US4681870A
Expired / Lapsed
US
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Protein A-silica immunoadsorbent and process for its production
Assignee: Cypress Bioscience Inc
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Filing: 1985-01-11
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Granted: 1987-07-21
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Expires: 2005-01-11
Abstract
Abstract An immunoadsorbent material for removing IgG and IgG-complexes from biological fluids is prepared by covalently binding protein A to a solid-phase silica matrix. It has been found that particularly stable, high-capacity immunoadsorbents are obtained by derivatizing the silica with amino and/or carboxyl groups, and reacting the protein A with a carbodiimide at a pH in the range from 3.5 to 4.5. Binding through free hydroxyl groups may be achieved with cyanogen halides at a pH in the range from 11.0 to 11.5. After acid washing (pH 2.0-2.5) to remove non-covalently bound protein A, the immunoadsorbent may be employed in a column for therapeutic treatment of various cancers and autoimmune disorders where IgG-complexes are implicated as suppressing factors in inhibiting a normal immune response.
Inventors
- 👤 Joseph P. Balint, Jr.
- 👤 Richard E. Hargreaves
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 21:08
CPC Classifications
B; B01; B01J; B01J20/00; B01J20/30; B01J20/32; B01J20/3202; B01J20/3204; A; A61; A61P; A61P35/00; A61P37/00; B01J20/02; B01J20/10; B01J20/103; B01J20/28; B01J20/28002; B01J20/28004; B01J20/3078; B01J20/3085; B01J20/3214; B01J20/3217; B01J20/3219; B01J20/3231; B01J20/3242; B01J20/3244; B01J20/3246; B01J20/3257; B01J20/3263; B01J20/3268; B01J20/3272; B01J20/3274; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/551; G01N33/552; A61M; A61M1/00; A61M1/36; A61M1/3679; B01J2220/00; B01J2220/50; B01J2220/58; Y; Y10; Y10S; Y10S128/00; Y10S128/03
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
92 words
A method for preparing an immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said method comprising: introducing free amino or carboxyl groups onto a silica matrix; reacting the silica matrix with purified protein A in the presence of a carbodiimide at a pH in the range from 3.5 to 4.5 to covalently link the protein A to the matrix through the amino or carboxyl groups; and washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.
Claim 2
Independent Claim
92 words
A method for preparing an immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said method comprising: introducing free amino or carboxyl groups onto a silica matrix; reacting the silica matrix with purified protein A in the presence of a carbodiimide at a pH in the range from 3.5 to 4.5 to covalently link the protein A to the matrix through the amino or carboxyl groups; and washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.
Claim 3
Dependent Claim
20 words
A method as in claim 1, wherein amino groups are introduced onto the silica matrix by reaction with a silane.
Claim 4
Dependent Claim
12 words
A method as in claim 2, wherein the amino silane is γ-aminoproplytriethoxysilane.
Claim 5
Dependent Claim
42 words
A method as in claim 1, wherein carboxyl groups are introduced by first reacting the silica matrix with an amino silane to introduce amino groups, followed by reacting the silica matrix with succinic anhydride to replace the amino groups with carboxyl groups.
Claim 6
Dependent Claim
13 words
A method as in claim 1, wherein the silica matrix is amorphous silica.
Claim 7
Dependent Claim
12 words
A method as in claim 5, wherein the amorphous silica is diatomite.
Claim 8
Dependent Claim
16 words
A method as in claim 6, wherein the diatomite is sized from 45 to 60 mesh.
Claim 9
Dependent Claim
22 words
A method as in claim 1, wherein the protein A is present at at least 0.05 weight percent of the silica matrix.
Claim 10
Dependent Claim
102 words
An immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said material comprising purified protein A covalently coupled to a silica matrix prepared by the method comprising: introducing free amino or carboxyl groups onto a silica matrix; reacting the silica matrix with purified protein A in the presence of a carbodiimide at a pH in the range from 3.3 to 3.7 to covalently link the protein A to the matrix through the amino or carboxyl groups; and washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.
Claim 11
Dependent Claim
102 words
An immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said material comprising purified protein A covalently coupled to a silica matrix prepared by the method comprising: introducing free amino or carboxyl groups onto a silica matrix; reacting the silica matrix with purified protein A in the presence of a carbodiimide at a pH in the range from 3.3 to 3.7 to covalently link the protein A to the matrix through the amino or carboxyl groups; and washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.
