Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 6 to 7 amino acid residues in defined sequence
Reexamination Certificate
1998-11-20
2001-02-20
Low, Christopher S. F. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
6 to 7 amino acid residues in defined sequence
C530S330000, C530S383000, C530S413000
Reexamination Certificate
active
06191256
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field
This invention is concerned generally with identifying protein-ligand interactions, and specifically with peptide ligands which bind rFVIII and which may be used in a method for the affinity purification of rFVIII.
2. Prior Art
Human Factor VIII, (antihemophilic factor) is a human plasma protein consisting of 2 polypeptides (light chain molecular weight of 80,000 and heavy chain molecular weight variable from 90,000 to 220,000). It is an essential cofactor in the coagulation pathway; required for the conversion of Factor X into its active form (Factor Xa). Factor VIII circulates in plasma as a non-covalent complex with von Willebrand Factor. Blood concentrations of Factor VIII below 20% of normal cause a bleeding disorder designated hemophilia A. Factor VIII blood levels less than 1% of normal result in a severe bleeding disorder, with spontaneous joint bleeding being the most common symptom. Recombinant DNA technology has allowed construction of plasmids that direct the expression of fusion products of Factor VIII protein in transfected mammalian cells. Factor VIII can be isolated from either a plasma derived source (cryoprecipitate) or from a genetically engineered recombinant source. The term Factor VIII is not meant to be a limitation but refers to a functional protein for treating bleeding disorders.
Several methods have been described for purification of Factor VIII from plasma sources (1,2,3). Several purification schemes utilize antibody affinity columns (4,5). To date, the most successful purifications of Factor VIII from plasma or from recombinant sources has been accomplished by using murine monoclonal antibodies specific to either Factor VIII or von Willibrand Factor.
Although monoclonal antibodies have been used successfully to obtain a relatively pure Factor VIII preparation, monoclonal antibodies can be present in the Factor VIII effluent because of leaching from the support matrix. This raises the possibility of antigenicity when the final preparation is introduced into animal systems, since murine monoclonal antibodies have been shown to be antigenic. A second disadvantage of the use of monoclonal antibodies is the requirement of cell culture facilities for producing the antibodies and the concomitant cost of purification and attachment onto a support matrix. Finally, the stability of the antibody binding site may not be amenable to the rigorous conditions necessary to sanitize the column.
Affinity chromatography is one of the most efficient techniques for purifying a protein from a complex mixture. With potential advantages including high stability, efficiency, selectivity, and low price, peptides have been investigated as affinity ligands in the pharmaceutical industry. A recent approach for identifying such ligands is to screen peptides from combinatorial peptide libraries (6,7,8,9).
Using the ‘divide-couple-recombine’ approach (10,11,12), millions of unique peptides of a defined length may be synthesized on resin beads. Each bead contains a unique peptide sequence. These library beads and their corresponding peptide sequences are then exposed to a target protein. Among these millions of peptide sequences, the target protein may bind to several unique bead-sequences. Those beads and their corresponding sequences must be detected, isolated, and identified. Several detection systems, including calorimetric two-step methods (7,12,13) as well as direct fluorescence detection methods (14,15,16) have been used.
Peptides disclosed in the U.S. patent application Ser. No. 08/595,718, incorporated herein by reference, also were found to bind Factor VIII. See also Necina et al. (23)
SUMMARY OF THE INVENTION
We have now discovered a group of peptides characterized by their ability to bind rFVIII. The sequences of the more preferred peptides having available rFVIII binding domains are Asn-Ala-Ile-Phe-Gln-Trp (SEQ ID NO:11), Ala-Phe-Val-Arg-Ser-Leu (SEQ ID NO:10), Gln-Arg-Leu-Ile-Gln-Phe (SEQ ID NO:12), Phe-Arg-Pro-His-Trp-Ala (SEQ ID NO:4), Arg-Pro-Arg-Trp (SEQ ID NO:8), and others of those presented in Table 1. A
TABLE 1
rFVIII Binding Peptide Sequences
Sequences discovered from screening of hexameric peptide library using
14
C-rFVIII with confirmed binding of rFVIII.
