Vaccine. 2013 Oct 1;31(42):4714-9.

A novel method for purification of Vi capsular polysaccharide produced by Salmonella enterica subspecies enterica serovar Typhi.

Sudeep Kothari, Neha Kothari, Jeong-Ah Kim, Eugene Lee, Yeon Kyung Yoon, So Jung An, Christopher Jones, Woo Seok Choe, Rodney Carbis.

Vaccine Development Section, International Vaccine Institute, SNU Research Park, San 4-8, Nakseongdae-dong, Gwanak-gu 151-919, Seoul, Republic of Korea; School of Chemical Engineering, Sungkyungkwan University, Suwon 440-746, Republic of Korea.

 

ABSTRACT

Vi capsular polysaccharide is the major component of Vi polysaccharide typhoid vaccines. Vi is synthesized during growth of Salmonella enterica subspecies enterica serovar Typhi and is released into the fermentation broth in large quantities. Along with the Vi considerable amounts of impurities consisting of bacterial protein, nucleic acid and lipopolysaccharide (LPS) as well as media components contaminate the fermentation broth. A purification method based on selective precipitation of Vi using the cationic detergent cetavlon was developed to separate impurities from Vi. A novel method for handling the Vi precipitate using 0.2 µm sterilizing grade filters to trap and wash the Vi and then, after re-solubilization, allow the Vi to pass through the filter was developed. Cetavlon selectively precipitates Vi and is the major purification step in the process, however, the conditions must be carefully controlled otherwise LPS will co-precipitate in large quantities. Various diafiltration steps help to remove contaminating protein, nucleic acid and fermentation media components as well as chemicals added during the process to induce precipitation of either Vi or contaminants. The final yield of purified Vi was approximately 45% and the bulk concentrate complied with the specifications defined in the WHO recommendations for Vi polysaccharide vaccine. Analysis of the Vi by size exclusion chromatography revealed a uniform peak with a narrow size distribution. The Nuclear Magnetic Resonance spectrum was similar to Vi produced by other methods. The method developed produces large quantities of Vi using low cost production methods translating into Vi based vaccines that can be produced at affordable prices for use in developing countries. Copyright © 2013 Elsevier Ltd.

KEYWORDS: Polysaccharide purification, Salmonella Typhi, Typhoid vaccine, Vi polysaccharide

PMID: 23994374

 

Supplement:

Typhoid fever caused by Salmonella Typhi continues to cause a considerable disease burden particularly in developing countries where sanitation is poor and good quality drinking water is not always available.  Improvements in sanitation and drinking water are a distant goal for many resource poor impoverished countries.  The best option to control typhoid fever is to vaccinate populations most at risk from infection with S. Typhi.

Funding for vaccination programs is limited and priority is given to vaccines delivered in routine EPI (Expanded Program of Immunization) programs and vaccines such as typhoid and cholera receive little funding.  As a result of this limited funding it is essential that vaccines be as low cost as possible so that as many at risk people as possible can benefit from being vaccinated.  Because of the low selling prices of vaccines targeted at diseases of developing countries large pharmaceutical companies are less interested in these low margin vaccines.  In order to address the cost and supply problems the International Vaccine Institute has been working on providing high yielding vaccine production processes to developing country vaccine manufacturers that are willing to provide high quality vaccines to their communities at affordable prices.

One such program is the development of a typhoid conjugate vaccine [1].  The protective antigen is the capsular polysaccharide known as Vi which S. Typhi secretes and surrounds itself with to evade the host defense mechanisms.  Antibody to the Vi protects humans from typhoid fever by facilitating neutralization of S. Typhi, and vaccines containing Vi induce anti-Vi protective responses.  To make a Vi based vaccine production and purification of Vi must first be developed.  Old methods used dangerous chemicals such as phenol to extract the Vi and also used expensive equipment, in order to simplify manufacture and keep the cost down we eliminated the use of phenol and minimized the use of capital equipment.

The first step was to maximize the yield of Vi produced by the bacterium, this was done by optimizing the growing conditions for S. Typhi and also to provide the right concentration of nutrients for Vi production [2].  Next the Vi had to be purified involving removal of potentially harmful contaminants.  The process that was developed involved a number of selective precipitation and washing steps.  What was novel about the process was the way in which the precipitates were handled, precipitates were trapped on very fine pore size filters (0.2 µm) washed with various solvents then dissolved and passed through the filter resulting in a final product that was sterile and very pure, with good yields of Vi.

This process has now been transferred to three manufacturers who have demonstrated capacity to scale up the process to production scale and have committed to provide the product to the public sector at affordable prices.   The greatest advantage for the manufacturers was that they did not need to pay for the development of a production process.   Also the process developed by IVI did not need to use dangerous chemicals and the cost of capital equipment is minimized.  The use of disposable 0.2 µm filters simplifies manufacture by reducing the amount of cleaning and validation that the cleaning was effective.

The end result being: high quality affordable vaccine for people living in impoverished conditions at risk of Typhoid fever.

 

Acknowledgements

This work was supported by grants from the Bill and Melinda Gates Foundation (VIVA-grant no. OPP417.01), the UBS-Optimus

foundation (Enteric Fever Vaccines for the Poor) and the Governments of Republic of Korea and Sweden (SIDA).

REFERENCE

[1] An SJ, Yoon YK, Kothari S, Kothari N, Kim JA, Lee E, et al. Physico-chemical properties of Salmonella typhi Vi polysaccharide-diphtheria toxoid conjugate vaccines affect immunogenicity. Vaccine 2011;29(44):7618-23.

[2] Jang H, Yoon YK, Kim JA, Kim HS, An SJ, Seo JH, Cui C, Carbis R. Optimization of Vi capsular polysaccharide production during growth of Salmonella enteric serotype Typhi Ty2 in a bioreactor. J Biotechnol. 2008;135(1):71-7.

Rodney Carbis fig1Fig 1. Separation of crude Vi from bacterial cells

 Rodney Carbis fig2Fig 2. Scale up of filtration process at manufacturer.  Trapping of precipitated Vi on 0.2 µm filter.

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