UNITED STATES PATENT AND TRADEMARK OFFICE
`
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`
`FRESENIUS KABI USA, LLC and FRESENIUS KABI SWISSBIOSIM GmbH
`Petitioners,
`v.
`
`AMGEN, INC. and AMGEN MANUFACTURING LIMITED
`Patent Owner.
`
`
`IPR2019-00971
`
`Patent No. 9,856,287
`
`Title: REFOLDING PROTEINS USING A CHEMICALLY CONTROLLED
`REDOX STATE
`
`
`
`PETITION FOR INTER PARTES REVIEW
`OF U.S. PATENT NO. 9,856,287 B1
`
`
`
`Mail Stop PATENT BOARD
`Patent Trial and Appeal Board
`United States Patent and Trademark Office
`P.O. Box 1450
`Alexandria, VA 22313-1450
`
`
`
`

`

`
`
`
`
`TABLE OF CONTENTS
`
`Page
`
`IV.
`
`B.
`
`
`
`I.
`INTRODUCTION ...............................................................................................................1
`II.
`GROUNDS FOR STANDING ............................................................................................3
`III. MANDATORY NOTICES ..................................................................................................4
`Real Parties In Interest (§ 42.8(b)(1)) ......................................................................4
`A.
`
`Related Matters (§ 42.8(b)(2)) .................................................................................4
`B.
`
`Identification of Counsel (§ 42.8(b)(3)) and Service Information
`C.
`
`(§ 42.8(b)(4))............................................................................................................5
`TECHNOLOGY BACKGROUND .....................................................................................5
`The Basic Science of Proteins..................................................................................5
`A.
`
`1.
`Protein Structure ..........................................................................................5
`2.
`Protein Synthesis in and out of the Lab .......................................................6
`Recovery and Refolding of Expressed Protein ........................................................7
`1.
`Unfolding and Refolding of Recombinant Proteins .....................................7
`2.
`Optimizing Redox Conditions .....................................................................8
`Additional Considerations in Commercial Production of Recombinant
`Proteins ..................................................................................................................10
`THE ’287 PATENT, PROSECUTION HISTORY, AND RELATED
`PROCEEDINGS ................................................................................................................11
`The ’287 Patent ......................................................................................................11
`A.
`
`1.
`The Known Problem in the Art and the Alleged Innovative
`Solution ......................................................................................................12
`2.
`The Scope of the Challenged Claims .........................................................14
`Prosecution History ................................................................................................16
`B.
`
`The Adello PGR .....................................................................................................18
`C.
`
`The Board’s Invalidation of Analogous Claims of the ’138 Patent .......................18
`D.
`
`VI.
`PERSON OF ORDINARY SKILL IN THE ART .............................................................19
`VII. CLAIM CONSTRUCTION ...............................................................................................19
`“Preparation”..........................................................................................................20
`A.
`
`“Is Calculated” .......................................................................................................20
`B.
`
`“Maintains Solubility” ...........................................................................................21
`C.
`
`D.
`Defined Claim Terms In Specification ..................................................................21
`
`
`C.
`
`
`
`V.
`
`
`
`- i -
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`

