Wyeth

Wei-Qiang Willie Sun

Mark Edward Ruppen

Justin Keith Moran

John W Simpson

Ling Chu

Earl Joseph Pursell

Yonghui Yuan

James Patch

Pamela S Fink

Pamela Fink Charbonneau

Methods for producing proteins, for example, recombinant meningococcal 2086 proteins, using fed-batch fermentation with continuous input of an inducer after achieving a threshold parameter, and continuous input of a carbon source, for example, a constant rate input, to improve protein yields, as well as high density protein compositions for use in the methods of the present invention, are provided.

High-cell density fed-batch fermentation process for producing recombinant protein

A shortened process for producing a solution containing substantially purified capsular polysaccharides from a cellularlysate broth is described. Ultrafiltering and diafiltering a clarifiedlysate 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, thecells 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).

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Methods for producing proteins, for example, recombinant meningococcal 2086 proteins, using fed-batch fermentation with continuous input of an inducer after achieving a threshold parameter, and optionally continuous input of a carbon source, for example, a constant rate input, to improve protein yields, as well as high density protein compositions and compositions for use in the methods of the present invention, are provided.

A shortened process for producing a solution containing substantially purified capsular polysaccharides from a cellularlysate broth is described. Ultrafiltering and diafiltering a clarifiedlysate 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-limitingserotypes 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, thecells 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).

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Morristown, NJ, United States of America

Wei-Qiang Willie Sun