US4689401A
Expired / Lapsed
US
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Method of recovering microbially produced recombinant ricin toxin a chain
Assignee: Novartis Vaccines and Diagnostics Inc
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Filing: 1986-09-09
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Granted: 1987-08-25
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Expires: 2006-09-09
Abstract
Abstract Substantially pure, intracellularly produced, soluble recombinant ricin toxin A (RTA) is recovered from transformed cells by disrupting the cell membrane, removing insoluble cell membrane materials from the disruptate, adjusting the pH of the cell membrane material-free solution to 6 to 6.5 and the conductivity to 1.25 to 1.75 millisiemens, passing the adjusted solution through a bed of SP-cellulose cation exchanger, and eluting the substantially pure RTA from the bed.
Inventors
- 👤 Robert Ferris
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 11:38
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/415; Y; Y10; Y10S; Y10S530/00; Y10S530/808; Y10S530/809
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
139 words
Method of recovering recombinant intracellularly produced, soluble ricin toxin A chain (RTA) from an aqueous suspension of transformed microorganisms containing the RTA comprising: (a) disrupting the cell membrane of the microorganisms at a pH that keeps the RTA in solution; (b) removing insoluble cell membrane materials from the disruptate at a pH that keeps the RTA in solution; (c) adjusting, if necessary, the pH of the solution resulting from step (b) to about 6 to about 6.5 and the conductivity of said solution to about 1.25 to about 1.75 millisiemens; (d) then passing said solution through a bed of an SP-cellulose cation exchanger, whereby RTA in the solution is retained by the exchanger; and (e) eluting RTA from the bed to produce a solution whose protein content is at least about 90% by weight RTA as determined by SDS-PAGE.
Claim 2
Dependent Claim
139 words
Method of recovering recombinant intracellularly produced, soluble ricin toxin A chain (RTA) from an aqueous suspension of transformed microorganisms containing the RTA comprising: (a) disrupting the cell membrane of the microorganisms at a pH that keeps the RTA in solution; (b) removing insoluble cell membrane materials from the disruptate at a pH that keeps the RTA in solution; (c) adjusting, if necessary, the pH of the solution resulting from step (b) to about 6 to about 6.5 and the conductivity of said solution to about 1.25 to about 1.75 millisiemens; (d) then passing said solution through a bed of an SP-cellulose cation exchanger, whereby RTA in the solution is retained by the exchanger; and (e) eluting RTA from the bed to produce a solution whose protein content is at least about 90% by weight RTA as determined by SDS-PAGE.
Claim 3
Dependent Claim
21 words
The method of claim 1 wherein step (a) is effected by high pressure cycling and is carried out under reducing conditions.
Claim 4
Dependent Claim
12 words
The method of claim 1 wherein step (b) is effected by diafiltration.
Claim 5
Dependent Claim
14 words
The method of claim 1 wherein said conductivity is about 1.45 to about 1.55.
Claim 6
Dependent Claim
38 words
The method of claim 1 wherein the cation exchanger is composed of cellulosic backbones crosslinked with vinyl polymer carrying pendant sulfopropyl functional groups and the bed is configured such that the solution is passed radially through the bed.
Claim 7
Dependent Claim
21 words
The method of claim 1 wherein said protein content is at least about 95% by weight RTA as determined by SDS-PAGE.
Claim 8
Dependent Claim
45 words
The method of claim 5 wherein step (a) is effected by high pressure cycling, step (b) is effected by diafiltration, the conductivity is about 1.45 to about 1.55 millisiemens, and the protein content is at least about 95% by weight RTA as determined by SDS-PAGE.
Claim 9
Dependent Claim
27 words
The method of claim 1 including (f) chromatographing the eluted RTA using a phenyl-sepharose column, whereby the purity of the RTA is increased to above about 99%.
Claim 10
Dependent Claim
24 words
The method of claim 1 wherein steps a-f are singly or in combination run at a temperature that reduces formation of precipitates comprising RTA.
