Purification of meningococcal group C polysaccharide by a procedure suitable for scale-up

Tanizaki, Martha M.; Garcia, Ligiane R.; Baruque-Ramos, Júlia; Leite, Luciana C. C.; Hiss, Haroldo; Furuta, Joana Akiko; Cabrera-Crespo, Joaquín; Raw, Isaı́as

doi:10.1016/0167-7012(96)00921-9

Cited by 21 publications

(20 citation statements)

References 9 publications

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“…In contrast, the 6B best puriWcation step for proteins and nucleic acid were the ethanol precipitation [14]. On the other hand, the Gram negative N. meningitidis behaves similar to the Gram negative H. inXuenzae and the last step of enzymatic treatment is the best step [12].…”

Section: Resultsmentioning

confidence: 99%

“…Our laboratory has developed an improved method for puriWcation of vaccines polysaccharides against Neisseria meningitidis serotype C [12] and Streptococcus pneumoniae serotype 23 and 6B [13,14]. This process reduces considerably the number of ethanol precipitation steps, phenol extraction was replaced by enzymatic treatment and the ultracentrifugation by ultraWltration in the presence of chelating agent and detergent.…”

Section: Introductionmentioning

confidence: 99%

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Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up

Takagi¹,

Lima²,

Albani³

et al. 2008

J Ind Microbiol Biotechnol

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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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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up

Takagi¹,

Lima²,

Albani³

et al. 2008

J Ind Microbiol Biotechnol

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“…[5,6,11] All other reagents of analytical grade were used without prior purification. The 18kDa-hsp and PSC were purified in our laboratory and at the CB I. Butantan as described.…”

Section: Methodsmentioning

confidence: 99%

Heat Shock Protein Micro-Encapsulation as a Double Tool for the Improvement of New Generation Vaccines

Costa¹,

Quintilio²,

Tanizaki³

et al. 2002

Journal of Liposome Research

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The modern vaccinology encompasses the recombinant DNA technology, protein and carbohydrate chemistry to obtain safe molecularly defined vaccines. Nevertheless most of the vaccines are poorly immunogenic because a large number of antigens are membrane proteins and consequently they are not present in their active conformation in the vaccine. Others are not as potent because they contain only B epitopes and therefore, cannot stimulate cellular memory. We have been studying the characteristics of the recombinant heat shock protein 18kDa-hsp from Mycobacterium leprae as an alternative carrier protein with a T epitope source to enhance the activity of these second generation vaccines. Here we proved that the 18kDa-hsp acted as carrier, without masking the activity of the carried antigen, with similar immune stimulatory effect when compared with ODN1668. Supramolecular aggregates of 18kDa-hsp and Mice serum albumin (MSA) were obtained using glutaraldehyde as cross linker. The Neisseria meningitides serogroup C polysaccharide (PSC, a B epitope) and the carrier protein 18kDa-hsp were co-encapsulated within Soybean phosphatidylcholine liposomes (SPC: Cho : alpha-Toc, 22 : 5 : 0.18 molar ratio, respectively). These liposomes were prepared in MPB buffer (20 mM phosphate, 295 mM mannitol pH 7.2) in the presence or absence of the ODN1668, TCCATGACGTTCCTGATGCT. When mice were injected with 18kDa-hsp-MSA no antibody against the MSA was observed. This means that the 18kDa-hsp acted as carrier, without masking the carried protein immune activity. Stable liposomes of 150 nm were obtained using mannitol as a cryoprotector. Genetically selected mice when injected with liposomes containing PSC and 18kDa-hsp displayed an antibody titer of 12. In contrast, in those mice injected with free PSC there was no response. The 18kDa-hsp adjuvant effect on the PSC liposomal formulation was comparable to that observed when ODN1668 was co-encapsulated with PSC. Confirming our expectations we observed that the formulation containing 18kDa-hsp conferred a memory response to the carried antigen--the Neisseria meningitidis serogroup C polysaccharide.

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“…This detergent lysis step breaks down cell membranes, releasing large amounts of intracellular contaminants and causing a negative impact on the purification process similar to that observed in purification 2. Tanizaki et al (1996) eliminated the use of phenol, replacing it by enzymatic treatment and ultrafiltration for the Neisseria meningitides serogroup C capsular PS. This process also proved to be efficient for capsular polysaccharide purification of other microorganisms (Gonçalves et al, 2003;Gonçalves et al, 2007;Takagi et al, 2008).…”

Section: Comparison Of Purification Strategiesmentioning

confidence: 99%

“…In our laboratory, purification processes were developed for purification of capsular polysaccharides of Neisseria meningitidis (Tanizaki et al, 1996), Streptococcus pneumoniae (Gonçalves et al, 2003;Gonçalves et al, 2007) and Haemophilus influenzae type b (Takagi et al, 2008). In these processes, the number of ethanol precipitation steps and centrifugations were reduced and the use of phenol was completely eliminated, being replaced by treatment with endonucleases and proteases, followed by tangential ultrafiltration, wherein the low molecular weight hydrolysate material was removed while PS was retained in the concentrate.…”

Section: Introductionmentioning

confidence: 99%

DEVELOPMENT OF A NEW PROCESS FOR PURIFICATION OF CAPSULAR POLYSACCHARIDE FROM Streptococcus pneumoniae SEROTYPE 14

Zanardo

Ferri

Figueiredo

et al. 2016

Braz. J. Chem. Eng.

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-The main virulence factor of Streptococcus pneumoniae is the capsular polysaccharide (PS), which is the antigen of all current vaccines that are prepared with PS purified from serotypes prevalent in the population. In this work, three purification strategies were evaluated and a new process was developed for purification of serotype 14 PS (PS14), responsible for 39.8% of diseases in children of 0-6 years old in Brazil. The developed method consists of cell separation by tangential microfiltration, concentration of the microfiltrate by tangential ultrafiltration (50 kDa), diafiltration in the presence of sodium dodecyl sulfate using a 30 kDa ultrafiltration membrane, precipitation with 5% trichloroacetic acid, precipitation with 20% and 60% ethanol, and anion exchange chromatography. The required purity regarding nucleic acids (≤ 2%) and proteins (≤ 3%) was achieved, resulting in a relative purity of 439 mg PS14/mg nucleic acids and 146 mg PS14/mg proteins. The final polysaccharide recovery was 65%, which is higher than the recovery of the majority of processes described in the literature.

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Purification of meningococcal group C polysaccharide by a procedure suitable for scale-up

Cited by 21 publications

References 9 publications

Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up

Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up

Heat Shock Protein Micro-Encapsulation as a Double Tool for the Improvement of New Generation Vaccines

DEVELOPMENT OF A NEW PROCESS FOR PURIFICATION OF CAPSULAR POLYSACCHARIDE FROM Streptococcus pneumoniae SEROTYPE 14

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