United States Patent
`
`[19]
`
`IlllllllllllllllllllllIlllllillllllllllllllllllIillllllllllllllllllllllllll
`
`USO05663304A
`[11] Patent Number:
`
`5,663,304
`Builder et al. [45] Date of Patent:
`Sep. 2, 1997
`
`
`
`[54] REFOLDING OF MISFOLDED INSULlN-
`L[KE GROWTH FAc'1'()R.[
`
`[75]
`
`Inventors: Stuart Builder. Belmont; Roger Hart.
`Burlingame; Philip Lester. San
`Lorenzo; David Reifsnyder. San
`Mate“ all 0f Calm
`
`_
`.
`.
`[73] Ass1gnee: Genentech, Inc. San Francisco. Calif.
`
`[21] App1_ NW 110,554
`
`[22] Filed:
`
`Aug. 20, 1993
`
`Int. Cl.“ ......................... C07K 14/475; C12N 15/18
`[51]
`[52] U.S. Cl.
`.......................... 530/399; 530/418; 530/420;
`530/422; 530/424; 435/69.4; 435/172.3;
`435/252.3; 435/320.1
`[58] Field of Search .................................. 435/69.4. 69.1.
`435/240.2. 172.1. 172.3. 252.3. 320.1; 530/350.
`399. 303. 418-420. 421-422. 424
`
`361330
`360937
`0433225/x1
`M13440”
`
`4/1990 Eumpean Pat 05- -
`4/1990 European Pat. OE. .
`11/1990 European Pat. on. .
`2/1991
`E“‘°P°"‘“ P3" 05- -
`gumpean gm‘
`‘
`8/1987 1;?” at‘
`’
`62490199
`63-204796 1211983
`Japan.
`63-294796 12/1933
`Japan.
`W036/05399 10/1935 wlpo.
`W088/08003 10/1988 WIPO .
`W088/08849 11/1988 WIPO .
`W091/00344
`1/1991 WIPO .
`W091/(T2089 W199l WIPO .
`W092/03477
`3/1992 WIPO .
`w093m240
`5,1993 WIPO A
`W093/19084
`9/1993 WIPO .
`
`‘
`
`OTHER PUBLICATIONS
`Callard & Gearing (1994). The Cytokine Facts Book. Aca-
`demic Press. Harcourt Brace & Co. Publishers pp. 2-3.
`Marston (1986). Biochemical
`Journal. vol. 240. pp.
`1-l2.Brems et al.. “Equilibrium Denaturation of Pituitar-
`y-and Recombinant—Derived Bovine Growth Hormone”.
`3:.:;6::;66§.5:;:::a::§.:;::.::.;::.:s..:38:......
`in Escherichia coli is Favored By Lower Growth Tempera-
`tures”. pp. 291-294. Biotechnology. vol. 6. Mar. 1988.
`Mizukami et al.. “Production of Active Human Interferon-B
`in E. coli I. Preperential Production By Lower Culture
`T
`19‘
`‘
`I.
`-
`.
`I
`Nzmgerfggrg Pp 605410 B‘°t°°h“°1°gy L°“°“ V°1 8
`.'
`'.
`'
`.
`.
`.
`.
`%’I‘:1"ak1F°‘F‘g1dmg9I_1]1t¢T1;I}°t“l:13lt1¢5i1nd
`
`011113 0" ~ PP-
`
`~
`
`10 C no 0837» V0 -
`
`~
`
`0 Y
`1989.
`Marston et al.. “[20] Solubilization of Protein Aggregates".
`pp. 264-276. Biochem. J. 240. 1 (1986).
`Wetzel. “Protein Aggregation in Vivo-Bacterial Inclusions
`-
`ammah
`-
`-v
`__
`-~
`glfgigzgggltliials Parf';3i°§l'qn3{};::§::fl’{V:§’,s§)i'.‘])S‘:abfl;:3;i;)If
`.
`'
`. '
`.
`.
`.
`gm
`and Strateg1es for Ptotem Stab1l1zat1on. New York. 1992.
`Raschdort et al.. “Location of Disulphide Bonds in Human
`1080110-Like Growth Factors (IGFS) Synthcs1zed by Recom-
`binant DNA Technolgy”. pp. 3-8. Biomedical and Environ-
`mental Mass Spectormetry. vol. 16. 1988.
