CA2237338C - Stable avidin composition and methods using same - Google Patents
Stable avidin composition and methods using same Download PDFInfo
- Publication number
- CA2237338C CA2237338C CA002237338A CA2237338A CA2237338C CA 2237338 C CA2237338 C CA 2237338C CA 002237338 A CA002237338 A CA 002237338A CA 2237338 A CA2237338 A CA 2237338A CA 2237338 C CA2237338 C CA 2237338C
- Authority
- CA
- Canada
- Prior art keywords
- composition
- avidin
- inert
- weight
- protectant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
- A61L2/0029—Radiation
- A61L2/0035—Gamma radiation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/106—Fibrin; Fibrinogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/75—Fibrinogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
- C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/966—Chemistry: molecular biology and microbiology involving an enzyme system with high turnover rate or complement magnified assay, e.g. multi-enzyme systems
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/967—Standards, controls, materials, e.g. validation studies, buffer systems
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/972—Modified antibody, e.g. hybrid, bifunctional
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
- Y10S530/813—Carrier is a saccharide
Abstract
Compositions and methods for avidin immobilized on an inert support material, e.g., agarose, are disclosed. The compositions have high activity levels of avidin and may further include a bulking agent, e.g., maltose, and a protectant to maintain the stability and integrity of the avidin agarose during lyophilization and terminal sterilization processes.
These compositions have applicability in any instance wherein avidin, agarose and/or the avidin/biotin technology are useful. In particular, the present compositions are useful in an enzyme capture system to prepare fibrin monomer useful for fibrin sealants.
These compositions have applicability in any instance wherein avidin, agarose and/or the avidin/biotin technology are useful. In particular, the present compositions are useful in an enzyme capture system to prepare fibrin monomer useful for fibrin sealants.
Description
STABLE AVIDIN COMPOSITION AND
METHODS USING SAME
BACKGROUND
The avidin-biotin affinity-based technology has found wide applicability in numerous fields of biology and biotechnology since the pioneering work by Dr. Edward Bayer and Dr. Meier Wilchek in the 1970's. The affinity constant between avidin and biotin is remarkably high and is not significantly lessened when biotin is coupled to a wide variety of biomolecules. This affinity is substantially maintained even when derivatized forms of the biotin are employed and numerous chemistries have been identified for coupling biomolecules to biotin with minimal or negligible loss in the activity or other desired characteristics of the biomolecule. Originally applied to purification and localization procedures for biologically active macromolecules, avidin-biotin technology today has widespread use in medical diagnostics. Newer applications which continue to be developed include affinity targeting, cell cytometry, blotting technology, drug delivery, hybridoma technology, human stem cell selection and reinfusion as well as several approaches to enzyme capture.
In some applications, avidin is immobilized onto an inert material over which a solution containing biotinylated biomolecules is passed. The affinity of the biotin for the avidin provides for the separation of the biomolecule from the solution. A review of the biotin-avidin technology can be found in Applications of Avidin-Biotin Technology to Affinity-Based Separation, Bayer, et al., J. of Chromatography, 1990, pgs. 3-11.
EP S92242 describes a novel fibrin sealant based on fibrin = monomer as opposed to the traditional fibrinogen-based sealants and involves subjecting fibrinogen to a thrombin-like enzyme which is preferably removed after such treatment. EP 592242 describes that the enzyme capture and removal can be accomplished by using biotinylated batroxobin which can be recaptured with an avidin material. The fibrin monomer sealant described in EP =
592242 is advantageously completely autologous. Since autologous fibrin sealants can not always be prepared in advance, autologous processes which provide such sealants in short periods of time (i.e., less than one hour or preferably less than 30 minutes) fr_om the patients' own blood provide a great advantage over current techniques and products. The speed with which such autologous processes can be carried out is dependent to a large degree on the activity of the biotin-and avidin-based reagents.
Commercially available immobilized avidin typically contains about 200 to 400 biotin binding units (BBU) of activity (where 1BBU will bind 1 E.cg of a-biotin) per gram of lyophilized powder (e.g., avidin on acrylic beads from Sigma) or about 20 to 50 BBU
per milliliter of slurry or gel (e.g., avidin on agarose available from Sigma and Pierce).
Also, the above fibrin monomer technology and other biological applications would benefit from more convenient forms of avidin-and biotin-based reagents. For example, the processing necessary to prepare such compositions can have an adverse effect on the activity levels since many of the coupling/immobilization techniques involve materials which can significantly reduce these activities. Additionally, systems which reduce or eliminate le.aching of avidin or of the avidin-biotin complexes would be advantageous in many applications. Further, many biological applications would be greatly enhanced by the availability of high activity avidin compositions which could be lyophilized and further, terminally sterilized while maintaining stability.
Clearly, avidin compositions having higher avidin activity levels with greater stability, especially in freeze dried powder forms capable of withstanding terminal sterilization, e.g., gamma irradiation, would be an advance in the art.
SUMMARY OF THE INVENTION
In accordance with the present invention, stable, highly active compositions of avidin and an inert, easily separable support material such as a water soluble polymer, e.g., polysaccharides selected from agars and alginates, and having an activity level of 1000 BBU or more per gram of lyophilized form and 50 BBU or more per milliliter of slurry or hydrated gel, are disclosed. Preferred compositions include a bulking agent selected from nonionic water soluble polymers, a protectant, and the avidin/inert support material. These compositions may also include one or more materials selected to adjust and/or maintain the pH of the composition and are useful in an aqueous suspension or preferably in lyophilized form.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Although avidin/inert support compositions are known, such compositions having the high levels of activity as described in this invention have not been heretofore disclosed. It has surprisingly been found that avidin compositions can now be prepared having activity levels multiples beyond those presently available without damaging the integrity and stability of the avidin or its inert support. Indeed, these compositions in any convenient form, i.e., slurry, suspension, hydrated gel, dehydrated gel, dried powders, etc., can be the basis of sterile, stable aqueous suspensions and remarkably stable lyophilized compositions capable of withstanding terminal sterilization e.g., gamma irradiation. The novel compositions herein have ~ activity levels for lyophilized forms in excess of 1000 BBU
(which is a measure of the biotin binding capability of the ~ - 3 -avidin composition as discussed above) and preferably between 1500 and 3000 BBU of activity and most preferably between 1800 and 2400 BBU of activity per gram of powder. In slurry, suspension or gel forms the novel avidin compositions of the present invention have activity levels in excess of S0 BBU and preferably between 7S and 150 BBU and more preferably between 90 and 110 BBU of activity per milliliter.
The preferred lyophilized avidin/inert support compositions of the present invention are stable, can be terminally sterilized, have low moisture uptake/moisture content, reswell completely and rapidly upon reconstitution, are non-leachable and pharmaceutically acceptable. The bulking agents used in conjunction with this invention protect the gel bead from damage during the freeze drying process and provide for rapid and complete reswelling of the gel beads upon reconstitution of the freeze dried material. The unique combination of components herein also protects the avidin/inert support component from any deleterious effects upon terminal sterilization of the composition. Thus, the affinity of the avidin for biotinylated biomolecules is maintained even after these rigorous processing steps, i.e., lyophilization and/or terminal sterilization.
Accordingly, in the various applications where the affinity characteristics of the avidin and the integrity of the inert support are both critical to the effectiveness of the avidin/biotin technology, a superior composition is provided by the invention.
The avidin component of the present invention refers to avidin, monomeric avidin, Streptavidin, other proteins having an affinity for biotin including derivatized forms of avidin and recombinant forms of any of the above. The avidin should be insoluble, which is preferred, or otherwise easily separable from the inert support using techniques such as emulsion/phase separation and the like. The inert support material is any material that has low reactivity, is hydrophilic, forms a porous or nonporous matrix which can be readily separated from a liquid phase of the reaction mixture when necessary. Such materials include, but are not limited to, dextran, cellulose, starch, carageenan, chitin, polyacrylamide, hydroxyethylmethacrylate, stryrenedivinylbenzene, oxiraneacrylic, silica, alumina, zirconia, glass, perfluorocarbons and polysaccharides from the agar family or the alginate family which form gel beads. These and other such materials are useful and well known as inert materials in separation columns. Most are commercially available, e.g., agarose which is preferred and which is available as Sepharose=' from Pharmacia.
