(12) United States Patent
`Pantoliano et al.
`
`I IIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111
`US006303322Bl
`US 6,303,322 Bl
`Oct. 16, 2001
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) METHOD FOR IDENTIFYING LEAD
`COMPOUNDS
`
`(75)
`
`Inventors: Michael W. Pantoliano, Avondale, PA
`(US); Alexander W. Rhind,
`Libertyville, IL (US); Francis R.
`Salemme, Yardley, PA (US)
`
`(73) Assignee: 3-Dimensional Pharmaceuticals, Inc.,
`Exton, PA (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`(21) Appl. No.: 09/478,216
`
`(22) Filed:
`
`Jan. 5,2000
`
`Related U.S. Application Data
`
`(62) Division of application No. 08/853,464, filed on May 9,
`1997, now Pat. No. 6,020,141.
`(60) Provisional application No. 60/017,860, filed on May 9,
`1996.
`
`Int. Cl.7 .............................. GOIN 33/53; C12Q 1/68
`(51)
`(52) U.S. Cl. .................................................. 435/7.l; 435/6
`(58) Field of Search ........................................... 435/7.1, 6
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,255,976
`5,260,207
`5,355,215
`5,463,564
`5,525,300
`5,585,277
`5,679,582
`6,020,141
`6,036,920
`
`10/1993 Connelly ................................ 374/31
`11/1993 Pantoliano et al. .................. 435/221
`10/1994 Schroeder et al. ................... 356/317
`10/1995 Agrafiotis et al. ................... 364/496
`6/1996 Danssaert et al. ..................... 422/99
`12/1996 Bowie et al.
`........................ 436/518
`10/1997 Bowie et al.
`........................ 436/518
`2/2000 Pantoliano et al. ................... 435/7.1
`3/2000 Pantoliano et al. .................... 422/67
`
`FOREIGN PATENT DOCUMENTS
`
`0 640 828 Al
`0 770 876 Al
`WO 93/14781
`WO 94/05394
`WO 97/09342
`WO 97/20952
`WO 98/39484
`
`3/1995 (EP) .
`5 /1997 (EP) .
`8/1993 (WO) .
`3/1994 (WO) .
`3/1997 (WO) .
`6/1997 (WO) .
`9/1998 (WO) .
`
`OIBER PUBLICATIONS
`
`Becktel, W.J., et al., "Protein Stability Curves," Bipolymers
`26:1859-1877 (1987).
`Brandts, G.F. and Lin, L.N., "Study of Strong to Ultra-Tight
`Protein Interactions Using Differential Scanning Calorim(cid:173)
`etry," Biochem. 29:6927-6940 (1990).
`Brandts, J.F., et al., "An instrument for rapid determination
`of binding constants for biomolecules," Am. Lab. 22:30-41
`(1990).
`Buchner, J., et al., "Alternatively Folded States of an Immu(cid:173)
`noglobulin," Biochem. 30:6922-6929 (1991).
`Chavan, A.J., et al., "Interaction of Nucelotides With Acidic
`Fibroblast
`Growth
`Factor
`(FGF-1),"
`Biochem.
`33:7193-7202 (1994).
`
`Clegg, R.M., et al., "Observing the helical geometry of
`double-stranded DNA in solution by fluoresence resonance
`energy transfer," Proc. Natl. Acad. Sci. USA 90:2994-2998
`(1993).
`Cox, M.J. and Weber, P.C., "An Investigation of Protein
`Crystallization Parameters Using Successive Automated
`Grid Searches (SAGS)," J. Crystal Growth 90:318-324
`(1988).
`Darynkiewicz, Z., et al., "DNA Denaturation In Situ: Effect
`of Divalent Cations and Alcohols," J. Cell Biol. 68:1-10
`(1976).
`Draper, D.E. and Gluick, T.C., "Melting Studies of RNA
`Unfolding and RNA-Ligand Interactions," Meth. Enzymol.
`259:281-305 (Sep. 1995).
`Eftink, N.R., "The Use of Fluorescent Methods to Monitor
`Unfolding Transitions in Proteins," Biophys. J. 66:482-501
`(1994).
`Elwell, M. and Schellman, J., "Phage T4 Lysozyme Physical
`Properties and Reversible Unfolding," Biochim. Biophys.
`Acta 386:309-323 (1975).
`Freire, E., "Thermal Denaturation Methods in the Study of
`Protein Folding," Meth. Enzymol. 259:144-168 (Sep. 1995).
`Gordon, E.M., et al., "Applications of Combinatorial Tech(cid:173)
`nologies to Drug Discovery. 2. Combinatorial Organic Syn(cid:173)
`thesis, Library Screening Strategies, and Future Directions,"
`J. Med. Chem. 37:1385-1401 (1994).
`Green, S.N., et al., "Roles of Metal Ions in the Maintenance
`of the Tertiary and Quaternary Structure of Arginase from
`cerevisiae, "
`Saccharomyces
`Biol.
`Chem.
`J.
`266:21474-21481 (1991).
`Hanna, M. and Szostak, J.W., Suppressions of mutations in
`the core of the Tetrahymena ribozyme by spermidine, etha(cid:173)
`nol and by substrate stabilization, Nucl. Acids Res.
`22:5326-5331 (1994).
`
`(List continued on next page.)
`
`Primary Examiner---Eggerton A. Campbell
`(74) Attorney, Agent, or Firm-Sterne, Kessler, Goldstein
`& Fox, PLLC
`
`(57)
`
`ABSTRACT
`
`The present invention is a method for ranking the affinity of
`each of a multiplicity of different molecules for a target
`molecule which is capable of denaturing due to a thermal
`change. The method comprises contacting the target mol(cid:173)
`ecule with one molecule of the multiplicity of different
`molecules in each of a multiplicity of containers, simulta(cid:173)
`neously heating the multiplicity of containers, measuring in
`each of the containers a physical change associated with the
`thermal denaturation of the target molecule resulting from
`the heating in each of the containers, generating a thermal
`denaturation curve for the target molecule as a function of
`temperature for each of the containers and determining a
`midpoint temperature (Tm) therefrom, comparing the Tm of
`each of the thermal denaturation curves with the Tm of a
`thermal denaturation curve obtained for the target molecule
`in the absence of any of the molecules in the multiplicity of
`different molecules, and ranking the affinities of the multi(cid:173)
`plicity of different molecules according the change in Tm of
`each of the thermal denaturation curves.
`
`16 Claims, 40 Drawing Sheets
`
`Agilent Exhibit 1221
`Page 1 of 75
`
`