Claim 11
Dependent Claim
24 words
The method of claim 9 wherein said temperature is in a range from one sufficient to keep solutions from freezing to about 10° C.
Claim 12
Dependent Claim
12 words
The method of claim 9 wherein said temperature is about 4° C.
Claim 13
Dependent Claim
136 words
Ricin toxin A chain (RTA) recovered from an aqueous suspension of transformed microorganisms containing the RTA by the steps comprising: (a) disrupting the cell membrane of the microorganisms at a pH that keeps the RTA in solution; (b) removing insoluble cell membrane materials from the disruptate at a pH that keeps the RTA in solution; (c) adjusting, if necessary, the pH of the solution resulting from step (b) to about 6 to about 6.5 and the conductivity of said solution to about 1.25 to about 1.75 millisiemens; (d) then passing said solution through a bed of an SP-cellulose cation exchanger, whereby RTA in the solution is retained by the exchanger; and (e) eluting RTA from the bed to produce a solution whose protein content is at least about 90% by weight RTA as determined by SDS-PAGE.
Claim 14
Dependent Claim
136 words
Ricin toxin A chain (RTA) recovered from an aqueous suspension of transformed microorganisms containing the RTA by the steps comprising: (a) disrupting the cell membrane of the microorganisms at a pH that keeps the RTA in solution; (b) removing insoluble cell membrane materials from the disruptate at a pH that keeps the RTA in solution; (c) adjusting, if necessary, the pH of the solution resulting from step (b) to about 6 to about 6.5 and the conductivity of said solution to about 1.25 to about 1.75 millisiemens; (d) then passing said solution through a bed of an SP-cellulose cation exchanger, whereby RTA in the solution is retained by the exchanger; and (e) eluting RTA from the bed to produce a solution whose protein content is at least about 90% by weight RTA as determined by SDS-PAGE.
Claim 15
Dependent Claim
23 words
Ricin toxin A chain of claim 12 wherein step (a) is effected by high pressure cycling and is carried out under reducing conditions.
Claim 16
Dependent Claim
14 words
Ricin toxin A chain of claim 13 wherein step (b) is effected by diafiltration.
Claim 17
Dependent Claim
16 words
Ricin toxin A chain of claim 12 wherein said conductivity is about 1.45 to about 1.55.
Claim 18
Dependent Claim
40 words
Ricin toxin A chain of claim 12 wherein the cation exchanger is composed of cellulosic backbones cross-linked with vinyl polymer carrying pendant sulfopropyl functional groups and the bed is configured such that the solution is passed radially through the bed.
Claim 19
Dependent Claim
23 words
Ricin toxin A chain of claim 12 wherein said protein content is at least about 95% by weight RTA as determined by SDS-PAGE.
Claim 20
Dependent Claim
47 words
Ricin toxin A chain of claim 16 wherein step (a) is effected by high pressure cycling, step (b) is effected by diafiltration, the conductivity is about 1.45 to about 1.55 millisiemens, and the protein content is at least about 95% by weight RTA as determined by SDS-PAGE.
Claim 21
Dependent Claim
29 words
Ricin toxin A chain of claim 12 including (f) chromatographing the eluted RTA using a phenylsepharose column, whereby the purity of the RTA is increased to above about 99%.
Claim 22
Dependent Claim
26 words
Ricin toxin A chain of claim 12 wherein steps a-f are singly or in combination run at a temperature that reduces formation of precipitates comprising RTA.
Claim 23
Dependent Claim
24 words
The method of claim 20 wherein said temperature is in a range from one sufficient to keep solutions from freezing to about 10° C.
Claim 24
Dependent Claim
12 words
The method of claim 20 wherein said temperature is about 4° C.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-08-24 | 1991-02-23 | 1991-08-25 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-08-24 | 1995-02-23 | 1995-08-25 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-08-24 | 1999-02-23 | 1999-08-25 | $7700.00 | Window Closed / Lapsed |