`

`
`
`
`
`IX.
`
`D.
`
`
`
`
`TABLE OF CONTENTS
`(Continued)
`
`Page
`
`B.
`
`
`
`IDENTIFICATION OF CHALLENGE AND RELIEF REQUESTED ............................22
`Ground 1: Claims 1, 4, 8-10, 12, 14-16, 19, 23-26, 29-30 are anticipated
`A.
`
`by Vallejo (Ex. 1031) .............................................................................................23
`1.
`Claims 1, 10, 16, and 26 ............................................................................24
`2.
`Claims 4, 12, 19 and 29 .............................................................................32
`3.
`Claims 8, 9, 14, 15, 23, 24, 25 and 30 .......................................................33
`4.
`Claim Chart ................................................................................................34
`Ground 2: Claims 16, 19-21, 23-26 and 29-30 are anticipated by Ruddon
`(Ex. 1025) ..............................................................................................................38
`1.
`Claims 16, and 26 are Anticipated by Ruddon ..........................................38
`2.
`Claims 19 and 29 Are Anticipated by Ruddon ..........................................42
`3.
`Claims 23, 24, 25 and 30 Are Anticipated By Ruddon .............................43
`4.
`Claim Chart ................................................................................................44
`Ground 3: Claims 1, 4-6, 8-10, 12, 14-16, 19-21, 23-26, 29-30 are obvious
`over Ruddon in view of Clark 1998 in light of Schafer or Gilbert ........................47
`2.
`Scope and Content of the Prior Art and Differences Between the
`Prior Art and the Challenged Claims .........................................................48
`3.
`Motivation To Combine and Expectation of Success ...............................54
`4.
`Obviousness of the Independent Claims ....................................................56
`5.
`Obviousness of Claims 4, 12, 19 and 29 ....................................................59
`6.
`Obviousness of Claims 5, 6, 20, and 21 .....................................................60
`7.
`Obviousness of Claims 8, 9, 14, 15, 23, 24, 25 and 30 ..............................61
`Ground 4: Claims 8, 9, 14, 15, 23, 24, 25 and 30 Would Have Been
`Obvious From Vallejo In Combination With Ruddon and Clark 1998, In
`Light Of Schafer or Gilbert ....................................................................................62
`Secondary Considerations ......................................................................................63
`E.
`
`CONCLUSION ..................................................................................................................64
`
`C.
`
`
`
`D.
`
`
`
`ii
`
`

`

`
`
`Cases
`
`TABLE OF AUTHORITIES
`
`
`
`Page(s)
`
`King Pharm., Inc. v. Eon Labs, Inc.,
`616 F.3d 1267 (Fed. Cir. 2010)................................................................................................34
`
`Phillips v. AWH Corp.,
`415 F.3d 1303 (Fed. Cir. 2005) (en banc) ...............................................................................19
`
`PTAB Decisions
`
`Apotex Inc. v. Amgen Inc., IPR 2016-01542, Paper 60 at 29 (P.T.A.B. Feb. 15,
`2018) (Ex. 1038) ...........................................................................................................2, 16, 18
`
`Statutes
`
`35 U.S.C. §§ 102 and 103 ..............................................................................................................22
`
`35 U.S.C. § 102(a)(1) .........................................................................................................23, 38, 48
`
`35 U.S.C. § 102(b) .............................................................................................................23, 38, 48
`
`35 U.S.C. § 112 ........................................................................................................................17, 20
`
`35 U.S.C. §§ 311-319 ......................................................................................................................1
`
`35 U.S.C. § 311(c) ...........................................................................................................................3
`
`35 U.S.C. § 314(a) ...........................................................................................................................3
`
`35 U.S.C. § 315(a) ...........................................................................................................................4
`
`35 U.S.C. § 325(d) ...........................................................................................................................3
`
`Pub. L. 112–274, § 1(d)(1), Jan. 14, 2013, 126 Stat. 2456 ..............................................................4
`
`Regulations
`
`37 C.F.R. § 42, et seq. ......................................................................................................................1
`
`37 C.F.R. § 42.15(a).........................................................................................................................3
`
`37 C.F.R. § 42.100(b) ....................................................................................................................19
`
`37 C.F.R. § 42.101 ...........................................................................................................................4
`
`
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`- iii -
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`

`

`
`
`37 C.F.R. § 42.102 ...........................................................................................................................4
`
`
`37 C.F.R. § 42.102 ...........................................................................................................................437 C.F.R. § 42.102 ...........................................................................................................................437 C.F.R. § 42.102 ...........................................................................................................................4
`
`37 C.F.R. § 42.103 ...........................................................................................................................3
`
`
`37 C.F.R. § 42.103 ........................................................................................................................... 337 C.F.R. § 42.103 ........................................................................................................................... 337 C.F.R. § 42.103 ........................................................................................................................... 3
`
`37 C.F.R. § 42.104(a).......................................................................................................................3
`
`
`37 C.F.R. § 42.104(a) ....................................................................................................................... 337 C.F.R. § 42.104(a) ....................................................................................................................... 337 C.F.R. § 42.104(a) ....................................................................................................................... 3
`
`37 C.F.R. § 42.8(b)(1)-(4) ............................................................................................................ 4-5
`
`
`37 C.F.R. § 42.8(b)(1)-(4) ............................................................................................................ 4-537 C.F.R. § 42.8(b)(1)-(4) ............................................................................................................ 4-537 C.F.R. § 42.8(b)(1)-(4) ............................................................................................................ 4-5
`
`
`
`
`
`iv
`
`
`iviviv
`
`