`

`US 6,303,322 Bl
`Page 2
`
`OIBER PUBLICATIONS
`
`Hei, D.J. and Clark, D.S., "Estimation of Meltin Curves
`From Enzymatic Activity-Temperature Profiles," Biotech(cid:173)
`nol. Bioeng. 42:1245-1251 (1993).
`Hoffman, A., et al., "A Sparse Matrix Screen to Establish
`Initial Conditions for Protein Renaturation," Anal. Biochem.
`230:8-15 (Sep. 1995).
`Hutton, J.R., "Renaturations kinetics and thermal stability of
`DNA in aqueous solutions of foramide and urea," Nucl.
`Acids Res. 4:3537-3555 (1977).
`Jensen, D.E., and von Rippel, P.H., "DNA single 'melting'
`proteins: I. Effects of Bovine pancreatic ribonuclease bind(cid:173)
`ing on the conformation and stabillity of DNA," J. Biol.
`Chem. 251:7198-7214 (1976).
`Johnson, C.M., et al., "Differential Scanning Calorimetry of
`Thermal Unfolding of the Methionine Repressor Protein
`(MetJ) from Escherichia coli," Biochem. 31:9717-9724
`(1992).
`Ma, C.-Y. and Harwalkar, V.R., "Effects of Medium and
`Chemical Modification on Thermal Characteristics of ~-lac(cid:173)
`toglobulin," J. Thermal Analysis 47:1513-1525 (Jan. 1996).
`Markovic-Housley, Z. and Garavito, R.M., "Effect of tem(cid:173)
`perature and low pH on structure and stability of matrix
`porin and micellar detergent solutions," Biochem. Biophys.
`Acta 869: 158-170 (1986).
`Merabet, E.K., et al., "Differential scanning calorimetric
`study of 5-enolpyruvoyl shikimate-3-phosphate synthase
`and its complexes with shikimate-3-phosphate and gly(cid:173)
`phosphate: irreversible thermal transitions," Biochem. Bio(cid:173)
`phys. Acta 1161:272-278 (1993).
`Morton, A, et al., "Energetic Origins of Specificity of
`Ligand Binding in an Interior Nonpolar Cavity of T4
`Lysozyme," Biochem. 34:8564-8575 (Jul. 1995).
`Muriana, F.J.G., et al., "Further thermal characterization of
`an aspartame aminotransferase from a halophilic organism,"
`Biochem. J. 298:465-470 (1994).
`Murray-Brelier, A and Goldberg, M.E., "A physical(cid:173)
`chemical and immunological comparison shows that native
`and renatured Escherichia coli tryptophan synthase ~2 sub(cid:173)
`units are identical," Biochimie 71:533-543 (1989).
`Otto, A. and Seckler, R., "Characerization, stability and
`refolding of recombinant hirudin," Eur. J. Biochem.
`202:67-73 (1991).
`Pantoliano, M.W., "Automated Receptor Screening By
`Thermal Physical Assay," Department of Health and Human
`Services Small Business Research Program Phase I Grant
`Application (May 15, 1995).
`Pantoliano, M.W., "Automated Receptor Screening By
`Thermal Physical Assays," Department of Health and
`Human Services Small Business Research Program Phase II
`Grant Application (Sep. 1996).
`Pilch, D.S., et al., "Characterization of a Triple Helix-Spe(cid:173)
`cific Ligand: BePI {3-methoxy-7H-8-methyl-11-[3'-ami(cid:173)
`no )propylamino ]benzo[ e ]pyrido[ 4,3-b ]indole} Intercalates
`into Both Double-helical and Triple-helical DNA," J. Mal.
`Biol. 232:926-946 (1993).
`Pilch, D.S. and Breslauer, K.J., "Ligand-induced formation
`of nucleic acid triple helices," Proc. Natl. Acad. Sci. USA
`91 :9332-9336 (1994).
`Piirschke, D. and Jung, M., "Stability decrease of RNA
`double helices by phenylalanine-, tyrosine- and tryptopha(cid:173)
`ne-amides. Analysis in terms of site binding and relation to
`melting proteins," Nucl. Acids Res. 10:6163-6176 (1982).
`
`Ramalingam, K., et al., "Conformational Studies of Anionic
`Melittin Analogues: Effect of Peptide Concentration, pH,
`Ionic Strength, and Temperature-Models for Protein Fold(cid:173)
`ing and Halophilic Proteins," Biopolymers 32:981-992
`(1992).
`Sagar, S.L. and Domach, M.M., "Thermostability of Drifted
`Oligomeric, Reduced and Refolded Proteins," Chapter 5 in:
`ACS Symposium Series: Protein Refolding, Gerorgiou, G.,
`Ed., American Chemical Society, publ., pp. 64--78 (1991).
`Schellman, J.A., "Macromolecular Binding," Biopolymers
`14:999-1018 (1975).
`Schellman, J.A., "Fluctuation and Linkage Relations in
`Macromolecular Solution," Biopolymers 29:215-224
`(1990).
`Schellman, J.A., "The relations between the free energy of
`interaction and binding," Biophys. Chem. 45:273-279
`(1993).
`Schwarz, F.P., et al., "Thermodynamics of the Binding of
`Galactopyranoside Derivatives to the Basic Lectin from
`Winged Bean (Psophocarpus tetrogonolobus)," J. Biol.
`Chem. 266:24344-24350 (1991).
`Shchyolkina, AK., et al., "The R-Form of DNA Does
`Exist," FEES Lett. 339:113-118 (1994).
`Shrake, A., et al., "Partial Unfolding of Dodecameric
`Glutamine Synthetase from Escherichia coli: Temperature(cid:173)
`Induced, Reversible Transitions of Two Domains," Bio(cid:173)
`chem. 28:6281-6294 (1989).
`Tachibana, H., et al., "Relationship Between the Optimal
`Temperature for Oxidative Refolding and the Thermal Sta(cid:173)
`bility of Refolded State of Hen Lysozyme Three-Disulfide
`Derivatives," Biochem. 33:15008-15016 (1994).
`Timasheff, S.N. and Arakawa, T., "Stabilizations of protein
`structure by solvents," Chapter 14 in Protein Structure, a
`practical approach, Creighton, T.E., Ed., IRLPress, Oxford,
`England, pp. 331-345 (1994).
`Tsai, P.K., "Formulation Design of Acidic Fibroblast Growth
`Factor," Pharm. Res. 10:649-659 (1993).
`Viguera, A.R., et al., "Thermodynamic and Kinetic Analysis
`of the SH3 Domain of Spectrin Shows a Two-State Folding
`Transition," Biochem. 33:2142-2150 (1994).
`Volkin, D.B., et al., "The Effect of Polyanions on the
`Stabilization of Acidic Fibroblast Growth Factor," in Har(cid:173)
`nessing Biotechnology for the 21st Century. Proceedings of
`the Ninth International Biotechnology Symposium and
`Exposition, Ladisch, M.R. and Bose, A., Eds., American
`Chemical Society, pp. 298-302 (1992).
`Volkin, D.B., et al., "Physical Stabilization of Acidic Fibro(cid:173)
`blast Growth Factor by Polyanions," Arch. Biochem. Bio(cid:173)
`phys. 300:30--41 (1993).
`Wagenhiifer, M., et al., "Thermal Denaturation of Engi(cid:173)
`neered Tet Repressor Proteins and Their Complexes with tet
`Operator and Tetracycline Studied by Temperature Gradient
`Gel Electrophoresis," Anal. Biochem. 175:422-432 (1988).
`Walz, F.G. and Kitareewan, S., "Spermine Stabilization of a
`Folded Ribonuclease T1 ," J. Biol. Chem. 265:7127-7137
`(1990).
`Waring, M.J. and Henley, S.M., "Stereochemical aspects of
`the interactions between steroidal diamine and DNA," Nucl.
`Acids Res. 2:567-586 (1975).
`Weber, P.C., et al., "Structure-Based Design of Synthetic
`Azobenzene Ligands for Streptavadin," J. Am. Chem. Soc.
`116:2717-2724 (1994).
`
`Agilent Exhibit 1221
`Page 2 of 75
`
`

