7) into immunization programs, serotype replacement has been observed after its widespread use. As there are many serotypes currently documented, the use of a conjugate vaccine relying on protective pneumococcal proteins as active carriers is a promising alternative to expand PCV coverage. In this study, capsular polysaccharide serotype 6B (PS6B) and recombinant pneumococcal surface protein A (rPspA), a well-known protective antigen from Streptococcus pneumoniae, were covalently attached by two conjugation methods. The conjugation methodology developed by our laboratory, employing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as an activating agent through carboxamide formation, was compared with reductive amination, a classical methodology. DMT-MM-mediated conjugation was shown to be more efficient in coupling PS6B to rPspA clade 1 (rPspA1): 55.0% of PS6B was in the conjugate fraction, whereas 24% was observed in the conjugate fraction with reductive amination. The influence of the conjugation process on the rPspA1 structure was assessed by circular dichroism. According to our results, both conjugation processes reduced the alpha-helical content of rPspA; reduction was more pronounced when the reaction between the polysaccharide capsule and rPspA1 was promoted between the carboxyl groups than the amine groups (46% and 13%, respectively). Regarding the immune response, both conjugates induced functional anti-rPspA1 and anti-PS6B antibodies. These results suggest that the secondary structure of PspA1, as well as its reactive groups (amine or carboxyl) involved in the linkage to PS6B, may not play an important role in eliciting a protective immune response to the antigens.
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Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/inmunología , Vacunas Neumococicas/inmunología , Polisacáridos Bacterianos/inmunología , Streptococcus pneumoniae/inmunología , Animales , Proteínas Bacterianas/química , Dicroismo Circular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Vacunas Neumococicas/química , Polisacáridos Bacterianos/química , Conformación Proteica , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunología , Vacunas Sintéticas/química , Vacunas Sintéticas/inmunologíaRESUMEN
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.
Asunto(s)
Cápsulas Bacterianas/aislamiento & purificación , Haemophilus influenzae tipo b/metabolismo , Ultrafiltración/métodos , Cápsulas Bacterianas/biosíntesis , Glucosa/farmacología , Haemophilus influenzae tipo b/efectos de los fármacos , Haemophilus influenzae tipo b/crecimiento & desarrolloRESUMEN
Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.
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Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Cromatografía/métodos , Escherichia coli/crecimiento & desarrollo , Proteínas Bacterianas/genética , Biotecnología/métodos , Medios de Cultivo/química , Escherichia coli/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/genéticaRESUMEN
New conjugated vaccines against Streptococcus pneumoniae are being developed using pneumococcal surfaceproteins as carriers. The pneumococcal surface protein A (PspA) was selected as carrier because it is indispensablefor virulence of S. pneumoniae. The PspA can be classified into 3 families according to the homology of proteinsequences, within each family there is immunological cross-reactivity and PspA from family 1 or 2 are present in99% of strains associated with pneumococcal invasive disease. Hence, the purpose of this work was to develop an industrial production and purification process of His-tagged recombinant fragment of PspA in E. coli BL21 (DE3),rfPspA245 from family 1. Fed-batch cultivations in 5-L bioreactors with defined medium were carried out using glycerol as carbon source. Itwas obtained circa 60 g/L of dry cell weight and 3.0 g/L of rfPspA. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. The clarification step was done by centrifugation. The results ofchromatographic steps were analyzed by densitometry of SDS-PAGE protein bands. Using the chromatographicsequence anion exchange (Q-Sepharose) followed by metal affinity (IMAC-Sepharose), the rfPspA245 was obtained with 67% and 97% of purity respectively for each step and final recovery of 23%. In conclusion, the purification process was developed and rfPspA245 was obtained with high purity, but the recovery should still be improved.
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Biomasa , Producción de Productos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/aislamiento & purificación , Vacunas Estreptocócicas/aislamiento & purificación , Vacunas Estreptocócicas/uso terapéutico , Cromatografía de Afinidad/métodos , Cromatografía/métodosRESUMEN
Haemophilus influenzae type b, an encapsulated bacterium, causes meningitis in infants worldwide. The capsular polysaccharide conjugated to a carrier protein is effective in the prevention of such infections. The traditional purification process of polysaccharide from bacterial cultures for vaccine production is based on several selective precipitations with solvents such as: ethanol, phenol, and cationic detergents. The separations of solid and liquid phases are based on continuous centrifugation in explosion proof installations. The lipopolysaccharides are separated by ultracentrifugation. A simple and efficient method that can easily be scaled-up was developed for purification of polysaccharides. The ethanol precipitation was reduced to only two steps. The phenol treatment was substituted by ultrafiltration and enzymatic digestion. Lipopolysaccharide was removed by ultrafiltration together with addition of detergent and chelating agent.