`

`
`
`LIST OF EXHIBITS
`
`EXHIBIT NO.
`
`DESCRIPTION
`
`1001
`
`1002
`
`1003
`
`1004
`
`1005
`
`1006
`
`1007
`
`1008
`
`1009
`
`1010
`
`1011
`
`1012
`
`1013
`
`1014
`
`1015
`
`
`
`United States Patent No. 9,856,287
`
`Declaration of Professor Paul A. Dalby, Ph.D.
`
`United States Patent No. 4,237,224
`
`United States Patent No. 4,468,464
`
`United States Patent No. 4,740,470
`
`Curriculum Vitae of Professor Paul A. Dalby, Ph.D.
`De Bernardez Clark, E. et al., “Oxidative Renaturation of Hen
`Egg-White Lysozyme. Folding vs Aggregation,” Biotechnology
`Progress 14(1):47-54 (1998)
`De Bernardez Clark, E., “Refolding of recombinant proteins,”
`Current Opinion in Biotechnology 9:157-163 (April 2001)
`Ejima, D. et al., “High Yield Refolding and Purification Process
`for Recombinant Human Interleukin-6 Expressed in Escherichia
`coli,” Biotechnology and Bioengineering, 62(3):301-310
`(February 1999)
`Excerpts of United States Patent No. 9,856,287 File History
`Ferrer-Miralles, N. et al., “Microbial factories for recombinant
`pharmaceuticals,” Microbial Cell Factories 8:17 (2009)
`Georgiou, G. & Valax, P., “Isolating Inclusion Bodies from
`Bacteria,” Methods in Enzymology 309:48-58 (1999)
`Gilbert, H., “Molecular and Cellular Aspects of Thiol-Disulfide
`Exchange,” in Advances in Enzymology and Related Areas of
`Molecular Biology, ed. Alton Meister, Vol. 63, pp. 69-172 (John
`Wiley & Sons 1990)
`Gilbert, H., “Thiol/Disulfide Exchange Equilibria and Disulfide
`Bond Stability,” in Methods in Enzymology, ed. Lester Packer,
`Vol. 251, pp. 8-28 (Academic Press 1995)
`Graumann, K. & Premstaller, A., “Manufacturing of recombinant
`therapeutic proteins in microbial systems,” Biotechnology
`Journal 1:164-186 (2006)
`
`- v -
`
`