`

`US 6,303,322 Bl
`Page 3
`
`Zahnley, J.C., "Effects of Manganese and Calcium on Con(cid:173)
`formational Stability of Concanaval in A: A Differential
`Scanning Calorimetric Study," J. Inorg. Biochem. 15:67-78
`(1981).
`Zolkiewski, M. and Ginsburg, A., "Thermodynamic Effects
`of Active-Site Ligands on the Reversible, Partial Unfolding
`of Dodecameric Glutamine Synthetase from Escherichia
`coli: Calorimetric Studies," Biochem. 31: 11991-12000
`(1992).
`Belevitch, G.V., et al., "Calicum antagonists, riodipin,
`niphedipine and verapamil, binding to model and biological
`membranes: fluorescence analysis," Biologicheskie Mem(cid:173)
`brany 5:768-776 (1988).
`Fukada, H., "Calorimetry of the Ligand Binding to Pro(cid:173)
`teins," Tanpakushitsu Kakusan Koso 33:328-336 (1988).
`Iida, K., "Structure and biological activity of complement
`receptors CRl, CR2 and CR3," Seikagaku 58:1471-1474
`(1986).
`Kogtev, L.S., et al., "Use of fluorescein-labeled lipid probes
`for analyzing Substance P and its derivatives binding to the
`rat brain tachykinin receptors," Biologicheskie Membrany
`6:34-41 (1989).
`English translation of Kogtev, L.S., et al., "Use of fluores(cid:173)
`cein-labeled lipid probes for analyzing Substance P and its
`derivatives binding to the rat brain tachykinin receptors,"
`Bilogicheskie Membrany 6: 34-41 (1989).
`Kuroki, R. and Inaka, K., "Stabilization of a Protein by
`Constructing a Ligand Binding Site," Tanpakushitsu Kaku(cid:173)
`san Koso 37: 314--321 (1992).
`Kuwajima, K., "Kinetic pathway of globular-protein fold(cid:173)
`ing," Seikagaku 62:117-121 (1990).
`Manevich, E. M., et al., "The binding of the B-chain of ricin
`to Burkitt lymphoma cells," FEES Lett. 194: 313-316
`(1986).
`Molotskovskaya, I.M., et al., "The concavalin A binding to
`the plasma membranes of young and old mouse lympho(cid:173)
`cytes: a fluorescent probe study," Biologicheskie Membrany
`9: 32-39 (1991).
`Molotkovskaya, I.M., et al., "Immunosuppressive activity of
`glycosphinoglipids: a study of the interaction of interleu(cid:173)
`kin-2 with gangliosides using cells and model systems,"
`Biologicheskie Membrany 9:143-151 (1992).
`Rakhamininova, A.B., et al., "Model for auxin receptor,"
`Biokhimiya 43:806-823 (1978).
`Rakhaminova, A.B., et al., "Construction of a model of the
`auxin receptor," Biorak 43:639-653 (1978).
`Smirnov, O.N., et al., "Study on the Interaction of the
`cholera toxin and its B-subunit with liposomes containing
`ganglioside GMl and fluorescent-labeled gangliosides,"
`Biologicheskie Membrany 12:174--184 (1995).
`Sominsky, V.N., et al., "Using a fluorescent probe for
`investigating ~-adrenoreceptive function of human erythro(cid:173)
`cytes," Biofizika 30:642-645 (1985).
`Surin, A.M., et al., "A study of the interaction of cholera
`toxin B-subunit with liposomes containing ganglioside
`GMl and fluorescein-labeled lipids," Biologicheskie Mem(cid:173)
`brany 9:495-508 (1992).
`Timofeev, A.A., et al., "Ispol'zovanie spektral'nykh khar(cid:173)
`akteristik riodipina dlia izucheniia digidropirinin-retseptor(cid:173)
`nogo kompleksa neironal'nykh membran (The use of spec(cid:173)
`ryodipine
`for
`the
`study of
`tral characterisitics of
`dihydropiridin receptor complex in neuronal membranes),"
`Biull. Eksp. Biol. Med. 114: 29-32 (1992).
`
`Takahashi, N., "Cyclophiin and FK506-binding protein,"
`Seikagaku 64: 325-331 (1992).
`Partial English translation of Fukada, H., "Calorimetry of
`the Ligand Binding to Proteins," Tanpakushitsu Kakusan
`Koso 33: 328-336 (1988).
`Partial English translation of Kuroki, R. and Inaka, K.,
`"Stabilization of a Protein by Constructing a Ligand Binding
`Site," Tanpakushitsu Kakusan Koso 37:314-321 (1992).
`Partial English translation of Kuwajima, K., "Kinetic path(cid:173)
`way of globular-protein folding," Seikagaku 62: 117-121
`(1990).
`Lennick, M., et al., "Changes in Protein Conformation and
`Stability Accompany Complex Formation Between Human
`Cl Inhibitor and Cls," Biochem. 24:2561-2568 (1985).
`Lapadat, M.A. and Spremulli, LL., "Effect of Guanine
`Nucleotides on the Conformation and Stability of Chloro(cid:173)
`plast Elongation Factor Tu," J. Biol. Chem. 264:5510-5514
`(1989).
`Pace, C.N. and Grimsley, G.R., "Ribonuclease T1 Is Stabi(cid:173)
`and Anion Binding," Biochem.
`lized by Cation
`27:3242-3246 (1988).
`Barcelo, F., et al., "A Scanning Calorimetric Study of
`Natural DNA and Antitumoral Anthracycline Antibiot(cid:173)
`ic-DNA Complexes," Chem.-Biol. Interactions 74:315-324
`(1990).
`Bone, R., et al., "Inhibition of Thymidine Kinase by
`P1 l(Adenosine-5')-P5
`-( thymidine-5')-pentaphosphate," J.
`Biol. Chem. 261:5731-5735 (1986).
`Bouvier, M. amd Wiley, D.C., "Importance of Peptide
`Amino and Carboxyl Termini to the Stability of MHC Class
`I Molecules," Science 265:398-402 (1994).
`Butour, J.-L., et al., "Effect of the amine non-leaving group
`on the structure and stability of DNA complexes with
`cis-{Pt(R-NH2)iN0 3 ) 2 ]," Eur. J. Biochem. 202:975-980
`(1991).
`Copeland, R.A., et al., "The Structure of Human Acidic
`Fibroblast Growth Factor and Its Interaction with Heparin,"
`Arch. Biochem. Biophys. 289:53-61 (1991).
`Elwell, M.L. and Schellman, J.A., "Stability of Phage T4
`Lysozymes," Biochim. Biophys. Acta 494:367-383 (1977).
`Lin. L.-N., et al., "Calorimetric Studies of Serum Transfer(cid:173)
`rin and Ovotransferrin. Estimates of Domain Interactions,
`and Study of the Kinetic Complexities of Ferric Ion Bind(cid:173)
`ing," Biochem. 33:1881-1888 (1994).
`Porter, P.N., et al., "Stabilization of SV40 transformed
`human fibroblast cytoplasmic thymidine kinase by ATP,"
`Mal. Cell. Biochem. 35:59-64 (1990).
`Privalov, P.L., "Thermal Investigations of Biopolymer Solu(cid:173)
`tions and Scanning Microcalorimetry," FEES Lett.
`40(Suppl.):s140-s153 (1974).
`Roy, K.B., et al., "Hairpin and Duplex Forms of a Self(cid:173)
`Complementary Dodecamer, d-AGATCTAGATCT, and
`Interaction of the Duplex Form with the Peptide KGWGK:
`Can a Pentapeptide Destabilize DNA?," Biochem.
`31:6241-6245 (1992).
`Schellman, J.A., "The Thermodyamic Stability of Proteins,"
`Ann. Rev. Biophys. Chem. 16:115-137 (1987).
`Schellman, J.A., "A simple model for salvation in mixed
`solvents. Applications to the stabilization and destabilization
`of macromolecular structures," Biophys. Chem. 37: 121-140
`(1990).
`Schellman, J.A., "The Thermodynamics of Solvent
`Exchange," Biopolymers 34:1015-1026 (1994).
`
`Agilent Exhibit 1221
`Page 3 of 75
`
`