The invention will be further described referring to avidin and agarose. It should be understood that avidin is meant to include any of the forms of avidin described hereinabove and agarose represents any of the inert support materials described hereinabove.
The bulking agent is a nonionic water soluble polymer.
Examples include, but are not limited to, simple sugars (e.g., mono- and di-saccharides), oligosaccharides, polysaccharides, polyvinylpyrrolidone, polyvinylalcohol or polyethyleneglycol.
Preferably, the bulking agent is a sugar ranging in molecular weight from that of glucose up to and including that of high molecular weight dextran. More preferably, the bulking agent is an oligosaccharide based on glucose such as dimeric glucose (i.e., maltose), trimeric glucose (maltotriose), maltotetraose, maltopentaose, maltohexaose, maltoheptaose, low molecular weight dextran, high molecular weight dextran including combinations of any of the above, with maltose being preferred.
= - 5 -The protectant of the present compositions is selected from antioxidants, free radical scavengers and reducing agents.
Preferred are antioxidants such as a-tocopherol, reduced glutathione, quinones, N, N-dimethyl-p-phenylenediamine, a-scorbylpalmitate, amino acids, tartaric acid, phosphoric acid and ascorbic acid/sodium ascorbate with ascorbic acid/sodium, ascorbate being most preferred.
The present compositions may also include an agent to adjust the pH to a desired level. For example, alkaline materials, e.g., sodium hydroxide can be added to adjust the pH which is preferably at about 4 for use with biotinylated batroxobin.
Further, buffers may be incorporated to maintain the pH level.
Buffers and agents to adjust the pH are well known in the art and any such materials are suitable depending upon the application.
In a preferred embodiment, the ascorbic acid protectant also serves as a buffer. However, it should be understood that any convenient buffer and pH can be utilized as required for the particular application.
The compositions of the present invention are conveniently in an aqueous slurry or suspension. Since these slurries or suspensions can either be prepared aseptically or can be terminally sterilized, they are also an integral part of this invention. Preferably, the aqueous slurries or suspensions of this invention are freeze dried since the lyophilized powders resulting therefrom are highly stable, terminally sterilizable (e.g., by gamma irradiation), non-hygroscopic and extremely easy to handle.
As the compositions of this invention deal, inter alia, with beaded gels in slurries or suspensions, it is important to clarify what some of these terms are understood to mean within this art. By way of example, agarose is commercially available in 4% and 6% gels. This refers to the fact that the hydrated gel bead material is, for example, 496 by weight of cross-linked agarose beads containing 96% by weight of water (i.e., within the bead) for the 4% gel and 6% by weight of cross-linked agarose beads containing 94% by weight of water for the 6% gel. The beads can be any convenient size and size range as are known and available in the art. The 4% and 6% gels available above typically comprise beads having a diameter range between 60 and 120 microns.
These gel beads, in turn, can be utilized in several forms.
For example, a "wet settled gel" is obtained when the gel beads in water are allowed to settle out under gravity, i.e., by draining off most of the water, leaving only the hydrated gel beads and interstitial water, i.e., water between the beads.
This typically results in a wet settled gel comprising 70-80%
hydrated bead volume and 20-30% interstitial water volume, preferably about 75% by volume of hydrated beads about 25% by volume of interstitial water. The wet settled gel form is convenient to use in processing because the material is mostly water providing a density close to 1. This, in turn, provides flexibility in relatively accurate measuring either by weight or volume, especially when using larger quantities, i.e., 10 grams and above.
A "moist" or "sucked" gel comprises the hydrated gel beads with the interstitial water removed and is a more accurate way to measure smaller amounts of gel.
In the processing discussed below and in the Examples which follow, the agarose gel, agarose gel beads, agarose or agarose beads refers to a wet settled gel unless otherwise noted.
The aqueous composition of the present invention preferably comprises avidin agarose gel beads (wet settled gel) in a slurry or suspension with a solution comprising:
1 to 50% by weight of the bulking agent;
0.01 to 50% by weight of the protectant; and 40 to 98.99% by weight of water.
More preferably, the aqueous composition according to the present invention comprises avidin agarose gel beads in a slurry or suspension with a solution comprising:
to 40% by weight and most preferably 10% by weight of a bulking agent, preferably a sugar, more preferably maltose;
0.1 to 10% by weight and most preferably 1% by weight of a protectant, preferably an antioxidant, more preferably ascorbic acid; and 50 to 94.9% by weight of water;
and optionally, in a preferred embodiment, further including:
an agent sufficient to adjust the pH to a desired level, preferably an alkaline material, e.g., sodium hydroxide to adjust the pH to about 4; and a buffer, which is preferably the ascorbic acid protectant.
Typically the slurry or suspension comprises about 10 to ,~ .
about 70% by volume of wet settled gel beads in about 30 to 90ic of one of the above "protectant" solutions, it being understood that compositions having 10% beads are in a suspension whereas those compositions having 70V beads are in the form of a slurry or even a gel.
in order to immobilize the avidin to a support, e.g., agarose, the support must be pre-activated prior to avidin coupling. A preferred process involves the use of epichlorohydrin as the activating agent, however, activation can be carried out by any suitable technique capable of providing an activated support which can form covalent bonds with avidin.
For example, various activation reagents available for derivatizing supports are: diazonium groups, isocyanate groups, acid chloride groups, acid anhydride groups, sulfonyi chloride groups, dinitro fluorophenyl groups, isothiocyanate groups, hydroxyl groups, amino groups, n-hydroxysuccinmide groups, triazine groups, hydrazide groups, carbodiimide groups, silane groups, aldehydes, 1, 4-butanediol diglycidyl ether, sodium metaperiodate, 1, 1-carbonyl diimidazole, divinylsulphone, 2-fluoro-1-methylpyridinium toluene-4-sulphonate and cyanogen bromide. See (a) Pentapharm Patent DT 2440 254 A1; (b) P.D.G.
Dean, W.S. Johnson and F.A. Middle (Editors) (1991) IRL Press Oxford - Affinity Chromatography - A practical approach - chapter 2 - Activation Procedures and (c) C.R. Lowe and P.D.G. Dean (1974) John Wiley and Sons Ltd., London, Affinity Chromatograph,y.
The preferred activation chemistry is by means of an epoxide group following activation with epichlorohydrin. The use of a support activated in this manner results in essentially no avidin leaching after-avidin bonding.
WO 97/17436 PC'T/US96/17268 Generally, the support is activated by a highly reactive compound, which subsequently reacts with a functional group of the ligand, e.g., -OH, -NHa1 -SH, -CHO, to form a covalent linkage. Remaining active groups, which have no avidin attached, can be, but it is not essential, blocked with compounds such as ethanolamine, acetic anhydride or glycine.
The preferred activation chemistries for use in the subject matter invention are:
(a) Activation of the support by epichlorohydrin or a bifunctional epoxide compound followed by coupling avidin via -NH2, -SH or -OH groups.
(b) Cyanogen bromide activation followed by direct coupling of avidin via -NH2 groups on the protein.
(c) Activation of the support with monochlorotriazine followed by coupling of avidin via -NHz, -OH or -SH
groups.
(d) Activation of the support with dichlorotriazine followed by coupling of avidin via -NHZ, -OH or -SH
groups.
(e) Tresyl chloride activation of the support followed by coupling of avidin via -NH21 -OH or -SH groups.
(f) Activation of the support with adipic acid hydrazide or hydrazide followed by coupling of oxidized avidin via -CHO groups.
(g) Activation of the support with an amino ligand followed by coupling of oxidized avidin via -CHO groups.
All the above preferred methodologies employ agarose as the support, however, it is possible to use other aforementioned supports as well- For example, when using silica, the preferred activation chemistries are:
(a) Activation of the support by epichlorohydrin or a bifunctional epoxide compound followed by coupling avidin. via -NH21 -SH or -OH groups.