`

`
`
`EXHIBIT NO.
`
`1016
`
`1017
`
`1018
`
`1019
`
`1020
`
`1021
`
`1022
`
`1023
`
`1024
`
`1025
`
`1026
`
`1027
`
`1028
`
`
`
`DESCRIPTION
`Hevehan, D, & Clark, E., “Oxidative Renaturation of Lysozyme
`at High Concentrations,” Biotechnology and Bioengineering,
`54(3):221-230 (May 1997)
`Horton, R. et al., Principles of Biochemistry ( Pearson Education,
`4th ed., 2006)
`Jungbauer, A. & Kaar, W., “Current status of technical protein
`refolding,” Journal of Biotechnology 128:587-596 (2007)
`Keire, D. et al., “Kinetics and Equilibria of Thiol/Disulfide
`Interchange Reactions of Selected Biological Thiols and Related
`Molecules with Oxidized Glutathione,” J. Org. Chem. 57(1):123-
`127 (1992)
`Neubauer, P. et al., “Protein Inclusion Bodies in Recombinant
`Bacteria,” in Inclusions in Prokaryotes, ed. J.M. Shively, pp. 237-
`292 (Springer-Verlag Berlin Heidelberg 2006)
`Palmer, I. & Wingfield, P., “Preparation and Extraction of
`Insoluble (Inclusion-Body) Proteins from Escherichia coli, Curr
`Protoc Protein Sci. Chapter: Unit-6.3 (November 2004)
`Panda, A., “Bioprocessing of Therapeutic Proteins from the
`Inclusion Bodies of Escherichia coli,” Adv Biochem
`Engin/Biotechnol 85:43-93 (2003)
`Patra, A. et al., “Optimization of Inclusion Body Solubilization
`and Renaturation of Recombinant Human Growth Hormone from
`Escherichia coli,” Protein Expression and Purification 18:182-
`192 (2000)
`Profacgen, “Inclusion body purification & protein refolding,”
`accessed at https://www.profacgen.com/inclusion-body-
`purification-protein-refolding.htm
`International Publication No. WO 95/32216
`Ryan, R. et al., “Structure-Function Relationships of
`Gonadotropins,” in Recent Progress in Hormone Research, Vol.
`43, pp. 383-429 (Academic Press 1987)
`Schafer, F. & Buettner, G., “Redox Environment of the Cell as
`Viewed Through the Redox State of the Glutathione
`Disulfide/Glutathione Couple,” Free Radical Biology & Medicine
`30(11):1191-1212 (June 2001)
`United States Patent Application Publication No. 2007/0238860
`
`vi
`
`

`

`
`
`EXHIBIT NO.
`
`1029
`
`1030
`
`1031
`
`1032
`
`1033
`
`1034
`
`1035
`
`1036
`
`1037
`
`1038
`
`1039
`
`1040
`
`1041
`
`1042
`
`1043
`
`
`
`DESCRIPTION
`Singh, S. & Panda, A., “Solubilization and Refolding of Bacterial
`Inclusion Body Proteins,” Journal of Bioscience and
`Bioengineering 99(4):303-310 (2005)
`Vallejo, L. & Rinas, U., “Strategies for the recovery of active
`proteins through refolding of bacterial inclusion body proteins,”
`Microbial Cell Factories 3:11 (2004)
`European Patent Application No. 1 449 848 A1, Method for the
`production of cystine-knot proteins (2004)
`Ventura, S. & Villaverde, A., “Protein quality in bacterial
`inclusion bodies,” Trends in Biotechnology 24(4):179-185 (April
`2006)
`Wetlaufer, D. et al., “The oxidative folding of proteins by
`disulfide plus thiol does not correlate with redox potential,”
`Protein Engineering 1(2):141-146 (1987)
`Whitford, D., Proteins: Structure and Function (John Wiley &
`Sons 2005)
`Peptides Guide, “What are Proteins?” accessed at
`http://www.peptidesguide.com/proteins.html
`Xie, Y. et al., “Recombinant Human Retinol-Binding Protein
`Refolding, Native Disulfide Formation, and Characterization,”
`Protein Expression and Purification 14:31-37 (1998)
`Patent Owner’s Preliminary Response Under 37 C.F.R. § 42.207,
`dated January 23, 2019
`Final Written Decision in IPR2016-01542, Patent 8,952,138,
`dated February 15, 2018
`Archer, D. et al., “Hen Egg White Lysozyme Expressed In, and
`Secreted from, Aspergillus Niger is Correctly Processed and
`Folded,” Bio/Technology 8:741-745 (August 1990)
`United States Patent No. 5,663,304
`
`United States Patent No. 8,952,138
`De Bernardez Clark, E., “Protein refolding for industrial
`processes,” Current Opinion in Biotechnology 12(2):202-207
`(1998)
`Atassi, M.Z., “Chemical Strategy for Studying the Antigenic
`Structures of Disulfide-Containing Proteins: Hen Egg-White
`
`vii
`
`