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Centrifugación/métodos , Haemophilus influenzae tipo b/química , Polisacáridos Bacterianos/aislamiento & purificación , Ultrafiltración/métodos , Reactores Biológicos/microbiología , Precipitación Química , Etanol/química , Haemophilus influenzae tipo b/metabolismo , Lipopolisacáridos/química , Lipopolisacáridos/aislamiento & purificación , Polisacáridos Bacterianos/químicaRESUMEN
Streptococcus pneumoniae is the agent responsible for infections such as pneumonia, otitis media, and meningitis. Among virulence factors, the Pneumococcal surface protein A (PspA) has been shown to be immunogenic and protective in mice, and is thus a good vaccine candidate. PspA has been classified into 6 clades and 3 families. Initially, pspA fragments, clades 1 and 3, were cloned into the pAE-6His expression vector. Proteins were expressed in Escherichia coli BL21(DE3) and purified by affinity and anion exchange chromatographies, with a yield of 11 mg/l of culture. Due to plasmid instability in E. coli, another construct using pspA1 was obtained based on pET-37b(+), which was shown to be stable in E. coli and increased the yield approximately 3-fold. Our results show good conditions for scale-up. Sera from immunized mice recognized PspA in total extracts of S. pneumoniae strains: anti-rPspA1p sera recognized native PspA clades 1 (+++), 2 (++) and 4 (+) and anti-rPspA3p sera recognized PspA clades 1 (+), 2 (+), 3 (+++) and 4 (+). The cross-reactivity pattern obtained confirms the notion that proteins from both families should be included for development of a broad-coverage vaccine; lower-cross reactivity between rPspAs of family 2 indicates that it may be necessary to include 2 proteins from this family.
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Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/metabolismo , Animales , Proteínas Bacterianas/genética , Reacciones Cruzadas/inmunología , Expresión Génica , Vectores Genéticos/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.
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Enterotoxinas/biosíntesis , Escherichia coli/metabolismo , Animales , Enterotoxinas/genética , Enterotoxinas/metabolismo , Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Calor , Humanos , ConejosRESUMEN
Haemophilus influenzae type b (Hib) causes invasive infections in infants and young children. Vaccines consisting of Hib capsular polysaccharide (polymer of ribosylribitol phosphate [PRP]) conjugated to a protein are effective in the prevention of such infections. The production of capsular polysaccharide type b was studied in three cultivation conditions: single, glucose pulse, and repeated batch. Specific polysaccharide production (Yp/x) was calculated for all experiments, showing the following values: 67 (single-batch cultivation), 71 (glucose pulse), 75 (repeated-batch cultivation, first batch), and 87 mg of PRP/g of dry cell weight (DCW) (repeated-batch cultivation, second batch). Biomass concentration reached approximately 1.8 g of DCW/L, while polysaccharide concentration was about approximately 132 mg/L in the three fermentation runs. Polysaccharide synthesis is associated with cell growth in all studied conditions as established by Kono's analysis and Luedeking-Piret's model.
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Infecciones por Haemophilus/metabolismo , Vacunas contra Haemophilus/biosíntesis , Haemophilus influenzae tipo b/metabolismo , Polisacáridos Bacterianos/biosíntesis , Cápsulas Bacterianas , Técnicas Bacteriológicas , Biomasa , Reactores Biológicos , División Celular , Fermentación , Glucosa/metabolismo , Haemophilus influenzae tipo b/crecimiento & desarrollo , Oxígeno/metabolismoRESUMEN
The therapeutic use of an antioxidant complex containing superoxide dismutase and catalase has been proposed for the treatment of several diseases in which reactive oxygen species have an important role. Although superoxide dismutase for human use is commercially available, methods for the production of catalase for human use have not been described. An industrial process was developed for the purification of catalase for human use as a by-product of albumin production from human placenta, comprising two parts: (1) albumin and catalase co-purification steps, including blood extraction from ground placentas, precipitation of haemoglobin with ethanol/chloroform, concentration/diafiltration by tangential filtration and anionic chromatography, by which non-adsorbed catalasewas separated from albumin; and (2) catalase purification steps after albumin separation, including a second anionic chromatography step and dye-affinitychromatography. This method provided a final recovery of 27% (70100% in each step) with 670-fold purification of catalase (85%pure) and a specific activity of 49000 units/mg, which is higher than that of commerciallyavailable human catalase. This processpermits the co-purification of catalase and albumin and can easily be scaled up.