(b) Gamma - glycidoxypropyltrimethoxysilane activation with direct coupling of the avidin via -NHa groups on the protein.
(c) Cyanogen bromide activation followed by direct coupling of avidin via -NHZ groups on the protein.
(d) Gamma - glycidoxytrimethoxysilane activation followed by opening of the epoxide ring to form a diol group, which can be subsequently activated with cyanogen bromide. Direct coupling of the avidin can be achieved via -NH2 groups on the protein.
(e) Gamma - giycidoxypropyltrimethoxysilane activation followed by preparation of amino-silica by treatment with ammonia solution.
The amino-silica can be subsequently activated with cyanuric chloride (triazine) and the avidin coupled via -NHa, -OH or -SH
groups.
= Coupling of the avidin to the activated support must be buffered at a certain pH to obtain optimal avidin binding.
Generally, with standard activation techniques such as gamma -glycidoxypropyltrimethoxysilane coupling of avidin to activated support and cyanogen bromide coupling of any protein to active groups requires buffering at a pH 1-2 units higher than the pKa of the primary and secondary amines of the avidin. However, the use of cyanuric chloride as the activator enables the use of much lower pH buffers (optimal coupling pH is 4-6). Another method of coupling avidin to an inert support is via its carbohydrate moieties. This involves first the oxidation of the sugar group to -CHO groups followed by direct coupling a acid pH to an amino group such as hydrazide. A wide range of coupling buffers can be used. See, for example, Table 1.
O
Ab w EXAMPLES OF COUPLING BUFFERS USED IN AVIDIN IMMOBILIZATION TO SILICA AND
AGAROSE
SUPPORTS
SUPPORT ACTIVATION METHOD COUPLING BUFFER
Silica gammaglycidoxypropyltrimethoxysilane 0.1M Sodium bicarbonate pH 8-9 10mM
HEPES pH 7.0 >
Silica Y-glycidoxypropyltrimethoxysaline + 0.1M Sodium bicarbonate pH 8-9 10mM
cyanogen bromide HEPES pH 7.0 Silica Cyanogen Bromide Water pH 7.0 0.1M Sodium bi-carbonate pH 7-9 10mM HEPES pH 7.0 Agarose Monochlorotriazine 50 mM Sodium Acetate/1MNaC1 pH 4.0 Agarose Dichlorotriazine 0.1M Potassium phosphate/1MNaC1 pH
8.0-9.0 Agarose Tresyl chloride 50 mM Potassium phosphate/0.5M NaCl pH 7.7 Agarose Hydrazide 50 mM Sodium Acetate pH 5.5 10 mM
NaBH, Agarose Amine 50 mM Sodium Acetate pH 5.5 10 mM
NaBH, Agarose Epoxide 20mM Sodium Bicarbonate/0.5M NaCl pH 10.0 OI
As described previously, these "high activity" compositions of avidin immobilized on an inert support, e.g., agarose, are useful in any and all chemical and biological applications where present avidin technology is useful. In a preferred embodiment, the avidin immobilized onto agarose is thereafter incorporated into composition of this invention conveniently by mixing the various components in water or by mixing the "protectant"
solution components in water and thereafter adding the avidin agarose gel. For situations requiring a sterile aqueous composition this can be carried out aseptically or preservatives can be added to the composition. Preferably, the aqueous composition is lyophilized into a powder form which can be terminally sterilized, e.g., by gamma irradiation. Any convenient freeze-drying process can be employed. A preferred process involves cooling the aqueous composition in a lyophilization apparatus to about -33 C and maintaining this while a vacuum is initiated and the composition is dried under a reduced pressure of about 0.3mbar. Thereafter, the composition is allowed to warm to room temperature.
The compositions of this invention involving the use of stable avidin compositions for the capture of a biotinylated form of thrombin or a thrombin-like enzyme, e.g., Batroxobin, are useful in methods to convert fibrinogen, or a fibrinogen-containing composition, into fibrin monomer, or a fibrin monomer-containing composition. Accordingly, the present invention further includes a novel method, to prepare a fibrin monomer useful, for example, in preparing a fibrin sealant. This novel method involves subjecting a source of fibrinogen to a biotinylated thrombin or thrombin-like enzyme composition to convert fibrinogen into fibrin monomer, "capturing" the biotinylated enzyme with an avidin composition of this invention to form a biotin/avidin complex, and removing the enzyme which is a part of the so-formed biotin/avidin complex.
The compositions of the present invention can further be incorporated into a processing unit, e.g., an automated centrifuge for preparing fibrin monomer as defined above. The avidin agarose composition can be preloaded into the processing unit in powder form or can be lyophilized in situ in the device or in a controlled release compartment of the device.
Approximately 3.5 liters of beaded agarose gel (grade 4XL
commercially available as Sepharose CL-4BT"' from Pharmacia Co.) was gravity settled in a filter funnel. Approximately 2.8 liters of the so-settled agarose gel was transferred to a reaction vessel. The agarose gel was washed 12 times with 2.8 liter volumes of water. The so-washed gel was thereafter mixed with 2016 milliliters of 0-11 Molar sodium hydroxide and then reacted with 202 milliliters of epichlorohydrin for about 3 hours while maintaining 40 C. This activated gel was then washed with water and thereafter coupled to 19.6 grams of avidin in the presence of a sodium chloride/sodium bicarbonate pH 10 buffer at about 40 C
for 48 hours. The avidin-agarose gel was thereafter washed several times with sodium chloride solution and any unreacted epoxide groups were blocked by treatment with 1M ethanolamine (pH
9.5) for 16 hours at 20 C. The avidin-agarose gel was next washed with water and thereafter mixed with an equal volume (28L) of a solution containing maltose (209,; w/v) and ascorbic acid (2%-w/v) at pH 4.0 and allowed to drain under gravity.
The end-product provided by the method of Example 1, above, was placed on trays and loaded into a EF6(S) lyophilization apparatus (available from Edwards High Vacuum Co.) in which the shelves had been pre-cooled to -37 C. The avidin agarose slurry was cooled to -33 C and the pressure was reduced (by vacuum) to 0.3 millibars. The product was maintained at this pressure and temperature until all of the ice had sublimed (about 70 hours).
The pressure was then adjusted to 0.08 millibars and the temperature was raised stepwise 5 C per hour to 30 C to provide the lyophilized.product.
METHODS USING SAME
BACKGROUND
The avidin-biotin affinity-based technology has found wide applicability in numerous fields of biology and biotechnology since the pioneering work by Dr. Edward Bayer and Dr. Meier Wilchek in the 1970's. The affinity constant between avidin and biotin is remarkably high and is not significantly lessened when biotin is coupled to a wide variety of biomolecules. This affinity is substantially maintained even when derivatized forms of the biotin are employed and numerous chemistries have been identified for coupling biomolecules to biotin with minimal or negligible loss in the activity or other desired characteristics of the biomolecule. Originally applied to purification and localization procedures for biologically active macromolecules, avidin-biotin technology today has widespread use in medical diagnostics. Newer applications which continue to be developed include affinity targeting, cell cytometry, blotting technology, drug delivery, hybridoma technology, human stem cell selection and reinfusion as well as several approaches to enzyme capture.
In some applications, avidin is immobilized onto an inert material over which a solution containing biotinylated biomolecules is passed. The affinity of the biotin for the avidin provides for the separation of the biomolecule from the solution. A review of the biotin-avidin technology can be found in Applications of Avidin-Biotin Technology to Affinity-Based Separation, Bayer, et al., J. of Chromatography, 1990, pgs. 3-11.
EP S92242 describes a novel fibrin sealant based on fibrin = monomer as opposed to the traditional fibrinogen-based sealants and involves subjecting fibrinogen to a thrombin-like enzyme which is preferably removed after such treatment. EP 592242 describes that the enzyme capture and removal can be accomplished by using biotinylated batroxobin which can be recaptured with an avidin material. The fibrin monomer sealant described in EP =
592242 is advantageously completely autologous. Since autologous fibrin sealants can not always be prepared in advance, autologous processes which provide such sealants in short periods of time (i.e., less than one hour or preferably less than 30 minutes) fr_om the patients' own blood provide a great advantage over current techniques and products. The speed with which such autologous processes can be carried out is dependent to a large degree on the activity of the biotin-and avidin-based reagents.