`

`
`
`EXHIBIT NO.
`
`1044
`
`1045
`
`1046
`
`
`DESCRIPTION
`Lysozyme as a Model,” in Protein Crosslinking, ed. M.
`Friedman, Vol. 6, pp. 89-137 (Plenum Press 1977)
`Table of categorized claims for United States Patent No.
`9,856,287
`“Glutathione” in The Merck Index, 12th Ed., pp. 4483-4484
`(Merck Research Laboratories 1996)
`Middleberg, A., “Preparative protein folding,” TRENDS in
`Biotechnology 20(10):437-443 (October 2002)
`
`
`
`viii
`
`

`

`
`
`I.
`
`INTRODUCTION
`
`Fresenius Kabi USA, LLC and Fresenius Kabi SwissBioSim GmbH,
`
`pursuant to 35 U.S.C. §§ 311-319 and 37 C.F.R. § 42, et seq.,1 petition for Inter
`
`Partes Review (“IPR”) of claims 1, 4-6, 8-10, 12, 14-16, 19-21, 23-26, 29-30 of
`
`U.S. Patent No. 9,856,287 (“the ’287 patent,” Ex. 1001). Petitioners’ request is
`
`supported by the Expert Declaration of Paul Dalby, Ph.D. (Ex. 1002) and the other
`
`exhibits submitted herewith.
`
`The challenged claims of the ’287 patent are generally directed to methods
`
`of refolding proteins expressed in non-mammalian cells. Unfolded proteins are
`
`incubated in a buffer containing, among other ingredients, amounts of an oxidant
`
`and a reductant that permit the proteins to refold into their native three-dimensional
`
`structure. This basic “redox” refolding method was in common use as of June 22,
`
`2009, the earliest possible filing date of the patent, and scientists routinely tailored
`
`the compositions of their redox buffers to optimize the yield of properly refolded
`
`proteins. In particular, it was understood that for a given protein, the yield could
`
`be optimized in part by varying the ratio and strength of the oxidant and reductant
`
`(i.e., thiol pair) to determine which combinations produced the highest yield at a
`
`
`1 Unless otherwise stated, all statutory and regulatory citations herein are to 35
`
`U.S.C. or 37 C.F.R.
`
`
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`- 1 -
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`

`
`
`given protein concentration. As explained by Dr. Dalby, this optimization was
`
`routine and well within the scope of ordinary skill in 2009.
`
`While the ’287 patent purports to disclose novel mathematical equations to
`
`calculate thiol pair ratio and buffer strength, it cannot be disputed that the specific
`
`ranges of thiol pair ratio and buffer strength that appear in the claims encompass
`
`ratios and strengths described in the prior art. Moreover, the equations themselves
`
`are not novel. They express basic redox chemistry principles and were expressly
`
`disclosed and used in the prior art. Even if the equations had not been written
`
`down in the prior art, a mathematical equation does not make a claim patentable
`
`where “its only contribution was to quantify into a previously unwritten equation
`
`relationships that were discernible to one of ordinary skill in the art from the prior
`
`art.” Apotex Inc. v. Amgen Inc., IPR 2016-01542, Paper 60 at 29 (P.T.A.B. Feb.
`
`15, 2018) (Ex. 1038).
`
`As described below, each of the challenged claims is anticipated by the prior
`
`art. To the extent a single reference does not disclose every element of every
`
`claim, every element was disclosed in the prior art and there was a motivation to
`
`combine these elements, rendering the claimed subject matter obvious from that art
`
`as a whole. Petitioners are not aware of any relevant secondary evidence of non-
`
`obviousness.
`
`
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`- 2 -
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`

