Joaquin Cabrera-Crespo

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

Abstract Neisseria meningitidis group C capsular polysaccharide is the antigen for the vaccine. An easier method has been developed for the purification of N. meningitidis group C capsular polysaccharide. In this method, two steps of the traditional procedure have been modified: the removal of protein and lipopolysaccharide. The phenol extraction for removal of contaminant protein was substituted by proteinases digestion using three different proteinases: proteinase K, nagarse and trypsin. Tangential ultrafiltration in hollowfiber 100 kDa cutoff was used instead of ultracentrifugation. Extensive diafiltration on a 100 kDa cutoff in Tris-HCl buffer containing 0.5% of deoxycholate was able to eliminate lipopolysaccharide as well as low molecular weight protein. The resultant purified polysaccharide contained around 2% of protein, 1.5% of nucleic acid, and it passed the pyrogen test for lipopolysaccharide in rabbit.

Albumin purification from human placenta

Albumin is the human protein used mainly for therapeutic purposes. Besides the traditionally used plasma, blood from placenta is an alternative source for albumin purification. We describe here an industrial process for purification of albumin from human placenta. The proposed albumin‐purification process, for 50 kg of placentas, comprises: (i) extraction of haemolysed blood with saline and solid/liquid separation by basket centrifugation; (ii) selective precipitation of haemoglobin by ethanol/chloroform and precipitate removal by filtration in a press filter; (iii) concentration/diafiltration of the filtrate in a 30 kDa cross‐flow ultrafiltration (CFUF) membrane; (iv) thermo‐coagulation at 70 °C with sodium octanoate/EDTA; (v) treatment with activated charcoal at pH 3; (vi) concentration/diafiltration of the filtrate in a 30 kDa CFUF membrane; (vii) anion‐exchange chromatography Q‐Sepharose; (viii) hydrophobic‐interaction chromatography with phenyl‐Sepharose; and (ix) conditioning and pasteurization. The process yields an average of 4.5 g of albumin/kg of placenta with a purity of 97.1% and A403 of 0.05 (1% protein). The final product passes pyrogen and toxicity tests in vivo and it does not contain polymers or aggregates, even after the accelerated stability test, as judged by gel filtration, as required by the Brazilian Pharmacopoeia.

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

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.

Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA conformation and its effect on the immune response

ABSTRACT 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.

Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al− eryR was originally cultured in Todd–Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al− eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al− kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al− kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

Characterization of Polysaccharide Production of Haemophilus influenzae Type b and Its Relationship to Bacterial Cell Growth

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 ∼1.8 g of DCW/L, while polysaccharide concentration was about ∼132 mg/L in the three fermentation runs. Polysaccharide synthesis is associated with cell growth in all studied conditions as established by Konos analysis and Luedeking-Pirets model.

Purification of catalase from human placenta

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 catalase was separated from albumin; and (2) catalase purification steps after albumin separation, including a second anionic chromatography step and dye‐affinity chromatography. This method provided a final recovery of 27% (70–100% 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 commercially available human catalase. This process permits the co‐purification of catalase and albumin and can easily be scaled up.

Purification of fibroblast growth factor-2 from human placenta using tri(n-butyl)phosphate and sodium cholate

Tri(n-butyl)phosphate (TNBP) and sodium cholate (SC) mixtures have been used to inactivate lipid-enveloped viruses like HIV and hepatitis B. We exploited the use of this combination to purify fibroblast growth factor-2 (FGF-2) from human placenta. Human placentas were extracted in the presence of 0.3% TNBP/0.2% SC and the clarified homogenate was adsorbed to S-Sepharose. The active fractions were further loaded onto a heparin-Sepharose column and purified FGF-2 was eluted with 2.0 M NaCl. FGF-2 purified this way was indistinguishable from FGF-2 purified without TNBP/SC in the extraction step in terms of yield, specific activity and biological response. The lipid-enveloped vaccinia virus was used in a parallel experiment to evaluate the inactivation capacity of our protocol. Under the conditions described here, the combined use of TNBP/SC did not eliminate but reduced significantly the number of vaccinia virus PFUs by log 2-3.

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.

Purification of porcine plasma factor VIII using chromatographic methods

Factor VIII was purified from porcine plasma using adsorption on aluminium hydroxide with CM-cellulose as a filtration aid, cold ethanol precipitation, and two anion-exchange (Q-Sepharose fast flow) chromatographies. The final product was purified 264-fold and had a specific activity of 10 U mg−1. The method is suitable to produce purified porcine FVIII by an easy process where all steps can be scaled up. The final product is free of von Willebrand factor that is responsible for the main side effects in patients. Finally, this method can be used to obtain purified porcine plasma FVIII for use in haemophilic patients with inhibitors.