`

`US 6,303,322 Bl
`Page 4
`
`Bagshaw, C.R. and Harris, D.A., "Chapter 4. Measurement
`of Ligand Binding to Proteins," in Spectrophotometry &
`spectrofluorimetry: a practical approach, Bashford, C.L.
`and Harris, D.A., Eds., IRL Press, Washington, DC, pp.
`91-113 (1987).
`Bell, J.E., "Chapter 4. Fluorescence: Solution Studies," in
`Spectroscopy in Biochemistry, vol. I, Bell, J.E., Ed., CRC
`Press, Inc., Boca Raton, FL, pp. 155-194 (1981).
`Brand, L. and Gohlke, J. R., "Fluorescence Probes for
`Structure," Ann. Rev. Biochem. 41 :843-868 (1972).
`Burke, C.J., et al., "Effect of Polyanions on the Unfolding of
`Acidic Fibroblast Growth Factor," Biochem. 32:6419-6426
`(1993).
`
`Davidson, A.R., et al., "Cooperatively folded proteins in
`random sequence libraries," Nature Struct. Biol. 2:856-864
`(1995).
`Eriksson, A.E., et al., "Similar Hydrophobic Replacements
`of Leu99 and Phe153 within the Core of T4 Lysozyme Have
`Different Structural and Thermodynamic Consequences," J.
`Mal. Biol. 229:747-769 (1993).
`Lee, M., et al., "In Vitro Cytotoxicity of GC Sequence
`Directed Alkylating Agents Related to Distamycin," J. Med.
`Chem. 36:863-870 (1993).
`Schellman, J.A., "The Effect of Binding on the Melting
`Temperature of Biopolymers," Biopolymers 15:999-1000
`(1976).
`
`Agilent Exhibit 1221
`Page 4 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 1 of 40
`
`US 6,303,322 Bl
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`Agilent Exhibit 1221
`Page 5 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 2 of 40
`
`US 6,303,322 Bl
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`u:) -
`-
`~
`a..
`0 ~
`
`0
`
`LO
`
`I
`
`I
`
`• •
`• I +
`•
`• I
`+
`•
`+
`
`I
`
`I
`
`0
`oe
`N
`@p_/
`:z:~
`~!
`§s <]
`
`(/)
`
`E
`
`-
`_J.
`~/
`LO .
`0 ...__.
`:::iE
`:::t.
`co
`N
`
`@_JP
`LL.~ ;t
`f2 ~ ~
`
`CU t-
`
`2i C)t-
`LO <J
`:z:
`c2
`~
`w
`::c:
`
`.
`
`LO
`0
`
`.
`+
`0
`
`rr)
`
`d
`
`N
`ci
`
`.
`..-
`0
`
`C)
`
`( wuog£ V) A~!P!qJnl
`
`0
`rr)
`
`..-
`d
`I
`
`Agilent Exhibit 1221
`Page 6 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 3 of 40
`
`US 6,303,322 Bl
`
`50
`
`TEMPERATURE ( °C)
`60
`
`55
`
`65
`
`1.5 104
`
`•
`.t.
`
`TH 1 µM
`TH 1 µM + 30P 4026
`
`70
`•
`
`1 104
`E
`C
`0 c.o
`-.r
`05000
`
`-5000
`
`TH@lµM
`Tm .., 57.4 °C
`
`TH + 30P 4026 @ 67 µM
`Tm "' 65.8 (6TM = 8.4)
`Kd N 6.5 nM @ 310°1<
`
`y(T) = Yu+
`
`(yr - Yu)
`
`I + exp (t'\H•][f- ~]+ [ Ll ?][ (7'-1) +In (W ])
`
`320
`
`325
`
`335
`330
`TEMPERATURE (°K)
`FIG.3
`
`340
`
`345
`
`Agilent Exhibit 1221
`Page 7 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 4 of 40
`
`US 6,303,322 Bl
`
`25
`
`30
`
`TEMPERATURE {°C)
`35
`40
`45
`
`60
`
`200 -E
`
`C
`
`-tt- D2_FGFR1_o
`___._ D2_FGFR1_b
`- e - HS_5000
`-.wr- PPS
`
`• •
`
`•
`
`•
`•
`HS_5000 @ 150 µM.
`Li Tm,., 5.1 °C
`Kd "' 13.6 µM @ 298°K •
`•
`
`~ en
`~ 100
`
`D2_FGFR1 @ 71 µM (1 mg/ml)
`+ 134 µM ANS
`Tm"' 39.7t0.3°C ~
`
`Lu
`(..)
`i:5
`~ 50
`~
`
`0:::
`
`0
`
`...__PPS @ 156 µM
`/J. Tm "' 6.6°C
`Kd "' 4.9 µM @ 298°K
`
`-c====-==--
`(yr - Yu)
`.
