| 摘要 |
A shortened process for producing a solution containing substantially purified capsular polysaccharides from a cellular Streptococcus pneumoniae lysate broth is described. Ultrafiltering and diafiltering a clarified S. pneumoniae lysate followed by pH adjustment to less than 4.5, preferably about 3.5, precipitated at least 98% of the protein in the solution without seriously affecting polysaccharide yield. Furthermore, following ultrafiltration and diafiltration and acidification to a pH of less than 4.5, filtration using activated carbon precipitated at least 90% of remaining protein without seriously affecting polysaccharide yield. Exemplary, non-limiting S. pneumoniae serotypes that can be purified using the shortened process of the invention are 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In one embodiment, the Streptococcus pneumoniae cells are lysed using deoxycholate sodium (DOC), while in another embodiment the lytic agent is a non-animal derived lytic agent such as N-lauryl sarcosine sodium (NLS). |
| 主权项 |
1. A process for producing a solution containing substantially purified capsular polysaccharides from a Streptococcus pneumoniae cell lysate, the process comprising the steps of:
(a) providing a fermentation broth comprising bacterial cells that produce a selected Streptococcus pneumoniae serotype; (b) lysing the bacterial cells in step (a) with a lytic agent, thereby producing a cell lysate comprising cell debris, soluble proteins, nucleic acids, and polysaccharides; (c) clarifying the cell lysate of step (b) using centrifugation or filtration to remove cell debris, thereby producing a clarified cell lysate; (d) ultrafiltering and diafiltering the clarified cell lysate of step (c) to remove low molecular weight impurities and increase polysaccharide concentration, thereby producing a retentate; (e) lowering the pH of the retentate of step (d) to less than 4.5 to precipitate protein and nucleic acids, thereby forming an acidified retentate solution; (f) holding the acidified retentate solution formed in step (e) for a time sufficient to allow settling of the precipitate, followed by filtration or centrifugation of the acidified retentate solution, thereby producing a clarified polysaccharide solution; (g) filtering the clarified polysaccharide solution of step (f) through an activated carbon filter; (h) ultrafiltering and diafiltering the filtered solution produced by step (g), thereby producing a concentrated purified polysaccharide solution; and (i) filtering the concentrated purified polysaccharide solution produced by step (h) using a sterile filter;whereby a solution containing substantially purified capsular polysaccharides is produced. |