`§,‘,$i,.f.E..‘:‘y‘;s.§’Z’.i’..?°1§.';.f..f"£.‘é‘ii‘.’::,§?.§‘.§.*“%.§‘.i‘.“§ZS§§
`pp. 803-806. Biochem. J. (1990) 268.
`.
`.
`0415‘ °°“‘"'“°d °" "W P3339
`primary Examiner____[ohn mm
`Assistant Egmml-ner__P1.ema Menz
`Attome); Agent, or Firm—Walter H. Dreger
`[57]
`ABSTRACT
`
`A composition is provided comprising about 0.1 to 15
`mymL of a polypeptide in a bufler having a pH of about
`7_
`-
`'
`-
`
`metal. or ammonium salt. about 0.1 to 9M of a chaotropic
`d
`1
`agent. an about.0.0. to 15 11M of a copper or manganese
`salt. The buffer is suitably used 1n a method for refoldmg
`Improperly folded polypeptides.
`
`12 Claims, 12 Drawing Sheets
`
`APOTEX EX1033
`
`Page 1
`
`[56l
`
`4,511,502
`4.511503
`4512322
`
`4,565,785
`4 72,79
`
`.......................... 530/417
`.- 530/422
`-- 530/403
`-
`
`Referellces Cited
`mm
`4/1985 Builder et al.
`4/1985 Olson et al.
`4/1935 101165 3‘ 31-
`(S__;:‘)‘l’:‘“z
`.
`.........
`1/1936 Gilbert et 21.
`2
`6
`ths
`.
`.7133. §;:..::_'.......
`
`
`
`435/317
`2:321;
`
`530/344
`....... 435/68
`S30/399
`..... 435/320
`
`..
`
`..
`.
`
`~-
`
`_
`
`3/1987 Bentle et al.
`4,652,630
`6/1987 Georgeet al.
`4,673,641
`11/1987 Yang et al.
`4,705,848
`4,710,473 12/1987 Morris ............
`I1?I:!1a8aJ€t<:l31«
`unge
`.
`.
`,
`,
`.
`5/1990 Bobbitt etal. ..
`4,923,967
`530/351
`1/1991 Yokoo et al.
`4,985,544
`530/399
`5/1991 Ieda at 31.
`5 019500
`435/69 1
`7/1991 Ueda gt 3].
`5:093531
`435/69:4
`5,153,375 10/1992 Miller et al.
`435/69.1
`5,191,o63
`3/1993 Inouye at a1_
`53o/324
`5,288,93l
`2/1994 Chang et al.
`........................... 530/399
`Foam: mm seams
`0128733
`6/1984 European Fat. 011".
`.
`0130166
`6/1984
`E
`P 1. Off.
`.
`123733 12/1984 Big: P::.o1r. .
`130166
`1/1985 European Pat. 01f. .
`0135094
`3/1985 European Pat. Off. .
`E:
`’
`196056 10/1986 European Pat. on. .
`219814
`4/1987 European Pat. Off. .
`0264074 10/1987 European Fat. 03..
`European :31»
`-
`uropean at.
`..
`mm on.
`0360937
`9,1988 Eumpean Pat‘ OE‘ '
`286345 10/1988 European Pat. Ofl‘.
`.
`238451
`lo/1983 European Pu OE _
`293793 12/1933 European pm‘ Ofig _
`302469
`2/1939 European Pat. on. .
`312358
`4/I989 European Pat. OE. .
`0361830A2
`4/1990 European Pat. Ofli .
`
`APOTEX EX1033
`
`Page 1
`
`

`
`5,663,304
`Page 2_j__
`
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`
`Toren et al.. “Determination of Interchain Crosslinkages in
`insulin B—Chain Dimers by Fast Atom Bombardment Mass
`Spectremetry”. pp. 287-299. Analytical Biochemistry 169.
`(1988).
`Green et al.. “Cheddar cheesemaking with recobinant calf
`chymosin synthesized in Escherichia coli”, pp. 281-286.
`Journal of Dairy Research. (1985).
`Frank et al.. “The Production of Human Proinsulin and Its
`Transformationto Human Insulin and C—Peptide”. pp.