`1 + exp([-~/"] [f- L] + [ ~;pu][ (';-t) tin~~)])
`
`(T) = Yu+
`
`295
`
`300
`
`305
`
`320
`315
`310
`TEMPERATURE {°K)
`FIG.4
`
`325
`
`330
`
`335
`
`Agilent Exhibit 1221
`Page 8 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 5 of 40
`
`US 6,303,322 Bl
`
`E
`C:
`
`TEMPERATURE ('C)
`
`45
`
`50
`
`55
`
`•
`
`FACTOR D
`
`60
`•
`
`•
`
`Tm =324.3 ± 0.3 'K
`
`C>
`(0
`~
`0
`i= 1000
`v,
`:z:
`~ :z:
`w
`~ 500
`~
`~
`0
`
`(_)
`
`(_)
`
`~
`
`•
`
`[FACTOR D] = 4 µM IN 10 ul DROPS(LID DIMPLES)
`[ 1,8 ANS] = 100 µM
`BUFFER = 50 mM HEPES pH 7.5, 0.1 NaCl
`Q_.__--r----r--~~-.---...---or--.--,--,,---,--,.-..-----.----.----.--T""---.----.---1-
`315
`320
`325
`330
`335
`
`TEMPERATURE ('K)
`
`FIG.5
`
`Agilent Exhibit 1221
`Page 9 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 6 of 40
`
`US 6,303,322 Bl
`
`~1000
`0
`U)
`~
`
`o 800
`~ en
`~ 600
`
`LaJ
`~ 400
`LaJ
`(.)
`~
`~ 200
`~
`
`0
`320
`
`50
`
`55
`
`TEMPERATURE {'C)
`60
`65
`
`70
`
`75
`
`•
`
`80
`•
`•
`
`• FXa
`
`FXa @ 1.4 µM
`68 µg/ml
`
`•
`
`Tm =337.9 ± 0.3 'K
`
`325
`
`330
`
`340
`335
`TEMPERATURE ('K)
`
`345
`
`350
`
`355
`
`FIG.6
`
`Agilent Exhibit 1221
`Page 10 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 7 of 40
`
`US 6,303,322 Bl
`
`TEMPERATURE ('C)
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`TH ALONE
`•
`TH + 4660
`•
`ASSAY VOLUME=5 µL (IN 96 WELL
`V BOTTOM)
`
`IThrombin ]=2 µM
`
`1,8 ANS]= 100 µM
`3DP-4660]=50 µM
`BUFFER=50 mM Hepes, pH 7 .5
`0.1 M NaCl
`
`• •
`
`Tm =323.9 t 0.4 °K
`
`Tm =332.2 t 0.5°K
`Kd @ Tm= 15 nM
`
`1000
`
`800
`
`600
`
`400
`
`200
`
`0
`
`E
`C
`
`C>
`(0
`~
`
`@
`~
`(/)
`:z: w
`1--:z:
`w
`(..)
`:z: w
`U1 w
`a:::
`0
`::>
`.....J
`LL.
`
`(..)
`
`•
`•
`
`-200-t-r--r--.--,-.-,---r-.....--,,.....-r-T""'"'T"'...,......,.-,-,-,--,--,.---,-......-,-.....--r---.-~-,-.,-l-
`340
`335
`320
`325
`330
`310
`315
`TEMPERATURE (' K)
`FIG.7
`
`Agilent Exhibit 1221
`Page 11 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 8 of 40
`
`US 6,303,322 Bl
`
`TEMPERATURE ('C)
`55
`35
`50
`40
`45
`30
`25
`1400Y....__._....__.__.___.___.___.__,.___.__,.___.___.__.__.___.___.__.__.__.__.__._-:::::::::::::======::::::!:::::::===r(cid:173)
`
`•
`•
`
`D2_FGFR1 H20
`APROSULATE
`
`[D2_FGFR1 ]=50 µM
`[ANS]=250 µM
`ASSAY VOLUME=lO µL
`[LIGAND ]=300 µ M
`BUFFER=50 mM Hepes, pH 7.5,
`0.1 M NaCl
`
`E
`C:
`~ 1200
`'V
`
`Cf)
`
`~ 1000
`~ 800
`z
`~ 600
`z
`LL.J
`(..)
`f:3 400
`a:::
`0
`:3
`LL 200
`
`Q-+-....-r-..--.--r--T--,--,.....,.....,--,--,--,-~~-.--.--.-..--.--,--,-........... -.--..-.-............ -r-T""'T"'"'t-
`295
`320
`325
`330
`300
`305
`310
`315
`TEMPERATURE ('K) FIG.BA
`
`OS03Na
`
`a
`
`OS03Na
`
`H~H
`H
`H
`N003SO
`
`OS03Na
`
`OS03Na
`
`OS03Na
`
`OS03Na
`
`FIG.BB
`
`OS03Na
`
`Agilent Exhibit 1221
`Page 12 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 9 of 40
`
`US 6,303,322 Bl
`
`20
`
`30
`
`40
`
`TEMPERATURE, (°C)
`50
`60
`70
`
`80
`
`90
`
`100
`
`E
`C:
`
`C)
`
`~ 800
`@
`w
`~ 600
`w
`(...)
`V) w
`a::: g 400
`.....J u....
`
`•
`
`pH 7 /0.1 NaCl
`
`ASSAY VOLUME=5 µl {IN 96 WELL
`V BOTTOM)
`[Urokinase/CMK]=74 µM