Commercially available immobilized avidin typically contains about 200 to 400 biotin binding units (BBU) of activity (where 1BBU will bind 1 E.cg of a-biotin) per gram of lyophilized powder (e.g., avidin on acrylic beads from Sigma) or about 20 to 50 BBU
per milliliter of slurry or gel (e.g., avidin on agarose available from Sigma and Pierce).
Also, the above fibrin monomer technology and other biological applications would benefit from more convenient forms of avidin-and biotin-based reagents. For example, the processing necessary to prepare such compositions can have an adverse effect on the activity levels since many of the coupling/immobilization techniques involve materials which can significantly reduce these activities. Additionally, systems which reduce or eliminate le.aching of avidin or of the avidin-biotin complexes would be advantageous in many applications. Further, many biological applications would be greatly enhanced by the availability of high activity avidin compositions which could be lyophilized and further, terminally sterilized while maintaining stability.
Clearly, avidin compositions having higher avidin activity levels with greater stability, especially in freeze dried powder forms capable of withstanding terminal sterilization, e.g., gamma irradiation, would be an advance in the art.
SUMMARY OF THE INVENTION
In accordance with the present invention, stable, highly active compositions of avidin and an inert, easily separable support material such as a water soluble polymer, e.g., polysaccharides selected from agars and alginates, and having an activity level of 1000 BBU or more per gram of lyophilized form and 50 BBU or more per milliliter of slurry or hydrated gel, are disclosed. Preferred compositions include a bulking agent selected from nonionic water soluble polymers, a protectant, and the avidin/inert support material. These compositions may also include one or more materials selected to adjust and/or maintain the pH of the composition and are useful in an aqueous suspension or preferably in lyophilized form.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Although avidin/inert support compositions are known, such compositions having the high levels of activity as described in this invention have not been heretofore disclosed. It has surprisingly been found that avidin compositions can now be prepared having activity levels multiples beyond those presently available without damaging the integrity and stability of the avidin or its inert support. Indeed, these compositions in any convenient form, i.e., slurry, suspension, hydrated gel, dehydrated gel, dried powders, etc., can be the basis of sterile, stable aqueous suspensions and remarkably stable lyophilized compositions capable of withstanding terminal sterilization e.g., gamma irradiation. The novel compositions herein have ~ activity levels for lyophilized forms in excess of 1000 BBU
(which is a measure of the biotin binding capability of the ~ - 3 -avidin composition as discussed above) and preferably between 1500 and 3000 BBU of activity and most preferably between 1800 and 2400 BBU of activity per gram of powder. In slurry, suspension or gel forms the novel avidin compositions of the present invention have activity levels in excess of S0 BBU and preferably between 7S and 150 BBU and more preferably between 90 and 110 BBU of activity per milliliter.
The preferred lyophilized avidin/inert support compositions of the present invention are stable, can be terminally sterilized, have low moisture uptake/moisture content, reswell completely and rapidly upon reconstitution, are non-leachable and pharmaceutically acceptable. The bulking agents used in conjunction with this invention protect the gel bead from damage during the freeze drying process and provide for rapid and complete reswelling of the gel beads upon reconstitution of the freeze dried material. The unique combination of components herein also protects the avidin/inert support component from any deleterious effects upon terminal sterilization of the composition. Thus, the affinity of the avidin for biotinylated biomolecules is maintained even after these rigorous processing steps, i.e., lyophilization and/or terminal sterilization.
Accordingly, in the various applications where the affinity characteristics of the avidin and the integrity of the inert support are both critical to the effectiveness of the avidin/biotin technology, a superior composition is provided by the invention.
The avidin component of the present invention refers to avidin, monomeric avidin, Streptavidin, other proteins having an affinity for biotin including derivatized forms of avidin and recombinant forms of any of the above. The avidin should be insoluble, which is preferred, or otherwise easily separable from the inert support using techniques such as emulsion/phase separation and the like. The inert support material is any material that has low reactivity, is hydrophilic, forms a porous or nonporous matrix which can be readily separated from a liquid phase of the reaction mixture when necessary. Such materials include, but are not limited to, dextran, cellulose, starch, carageenan, chitin, polyacrylamide, hydroxyethylmethacrylate, stryrenedivinylbenzene, oxiraneacrylic, silica, alumina, zirconia, glass, perfluorocarbons and polysaccharides from the agar family or the alginate family which form gel beads. These and other such materials are useful and well known as inert materials in separation columns. Most are commercially available, e.g., agarose which is preferred and which is available as Sepharose=' from Pharmacia.
The invention will be further described referring to avidin and agarose. It should be understood that avidin is meant to include any of the forms of avidin described hereinabove and agarose represents any of the inert support materials described hereinabove.
The bulking agent is a nonionic water soluble polymer.
Examples include, but are not limited to, simple sugars (e.g., mono- and di-saccharides), oligosaccharides, polysaccharides, polyvinylpyrrolidone, polyvinylalcohol or polyethyleneglycol.
Preferably, the bulking agent is a sugar ranging in molecular weight from that of glucose up to and including that of high molecular weight dextran. More preferably, the bulking agent is an oligosaccharide based on glucose such as dimeric glucose (i.e., maltose), trimeric glucose (maltotriose), maltotetraose, maltopentaose, maltohexaose, maltoheptaose, low molecular weight dextran, high molecular weight dextran including combinations of any of the above, with maltose being preferred.
= - 5 -The protectant of the present compositions is selected from antioxidants, free radical scavengers and reducing agents.
Preferred are antioxidants such as a-tocopherol, reduced glutathione, quinones, N, N-dimethyl-p-phenylenediamine, a-scorbylpalmitate, amino acids, tartaric acid, phosphoric acid and ascorbic acid/sodium ascorbate with ascorbic acid/sodium, ascorbate being most preferred.
The present compositions may also include an agent to adjust the pH to a desired level. For example, alkaline materials, e.g., sodium hydroxide can be added to adjust the pH which is preferably at about 4 for use with biotinylated batroxobin.
Further, buffers may be incorporated to maintain the pH level.
Buffers and agents to adjust the pH are well known in the art and any such materials are suitable depending upon the application.
In a preferred embodiment, the ascorbic acid protectant also serves as a buffer. However, it should be understood that any convenient buffer and pH can be utilized as required for the particular application.
The compositions of the present invention are conveniently in an aqueous slurry or suspension. Since these slurries or suspensions can either be prepared aseptically or can be terminally sterilized, they are also an integral part of this invention. Preferably, the aqueous slurries or suspensions of this invention are freeze dried since the lyophilized powders resulting therefrom are highly stable, terminally sterilizable (e.g., by gamma irradiation), non-hygroscopic and extremely easy to handle.
As the compositions of this invention deal, inter alia, with beaded gels in slurries or suspensions, it is important to clarify what some of these terms are understood to mean within this art. By way of example, agarose is commercially available in 4% and 6% gels. This refers to the fact that the hydrated gel bead material is, for example, 496 by weight of cross-linked agarose beads containing 96% by weight of water (i.e., within the bead) for the 4% gel and 6% by weight of cross-linked agarose beads containing 94% by weight of water for the 6% gel. The beads can be any convenient size and size range as are known and available in the art. The 4% and 6% gels available above typically comprise beads having a diameter range between 60 and 120 microns.
These gel beads, in turn, can be utilized in several forms.
For example, a "wet settled gel" is obtained when the gel beads in water are allowed to settle out under gravity, i.e., by draining off most of the water, leaving only the hydrated gel beads and interstitial water, i.e., water between the beads.
This typically results in a wet settled gel comprising 70-80%
hydrated bead volume and 20-30% interstitial water volume, preferably about 75% by volume of hydrated beads about 25% by volume of interstitial water. The wet settled gel form is convenient to use in processing because the material is mostly water providing a density close to 1. This, in turn, provides flexibility in relatively accurate measuring either by weight or volume, especially when using larger quantities, i.e., 10 grams and above.