`
`
`The Board should institute review because there is at least a reasonable
`
`likelihood that Petitioners will prevail with respect to at least one challenged claim.
`
`§ 314(a). Moreover, there are no persuasive grounds for denying institution under
`
`§ 314(a) or § 325(d). This is Petitioners’ first petition challenging any claim of the
`
`’287 patent, and the petition in part raises arguments that have not previously been
`
`presented to the Office.
`
`The required fee set forth in § 42.15(a) is paid pursuant to § 42.103, and the
`
`Commissioner is hereby authorized to charge all fees due in connection with this
`
`matter to Attorney Deposit Account 506989.
`
`II. GROUNDS FOR STANDING
`Pursuant to § 42.104(a), Petitioners certify that the ’287 patent is available
`
`for IPR and that Petitioners are not barred or estopped from requesting IPR on the
`
`grounds raised in this petition. In accordance with § 311(c), more than 9 months
`
`have passed since issuance of the ’287 patent and no post-grant review (“PGR”)
`
`has been instituted.2 Moreover, neither Petitioners nor their privies or the real
`
`2 As disclosed in section III(b), infra, the ’287 patent is the subject of a petition for
`
`PGR filed in PGR2019-00001 by Adello Biologics, LLC and others. Because the
`
`Board has not instituted review, Petitioners need not wait until termination of
`
`PGR2019-0001 before filing this petition for IPR. Cf. § 311(c) (“if a post-grant
`
`review is instituted . . . , [a petition for IPR shall be filed after . . . ] the date of the
`
`
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`- 3 -
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`

`
`
`parties in interest have filed or been served with any complaint alleging
`
`infringement or invalidity of the ’287 patent, and therefore are not subject to any
`
`bar under § 315(a) or (b).
`
`III. MANDATORY NOTICES
` Real Parties In Interest (§ 42.8(b)(1)) A.
`
`The real parties in interest are Fresenius Kabi USA, LLC, Fresenius Kabi
`
`SwissBioSim GmbH, Fresenius Kabi AG, Fresenius Kabi Pharmaceuticals
`
`Holding, Inc., Fresenius Kabi Deutschland GmbH, Fresenius SE & Co. KGaA, Dr.
`
`Reddy’s Laboratories, Ltd., Dr. Reddy’s Laboratories, S.A., and Dr. Reddy
`
`Laboratories Inc.
`
`
` Related Matters (§ 42.8(b)(2)) B.
`The ’287 Patent is currently the subject of the following litigations and post-
`
`grant proceedings: Amgen Inc. et al. v. Adello Biologics LLC, 2:18-cv-03347
`
`D.N.J.; Amgen Inc. et al. v. Apotex Inc. et al., 19-cv-61828, S.D. Fla; and Adello
`
`Biologics, LLC. et al. v. Amgen Inc., PGR-2019-00001 (P.T.A.B.).
`
`
`termination of such post-grant review”); see also § 42.101. Moreover, if the ’287
`
`patent does not fall under section 2(n)(1) of the Leahy-Smith America Invents Act,
`
`as the Board must determine in PGR2019-0001, then an IPR may be filed at any
`
`time after the date of the grant of the patent. See Pub. L. 112–274, § 1(d)(1), Jan.
`
`14, 2013, 126 Stat. 2456; 37 C.F.R. § 42.102.
`
`
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`- 4 -
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`

`
`
`In addition, U.S. Patent Application No. 15/889,559 is pending and claims
`
`priority to the ’287 Patent.
`
`C.
`
`
`Identification of Counsel (§ 42.8(b)(3)) and Service Information
`(§ 42.8(b)(4))
`
`Lead Counsel
`Huiya Wu
`(Reg. No. 44,411)
`Goodwin Procter LLP,
`620 Eighth Avenue,
`New York, NY 10018,
`T: (212) 813-7295
`Fax: (212) 355-3333
`hwu@goodwinlaw.com
`
`Back-Up Counsel
`Robert V. Cerwinski
`(to seek pro hac vice
`admission)
`Goodwin Procter LLP,
`620 Eighth Avenue,
`New York, NY 10018,
`T: (212) 813-8800
`Fax: (212) 355-3333
`rcerwinski@goodwinlaw.com
`
`Back-Up Counsel
`Linnea Cipriano
`(Reg. No. 67,729)
`Goodwin Procter LLP,
`620 Eighth Avenue,
`New York, NY 10018,
`T: (212) 813-7295
`Fax: (212) 355-3333
`lcipriano@goodwinlaw.com
`
`
`
`Please direct all correspondence to lead counsel and back-up counsel at the
`
`contact information above. Petitioners consent to electronic mail service at the
`
`following addresses: hwu@goodwinlaw.com; rcerwinski@goodwinlaw.com;
`
`lcipriano@goodwinlaw.com.
`
`IV. TECHNOLOGY BACKGROUND
` The Basic Science of Proteins A.
`
`Protein Structure
`1.
`Protein molecules must fold into precise three-dimensional shapes in order
`
`to be biologically active. Ex. 1002 ¶ 42. The biologically-active form of a protein
`
`is known as the “native” form. Id. Usually the native form is the most
`
`thermodynamically stable way of folding the particular sequence of amino acids
`
`
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`- 5 -
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`