`729-738 Rich and Gross. (1981).
`Cleland et al.. “Refolding an Deggregation of Bovine Car-
`bonic Anhydrase B: Quasi-Elastic Light Scattering Analy-
`sis”. pp. 11072-11078. Biochemistry. 29. 1990.
`Halenbeck et al.. “Renaturation and Purification of Biologi-
`cally
`Active
`Recombinant
`Human Macrophage
`Colony—Stirnu1ating Factor Expressed in E‘. Coli”, pp.
`710-715. Biotechnology. vol. 7. Jul. 1989.
`Tsuji et al.. “Characterization of Disulfide Bonds in Recom-
`binant Proteins: Reduced Human Interleukin 2 in inclusion
`Bodies and Its Oxidative Refolding”. pp. 3129-3134. Bio-
`chemistry. 26. 1987.
`George et al.. “High-Level Expression in Escherichia coli
`of Biologically Active Bovine Growth Hormone". pp.
`273-281. Mary Ann Liebert. Inc. Publishers, 1985.
`Gill et al.. “Recombinant Chicken and Bovine Growth
`Hormones Accelerate Growth in Aquacultured Juvenile
`Pacific Salmon Oncorhynchus Kisutch”. pp. 643-646. Bio-
`technology. vol. 3. 1985.
`Sekine et al.. “Cloning and expression of cDNA for salmon
`growth hormone in Escherichia coli ”, pp. 4306-43 10. Proc.
`Natl. Acad. Sci. USA. vol. 82. 1985.
`Winkler et al.. “Purification and Characterization of Recom-
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`Biotechnology. vol. 3. Nov. 1985.
`Kohnert et al. . “Production of a Recombinant Human Tissue
`Plasminogen Activator Variant (BM 06.022) from Escheri-
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`Posters 1. 1990.
`
`‘The pmiiication of eukaryotic polypeptides
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`Boss et al.. “Assembly of functional antibodies from immu-
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`
`Cabilly et al.. “Generation of antibody activity from immu-
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`
`Marston et al.. “Pm1'.fication of Calf Prochymosin (Proreen-
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`Hoppe
`et
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`Biochemistry. 28. 1989.
`Bowden et al.. “Structure and Morphology of Protein Inclu-
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`Wetzel et al.. “Mutations in Human Interferon Gamma
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`vol. 9. 1991.
`
`Hejnaes et al.. “Development of an optimized refolding
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`Protein Engineering. vol. 5. 1992.
`Obukowicz et al.. “Secretion and Export of IGF—1 in
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`
`Wong et al.. “Expression of secreted insulin-Like growth
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`Saito et al.. “Direct Expression of a Synthetic Somatomedin
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`Schulz et al.. “Increased Expression in Escherichia coli of a
`Synthetic Gene Encoding Human Somatiomedin C after
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`Bacteriology. Dec. 1987.
`Saito et al.. “Production and Isolation of Recombinant
`Somatomedin C”. pp. 123-134. J. Biochorn. 101. 1987.
`Niwa et al.. “Chemical Synthesis. Cloning. and Expression
`of Genes for Human Somatomedin C (Insulin-Like Growth
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`Hober et al.. “Disulfide Exchange Folding of Insulin-Like
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`
`Funakoshi et al.. “Isolation of a tumor—derived 186-residue
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`Lustig et al.. ‘The thermal denaturation of ribonuctease A in
`aqueous-methanol solvents”. pp. 205-210. Biochimica et
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`Bryant et al.. “Detection of an Equilibrium intermediate in
`the Folding of a Monomeric Insulin Analog”. pp.
`5692-5698. Biochemistry. vol. 31. No. 25 (1992).
`of
`Hua
`et
`al..
`"I‘wo—dimensional NMR studies
`Des-(B26-B30)-insulin:
`sequence-specific
`resonance
`assignments and efiects of solvent composition”. pp.
`101-110. Biochimica et Biophysica Acta. 1078 (1991).
`Brems et al.. “Equilibrium Denaturation of Insulin and
`Proinsulin”. Biochemistry. vol. 29. (1990).