A "moist" or "sucked" gel comprises the hydrated gel beads with the interstitial water removed and is a more accurate way to measure smaller amounts of gel.
In the processing discussed below and in the Examples which follow, the agarose gel, agarose gel beads, agarose or agarose beads refers to a wet settled gel unless otherwise noted.
The aqueous composition of the present invention preferably comprises avidin agarose gel beads (wet settled gel) in a slurry or suspension with a solution comprising:
1 to 50% by weight of the bulking agent;
0.01 to 50% by weight of the protectant; and 40 to 98.99% by weight of water.
More preferably, the aqueous composition according to the present invention comprises avidin agarose gel beads in a slurry or suspension with a solution comprising:
to 40% by weight and most preferably 10% by weight of a bulking agent, preferably a sugar, more preferably maltose;
0.1 to 10% by weight and most preferably 1% by weight of a protectant, preferably an antioxidant, more preferably ascorbic acid; and 50 to 94.9% by weight of water;
and optionally, in a preferred embodiment, further including:
an agent sufficient to adjust the pH to a desired level, preferably an alkaline material, e.g., sodium hydroxide to adjust the pH to about 4; and a buffer, which is preferably the ascorbic acid protectant.
Typically the slurry or suspension comprises about 10 to ,~ .
about 70% by volume of wet settled gel beads in about 30 to 90ic of one of the above "protectant" solutions, it being understood that compositions having 10% beads are in a suspension whereas those compositions having 70V beads are in the form of a slurry or even a gel.
in order to immobilize the avidin to a support, e.g., agarose, the support must be pre-activated prior to avidin coupling. A preferred process involves the use of epichlorohydrin as the activating agent, however, activation can be carried out by any suitable technique capable of providing an activated support which can form covalent bonds with avidin.
For example, various activation reagents available for derivatizing supports are: diazonium groups, isocyanate groups, acid chloride groups, acid anhydride groups, sulfonyi chloride groups, dinitro fluorophenyl groups, isothiocyanate groups, hydroxyl groups, amino groups, n-hydroxysuccinmide groups, triazine groups, hydrazide groups, carbodiimide groups, silane groups, aldehydes, 1, 4-butanediol diglycidyl ether, sodium metaperiodate, 1, 1-carbonyl diimidazole, divinylsulphone, 2-fluoro-1-methylpyridinium toluene-4-sulphonate and cyanogen bromide. See (a) Pentapharm Patent DT 2440 254 A1; (b) P.D.G.
Dean, W.S. Johnson and F.A. Middle (Editors) (1991) IRL Press Oxford - Affinity Chromatography - A practical approach - chapter 2 - Activation Procedures and (c) C.R. Lowe and P.D.G. Dean (1974) John Wiley and Sons Ltd., London, Affinity Chromatograph,y.
The preferred activation chemistry is by means of an epoxide group following activation with epichlorohydrin. The use of a support activated in this manner results in essentially no avidin leaching after-avidin bonding.
WO 97/17436 PC'T/US96/17268 Generally, the support is activated by a highly reactive compound, which subsequently reacts with a functional group of the ligand, e.g., -OH, -NHa1 -SH, -CHO, to form a covalent linkage. Remaining active groups, which have no avidin attached, can be, but it is not essential, blocked with compounds such as ethanolamine, acetic anhydride or glycine.
The preferred activation chemistries for use in the subject matter invention are:
(a) Activation of the support by epichlorohydrin or a bifunctional epoxide compound followed by coupling avidin via -NH2, -SH or -OH groups.
(b) Cyanogen bromide activation followed by direct coupling of avidin via -NH2 groups on the protein.
(c) Activation of the support with monochlorotriazine followed by coupling of avidin via -NHz, -OH or -SH
groups.
(d) Activation of the support with dichlorotriazine followed by coupling of avidin via -NHZ, -OH or -SH
groups.
(e) Tresyl chloride activation of the support followed by coupling of avidin via -NH21 -OH or -SH groups.
(f) Activation of the support with adipic acid hydrazide or hydrazide followed by coupling of oxidized avidin via -CHO groups.
(g) Activation of the support with an amino ligand followed by coupling of oxidized avidin via -CHO groups.
All the above preferred methodologies employ agarose as the support, however, it is possible to use other aforementioned supports as well- For example, when using silica, the preferred activation chemistries are:
(a) Activation of the support by epichlorohydrin or a bifunctional epoxide compound followed by coupling avidin. via -NH21 -SH or -OH groups.
(b) Gamma - glycidoxypropyltrimethoxysilane activation with direct coupling of the avidin via -NHa groups on the protein.
(c) Cyanogen bromide activation followed by direct coupling of avidin via -NHZ groups on the protein.
(d) Gamma - glycidoxytrimethoxysilane activation followed by opening of the epoxide ring to form a diol group, which can be subsequently activated with cyanogen bromide. Direct coupling of the avidin can be achieved via -NH2 groups on the protein.
(e) Gamma - giycidoxypropyltrimethoxysilane activation followed by preparation of amino-silica by treatment with ammonia solution.
The amino-silica can be subsequently activated with cyanuric chloride (triazine) and the avidin coupled via -NHa, -OH or -SH
groups.
= Coupling of the avidin to the activated support must be buffered at a certain pH to obtain optimal avidin binding.
Generally, with standard activation techniques such as gamma -glycidoxypropyltrimethoxysilane coupling of avidin to activated support and cyanogen bromide coupling of any protein to active groups requires buffering at a pH 1-2 units higher than the pKa of the primary and secondary amines of the avidin. However, the use of cyanuric chloride as the activator enables the use of much lower pH buffers (optimal coupling pH is 4-6). Another method of coupling avidin to an inert support is via its carbohydrate moieties. This involves first the oxidation of the sugar group to -CHO groups followed by direct coupling a acid pH to an amino group such as hydrazide. A wide range of coupling buffers can be used. See, for example, Table 1.
O
Ab w EXAMPLES OF COUPLING BUFFERS USED IN AVIDIN IMMOBILIZATION TO SILICA AND
AGAROSE
SUPPORTS
SUPPORT ACTIVATION METHOD COUPLING BUFFER
Silica gammaglycidoxypropyltrimethoxysilane 0.1M Sodium bicarbonate pH 8-9 10mM
HEPES pH 7.0 >
Silica Y-glycidoxypropyltrimethoxysaline + 0.1M Sodium bicarbonate pH 8-9 10mM
cyanogen bromide HEPES pH 7.0 Silica Cyanogen Bromide Water pH 7.0 0.1M Sodium bi-carbonate pH 7-9 10mM HEPES pH 7.0 Agarose Monochlorotriazine 50 mM Sodium Acetate/1MNaC1 pH 4.0 Agarose Dichlorotriazine 0.1M Potassium phosphate/1MNaC1 pH
8.0-9.0 Agarose Tresyl chloride 50 mM Potassium phosphate/0.5M NaCl pH 7.7 Agarose Hydrazide 50 mM Sodium Acetate pH 5.5 10 mM
NaBH, Agarose Amine 50 mM Sodium Acetate pH 5.5 10 mM
NaBH, Agarose Epoxide 20mM Sodium Bicarbonate/0.5M NaCl pH 10.0 OI
As described previously, these "high activity" compositions of avidin immobilized on an inert support, e.g., agarose, are useful in any and all chemical and biological applications where present avidin technology is useful. In a preferred embodiment, the avidin immobilized onto agarose is thereafter incorporated into composition of this invention conveniently by mixing the various components in water or by mixing the "protectant"
solution components in water and thereafter adding the avidin agarose gel. For situations requiring a sterile aqueous composition this can be carried out aseptically or preservatives can be added to the composition. Preferably, the aqueous composition is lyophilized into a powder form which can be terminally sterilized, e.g., by gamma irradiation. Any convenient freeze-drying process can be employed. A preferred process involves cooling the aqueous composition in a lyophilization apparatus to about -33 C and maintaining this while a vacuum is initiated and the composition is dried under a reduced pressure of about 0.3mbar. Thereafter, the composition is allowed to warm to room temperature.