`
`
`that make up the protein. Id. Thus, under appropriate conditions, proteins will
`
`automatically fold into their native forms. Id.
`
`For many proteins, their native three-dimensional structure is stabilized by
`
`“disulfide” bonds that cross-link different parts of the folded polypeptide chain.
`
`Disulfide bonds form between particular amino acids called “cysteines” when they
`
`come into close proximity during refolding and help lock the protein into its native
`
`shape . Ex. 1002 ¶ 43; Ex. 1006 at 32-33. However, if disulfide bonds form in
`
`improper locations, the proteins can misfold. Misfolded proteins can be inactiv
`
`Protein Synthesis in and out of the Lab
`
`2.
`In nature, organisms create proteins through the processes of transcription
`
`(DNA is used to make RNA) and translation (RNA is used to make the protein)
`
`Ex. 1002 ¶ 44; Horton (Ex. 1017) at 683-711. This natural machinery can be
`
`harnessed to make commercial amounts of protein using “recombinant” DNA
`
`technology, which has been known in the art since at least the 1970s. Ex. 1002 ¶
`
`45. In these methods, host cells in a cell culture are turned into “factories” for
`
`manufacturing proteins of interest by inserting a segment of recombinant DNA that
`
`encodes the protein into the host cells. Ex. 1002 ¶ 46; Ex. 1017 at 719-23.
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`Recombinant DNA technology can be used with both mammalian and non-
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`mammalian cell cultures (often referred to as “expression systems”), but scientists
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`have generally turned to high-yield bacterial expression systems to express
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`recombinant proteins at a lower cost. Id.; Ex. 1002 ¶ 47. One well-established
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`bacterial expression system is Escherichia coli, commonly referred to as E. coli.
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`Ex. 1002 ¶ 48.
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` Recovery and Refolding of Expressed Protein B.
`A bacterial host cell expressing recombinant proteins often produces
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`misfolded proteins that aggregate together into “inclusion bodies” in the cell. In
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`particular, recombinant proteins expressed in E. coli were known to form inclusion
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`bodies. Neubauer (Ex. 1020) at 244-47; Georgious (Ex. 1012) at 57-58. To make
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`the proteins more usable, techniques for recovering native, folded proteins in a
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`bioactive and stable form from those inclusion bodies were developed. The most
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`common techniques follow a process in which proteins that are isolated and
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`purified from inclusion bodies are (1) solubilized, causing the proteins to unfold;
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`and (2) refolded in a refolding buffer. See Ex. 1002 ¶ 52.
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`Unfolding and Refolding of Recombinant Proteins
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`1.
`During solubilization, the inclusion bodies are “denatured.” The bonds and
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`other forces holding the aggregated proteins together are disrupted by chemical
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`denaturants, which causes the proteins to unfold into single strands of polypeptide.
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`Ex. 1002 ¶ 53; Ex. 1015 at 267-68; Ex. 1021 at 12-13. Disulfide bonds are often
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`broken or “reduced” via a redox reaction. For example, two molecules of reduced
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`glutathione (“GSH”) will reduce a disulfide bond to give two “free thiols” and a
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`molecule of oxidized glutathione (“GSSG”), as shown in the reaction from right to
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`left in the below diagram.
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`
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`Ex. 1002 ¶ 54. Importantly, this process can be reversed during refolding of the
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`protein: the GSSG can oxidize the free thiols and re-form the disulfide bond, as
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`shown in the reaction from left to right in the above diagram.
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`Refolding of the denatured solubilized proteins is generally accomplished
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`with a “refold buffer.” Ex. 1002 ¶ 57. A refold buffer generally includes a number
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`of components, including denaturants, aggregate suppressors, protein stabilizers,
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`oxidants, and reductants, each of which may be adjusted to optimize the efficiency
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`of the refolding. Ex. 1002 ¶ 57. This includes the relative and total concentrations
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`of the components and redox systems. Ex. 1002 ¶¶ 62-70; Ex. 1020 at 269-73;