`[1,8 ANS]=200 µM
`BUFFER=50 mM MOPS pH 7.0,
`100 mM NaCl
`
`~ 200
`
`LaJ
`0:::
`
`•
`•••
`0 -t-T"""T"""T-.-.-"'T""T'""l'"""T""~'T"""T'"".--r-T-.--.-'"'T""'T"""T""'T""'T""T'"T""'T""'T"""T""'T"""T'""T""'T"""...........,....,............,........~
`300
`310
`320
`330
`340
`350
`360
`370
`FIG. g
`TEMPERATURE, fK)
`
`•
`
`TEMPERATURE, fC)
`70
`60
`40
`50
`30
`20
`~ 550 ""T""-....._.__.__...___._......__.___.__.__._ .......................... ___.__ ......................... _._.___.__....__.__.__..__,_
`--- 2ul 11 321.74 +/- 0.95
`C) c.o v
`--e- 2ul 2 323.44 +/- 1.16
`-.- 2uL 3 323.11 +/- 0.42
`e
`w
`MEAN = 322.76 °K; s.d. + /- 0.90
`{49.61 °c)
`~450
`w
`(...)
`V) w
`0:::
`0 =>
`~
`~350
`~ w
`
`0
`
`0:::
`
`300
`
`320
`310
`TEMPERATURE, \K)
`
`330
`
`340
`
`FI G • 1 o
`
`Agilent Exhibit 1221
`Page 13 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 10 of 40
`
`US 6,303,322 Bl
`
`E
`C
`0 550
`~
`a..J
`as
`C...)
`u 450
`f3
`a:::
`0
`::::,
`~
`~ 350
`i
`
`20
`
`TEMPERATURE, (°C)
`30
`40
`50
`Sul 1 323.74 +/- 0.49
`•
`o Sul 2 321.04 + /- 1.22
`• Sul 3 322.38 +/- 1.13
`5ul 4 325.58 +/- 2.42
`•
`--a--- Sul 5 324-.06 +/- 0.99
`MEAN = 323.36 °K; s.d. +/- 1.73 o
`(50.21 ·c)
`•
`
`60
`
`70
`•
`
`'wJ
`
`300
`
`320
`310
`TEMPERATURE, {°K)
`
`330
`
`340
`
`FIG.11
`
`Agilent Exhibit 1221
`Page 14 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 11 of 40
`
`US 6,303,322 Bl
`
`1.2-.-----r------...-----.--------(cid:173)
`
`.--..
`~
`0
`.
`l .0 -
`..-
`cri E
`~ Q) 0.8
`-E
`u C:
`0
`0
`LOte
`;~ 0.4
`>< w Cl)
`~E
`(.)0 ~---~--~~-~~~--~~-~_J
`0
`w C:
`~tO
`~~-0.2
`:::,
`ii
`
`CONTROL
`
`4076
`
`4660
`
`3811
`
`3959
`COMPOUND
`
`FIG.12
`
`Agilent Exhibit 1221
`Page 15 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 12 of 40
`
`US 6,303,322 Bl
`
`TEMPERATURE ('C)
`50
`55
`
`60
`
`65
`
`45
`
`• TH
`• BLANK
`
`E
`C:
`~ 650
`
`I"')
`
`•
`• •
`
`•
`• •
`
`•
`
`•
`
`[THROMBIN]=2 µ.M
`ASSAY VOLUME=100 µ.L
`BLANK = NO PROTEIN
`
`•
`
`•
`
`• •
`•
`•
`
`•
`•
`
`0
`
`@
`~ 600
`en :z w
`~
`~ 550
`• • •
`(...) z w
`c:..:>
`•
`•
`~ 500
`• •
`•
`~
`450--1----r--,---,.---.---.-...--.---.-~~~~~~~~~~~~-1-
`340
`315
`320
`335
`325
`330
`TEMPERATURE {'K)
`FIG.13
`
`Agilent Exhibit 1221
`Page 16 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 13 of 40
`
`US 6,303,322 Bl
`
`E
`C
`
`50
`
`C>
`LO
`I"") 100
`@
`~
`U) z w
`t-:z
`w
`CJ z w
`CJ
`V> w
`a:::
`0
`:::>
`--' LL.
`
`0
`
`-50
`315
`
`TEMPERATURE {'C)
`50
`55
`
`60
`
`65
`
`45
`
`• B
`
`[THROMBIN]=250 µ M
`ASSAY VOLUME= 100 µL
`BLANK IS SUBTRACTED
`
`• •
`
`320
`
`•
`
`330
`325
`TEMPERATURE {'K)
`
`FIG.14
`
`335
`
`340
`
`Agilent Exhibit 1221
`Page 17 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 14 of 40
`
`US 6,303,322 Bl
`
`c;
`
`60
`
`70
`
`TEMPERATURE, ° C
`50
`40
`
`30
`• THROMBIN
`• + HEPARIN
`• + 4660
`• + Hir. 53-65
`
`E 1200
`0
`...,.
`(0 1000
`L.,,j
`(.)
`:z 800
`lJ.J
`(.)
`{{}
`a:::
`0
`::::>
`ci
`~
`~
`LLJ 200
`a:::
`
`600
`
`400
`
`0
`
`300
`
`320
`310
`TEMPERATURE. °K
`
`330
`
`340
`
`FIG.15
`
`Agilent Exhibit 1221
`Page 18 of 75
`
`