`Jackson et al.. “Hatogenated alcohols as solvents for pro-
`teins: FI'lR spectroscopic studies”. pp. 139-143. Bio-
`chimica et Biophysica Acta. 1118. )1992.
`Shibata et al.. “Biphasic Eifects of Alcohols on the Phase
`Transition of Poly(L-Lysine) between a-Heliz and B—Sheet
`Confonnations". pp. 5728-5733. Biochemistry. vol. 31.
`(1992).
`
`Zhong et al.. “Enviromnent aifects amino acid preference for
`secondary structure”. pp. 4462-4465. Proc. Natl. Acad Sci.
`USA. May 1992.
`
`Wetzel. “Principles of protein stability. Part 2-enhanced
`folding and stabilization of proteins by suppression of
`aggregation in vitro and in vivo”. pp. 191-219. Protein
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`Gold.
`“Purification of Biosynthetic Human Relaxin
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`
`Page 2
`
`Page 2
`
`

`
`5,663,304
`Page 3
`
`of Recombinant Human
`“Production
`Rinderknecht.
`Relaxin". Twelfth international Symposium on HPLC of
`Proteins. Peptides and Polynucteotides”. Sydney. Australia.
`1992.
`
`Boss et al.. “Assembly of functional antibodies from immu-
`noglobulin heavy and light chains synthesised in E. coli”
`Nucleic Acids Research 12(9):3791-3806 (1984).
`Bowden et al.. “Structure and Morphology of Protein Inclu-
`sion Bodies in Escherichia Coii” Bio/Technology 91725-730
`(Aug. 1991).
`Brems D. et al.. “Equilibrium denaturation of pituitary-—
`and recombinant—derived bovine growth hormone” Bio-
`chemistry 24(26):7662—7668 (1985).
`Brerns et al.. “Equilibrium Denaturation of Insulin and
`Proinsulin” 29:9289—9293 (1990).
`Bryant et al.. “Detection of an Equilibrium Intermediate in
`the Folding of a Monomeric Insulin Analog" Biochemistry
`31(25):5692—5698 (1992).
`Cabilly et z«fl.. “Generation of antibody activity from immu-
`noglobulin polypeptide chains produced in Escherichia
`coli” Proc. Natl. Acad. Sci. USA 813273-3277 (1984).
`Cleland et al.. “Refolding and Aggregation of Bovine Car-
`bonic Anhydrase B: Quasi—Elastic Light Scattering Analy-
`sis” Biochemistry 29:11072—11078 (1990).
`Frank et al.. ‘The Production of Human Proinsulin and its
`Transformation to Human Insulin and C—Pepti
`" Peptides:
`Synthesis. Structure. Function, Rich and Gross pp. 729-738
`(1981).
`Funkakoshi et al.. “Isolation of a 'l‘umor—Derived 186—Resi-
`due Peptide Amide Related to Human Chromogranin A and
`its in vitro Conversion to Human Pancreastatin—48” Recent
`Bioactive Peptides and Biology pp. 512-514 (1989).
`George et al.. “I-Iigh—Level Expression in Escherichia coli
`of Biologically Active Bovine Growth Hormone” DNA
`4:273—28l (1985).
`Gill et al.. “Recombinant Chicken and Bovine Growth
`Hormones Accelerate Growth in Aquacultured Juvenile
`Pacific Salmon Onoorhynchus Kisutch” Bio/Technology
`3:643-646 (1985).
`Gold.
`“Purification of Biosynthetic Human Relaxin
`A—Chain and B—Chain and Scaleup of the Chain Combina-
`tion Reaction” Protein Folding and Recovery Symposium
`(1992).
`
`Green et al.. “Cheddar cheesemaldng with recombinant calf
`chymosin synthesized in Escherichia coli"
`J. Dairy
`Research 52281-286 (1985).
`
`Halenbeck et al.. “Renaturation and Purification of Biologi-
`cally
`Active
`Recombinant
`Human Macrophage
`Colony—Stimulating Factor Expressed in E. Coli” Bio/I'ech-
`nology 7:7 10-7 15 (1989).
`
`Hejnaes et al.. “Development of an optimized refolding
`process for recombinant Ala-Glu—IGF—1” Protein Engi-
`neering 5(8):797—806 (1992).
