Patent US5936941A successfully refreshed from Google Patents.
US5078985A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Radiolabeling antibodies and other proteins with technetium or rhenium by regulated reduction
Assignee: Rhomed Inc
•
Filing: 1989-08-09
•
Granted: 1992-01-07
•
Expires: 2009-08-09
Abstract
Abstract Proteins are radiolabeled with radionuclides of technetium or rhenium by a process in which the disulfide bonds of the protein are first partially reduced with stannous salts or other disulfide reducing agents, all substances other than the desired reduced protein removed, by size exclusion chromatography or other purification means, and a specified, smaller amount of pertechnetate or perrhenate reducing agent, such as a stannous salt, is added to the reduced protein in a manner such that further reduction of the protein is limited. Pertechnetate or perrhenate is then added to the mixture of the reduced protein and the pertechnetate or perrhenate reducing agent; the pertechnetate or perrhenate is reduced and becomes strongly bonded to the protein via the sulfhydryl groups previously exposed by reduction of disulfide groups. The reduced protein and pertechnetate or perrhenate reducing agent can be idefinitely stored frozen or lyophilized.
Inventors
- 👤 Buck A. Rhodes
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-08 05:38
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; A; A61; A61K; A61K51/00; A61K51/02; A61K51/04; A61K51/08; A61K51/10; C07K14/575; C07K14/655; C07K14/78; A61K2121/00; A61K2123/00; A61K38/00
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
135 words
A method of radiolabeling a protein containing disulfide bonds with a radionuclide selected from the group consisting of technetium and rhenium to obtain stable labeling, comprising the steps of: a) incubating the protein containing disulfide bonds with a first reducing agent, the period of incubation being sufficient to reduce available disulfide bonds to sulfhydryl groups while preventing excessive fragmentation of the protein; b) purifying the reduced protein to substantially remove the first reducing agent and impurities; c) adding a source of stannous ion agent to the reduced protein in a sufficient amount to reduce the radionuclide, the radionuclide to be added in a subsequent step; d) radiolabeling the purified reduced protein by adding the radionuclide, whereby the stannous ion agent reduces the radionuclide and the reduced radionuclide and reduced protein forms radionuclide-containing and sulfur-containing complexes.
Claim 2
Independent Claim
135 words
A method of radiolabeling a protein containing disulfide bonds with a radionuclide selected from the group consisting of technetium and rhenium to obtain stable labeling, comprising the steps of: a) incubating the protein containing disulfide bonds with a first reducing agent, the period of incubation being sufficient to reduce available disulfide bonds to sulfhydryl groups while preventing excessive fragmentation of the protein; b) purifying the reduced protein to substantially remove the first reducing agent and impurities; c) adding a source of stannous ion agent to the reduced protein in a sufficient amount to reduce the radionuclide, the radionuclide to be added in a subsequent step; d) radiolabeling the purified reduced protein by adding the radionuclide, whereby the stannous ion agent reduces the radionuclide and the reduced radionuclide and reduced protein forms radionuclide-containing and sulfur-containing complexes.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein the radionuclide is technetium-99m in the form of sodium pertechnetate.
Claim 4
Dependent Claim
35 words
The method of claim 1 wherein the first reducing agent comprises at least one member selected from the group consisting of 2-mercaptoethanol; 1,4 dithiothreitol; 2,3 dihydroxybutane-1; 4 dithiol; 2-aminoethanethiol HCl; 2-mercaptoethylamine; thioglycolate; cyanide; and cysteine.
Claim 5
Dependent Claim
31 words
The method of claim 1 wherein the source of the stannous ion agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0.
Claim 6
Dependent Claim
29 words
The method of claim 1 wherein the source of the stannous ion agent comprises a member selected from the group consisting of stannous glucoheptonate, stannous gluconate and stannous phosphonate.
Claim 7
Dependent Claim
25 words
The method of claim 1 wherein the protein comprises a member selected from the group consisting of monoclonal antibodies, monoclonal antibody fragments and polyclonal antibodies.
Claim 8
Dependent Claim
58 words
The method of claim 1 wherein following step c), and prior to step d), the purified reduced protein with stannous ion agent is lyophilized in a vial, whereby the lyophilized purified reduced protein with stannous ion agent can be maintained for an indefinite period before radiolabeling in step d) by the addition of the radionuclide to the vial.
Claim 9
Dependent Claim
87 words
The method of claim 1 wherein following step b), and prior to step c), the purified reduced protein is frozen in a vial, and wherein following step c), and prior to step d), the stannous ion agent is frozen, whereby essentially no chemical reaction occurs between the frozen purified reduced protein and the stannous ion agent and the frozen purified reduced protein and stannous ion agent can be maintained for an indefinite period before radiolabeling in step d) by the addition of the radionuclide to the vial.