Claim 12
Dependent Claim
21 words
An immunoadsorbent material as in claim 9, wherein amino groups are introduced onto the silica matrix by reaction with a silane.
Claim 13
Dependent Claim
13 words
An immunoadsorbent material as in claim 10, wherein the amino silane is γ-aminoproplytriethoxysilane.
Claim 14
Dependent Claim
43 words
An immunoadsorbent material as in claim 9, wherein carboxyl groups are introduced by first reacting the silica matrix with an amino silane to introduce amino groups, followed by reacting the silica matrix with succinic anhydride to replace the amino groups with carboxyl groups.
Claim 15
Dependent Claim
14 words
An immunoadsorbent material as in claim 9, wherein the silica matrix is amorphous silica.
Claim 16
Dependent Claim
13 words
An immunoadsorbent material as in claim 13, wherein the amorphous silica is diatomite.
Claim 17
Dependent Claim
23 words
An immunoadsorbent material as in claim 9, wherein the protein A is present at at least 0.05 weight percent of the silica matrix.
Claim 18
Independent Claim
90 words
A method for preparing an immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said method comprising: introducing free hydroxyl groups onto a silica matrix; activating the silica matrix in the presence of cyanogen bromide at a pH in the range from 11.0 to 11.5, and covalently linking the protein A to the matrix at a pH in the range from 8.5 to 9.0; and washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.
Claim 19
Independent Claim
90 words
A method for preparing an immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said method comprising: introducing free hydroxyl groups onto a silica matrix; activating the silica matrix in the presence of cyanogen bromide at a pH in the range from 11.0 to 11.5, and covalently linking the protein A to the matrix at a pH in the range from 8.5 to 9.0; and washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.
Claim 20
Dependent Claim
20 words
A method as in claim 16, wherein hydroxyl groups are introduced onto the silica matrix by reaction with a silane.
Claim 21
Dependent Claim
11 words
A method as in claim 17, wherein the silane is γ-glycidoxypropyltrimethoxysilane.
Claim 22
Dependent Claim
13 words
A method as in claim 16, wherein the silica matrix is amorphous silica.
Claim 23
Dependent Claim
12 words
A method as in claim 19, wherein the amorphous silica is diatomite.
Claim 24
Dependent Claim
97 words
An immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said material comprising purified protein A covalently coupled to a silica matrix prepared by the method comprising: introducing free hydroxyl groups onto a silica matrix; activating the silica matrix in the presence of cyanogen bromide at a pH in the range from 11.0 to 11.5, and covalently linking the protein A at a pH in the range from 8.5 to 9.0; and washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.
Claim 25
Dependent Claim
97 words
An immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said material comprising purified protein A covalently coupled to a silica matrix prepared by the method comprising: introducing free hydroxyl groups onto a silica matrix; activating the silica matrix in the presence of cyanogen bromide at a pH in the range from 11.0 to 11.5, and covalently linking the protein A at a pH in the range from 8.5 to 9.0; and washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.
Claim 26
Dependent Claim
21 words
An immunoadsorbent material as in claim 21, wherein hydroxyl groups are introduced onto the silica matrix by reaction with a silane.
Claim 27
Dependent Claim
12 words
An immunoadsorbent material as in claim 22, wherein the silane is γ-glycidoxypropyltrimethoxysilane.
Claim 28
Dependent Claim
14 words
An immunoadsorbent material as in claim 21, wherein the silica matrix is amorphous silica.
Claim 29
Dependent Claim
13 words
An immunoadsorbent material as in claim 24, wherein the amorphous silica is diatomite.
Claim 30
Dependent Claim
17 words
An immunoadsorbent material as in claim 25, wherein the diatomite is sized from 45 to 60 mesh.
References Cited (Backward Citations)
GB2014727A
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JPS5926066A
Cited
JPS5984828A
Cited
EP0110409A2
Cited
JPS59139936A
Cited
JPS59193135A
Cited
US4614513A
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-07-20 | 1991-01-19 | 1991-07-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-07-20 | 1995-01-19 | 1995-07-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-07-20 | 1999-01-19 | 1999-07-21 | $7700.00 | Window Closed / Lapsed |