`Hober et al.. “Disulfide Exchange Folding of Insulin—Like
`Growth Factor I” Biochemistry 3l:l749-1756 (1992).
`Hoppe
`et
`al..
`“Preparation of Biologically Active
`Platelet—Derived Growth Factor Type BB from a Fusion
`Protein Expressed in Escherichia coli” Biochemistry
`28 :2956—2960 (1989).
`of
`Hua
`et
`al..
`“'l‘wo—dimensional NMR studies
`Des-(B26—B30)-insulin:
`sequence-specific
`resonance
`assignments and efiects of solvent composition” Biochimica
`et Biophysica Acta l078:l0l—110 (1991).
`
`Jackson et al.. “Halogenated alcohols as solvents for pro-
`teins: FUR spectroscopic studies” et Biochimica et Bio-
`physica Acta l118:139—l43 (1992).
`Kohnert et al.. “Production of a Recombinant Human Tissue
`Plasminogen Activator Variant (BM 06.022) from Escheri-
`chia Cali Using a Novel Renaturation Technology” Fibrin-
`olysis 4(Suppl. 3. Abs. 116):44 (1990).
`Lustig et al.. ‘The Thermal Denaturation of Ribonuclease A
`in Aqueous—Methanol Solvents" Biochimica et Biophysica
`Acta 1ll9:205—2l0 (1992).
`Marston. ‘The purification of eukaryotic polypeptides syn-
`thesized in Escherichia Cali” Biochemical
`Journal
`240:l—12 (1986).
`Marston et al.. “Purification of Calf Prochymosin (Proteu-
`nin) Synthesized in Escherichia Cali" Bio/Technology
`2:800—804 (Sep. 1984).
`Marston et al.. “Solubilization of Protein Aggregates” Meth
`Enzymol. 182(1):264—276 (1990).
`Mitraki et al.. “Protein Folding Intermediates and Inclusion
`Body Fonnation” Bio/Technology 7:690—697 (1989).
`Mizukami et al.. “Production of Active Human Interferon—|3
`in E. coli, 1. Preferential Production by Lower Culture
`Temperature" Biotechnology Letters 8(9):605-610 (1986).
`Morris et al.. “Protein folding/refolding analysis by mass
`spectrometry—Scrambling of disulphide bridges in insulin”
`Biochemical Journal 268:803—806 (1990).
`Niwa et al.. “Chemical Synthesis. Cloning. and Expression
`of Genes for Human Somatomedin C (Insulin-like Growth
`Factor 1) and 59Val—Somatomedin C” Annals New York
`Academy of Sciences 469:3l—52 (1986).
`Obukowicz et al.. “Secretion and Export of IGF—1 in
`Escherichia coli strain JMIOI” Mol. Gen. Genet. 2l5:19—25
`(1988).
`Raschdorf et al.. “Location of disulphide bonds in human
`insulin like growth factors (IGFs) synthesized by recombi-
`nant DNA technology” Biomedical and Environmental Mass
`Spectromemr 16:3—8 (1988).
`of Recombinant Human
`Rindcrknecht.
`“Production
`Relaxin” Twelfih International Symposium on HPLC of
`Proteins, Peptides and Polynucleotides (Sydney. Australia)
`(1992).
`Saito et al.. “Direct Expression of a Synthetic Somatomedin
`C Gene in Escherichia coli by Use of a 'I‘wo—Cist:ron
`System” J. Biochem. 101:1281—1288 (1987).
`Saito et al.. “Production and Isolation of Recombinant
`Somatomedin C” J. Biochem. 101:123—134 (1987).
`Schein et al.. “Formation of Soluble Recombinant Proteins
`in Escherichia coli is Favored by Lower Growth Tempera-
`ture” Bio/Technology 6:291—294 (1988).
`Schulz et al.. “Increased Expression in Escherichia coli of a
`Synthetic Gene Encoding Human Somatomedin C after
`Gene Duplication and Fusion” Journal of Bacteriology
`169:5385-5392 (Dec. 1987).
`Sekine et al.. “Cloning and expression of cDNA for salmon
`growth hormone in Escherichia coli” Proc. Natl. Acad. Sci.
`USA 82143064310 (1985).