Claim 10
Dependent Claim
26 words
The method of claim 8 wherein following the freezing of the stannous ion agent the frozen purified reduced protein and frozen stannous ion agent are lyophilized.
Claim 11
Independent Claim
141 words
A method of radiolabeling a protein containing disulfide bonds with a radionuclide selected from the group consisting of technetium and rhenium to obtain stable labeling, comprising the steps of: a) incubating the protein containing disulfide bonds with a source of stannous (II) ion, the period of incubation being sufficient to reduce available disulfide bonds to sulfhydryl groups while preventing excessive fragmentation of the protein; b) purifying the reduced protein to substantially remove stannous salts and impurities; c) adding a second Sn (II) agent to the purified reduced protein in a sufficient amount to reduce the radionuclide yet not significantly degrade the purified reduced protein, the radionuclide to be added in a subsequent step; and d) radiolabeling the reduced purified protein by adding the radionuclide, whereby the Sn (II) agents reduce the radionuclide and the reduced radionuclide forms radionuclide-containing and sulfur-containing complexes.
Claim 12
Independent Claim
141 words
A method of radiolabeling a protein containing disulfide bonds with a radionuclide selected from the group consisting of technetium and rhenium to obtain stable labeling, comprising the steps of: a) incubating the protein containing disulfide bonds with a source of stannous (II) ion, the period of incubation being sufficient to reduce available disulfide bonds to sulfhydryl groups while preventing excessive fragmentation of the protein; b) purifying the reduced protein to substantially remove stannous salts and impurities; c) adding a second Sn (II) agent to the purified reduced protein in a sufficient amount to reduce the radionuclide yet not significantly degrade the purified reduced protein, the radionuclide to be added in a subsequent step; and d) radiolabeling the reduced purified protein by adding the radionuclide, whereby the Sn (II) agents reduce the radionuclide and the reduced radionuclide forms radionuclide-containing and sulfur-containing complexes.
Claim 13
Dependent Claim
16 words
The method of claim 10 wherein the radionuclide is technetium-99m in the form of sodium pertechnetate.
Claim 14
Dependent Claim
32 words
The method of claim 10 wherein the source of the first Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0.
Claim 15
Dependent Claim
32 words
The method of claim 10 wherein the source of the second Sn (II) agent is present in a solution comprising alkali metal tartrate having a pH of between approximately 5.0 and 6.0.
Claim 16
Dependent Claim
32 words
The method of claim 10 wherein the source of the second Sn (II) agent comprises at least one member selected from the group consisting of stannous glucoheptonate, stannous gluconate and stannous phosphonate.
Claim 17
Dependent Claim
25 words
The method of claim 10 wherein the protein comprises a member selected from the group consisting of monoclonal antibodies, monoclonal antibody fragments and polyclonal antibodies.
Claim 18
Dependent Claim
58 words
The method of claim 10 wherein following step c), and prior to step d), the purified reduced protein with stannous ion agent is lyophilized in a vial, whereby the lyophilized purified reduced protein with stannous ion agent can be maintained for an indefinite period before radiolabeling in step d) by the addition of the radionuclide to the vial.
Claim 19
Dependent Claim
87 words
The method of claim 10 wherein following step b), and prior to step c), the purified reduced protein is frozen in a vial, and wherein following step c), and prior to step d), the stannous ion agent is frozen, whereby essentially no chemical reaction occurs between the frozen purified reduced protein and the stannous ion agent and the frozen purified reduced protein and stannous ion agent can be maintained for an indefinite period before radiolabeling in step d) by the addition of the radionuclide to the vial.
Claim 20
Dependent Claim
26 words
The method of claim 17 wherein following the freezing of the stannous ion agent the frozen purified reduced protein and frozen stannous ion agent are lyophilized.
References Cited (Backward Citations)
US4323546A
Cited
US4305922A
Cited
US4424200A
Cited
US4311688A
Cited
US4472371A
Cited
US4478815A
Cited
US4331647A
Cited
US4348376A
Cited
US4421735A
Cited
US4668503A
Cited
US4479930A
Cited
WO1985003231A1
Cited
US4647445A
Cited
US4652440A
Cited
US4670545A
Cited
WO1987004164A1
Cited
EP0237150A2
Cited
US4877868A
Cited
EP0271806A2
Cited
WO1988007382A2
Cited
EP0336678A2
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1995-01-06 | 1995-07-08 | 1996-01-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-01-06 | 1999-07-08 | 2000-01-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-01-06 | 2003-07-08 | 2004-01-07 | $7700.00 | Window Closed / Lapsed |