Seq ID
1
Lys
Pro
Asn
Pro
Leu
Ala
2
Arg
Asn
Pro
Pro
Asn
Asn
3
Tyr
Val
Gln
Gly
Leu
Trp
4
Phe
Arg
Pro
His
Trp
Ala
5
Leu
Asn
Trp
Lys
Tyr
Gly
6
His
Tyr
Trp
Phe
Tyr
Lys
7
Ile
Arg
Phe
Tyr
Ser
Glu
8
Arg
Pro
Arg
Trp
9
Phe
Ala
Leu
Pro
Gly
Arg
10
Ala
Phe
Val
Arg
Ser
Leu
11
Asn
Ala
Ile
Phe
Gln
Trp
12
Gln
Arg
Leu
Ile
Gln
Phe
13
Lys
Ala
Gln
Glu
Thr
Trp
14
Glu
Pro
Arg
Val
Ile
Gly
15
Val
Tyr
Gly
Val
Gly
Gly
16
Trp
Arg
Arg
His
Arg
Tyr
17
Phe
Tyr
Arg
Phe
Trp
Asn
18
Trp
Leu
Trp
Ser
His
Asn
19
Phe
His
Phe
Gly
Leu
Gln
20
Trp
His
His
His
Arg
Gly
21
His
Phe
Gln
Ile
Phe
Gly
22
Phe
Val
Phe
Leu
Val
Arg
method of rFVIII binding assay using the peptides deduced from a combinatorial library in a filtration plate process is described. We also describe a method of using the peptides in an affinity chromatography process to purify rFVIII. The method comprises passing a rFVIII containing solution over a substrate which has bound thereupon peptides disclosed herein, and then eluting the rFVIII.
As used herein, an available rFVIII binding domain means a peptide sequence that is sterically available to bind with rFVIII in the surrounding solution and which adopts a conformation that ligates rFVIII with moderate to strong avidity under controlled conditions of pH, ionic strength, and solvent composition. The affinity of binding may be increased or decreased by altering the amino acids adjacent to the above listed sequences. The avidity may be modified further by altering the above mentioned conditions of solvent composition and temperature.
The peptides were isolated and identified using a modified version of a radiological screening technique (17,18,19). We also describe a method of rFVIII binding assay using the identified peptides in a filtration plate format, wherein the method comprises incubating a
14
C-rFVIII containing solution over a substrate that has bound thereupon peptides disclosed herein, and then washing the substrates with appropriate buffer conditions. Finally, a method of rFVIII binding assay using the identified peptides in a column format is also described, wherein the method comprises passing a rFVIII containing solution over a substrate that has bound thereupon peptides disclosed herein, and then eluting the rFVIII with appropriate buffer conditions. Ultimately, this method leads to an affinity chromatography process to purify rFVIII by adopting appropriate elution conditions.
REFERENCES:
patent: 4518584 (1985-05-01), Mark et al.
patent: 4913902 (1990-04-01), Kilpatrick et al.
patent: WO 93/00365 (1993-01-01), None
Necina et al.,Journal of Chromatography B,vol. 715, pp. 191-201, 1998.
Mazo et al.,Proc. Natl. Acad. Sci. USA,vol. 87, pp. 2112-2116, Mar. 1990.
Kunst et al.,Nature,vol. 390, pp. 249-256, Nov. 20, 1997.
Fleischmann et al.,Science,vol. 269, pp. 496-512, Jul. 28, 1995.
Loeber et al.,Biochem. J.,vol. 304, pp. 687-692, 1994.
Klenk et al.,Nature,vol. 390, pp. 364-370, Nov. 27, 1997.
Necina et al.,Journal of Chromatography B,vol. 715, pp. 191-201, 1998.
Buettner Joseph A.
Carbonell Ruben G.
Chen Li Ang
Bayer Corporation
Beck Michael J.
Giblin James A.
Low Christopher S. F.
Mohamed Abdel A.
LandOfFree
Recombinant factor VIII binding peptides does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant factor VIII binding peptides, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant factor VIII binding peptides will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2606607