`Shibata et al.. “Biphasic Eifects of Alcohols on the Phase
`Transition of Poly(L—lysine) between ot—Helix and [5-Sheet
`Conformations” Biochemistry 31:5728—5733 (1992).
`Snyder. "Free Energy Relationships for Thiol—Disulfide
`Interchange Reactions between Charged Molecules in 50%
`Methanol”
`Journal
`of
`Biological
`Chemistry
`259(12):7468—7472 (1984).
`
`Page 3
`
`Page 3
`
`

`
`5,663,304
`Page 4
`
`Toren et al.. “Determination of Interchain Crosslinkages in
`Insulin B—Chain Dimers by Fast Atom Bombardment Mass
`Spectrometry” Analytical Biochemistry
`169:287—299
`(1988).
`Tsuji et al.. “Characterization of Disulfide Bonds in Recom-
`binant Proteins: Reduced Human Interleukin 2 in Inclusion
`Bodies
`and Its Oxidative Refolding” Biochemistry
`26:3129-3 134 (1987).
`Wetzel. “Principles of protein stability. Part 2-enhanced
`folding and stabilization of proteins by suppression of
`aggregation in vitro and in vivo” Protein Engineering: A
`Practical Approach, Rees et al.. ed.. Chapter 8. pp. 191-219
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`Wetzel. “Protein Aggregation in vivo—Bacterial Inclusion
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`Wetzel et al.. “Mutations in Human Interferon Gamma
`Affecting Inclusion Body Formation Identified by a General
`Imrnunochemical
`Screen” Bio/I'echnology
`9:731—737
`( 1991).
`Winkler et al.. “Purification and Characterization of Recom-
`binant Urokinase from Escherichia Coli” Bio/Technology
`3:990-1000 (1985).
`Wong et al.. “Expression of secreted insulin—like growth
`factor-1 in Escherichia coli” Gene 68:193-203 (1988).
`Zhong et al.. “Environment affects amino acid preference for
`secondary
`structure” Proc. Natl. Acad.
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`5:1047—1051 (1987).
`
`Iwai et al.. “Direct Identification of Disulfide Bond Linkages
`in Human Insulin-Like Growth Factor I (IGF—I) by Chemi-
`cal Synthesis" J. Biochem. lO6:949—951 (1989).
`Meng et al., “Reduction Studies on Bacterial Recombinant
`Somatomedin C/Insulin—like Growth Factor—l” J. Chr-
`maotgraphy 443 : 183-192 (1988).
`
`Murphy et al.. “Common Features of Protein Unfolding and
`Dissolution
`of Hydrophobic Compounds”
`Science
`247:559-561 (1990).
`Pocket and Biswas. “Self—Association of Insulin and the
`Role of Hydrophobic Bonding: AThermodynamic Model of
`Insulin Dimerization” Biochemistry 20:4354-4361 (1981).
`Rudolph et al.. “Reactivation of Microbially Produced
`Human Tissue-Type Plasminogen Activator” Senior Advi-
`sory Group on Biotechnology; Biotechnology International,
`Londonzcentury Press pp. 321-322. 324-325 (1991).
`Samuelsson et al.. “Facilitated In Vitro Refolding of Human
`Recombinant Insulin-Like Growth Factor I Using a Solu-
`bilizing Fusion Partner” Bio/Technology 91363-366 ( 1991).
`Smith et al.. “Structure and Activity Dependence of Recom-
`binant Human Insulin-Like Growth Factor 11 on Disulfide
`Bond
`Pairing”
`Journal
`of Biological Chemistry
`264(l6):93l4—9321 (1989).
`Spolar et al.. “Hydrophobic elfect in protein folding and
`other noncovalent processes involving Proteins” Proc. Natl.
`Acad. Sci. USA 86:8332-8385 (1989).
`Wetzel
`et
`al..
`“Production of Biologically Active
`N“-Desacetyl Thyrnosin oz, in Escherichia coli Through
`Expression of a Chemically Synthesized Gene" Cellular
`Responses to Molecular Modulators 251-270 (1981).
`Zettlmeissl et al.. “Reconstitution of Lactic Dehydrogenase.
`Noncovalent Aggregation vs. Reactivation.
