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Botulinum antitoxin compositions and methods

a technology of botulinum and compositions, applied in the field of botulinum antitoxin compositions and methods, can solve the problems of severe flaccid paralysis, serious threat to human safety, respiratory collapse and death,

Inactive Publication Date: 2005-02-24
INTRACEL RESOURCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The resulting de-speciated heptavalent antitoxin composition preferably has a protein concentration of about 30-70 mg/ml; a pH of about 6-8; and preferably

Problems solved by technology

Clostridium botulinum produces one of the most toxic substances known to man and presents a serious threat to human safety.
Botulinum toxin works by blocking conduction at the neuromuscular junction and preventing the release of acetylcholine, which results in severe flaccid paralysis.
Eventually, the cholinergic autonomic blockade leads to respiratory collapse and death.
Furthermore, the prevalence and lethality of botulinum toxin give rise to potential risks by terrorist organizations.
Second, large segments of the population may become infected simultaneously.
Previous methods of manufacturing botulinum antitoxin compositions have produced unreliable results.
In particular, it was difficult to obtain a high titer response to botulinum toxins F and G through such immunization.
Furthermore, previous immunization methods used alum-precipitated toxoids, which resulted in low titered antitoxins inadequate to meet the requirements of a clinically useful antitoxin produced in sufficient quantities for treating large numbers of patients.
However, this method of production was unreliable because there was no control over the various concentrations of the different antitoxins produced in the horse.
Furthermore, previous methods were not suitable for large scale production of botulinum antitoxin.
The use of Cohn fractionation, however, is less effective in large scale manufacturing because large volumes of plasma must be subjected to centrifugation and the method produces antibodies with relatively low purity.
Additionally, previous methods of de-speciating equine botulinum antibodies were not particularly effective, and were quite ineffective for producing botulinum antitoxin on a large scale basis.
Such methods of digestion included the use of pepsin at 37° C. over a period of 4 hours, which often resulted in incomplete digestion.
To increase the rate of incubation and decrease incubation time, the incubation temperature may be increased to 70° C. However, that temperature is not compatible with achieving an active antitoxin preparation.
However, utilizing such a low pH causes aggregation and precipitation of equine IgG.

Method used

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  • Botulinum antitoxin compositions and methods
  • Botulinum antitoxin compositions and methods

Examples

Experimental program
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Effect test

example 1

Immunization Regimen

In order to obtain high polyclonal antibody titers, we used a multi-step immunization procedure. Several horses were each immunized with a single botulinum serotype. First, the horses were immunized with the toxoid form, i.e., inactivated form of the toxin, of the specific toxin serotype to be used for that horse. The first inoculation contained 2 mg of toxoid in Complete Freund's Adjuvant injected intradermally using up to 30 sites at approximately 0.1 mL per site. After 14 days, the horses received a second inoculation of 0.5 mg toxoid in Incomplete Freund's Adjuvant injected intradermally at multiple sites at approximately 0.1 mL per site. After another 14 days, antitoxin titers were determined using a mouse neutralization assay and, if necessary, a subsequent priming dose of 0.5 mg of toxoid in RIBI's adjuvant system (MPL+TDM=CWS emulsion) was administered.

The injection site for the toxoid inoculations was a rectangular area measuring approximately 24×8 i...

example 2

Purification and De-Speciation of Equine Botulinum Antitoxins

Eighteen bags of frozen plasma, containing antitoxin to serotype B, were thawed, pooled together and clarified by filtration into a 110 L vessel. The total volume of plasma processed was 98 L. After clarification and rinsing of the filters with phosphate buffered solution (“PBS”), the volume of the sample was 103 L. This material was loaded onto 30 L protein G affinity columns in four cycles of 25.75 L per cycle. During each cycle, the antitoxin was eluted with 10 mM sodium bicarbonate / carbonate buffer in an average volume of 31.4 L. The total volume of antibody collected was 126.7 L with a protein concentration of 9.3 mg / mL. The antitoxin was collected into a 300 L vessel and diluted with 1M sodium acetate to a concentration of 50 mM sodium acetate, such that the pH was 4.5.

Pepsin was then added to the antibody solution at a concentration of 4% w / v and the temperature was adjusted to 58° C. After two hours, the digest...

example 3

Formulation of the Heptavalent Antitoxin

Various monovalent batches of each of the seven antitoxin serotypes were processed as described above. The batches were pooled under aseptic conditions and based on the titers of the individual batches of antitoxin, the following volumes were combined: A-7.38 L; B-18.57 L; C-18.18 L; D-4.86 L; E-3.11 L; F-24.82 L; and G-12.62 L. To this pool was added 120.79 L of phosphate buffered saline (PBS) and then 7.212 kilograms of solid lactose. The mixture was stirred for 16 hours at 2-8° C. The final lactose concentration was approximately 5%. The mixture was then filtered through a 0.22 micron filter and release tests described above were performed. This final purified bulk product was then vialed at approximately 23 mL (5-100 mL depending on the size of the lyophilization vial) per vial and lyophilized.

For lyophilization, the optimal cycle time is 96-110 hours (range could be 80-125 hours). The temperature of the trays when loading the vials in...

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Abstract

This invention provides botulinum antitoxin compositions and methods of production, and methods of treating animals and humans prophylactically and also those suspected of having contacted botulism toxin. The botulinum antitoxin is prepared by inoculating an animal with a monovalent botulinum toxoid and toxin. The animal's plasma is collected and purified at a high pH by affinity chromatography. The resulting monovalent immunoglobulins are de-speciated by digestion with pepsin. Monovalent antitoxins for all seven botulinum serotypes are then combined to produce a high titered heptavalent botulinum antitoxin composition.

Description

FIELD OF THE INVENTION The present invention is directed to botulinum antitoxin compositions and methods for their production and use. More particularly, the present invention provides in one embodiment large scale purification and de-speciation procedures and the controlled combination of different antitoxins to produce consistently high titered heptavalent antitoxin compositions. BACKGROUND OF THE INVENTION Clostridium botulinum produces one of the most toxic substances known to man and presents a serious threat to human safety. The botulinum toxin is so potent that a lethal dose for an adult may be as low as 1 μg. Botulinum toxin works by blocking conduction at the neuromuscular junction and preventing the release of acetylcholine, which results in severe flaccid paralysis. Eventually, the cholinergic autonomic blockade leads to respiratory collapse and death. There are seven different serotypes of C. botulinum and consequently seven imunologically distinct toxins. These seven ...

Claims

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Application Information

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IPC IPC(8): A61K39/40C07K16/12
CPCA61K39/40C07K16/1282A61K2039/505A61P31/04Y02A50/30
Inventor POMATO, NICHOLASHASPEL, MARTIN V.RANSOM, JANET H.
Owner INTRACEL RESOURCES
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