Purification, potency and immunogenicity analysis of Vero cell culture-derived rabies vaccine: a comparative study of single-step column chromatography and zonal centrifuge purification.
Continuous Vero cell lines are more suitable for large-scale production of rabies vaccine. The purification of Vero cell-derived rabies vaccine is critical because of the residual cellular DNA and serum proteins. The perfection of techniques using column chromatography with different matrix material, gel filtration and zonal centrifugation is of paramount importance for the optimal purification of rabies vaccine, leaving minimal residual cellular DNA, below the permissible level of 100 pg per dose and serum protein content of 1 ppm. In this study the potency, immunogenicity and safety of Vero cell-derived rabies vaccines were compared following purification by densely or loosely packed DEAE-sepharose CL-6B columns with different bed heights or by zonal centrifugation. The optimal virus recovery and maximum removal of substrate DNA and serum proteins were achieved only when the sepharose CL-6B column bed height was maintained at a thickness of 2-2.5 cm. The rabies virus material was purified by layering over the matrix without applying pressure. DEAE-sepharose CL-6B column purification using a simplified, cost effective technique as described in this study enhances the antigen yield by 50% in comparison with zonal purification.
Prem Kumar
Ananda Arone
AA
Pasteur Institute of India, Coonoor 643103, Nilgiris, Tamil Nadu, India. apkumar_piic@yahoo.co.in
Mani
Kavaratty Raju
KR
Palaniappan
Chitrambalam
C
Bhau
Lakshman Narasingha Rao
LN
Swaminathan
Krishnaswami
K
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000956:Antigens, Viral / Q000032:analysis; D001798:Blood Proteins / Q000032:analysis; D002501:Centrifugation, Zonal / Q000379:methods*; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis; D006168:Guinea Pigs; D051379:Mice; D023421:Models, Animal; D009500:Neutralization Tests; D011819:Rabies Vaccines / Q000276:immunology* / Q000302:isolation & purification*; D011820:Rabies virus / Q000276:immunology; D012685:Sepharose / Q000031:analogs & derivatives; D014709:Vero Cells; D014764:Viral Proteins / Q000032:analysis
D000914:Antibodies, Viral; D000956:Antigens, Viral; D001798:Blood Proteins; D011819:Rabies Vaccines; D014764:Viral Proteins; C043982:DEAE-Sepharose CL-6B; D004247:DNA; D012685:Sepharose