`1. Physical
`Properties and Kinetics of Aggregation" Biochemistry
`18(25):5S67—5571 (1979).
`
`Hermann, R.. “Standard Techniques for Refolding” Protein
`Folding (Chapter 2). 1st edition, Netherlands:European
`Patent Oflice p. 1 (1993).
`
`Page 4
`
`Page 4
`
`

`
`U.S. Patent
`
`Sep. 2, 1997
`
`Sheet 1 of 12
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`5,663,304
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`U.S. Patent
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`

`
`U.S. Patent
`
`Sep.2, 1997
`
`Sheet 11 of 12
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`5,663,304
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`Page 15
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`

`
`U.S. Patent
`
`Sep.2, 1997
`
`Sheet 12 of 12
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`5,663,304
`
`40
`
`8
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`YIELDCORRECTLYFOLDEDIGF"1("/o) B
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`
`to
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`
`Page 16
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`

`
`Sbfllflfl
`
`1
`REFOLDING OF MISFOLDED INSULIN-
`LIKE GROWTH FACTOR-I
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`
`This invention relates to special buffer solutions and their
`use for refolding polypeptides.
`2. Description of Related Art
`For commercial production of many polypeptides and
`proteins. recombinant DNA techniques have become the
`method of choice because of the large quantities that can be
`produced in bacteria and other host cells. Manufacturing
`recombinant protein involves transfecting or transforming
`host cells with DNA encoding the desired exogenous protein
`and growing the cells under conditions favoring expression
`of the recombinant protein. E. coli and yeast are favored as
`hosts because they can be made to produce recombinant
`proteins at high titers.
`Numerous U.S. patents on general bacterial expression of
`recombinant-DNA-encoded proteins exist. including U.S.
`Pat. No. 4.565.785 on a recombinant DNA molecule com-
`prising a bacterial gene for an extracellular or periplasmic
`carrier protein and non-bacterial gene; U.S. Pat. No. 4.673.
`641 on coproduction of a foreign polypeptide with an
`aggregate-forrning polypeptide; U.S. Pat. No. 4.738.921 on
`an expression vector with a up promota/operator and up LE
`fusion with a polypeptide such as insulin-like growth factor
`(IGF-I); U.S. Pat. No. 4.795.706 on expression control
`sequences to include with a foreign protein; and U.S. Pat.
`No. 4.710.473 on specific circular DNA plasmids such as
`those encoding IGF-I.
`
`Under some conditions. certain heterologous proteins
`expressed in large quantities from bacterial hosts are pre-
`cipitated within the cells in dense aggregates. recognized as
`bright spots visible within the enclosure of the cells under a
`phase-contrast microscope. These aggregates of precipitated
`proteins are referred to as “refractile bodies.” and constitute
`a significant portion of the total cell protein. Brems et al..
`Biochemistry, 24: 7662 (1985). On the other hand.
`the
`aggregates of protein may not be visible under the phase
`contrast microscope. and the term “inclusion body” is often
`used to refer to the aggregates of protein whether visible or
`not under the phase-contrast microscope.
`It has been found that the soluble proportion of high-level
`expressed protein in E. coli has been dramatically increased
`by lowering the temperature of fermentation to below 30° C.
`A considerable fraction of various foreign proteins. i.e.,
`human interferon-alpha (IFN-(12). interferon-gamma (IFN-
`7). and murine MX protein [Schein and Notebom. Bio/
`Technology, 6: 291-294 (1988)) and human IFN-[3
`[Mizukami et al.. Biotechnol. Lett., 8: 605-610 (1986)].
`stayed in solution. This procedure represents an alternative
`to renaturation of proteins recovered from refractile bodies.
`but requires an expression system that is efliciently induced
`at temperatures below 30° C. The procedure is therefore not
`effective for all proteins.
`For general review articles on refractile bodies. see
`Marston. supra; Mitralci and King. Bio/Technology, 7: 690
`(1989); Marston and Hartley. Methods in Enzymol., 182:
`264-276 (1990); Wetzel. “Protein Aggregation In Vivo:
`Bacterial Inclusion Bodies and Mammalian Amyloi
`.” in
`Stability of Protein Phamzaceuticals: In Vivo Pathways of
`Degradation and Strategies for Protein Stabilization, Ahem
`and Manning (eds.) (Plenum Press. 1991); and Wetzel.