The compositions of this invention involving the use of stable avidin compositions for the capture of a biotinylated form of thrombin or a thrombin-like enzyme, e.g., Batroxobin, are useful in methods to convert fibrinogen, or a fibrinogen-containing composition, into fibrin monomer, or a fibrin monomer-containing composition. Accordingly, the present invention further includes a novel method, to prepare a fibrin monomer useful, for example, in preparing a fibrin sealant. This novel method involves subjecting a source of fibrinogen to a biotinylated thrombin or thrombin-like enzyme composition to convert fibrinogen into fibrin monomer, "capturing" the biotinylated enzyme with an avidin composition of this invention to form a biotin/avidin complex, and removing the enzyme which is a part of the so-formed biotin/avidin complex.
The compositions of the present invention can further be incorporated into a processing unit, e.g., an automated centrifuge for preparing fibrin monomer as defined above. The avidin agarose composition can be preloaded into the processing unit in powder form or can be lyophilized in situ in the device or in a controlled release compartment of the device.
Approximately 3.5 liters of beaded agarose gel (grade 4XL
commercially available as Sepharose CL-4BT"' from Pharmacia Co.) was gravity settled in a filter funnel. Approximately 2.8 liters of the so-settled agarose gel was transferred to a reaction vessel. The agarose gel was washed 12 times with 2.8 liter volumes of water. The so-washed gel was thereafter mixed with 2016 milliliters of 0-11 Molar sodium hydroxide and then reacted with 202 milliliters of epichlorohydrin for about 3 hours while maintaining 40 C. This activated gel was then washed with water and thereafter coupled to 19.6 grams of avidin in the presence of a sodium chloride/sodium bicarbonate pH 10 buffer at about 40 C
for 48 hours. The avidin-agarose gel was thereafter washed several times with sodium chloride solution and any unreacted epoxide groups were blocked by treatment with 1M ethanolamine (pH
9.5) for 16 hours at 20 C. The avidin-agarose gel was next washed with water and thereafter mixed with an equal volume (28L) of a solution containing maltose (209,; w/v) and ascorbic acid (2%-w/v) at pH 4.0 and allowed to drain under gravity.
The end-product provided by the method of Example 1, above, was placed on trays and loaded into a EF6(S) lyophilization apparatus (available from Edwards High Vacuum Co.) in which the shelves had been pre-cooled to -37 C. The avidin agarose slurry was cooled to -33 C and the pressure was reduced (by vacuum) to 0.3 millibars. The product was maintained at this pressure and temperature until all of the ice had sublimed (about 70 hours).
The pressure was then adjusted to 0.08 millibars and the temperature was raised stepwise 5 C per hour to 30 C to provide the lyophilized.product.
Claims (33)
1. A composition in the form of a solid or powder comprising:
an inert support material that is a substantially inert hydrophilic material capable of forming a polymer matrix and which is separable from a liquid phase; and avidin immobilized on said inert support material wherein said composition has at least 1000 biotin binding units of activity per gram of composition.
an inert support material that is a substantially inert hydrophilic material capable of forming a polymer matrix and which is separable from a liquid phase; and avidin immobilized on said inert support material wherein said composition has at least 1000 biotin binding units of activity per gram of composition.
2. The composition of claim 1 having between 1500 and 3000 units of biotin binding activity per gram.
3. The composition of claim 1 having between 1800 and 2400 units of biotin binding activity per gram.
4. A composition in the form of a gel, slurry or suspension comprising:
a liquid carrier;
an inert support material that is a substantially inert hydrophilic material capable of forming a polymer matrix and which is separable from a liquid phase; and avidin immobilized on said inert support material wherein said composition has at least 50 units of biotin binding activity per milliliter of composition.
a liquid carrier;
an inert support material that is a substantially inert hydrophilic material capable of forming a polymer matrix and which is separable from a liquid phase; and avidin immobilized on said inert support material wherein said composition has at least 50 units of biotin binding activity per milliliter of composition.
5. The composition of claim 4 having between 75 and 150 units of biotin binding activity per milliliter.
6. The composition of claim 4 having between 90 and 110 units of biotin binding activity per milliliter.
7. A stable composition comprising:
an inert support material that is a substantially inert hydrophylic material capable of forming a polymer matrix and which is separable from a liquid phase;
avidin immobilized on said inert support material;
a bulking agent selected from simple sugars, oligosaccharides, polysaccharides, polyvinylpyrrolidone, polyvinylalcohol and polyethyleneglycol; and a protectant.
an inert support material that is a substantially inert hydrophylic material capable of forming a polymer matrix and which is separable from a liquid phase;
avidin immobilized on said inert support material;
a bulking agent selected from simple sugars, oligosaccharides, polysaccharides, polyvinylpyrrolidone, polyvinylalcohol and polyethyleneglycol; and a protectant.
8. The composition of claim 7 in the form of an aqueous slurry or suspension.
9. The composition of claim 7 optionally including an agent to adjust the pH
of the composition to a desired level.
of the composition to a desired level.
10. The composition of claim 7 optionally including a buffer agent to maintain the desired pH
of the composition.
of the composition.
11. The composition of claim 10 wherein said protectant also functions as a buffer.
12. The composition of claim 7 wherein avidin is monomeric avidin, Streptavidin, an avidin derivative, or recombinant forms of any of these.
13. The composition of claim 7 wherein said inert material is selected from dextran, cellulose, starch, carageenan, chitin, polyacrylamide, hydroxyethylmethacrylate, stryrenedivinylbenzene, oxiraneacrylic, silica, alumina, zirconia, glass, perfluorocarbons and polysaccharides from agar or alginate families.
14. The composition of claim 7 wherein said inert material is a polysaccharide selected from agars and alginates.
15. The composition of claim 13 wherein said inert material is agarose.
16. The composition of claim 15 wherein said agarose is in the form of gel beads.
17. The composition of claim 7 wherein said simple sugars are selected from monosaccharides and disaccharides.
18. The composition of claim 17 wherein said bulking agent is a sugar ranging in molecular weight from that of glucose up to and including that of high molecular weight dextran.
19. The composition of claim 18 wherein said bulking agent is an oligosaccharide based on glucose.
20. The composition of claim 19 wherein said bulking agent is selected from dimeric glucose, trimeric glucose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, low molecular weight dextran, high molecular weight dextran, and combinations of any of the above.
21. The composition of claim 7 wherein said protectant is selected from antioxidants, free radical scavengers and reducing agents.
22. The composition of claim 21 wherein said protectant is an antioxidant selected from .alpha.-tocopherlol, reduced glutathione, quinones, N, N-dimethyl-p-phenylenediamine, ascorbylpalmitate, amino acids, tartaric acid, phosphoric acid and ascorbic acid/sodium ascorbate.
23. The composition of claim 9 including an alkaline material to adjust the pH
to about 4.
to about 4.
24. The composition of claim 23 wherein said alkaline material is sodium hydroxide.
25. The composition of claim 11 wherien the buffer/protectant is ascorbic acid.
26. The aqueous composition of claim 8 comprising avidin agarose gel particles in a slurry or suspension with a solution comprising:
2 to 50% by weight of a bulking agent;
0.01 to 50% by weight of a protectant; and 40 to 98.99% by weight of water; and optionally, materials to adjust and/or maintain a desired pH.
2 to 50% by weight of a bulking agent;
0.01 to 50% by weight of a protectant; and 40 to 98.99% by weight of water; and optionally, materials to adjust and/or maintain a desired pH.
27. The composition of claim 26 comprising avidin agarose gel particles in a slurry or suspension with a solution comprising:
to 40% by weight of said bulking agent;
0.1 to 10% by weight of said protectant; and 50 to 94.9% by weight of water.
to 40% by weight of said bulking agent;
0.1 to 10% by weight of said protectant; and 50 to 94.9% by weight of water.
28. The composition of claim 27 comprising:
about 10% by weight of maltose;
about 1% by weight of ascorbic acid; and sodium hydroxide sufficient to provide a pH of said composition of about 4.
about 10% by weight of maltose;
about 1% by weight of ascorbic acid; and sodium hydroxide sufficient to provide a pH of said composition of about 4.
