Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1995-11-09
2003-04-22
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C424S001690, C435S007500, C435S013000, C435S111000, C435S174000, C435S177000, C514S002600, C530S381000, C530S382000, C548S303700
Reexamination Certificate
active
06551794
ABSTRACT:
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 in coupled to a wide variety of biomolecules. Further, this affinity is substantially maintained even when cerivatized 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. In certain 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 592242 describes a novel fibrin sealant based on fibrin monomer as opposed to the traditional fibrinogen-based sealants andinvolves subjecting fibrinogen to a thrombin-like enzyme which is preferably removed after such treatment. EP 592242 describes that the enzyme captureand removal can be accomplished by using biotinylated batroxobin which can be recaptured with an avidin material. This and other applications would benefit by more convenient forms of biotinylated biomolecule and avidin materials. Presently, these materials are sometimes difficult to work with, can be unstable, can lose enzyme activity in processing such as lyophilization, may be unduly hygroscopic and do not withstand sterilization processes.
SUMMARY OF THE INVENTION
In accordance with the present invention, novel compositions and methods for biotinylated biomolecules and the biotin/avidin affinity technology are described. The novel composition involving biotin comprises:
i) a biotinylated biomolecule;
ii) a biomolecule protectant;
iii) buffer means to maintain a desired pH; and
iv) one or more bulking agents selected from water soluble, nonionic polymers.
This composition is conveniently an aqueous solution and preferably includes an agent to protect the composition against instability during terminal sterilization. Most preferably, this composition is freeze-dried to provide a stable, irradiatable powder form of the biotinylated biomolecule. Methods for making a fibrin monomer material, useful, for example, in a fibrin sealant, are also disclosed.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention discloses novel, stable compositions of biotin-biomolecule. The preferred compositions are freeze-dried and are stable, easy to handle, and can be terminally sterilized, e.g., by gamma irradiation without damage to the compositions. This is especially advantageous when the composition is a biotinylated biomolecule because it has been found to be very efficient to be able to terminally sterilize the lyophilized biotinylated biomolecule without damage to its activity. The present lyophilized biotin-based compositions have wide applicability wherever the avidin-biotin technology is useful because these compositions are water soluble, have low moisture uptake, have low bioburden, can be terminally sterilized (e.g., irradiated), remain stable and are pharmacologically acceptable. These advantages are provided by the unique combination of protectants and bulking agents as described herein.
These novel compositions include, along with the biotinylated biomolecule, a biomolecule protectant, buffer means to maintain the desired pH and one or more water soluble, nonionic polymer bulking agents. Preferably, the composition further includes an agent to protect the composition against deleterious effects of terminal sterilization, e.g., gamma irradiation. The biomolecule can be any desired enzyme or protein which is to be used in a biotinylated form. Numerous biotinylated biomolecules exist in the prior art and all of those prior biomolecules are useful herein as well. With regard to the novel fibrin monomer process in the above-referenced EP 592242, thrombin-like enzymes are useful in a biotinylated form. Such thrombin-like enzymes include thrombin or a thrombin-like enzyme selected from Acutin, Venzyme, Ancrod, Asperase, Batroxobin (from B. Altrox, B. Moojeni or B. Maranhao), Botropase, Crotolase, Flavoxogin and Gabonase. Nonlimiting examples of other biotinylated biomolecules include biotinylated lectins, antibodies, mitogens, DNA, RNA, tRNA, rRNA fragments, nucleosomes, membranes, membrane proteins, glycoproteins and synthetic peptides.
The biotin component of the biotinylated biomolecule can be biotin or any derivatized form or analog thereof, or any molecule having an affinity for avidin including monomeric avidin, Strept avidin, or any protein having biotin-binding properties including recombinant forms of any of the above. Patents and literature are replete with the various biotin compounds including various spacers, linking groups and the like, for use in the present applications. Nonlimiting examples can be found in M. D. Savage, et al. (1992), Pierce Chemical Co.,
Avidin
-
Biotin Chemistry: A Handbook
; DE 3629194, U.S. Pat. Nos. 5,180,828, 4,709,037 and 5,252,743, 4,798,795, 4,794,082, WO 85/05638 incorporated herein by reference.
The biomolecule protectant of the novel biotin compositions is any agent capable of protecting the desired activity of the biomolecule and thereby imparting stability to the biomolecule composition. Biomolecule protectants include, but are not limited to, trehalose, glycerol, ammonium sulphate and amino acids. Preferably the biomolecule protectant is an amino acid and, more preferably, the amino acid is a simple zwitterion such as glycine, alanine and valine with glycine being most preferred.
The buffer means of the present biotin compositions can be any convenient buffer suitable for maintaining the pH of the composition at a desired level. In the fibrin monomer process of EP 592242 it is desired to maintain the biotinylated biomolecule at about pH7, therefore sodium barbital, citrate, sodium barbital phosphate, potassium phosphate, imidazole-HCI, piperazine, sodium bicarbonate-5% CO
2
, triethano amine-HCI—NaOH, tris (hydroxymethyl)aminomethane and sodium phosphate bufferare useful with sodium phosphate being preferred.
The bulking agent of the present biotin-biomolecule compositions is selected from water soluble, nonionic polymers. The bulking agent provides both chemical and physical stability to the present compositions and, for example, it presents the novel compositions when in the form of a freeze-dried cake from collapsing. The nonionic water soluble polymers also provide protection to the biomolecule. Dextran and similar polysaccharides have been found to enhance the stability of the present compositions. Nonlimiting examples of such bulking agents include dextran, polyvinylpyrrolidone, polyvinylalcohol, polyethyleneglycol, hydrolyzed starch and polysaccharides (e.g., lactose, glucose, maltose, mannitol, etc.) with dextran, especially dextrans having a molecular weight between 50,000 and 100,000 Daltons (e.g., Dextran T-70 from Pharmacia Co.) being preferred.
The optional terminal sterilization protectant is selected from antioxidants, free radical scavengers and reducing agents. Preferred are antioxidants such as reduced glutathione, &agr;-tocopherol, N,N-dimethyl-p-phenylenediamine and sodium ascorbate with sodium ascorbate being most preferred.
Preparation of the biotinylated molecule is accomplished by known techniques. For example, a biotin derivative (which can be any desired biotin compound with spacer arm and/or leaving groups as discussed above) such as N-hydroxysuccinimide-biotin (NHS-biotin) can be reacted with the desired biomolecule, e.g., the soluble enzyme Batroxobin, in a solvent and in the presence of a buffer. The NHS functions as a leaving group to prov
Burton Steven James
Edwardson Peter A. D.
Menzies Alan
Pearson James C.
E. R. Squibb & Sons Inc.
Kilcoyne John M.
Naff David M.
Ware Deborah K.
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