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`Panda (Ex. 1022) at 73-76; Vallejo 2004 (Ex. 1030) at 7-8.
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`2. Optimizing Redox Conditions
`It was known before 2009 that proteins solubilized from inclusion bodies
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`must be placed in an environment that facilitates the formation of the desired
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`native protein structure. Ex. 1002 ¶¶ 55-58; Ex. 1007. In particular, if the desired
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`protein in its native state contains disulfide bonds, the unfolded protein must be
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`placed in appropriate redox conditions that favor the formation of the correct
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`disulfide bonds. Ex. 1002 ¶ 61. The redox conditions must be balanced so that
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`bond formation (oxidation) is favored, but not so favored that mis-formed bonds
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`cannot “reshuffle”—break and reform—as thermodynamics drive the protein
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`chains to fold into their native, most-stable conformation over time. Ex. 1002 ¶ 62;
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`Ex. 1042 at 158-59. Scientists generally used redox systems consisting of a
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`mixture of reduced and oxidized thiols to refold the protein. Id. This mixture is
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`sometimes called a “thiol pair.”
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`The “equilibrium” of oxidation and reduction that permitted optimal
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`“reshuffling” and refolding was known to be controlled by the ratio and relative
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`concentrations of the thiol pair oxidant and reducant in the buffer. Ex. 1008 at
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`205. While the example in Ex. 1002 ¶ 64, uses GSH/GSSG, other thiol pairs of
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`choice included cysteine/cystine and cysteamine/cystamine. Ex. 1002 ¶ 71; Ex.
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`1007. The refolding reaction was often optimized by selecting an appropriate
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`redox system and adjusting the ratios and concentrations of the oxidant and
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`reductant, until the yield of properly-refolded proteins was maximized. Ex. 1020
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`at 270-72.
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`It was also well known that the optimal redox conditions had to be identified
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`experimentally, and differed for each protein. Ex. 1002 at 68. The yield of
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`properly-refolded protein can vary greatly depending on the particular protein
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`being refolded, the concentration of the protein, the ingredients of the refold buffer,
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`and other parameters such as pH, temperature, incubation time, and purification
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`method. Ex. 1002 at 70; Ex. 1008; Ex. 1030. Thus, scientists routinely used
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`multifactorial matrix screens to test various physical and chemical parameters to
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`optimize the yield of properly-refolded proteins. Ex. 1002 at 57; Ex. 1046 at 441.
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`Moreover, refolding is never 100% efficient; even optimized yields were always
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`less than 100% and often much lower. Ex. 1002 at 59.
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`
` Additional Considerations in Commercial Production of C.
`Recombinant Proteins
`
`As of 2009, a POSA would have recognized that “[t]he ultimate goal of
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`recombinant fermentation research,” was “to obtain the highest amount of protein
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`in a given volume in the least amount of time.” Ex. 1002 ¶ 59; Ex. 1023 at 182.
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`POSAs also sought to decrease the size of refolding vessels by increasing the
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`concentration of protein before and during refolding. Ex. 1028 at [0019].
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`However, it was recognized that at higher concentrations, refolding proteins were
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`more prone to associate in unproductive ways, leading to misfolded proteins called
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`“aggregates.” Id. at [0008]. This process of “aggregation” competed with the
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`desired native folding pathway, lowering the yield of properly folded proteins. Id.
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`at [0008]-[0009]; Ex. 1002 ¶¶ 58-59.
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`Those ski