29. The composition of claim 7 in lyophilized form.
30. Use of the composition of claim 7 in a method comprising the step of forming a complex of a biotinylated molecule and the immobilized avidin.
31. In a method for producing a fibrin monomer comprising:
subjecting a fibrinogen-containing composition to a biotinylated enzyme to convert said fibrinogen to fibrin monomer;
introducing a material having an affinity for biotin into the so-formed fibrin monomer/biotinylated enzyme mixture so that a complex of the affinity material and biotinylated enzyme are formed; and separating the so-formed complex, and thereby said enzyme, from the fibrin monomer product;
the improvement comprising use of the composition of claim 7 as said material having an affinity for biotin.
subjecting a fibrinogen-containing composition to a biotinylated enzyme to convert said fibrinogen to fibrin monomer;
introducing a material having an affinity for biotin into the so-formed fibrin monomer/biotinylated enzyme mixture so that a complex of the affinity material and biotinylated enzyme are formed; and separating the so-formed complex, and thereby said enzyme, from the fibrin monomer product;
the improvement comprising use of the composition of claim 7 as said material having an affinity for biotin.
32. A method for the terminal sterilization of avidin immobilized on an inert support material that is a substantially inert hydrophylic material capable of forming a polymer matrix and which is separable from a liquid phase which provides sterility for the composition while maintaining stability and biotin affinity which method comprises subjecting the composition of claim 7 to a terminal sterilization process.
33. The use according to claim 30 wherein said avidin immobilized on said inert support material is in a lyophilized form.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/556,244 US5691152A (en) | 1995-11-09 | 1995-11-09 | Stable avidin composition |
US08/556,244 | 1995-11-09 | ||
PCT/US1996/017268 WO1997017436A1 (en) | 1995-11-09 | 1996-10-28 | Stable avidin composition and methods using same |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2237338A1 CA2237338A1 (en) | 1997-05-15 |
CA2237338C true CA2237338C (en) | 2007-09-25 |
Family
ID=24220509
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002237338A Expired - Fee Related CA2237338C (en) | 1995-11-09 | 1996-10-28 | Stable avidin composition and methods using same |
Country Status (9)
Country | Link |
---|---|
US (4) | US5691152A (en) |
EP (1) | EP0902834A4 (en) |
JP (2) | JP2000502883A (en) |
AU (1) | AU719834B2 (en) |
BR (1) | BR9612698A (en) |
CA (1) | CA2237338C (en) |
IL (1) | IL124376A (en) |
NZ (1) | NZ321820A (en) |
WO (1) | WO1997017436A1 (en) |
Families Citing this family (43)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040067157A1 (en) * | 1993-07-22 | 2004-04-08 | Clearant, Inc. | Methods for sterilizing biological materials |
US5362442A (en) | 1993-07-22 | 1994-11-08 | 2920913 Canada Inc. | Method for sterilizing products with gamma radiation |
US5691152A (en) * | 1995-11-09 | 1997-11-25 | E. R. Squibb & Sons, Inc. | Stable avidin composition |
US6551794B1 (en) * | 1995-11-09 | 2003-04-22 | E. R. Squibb & Sons, Inc. | Stable biotinylated biomolecule composition |
JP2001526027A (en) * | 1997-12-09 | 2001-12-18 | ブリストル−マイヤーズ スクイブ カンパニー | Fibrinogen converting enzyme hybrid |
DE69933310T2 (en) * | 1998-10-16 | 2007-05-10 | Ge Healthcare Bio-Sciences Ab | STABILIZED COMPOSITION CONTAINING SQUARE ACTION ACTIVATED CARRIER WHICH CAN BE USED FOR THE IMMOBILIZATION OF AMINGRUPPEN CONTAINING SUBSTANCES |
US6463335B1 (en) | 1999-10-04 | 2002-10-08 | Medtronic, Inc. | Temporary medical electrical lead having electrode mounting pad with biodegradable adhesive |
US20040086420A1 (en) * | 2000-03-23 | 2004-05-06 | Macphee Martin J. | Methods for sterilizing serum or plasma |
AU2002245107A1 (en) * | 2000-12-12 | 2002-07-30 | Autogenomics, Inc. | Improved biochip |
CA2434139C (en) * | 2001-01-23 | 2014-05-27 | President And Fellows Of Harvard College | Nucleic-acid programmable protein arrays |
WO2002062957A2 (en) * | 2001-02-07 | 2002-08-15 | Invitrogen Corporation | Ter sites and ter binding proteins |
US6682695B2 (en) * | 2001-03-23 | 2004-01-27 | Clearant, Inc. | Methods for sterilizing biological materials by multiple rates |
US6696060B2 (en) * | 2001-06-14 | 2004-02-24 | Clearant, Inc. | Methods for sterilizing preparations of monoclonal immunoglobulins |
US6946098B2 (en) | 2001-08-10 | 2005-09-20 | Clearant, Inc. | Methods for sterilizing biological materials |
US20030031584A1 (en) * | 2001-08-10 | 2003-02-13 | Wilson Burgess | Methods for sterilizing biological materials using dipeptide stabilizers |
US7252799B2 (en) * | 2001-08-31 | 2007-08-07 | Clearant, Inc. | Methods for sterilizing preparations containing albumin |
US6749851B2 (en) | 2001-08-31 | 2004-06-15 | Clearant, Inc. | Methods for sterilizing preparations of digestive enzymes |
US6783968B2 (en) | 2001-09-24 | 2004-08-31 | Clearant, Inc. | Methods for sterilizing preparations of glycosidases |
US20110091353A1 (en) * | 2001-09-24 | 2011-04-21 | Wilson Burgess | Methods for Sterilizing Tissue |
US20030185702A1 (en) * | 2002-02-01 | 2003-10-02 | Wilson Burgess | Methods for sterilizing tissue |
US20030095890A1 (en) * | 2001-09-24 | 2003-05-22 | Shirley Miekka | Methods for sterilizing biological materials containing non-aqueous solvents |
US20030124023A1 (en) * | 2001-12-21 | 2003-07-03 | Wilson Burgess | Method of sterilizing heart valves |
US20030180181A1 (en) * | 2002-02-01 | 2003-09-25 | Teri Greib | Methods for sterilizing tissue |
US6908591B2 (en) * | 2002-07-18 | 2005-06-21 | Clearant, Inc. | Methods for sterilizing biological materials by irradiation over a temperature gradient |
US20040013562A1 (en) * | 2002-07-18 | 2004-01-22 | Wilson Burgess | Methods for sterilizing milk. |
AU2003257109A1 (en) * | 2002-08-05 | 2004-02-23 | Invitrogen Corporation | Compositions and methods for molecular biology |
SE0300823D0 (en) * | 2003-03-23 | 2003-03-23 | Gyros Ab | Preloaded Microscale Devices |
AU2004236740A1 (en) * | 2003-05-02 | 2004-11-18 | Sigma-Aldrich Co. | Solid phase cell lysis and capture platform |
US7077922B2 (en) * | 2003-07-02 | 2006-07-18 | Owens Corning Composites S.P.R.L. | Technique to fill silencers |
EP1571204B1 (en) * | 2004-03-04 | 2009-11-18 | LeukoCare AG | Leukocyte stimulation matrix |
CA2563168A1 (en) * | 2004-04-14 | 2005-11-17 | President And Fellows Of Harvard College | Nucleic-acid programmable protein arrays |
WO2006026248A1 (en) | 2004-08-25 | 2006-03-09 | Sigma-Aldrich Co. | Compositions and methods employing zwitterionic detergent combinations |
JP2009529356A (en) * | 2006-02-28 | 2009-08-20 | タイコ ヘルスケア グループ リミテッド パートナーシップ | Tissue adhesives and sealants and methods of use thereof |
EP1852443A1 (en) * | 2006-05-05 | 2007-11-07 | Leukocare AG | Biocompatible three dimensional matrix for the immobilization of biological substances |