`

`i,-
`~
`N
`N
`~
`~
`Q
`~
`O'I
`r.,J.
`
`e
`
`C
`,i;;..
`....,
`0
`"'""
`Ul
`~ ....
`=-~
`
`'JJ.
`
`"'""
`C
`C
`N
`"'""
`O'I
`:-'"
`I")
`0
`
`~
`
`=
`.....
`~
`.....
`~
`~
`•
`r:JJ.
`d •
`
`I [
`
`70
`
`-+-+Hir. 53-65 -0-+Hep./ 4660 Hir.
`-0-+4660
`-e-+HEPARIN
`-0-THROMBIN
`
`-o-+4660/Hir.
`-6-+Hep./Hir
`-+-+Hep./ 4660
`
`340
`
`330
`
`FIG.16
`
`TEMPERATURE, °K
`320
`
`310
`
`+
`
`300
`
`~
`
`-200
`
`• ..-®
`
`"
`
`0
`
`y
`+
`
`fl)
`/
`,
`
`~·
`-.
`
`I
`
`I
`
`I
`
`I
`
`I
`60
`
`I
`
`I
`
`I
`
`I
`
`I
`50
`
`I
`40
`TEMPERATURE, °C
`
`I
`
`I
`
`I
`
`I
`
`Th. : 50.6 °C
`
`I
`
`I
`
`I
`
`I
`
`I
`30
`
`I
`
`I
`
`I
`
`1200]
`
`• 0
`
`4
`
`0-•"
`
`-------------------
`
`r-.
`
`+Hep./ 4660/Hir. : 63.5 ° C
`+4660/Hir. : 62.82 °C
`+Hep./Hir. : 57.42 °C
`+Hep./ 4660 : 60.05 °C
`+Hir. : 56.37 ° C
`+4660 : 58.25 ° C
`+Hep. : 54.8 °C
`
`0
`
`200
`
`400
`
`g
`>
`u.J
`~
`
`et:::
`u.J
`
`=> _J
`0
`~ e::::
`
`1000
`
`600
`
`(..)
`
`(..)
`u.J
`z
`w
`-.;I-
`co
`C) 800
`C:
`E
`
`Agilent Exhibit 1221
`Page 19 of 75
`
`

`

`i,-
`~
`N
`N
`~
`~
`Q
`~
`O'I
`rJ'J.
`e
`
`C
`,i;;..
`0 ....,
`"'"" O'I
`....
`=-~
`
`'JJ.
`
`~
`
`"'""
`C
`C
`N
`"'""
`O'I
`:-'"
`I")
`0
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`= .....
`~ .....
`~
`•
`r:JJ.
`d •
`
`~
`
`9
`
`6 pJ
`
`5
`
`~
`
`0.5 M NaCl
`
`A
`i
`I
`
`A
`
`•
`
`0 M Nac1--A
`
`0.1 M NaCl/•
`
`I It
`
`I 111 I
`
`I 111 I
`FIG.178
`8
`I I I
`
`I I~
`
`I
`
`9
`I I I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I at ii ti I
`
`I
`
`I
`
`b,s-ANS
`
`48
`j I•.• I
`
`I
`
`pH
`7
`I I I
`
`I
`
`I
`
`6
`I I
`
`I
`
`I
`
`I
`
`5
`I I
`
`I
`
`I
`
`I
`
`401 I
`
`,,~
`
`~O.lMNaCI
`o 0.5 M NaCl ~
`
`0
`
`I-
`
`---
`
`I ~
`
`I
`
`I
`
`I I I
`
`I
`
`I
`
`I I I
`
`I
`
`I
`
`I I
`
`I
`
`I
`
`I
`
`I I
`
`I
`
`I
`
`70 ~ I 216~A~S I
`
`FIG.170
`8
`3611 I I., I ••• , •••• , •• I., •••• , •••• r
`
`1-40
`
`~E 441
`
`FIG.17C
`8
`
`• I,. I. Ir
`
`9
`
`I
`
`• I, •• I I, I
`
`pH
`7
`
`I
`
`6
`4011 ... , .... , I
`
`5
`
`50
`
`j 60]
`
`I I I~ I ~ I ~ ~ ·l: ~~;, I ~
`
`I
`
`I
`
`I I
`
`I
`
`I
`
`70 ~ I 2,6~ T~S I
`
`FIG.17A
`8
`I I I
`
`I IF
`
`I
`
`9
`I I I
`
`I
`
`I
`
`1-50 -1
`
`~ 601
`
`~M NaClt
`
`I
`
`I
`
`pH
`7
`I I I
`
`I
`
`I
`
`6
`I I
`
`I
`
`I
`
`I
`
`5
`I I
`
`I
`
`I
`
`I
`
`401 I
`
`~E soj 1
`
`50
`
`I ~
`
`I
`
`I
`
`I
`
`,
`
`I
`
`I
`
`I
`
`I
`
`,
`
`I
`
`I
`
`I
`
`I
`
`,
`
`I
`
`I
`
`I
`
`I
`
`,
`
`I
`
`I
`
`70 ~ I ~ .B~A~S I
`
`Agilent Exhibit 1221
`Page 20 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 17 of 40
`
`US 6,303,322 Bl
`
`101--,-------,,---------,.-----------
`• CaCl2
`l2"LI EDTA
`[Z) on
`D PEG 6000
`IS1 GLYCEROL
`
`5
`
`~
`0
`
`-51-t--~--L-------L-----.l.----!:::=---..J-
`2,6-ANS
`2,6-TNS
`bisANS
`1,8-ANS
`FIG.18
`
`Agilent Exhibit 1221
`Page 21 of 75
`
`