`“Enhanced Folding and Stabilization of Proteins by Sup-
`
`2
`pression of Aggregation In Vitro and In Vivo." in Protein
`Engineering—A Practical Approach, Rees. A. R. et al. (eds.)
`(lRL Press at Oxford University Press. Oxford. 1991).
`Recovery of the protein from these bodies has presented
`numerous problems. such as how to separate the protein
`encased within the cell from the cellular material and
`proteins harboring it. and how to recover the inclusion body
`protein in biologically active form. The recovered proteins
`are often predominantly biologically inactive because they
`are folded into a three-dimensional conformation different
`from that of active protein. For example. rnisfolded IGF-I
`with different disulfide bond pairs than found in native IGF-I
`has significantly reduced biological activity. Raschdorf et
`al.. Biomedical and Environmental Mass Spectroscopy, 16:
`3-8 (1988). Misfolding occurs either in the cell during
`fermentation or during the isolation procedure. Methods for
`refolding the proteins into the correct. biologically active
`conformation are essential for obtaining functional proteins.
`Another property experienced by proteins during refold-
`ing is the tendency to produce disulfide-linked dimers.
`trimers. and multimers. Morris et al.. Biochem. J., 268:
`803-806 (1990); Toren et al.. Anal. Biochem., 169: 287-299
`(1988); Frank et al..
`in “Peptides:
`.s'ynthesis-structure-
`function, ”ed. D. H. Rich and E. Gross. pp. 729-738 (Pierce
`Chemical Company: Rockford. Ill.. 1981). This association
`phenomenon is very common during protein refolding.
`particularly at higher protein concentrations. and appears
`often to involve association through hydrophobic interaction
`of partially folded intermediates. Cleland and Wang.
`Biochemistry, 29: 11072-11078 (1990).
`Protein folding is influenced by the nature of the medium
`containing the protein and by a combination of weak attrac-
`tive or repellent intramolecular forces involved in hydrogen
`bonding.
`ionic bonding. and hydrophobic interactions.
`When pairs of cysteine residues are brought
`into close
`proximity as the peptide backbone folds. strong covalent
`disulfide bonds form between cysteine residues. serving to
`lock the tertiary conformation in place. Refolding protocols
`have been designed to break incorrect disulfide bonds. block
`random disulfide bonding. and allow refolding and correct
`disulfide bonding under conditions favorable to the forma-
`tion of active conformer.
`
`l0
`
`71)
`
`25
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`One series of techniques for recovering active protein
`from inclusion bodies involves solubilizing the inclusion
`bodies in strongly denaturing solutions and then optionally
`exchanging weakly denaturing solutions for the strongly
`denaturing solutions (or diluting the strongly denaturing
`solution). or using molecular sieve or high-speed centrifu-
`gation techniques. Such recovery methods. described. e.g..
`in U.S. Pat. Nos. 4.512.922; 4.518.256; 4511.502; and
`4.511.503, are regarded as being universally applicable. with
`only minor modifications. to the recovery of biologically
`active recombinant proteins from inclusion bodies. These
`methods seek to eliminate random disulfide bonding prior to
`coaxing the recombinant protein into its biologically active
`conformation through its other stabilizing forces.
`In one method for recovering protein from inclusion
`bodies. the denatured protein desired to be refolded is further
`purified under reducing conditions that maintain the cysteine
`moieties of the protein as free sulfhydryl groups. The
`reducing agent is then diluted into an aqueous solution to
`enable the refolded protein to form the appropriate disulfide
`bonds in the presence of air or some other oxidizing agent.
`This enables refolding to be easily incorporated into the
`overall purification process.
`In another approach. refolding of the recombinant protein
`takes place in the presence of both the reduced (R-S-E) and
`
`Page17
`
`Page 17
`
`

`
`5fi6lKM
`
`3
`oxidized (R-S-S-R) fortns of a sulfhydryl compound. This
`allows free sulfhydryl groups and disulfides to be formed
`and reformed constantly throughout the purification process.
`The reduced and oxidized forms of the sulfhydryl compound
`are provided in a buifer having sufficient denaturing power
`that all of t