US8178316B2 (en) * | 2006-06-29 | 2012-05-15 | President And Fellows Of Harvard College | Evaluating proteins |
ES2319061B1 (en) * | 2007-09-11 | 2010-02-10 | Biomedal, S.L. | METHOD OF CONSERVATION OF PEPTIDES OR PROTEINS. |
EP2058335B1 (en) * | 2007-11-07 | 2020-06-24 | Leukocare Ag | Biocompatible three dimensional matrix for the immobilization of biological substances |
DE102008053892A1 (en) | 2008-10-30 | 2010-05-06 | Fachhochschule Gelsenkirchen | Medical implant with biofunctionalized surface |
AU2010334412B2 (en) | 2009-12-22 | 2016-02-04 | Lifebond Ltd | Modification of enzymatic crosslinkers for controlling properties of crosslinked matrices |
WO2015109246A1 (en) * | 2014-01-17 | 2015-07-23 | Repligen Corporation | Sterilizing chromatography columns |
TWI709569B (en) * | 2014-01-17 | 2020-11-11 | 美商健臻公司 | Sterile chromatography resin and use thereof in manufacturing processes |
TWI709570B (en) | 2014-01-17 | 2020-11-11 | 美商健臻公司 | Sterile chromatography and manufacturing processes |
SG11202101860UA (en) | 2018-08-31 | 2021-03-30 | Genzyme Corp | Sterile chromatography resin and use thereof in manufacturing processes |
Family Cites Families (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3779706A (en) * | 1971-10-04 | 1973-12-18 | Energy Sciences Inc | Process for bulk sterilization, minimizing chemical and physical damage |
GB8331514D0 (en) * | 1983-11-25 | 1984-01-04 | Janssen Pharmaceutica Nv | Visualization method |
WO1986002077A1 (en) * | 1984-10-02 | 1986-04-10 | Meade Harry M | Production of streptavidin-like polypeptides |
IL76234A0 (en) * | 1985-08-28 | 1986-01-31 | Yeda Res & Dev | New biotinylation reagents |
US5068198A (en) * | 1986-03-26 | 1991-11-26 | Syntex (U.S.A.) Inc. | Liquid single reagent for assays involving confining gels |
US4794082A (en) * | 1987-02-17 | 1988-12-27 | Hoechst Celanese Corporation | Biotinylating agents |
US4798795A (en) * | 1987-02-17 | 1989-01-17 | Hoechst Celanese Corporation | Biotinylating agents |
US4709037A (en) * | 1987-02-17 | 1987-11-24 | Hoechst Celanese Corporation | Biotinylating agents |
JPS63246382A (en) * | 1987-04-02 | 1988-10-13 | Karupisu Shokuhin Kogyo Kk | Biotinyl reagent and biotinylation using said reagent |
US5043288A (en) * | 1988-06-20 | 1991-08-27 | Motsenbocker Marvin A | Immobilize molecular binding partners to contact activating supports |
DE3834766A1 (en) * | 1988-10-12 | 1990-04-26 | Boehringer Mannheim Gmbh | METHOD FOR DETERMINING A SPECIFICALLY BINDABLE SUBSTANCE |
FR2638099B1 (en) * | 1988-10-20 | 1991-01-25 | Immunotech Sa | LOW RELEASE RATE IMMUNOADSORBENT AND PROCESS FOR OBTAINING SAME |
US5026785A (en) * | 1989-05-12 | 1991-06-25 | The United States Of America As Represented By The Department Of Health And Human Services | Avidin and streptavidin modified water-soluble polymers such as polyacrylamide, and the use thereof in the construction of soluble multivalent macromolecular conjugates |
DK0423938T3 (en) * | 1989-09-29 | 1994-03-28 | Rohm & Haas | Ligand-containing medium for chromatographic separation, method for preparing the medium and using the medium for isolating synthetic or natural molecules from a fluid mixture |
CA2061519C (en) * | 1991-02-20 | 2004-05-18 | Naoki Asakawa | Packings combining protein to a support via a spacer |
US5306824A (en) * | 1992-04-15 | 1994-04-26 | Georgia Tech Research Corporation | Biotinylated isocoumarins |
US5306854A (en) * | 1992-07-10 | 1994-04-26 | Council Of Scientific & Industrial Research | Two step process for production of liquid hydrocarbons from natural gas |
CN1091315A (en) * | 1992-10-08 | 1994-08-31 | E·R·斯奎布父子公司 | Fibrin sealant compositions and using method thereof |
US5691152A (en) * | 1995-11-09 | 1997-11-25 | E. R. Squibb & Sons, Inc. | Stable avidin composition |
-
1995
- 1995-11-09 US US08/556,244 patent/US5691152A/en not_active Expired - Fee Related
-
1996
- 1996-10-28 EP EP96937780A patent/EP0902834A4/en not_active Withdrawn
- 1996-10-28 AU AU75244/96A patent/AU719834B2/en not_active Ceased
- 1996-10-28 CA CA002237338A patent/CA2237338C/en not_active Expired - Fee Related
- 1996-10-28 WO PCT/US1996/017268 patent/WO1997017436A1/en active Search and Examination
- 1996-10-28 BR BR9612698A patent/BR9612698A/en not_active Application Discontinuation
- 1996-10-28 IL IL12437696A patent/IL124376A/en not_active IP Right Cessation
- 1996-10-28 NZ NZ321820A patent/NZ321820A/en unknown
- 1996-10-28 JP JP9518221A patent/JP2000502883A/en active Pending
-
1997
- 1997-08-01 US US08/904,367 patent/US5942406A/en not_active Expired - Fee Related
- 1997-08-01 US US08/904,368 patent/US5998155A/en not_active Expired - Fee Related
- 1997-08-01 US US08/904,369 patent/US6046024A/en not_active Expired - Fee Related
-
2006
- 2006-07-26 JP JP2006203440A patent/JP2006300965A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
US6046024A (en) | 2000-04-04 |
IL124376A0 (en) | 1998-12-06 |
EP0902834A1 (en) | 1999-03-24 |
AU719834B2 (en) | 2000-05-18 |
CA2237338A1 (en) | 1997-05-15 |
US5691152A (en) | 1997-11-25 |
JP2000502883A (en) | 2000-03-14 |
AU7524496A (en) | 1997-05-29 |
WO1997017436A1 (en) | 1997-05-15 |
EP0902834A4 (en) | 2001-09-26 |
JP2006300965A (en) | 2006-11-02 |
NZ321820A (en) | 1999-11-29 |
US5942406A (en) | 1999-08-24 |
BR9612698A (en) | 1999-08-03 |
IL124376A (en) | 2000-10-31 |
US5998155A (en) | 1999-12-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2237338C (en) | Stable avidin composition and methods using same | |
US5750657A (en) | Methods and compositions using fibrin monomer to make a fibrin sealant | |
US5092992A (en) | Polyethyleneimine matrixes for affinity chromatography | |
US4464468A (en) | Immobilization of active protein by cross-linking to inactive protein | |
CA1040561A (en) | Cyanogen halide activation of carbohydrates and protein binding thereto | |
US5085779A (en) | Polyethyleneimine matrixes for affinity chromatography | |
JPS59225064A (en) | Carrier for immobilizing physiologically active substance | |
EP0865486B1 (en) | Stable biotinylated biomolecule composition and methods | |
CA1250276A (en) | Sterilization of adsorbent and column having improved storability including sterilized adsorbent for use in extracorporeal circulation treatment | |
EP0403700B1 (en) | Polyethyleneimine matrixes for affinity chromatography | |
US4411999A (en) | Immobilization of enzymes on granular gelatin | |
Kim et al. | Immobilization of urokinase on agarose matrices | |
JPS63258579A (en) | Production of carrier for immobilization of physiologically active substance | |
Keyes et al. | Immobilized enzymes | |
JPH0662680B2 (en) | Method for producing carrier for immobilizing antibody | |
JPS60156468A (en) | Protein decomposable wound cover | |
IE63044B1 (en) | Polyethyleneimine matrixes for affinity chromatography | |
JPS60224618A (en) | Carrier for immobilizing physiologically active substance | |
CS275662B6 (en) | Carrier for enzyme stabilization and process for preparing thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
MKLA | Lapsed | ||
MKLA | Lapsed |
Effective date: 20091028 |