`

`i,-
`~
`N
`N
`~
`~
`Q
`~
`O'I
`rJ'J.
`e
`
`C
`,i;;..
`0 ....,
`"'"" 00
`~ ....
`'JJ. =-~
`
`"'""
`C
`C
`N
`"'"" O'I
`:-'"
`I")
`0
`
`~
`
`~ = .....
`~ .....
`~
`•
`r:JJ.
`d •
`
`330
`
`FIG.19
`325
`
`320
`
`TEMPERATURE, ° K
`310
`315
`
`305
`
`300
`
`~
`
`55
`
`-~
`
`50
`
`rL
`
`:5
`u.J >
`::3 L&..
`0
`~ 1000
`
`500
`
`0:::
`u.J
`
`315.14/41.99
`311.70/38.55
`316.78/43.63
`312. 71 /39.56
`314.26/41.11
`310.86/37.71
`
`...
`• pH 8/0.5 NaCl
`B-pH 8/0.1 NaCl
`e--pH 7 /0.5 NaCl
`e---pH 7 /0.1 NaCl
`
`pH 9/0.5 NaCl
`pH 9/0.1 NaCl
`
`8
`
`~ 1500
`C:
`E
`
`(...)
`u.J
`(...) :z
`L.J
`~
`
`45
`
`40
`
`35
`
`30
`
`25
`
`Agilent Exhibit 1221
`Page 22 of 75
`
`

`

`i,-
`~
`N
`N
`~
`~
`Q
`~
`O'I
`rJ'J.
`e
`
`C
`,i;;..
`0 ....,
`"""
`\C
`~ ....
`'JJ. =-~
`
`""" ~~
`:-'"
`I")
`0
`
`"""
`
`C
`C
`N
`
`~ = .....
`~ .....
`~
`•
`r:JJ.
`d •
`
`~
`
`FIG.20
`
`TEMPERATURE, °K
`
`325
`
`320
`
`315
`
`310
`
`305
`
`300
`
`-
`
`Lu > 3 500
`
`0::
`LL.I
`
`~
`
`e
`
`~o
`
`6 ARGININE 311.76/38.61
`312.12/38.97
`311.05/37.90
`311.05/37. 90
`310.81/37.66
`
`D MgCl2
`e--CaCl2
`e-EDTA
`
`• MgS04
`
`1500
`
`~
`::::,
`0
`0:::
`~ 1000
`0
`i:5
`0
`LJ
`~
`co
`0
`
`C:
`E
`
`55
`
`50
`
`45
`
`40
`
`35
`
`30
`
`25
`
`Agilent Exhibit 1221
`Page 23 of 75
`
`

`

`i,-
`~
`N
`N
`~
`~
`Q
`~
`O'I
`rJ'J.
`e
`
`~ ....
`'JJ. =-~
`
`C
`,i;;..
`0 ....,
`C
`N
`
`'"""
`C
`C
`N
`'""" ~~
`:-'"
`I")
`0
`
`~ = .....
`~ .....
`~
`•
`r:JJ.
`d •
`
`55
`
`50
`
`45
`
`40
`
`35
`
`30
`
`25
`
`D Na2S04
`
`(NH4)2S04
`
`0
`
`• GLYCEROL
`• LiS04
`
`,,,---
`
`/
`
`/
`
`310.86/37.71
`310.69/37.54
`315.93/42.78
`316.21/43.06
`315.79/42.64
`316.80/43.65
`
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`/
`
`FIG.21
`320
`325
`
`TEMPERATURE, °K
`315
`310
`
`305
`
`300
`
`/
`
`j
`
`•
`
`---pH 7/0.1 NaCl
`
`PEG 6000
`
`l::,.
`
`a:::
`Lu
`~ 500
`:2:::
`u..J
`~
`0
`~ 1000
`
`~ 1500
`C:
`E
`
`(..)
`u..J
`(..) z
`LJ
`~
`
`Agilent Exhibit 1221
`Page 24 of 75
`
`

`

`i,-
`
`~
`~
`
`~
`
`O'I
`rJ'J.
`e
`
`C
`
`:
`~
`a
`
`N
`
`g2
`
`~
`g
`~
`~
`~
`
`S.
`~
`~
`~
`r'1
`d •
`
`FIG. 2 2
`
`325
`
`320
`
`TEMPERATURE, °K
`315
`310
`
`305
`
`300
`
`~
`F
`
`310.76/37.71 (pH 7 /0.1 NaCl)
`307.10/33.95 (DTT)
`305.29/32.14 (pH 6; 0.5 NaCl)
`
`55
`
`50
`
`45
`
`40
`
`35
`
`30
`
`25
`
`.A
`
`~ A
`•
`
`A A
`
`.A
`
`_ __:1 _ _(]_______
`D
`
`W
`
`--
`
`--
`
`.,,.
`
`,,.'
`
`pH 6/0.5 NaCl
`pH 6/0.1 NaCl
`pH 5/0.5 NaCl
`pH 5/0.1 NaCl
`
`• on
`AZnS04
`~ NiS04
`•
`0
`.A A e
`O
`
`---pH 7/0.1 NaCl
`
`e
`
`•
`
`A
`
`-.,;
`
`.,,.
`
`n ~ f
`
`1000-=l
`
`D O
`
`t:::.
`~ g O
`o
`•
`•.
`
`~
`Q
`
`e
`
`A
`
`7000
`
`~
`3
`~
`ci 3000
`g
`~
`~
`~
`~ 5000
`~
`o
`c
`E
`
`~
`
`Agilent Exhibit 1221
`Page 25 of 75
`
`

`

`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 22 of 40
`
`US 6,303,322 Bl
`
`• 0.1 M NaCl
`• 0.5 M NaCl
`
`FGFR1 DII
`
`45
`
`(.)
`0
`
`- 40 E
`..._
`
`35
`
`6
`
`7
`
`pH
`FIG.23
`
`8
`
`9
`
`Agilent Exhibit 1221
`Page 26 of 75
`
`

`

`U.S. Patent
`
`Oct.