Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Patent
1994-07-11
1997-04-01
Housel, James C.
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
4241841, 4242581, 4242401, 530420, 530412, A61K 39112, A61K 39108, A61K 3910, C07K 100
Patent
active
056163282
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention relates to the field of Gram-negative bacterial vaccines and their method of production.
BACKGROUND OF THE INVENTION
Vaccines made from Gram-negative bacteria have a well known tendency to cause endotoxic shock. This may result in abortion or death. Gram-negative bacteria release endotoxin from their outer membrane to a slight extent while they are alive, and dividing, but to a far greater extent during and after their death. Bacterial endotoxin is naturally present in tap water, where it is called pyrogen. To avoid the induction of fever, pyrogen-free water for injection is prepared by distillation or other methods of purification. In man the injection of as little as one endotoxin unit (EU) (approximate 0.1 nanogram, or 10.sup.-10 gram) may cause a transient rise in body temperature. In man and other animal, larger doses cause endotoxic shock and death.
The rabbit is similar to man in sensitivity to endotoxin, and the rabbit traditionally has been used to test human injectable products for pyrogenicity. Most other animal species are less sensitive. Horses and pigs are considerably more sensitive than most laboratory rodents. Thus, in the laboratory, only the rabbit is suitable for testing the endotoxic activity of veterinary injectables, although mice can be made relatively sensitive with drugs that alter macrophage function.
In endotoxin assays, the rabbit has largely been replaced by an in vitro test that is even more sensitive. This depends on the action of endotoxin on a fluid extracted from the horseshoe crab (Limulus amebocyte lysate, or LAL). The addition of endotoxin in trace amounts causes LAL to gel. Before the gel develops, the transparency of LAL changes in a way that can be measured by a spectrophotometer as increasing optical density.
The free-endotoxin content of cultures of Gram-negative bacteria, killed to make vaccines, varies according to the skill of the manufacturer. One ml. may contain as little as 20 micrograms (2.times.10.sup.-5 gram) or as much as one milligram (10.sup.-3 gram). Vaccine makers have used several methods to decrease free endotoxin. One involves harvesting the bacterial cells, by centrifuging or filtering, and discarding the endotoxin-rich culture fluid. Another involves adsorbing the cultures with insoluble aluminum (Al) or calcium compounds (carriers) such as aluminum hydroxide gel (Al gel).
Adsorbing with Al gel tends to remove most of the free-endotoxin from solution. Some manufacturers allow the gel, with the endotoxin and other culture products that are absorbed to it, to sediment before decanting the supernatant fluid to remove the remaining free-endotoxin. The bacteria may not be adsorbed to the gel, in which case they usually sediment, leaving the supernatant fluid clear. Decanting has to be delayed until everything has settled and the fluid is clear.
Whether the bacteria have been harvested from the culture, or sedimenting has taken place after adsorbing with an aluminum or calcium compound, the materials are then usually resuspended in a simple aqueous fluid (water, saline, or a buffer solution). Assays will then usually show a disappointing decrease in free-endotoxin content; the change is much less than calculated from the dilution factor on resuspension. This is because endotoxin continues to escape from the bacterial surface, and loosely bound endotoxin becomes desorbed from the carrier.
The two processes are sometimes combined; the bacteria are harvested from the culture medium, resuspended in aqueous fluid, and then adsorbed. It has been shown that adsorbing aqueous suspensions of harvested bacteria with conventional amounts of Al gel produces an undesirable result. For example, when an aqueous suspension of Salmonella choleraesuis was adsorbed with Al gel, 25% v/v, there was no detectable free-endotoxin but the ability of the preparation to immunize mice against S. choleraesuis was almost completely eliminated. As soon as the gel began to settle, the supernatant fluid was crystal clear, denoting total adsorp
REFERENCES:
patent: 4455297 (1984-06-01), Syukuda et al.
patent: 4465665 (1984-08-01), Dobrescu
patent: 4705686 (1987-11-01), Scott et al.
patent: 4789544 (1988-12-01), Nelson et al.
patent: 5019388 (1991-05-01), Brown et al.
patent: 5101019 (1992-03-01), Fujita et al.
patent: 5536496 (1996-07-01), Frantz
Beecham Laboratories 1989 Atrobac 3 product literature.
Dearwester Donald A.
Roberts David S.
Swearingin Leroy A.
Ginsburg Paul H.
Housel James C.
Koller Alan L.
Minnifield N. M.
Pfizer Inc.
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