UNITED STATES PATENT AND TRADEMARK OFFICE
`_____________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`_____________________
`
`BIOEQ IP AG
`
`
`
`Petitioner
`
`v.
`
`GENENTECH, INC.
`
`Patent Owner
`
`_____________________
`
`Case No. Unassigned
`
`U.S. Patent No. 6,716,602
`_____________________
`
`
`PETITION FOR INTER PARTES REVIEW OF U.S. PATENT NO. 6,716,602
`UNDER 35 U.S.C. §§ 311-319 AND 37 C.F.R. §§ 42.1-.80, 42.100-.123
`
`
`
`Mail Stop “PATENT BOARD”
`Patent Trial and Appeal Board
`U.S. Patent and Trademark Office
`P.O. Box 1450
`Alexandria, VA 22313-1450
`
`

`
`
`
`
`I. 
`
`II. 
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`TABLE OF CONTENTS
`
`Introduction ...................................................................................................... 1 
`
`Grounds for standing (37 C.F.R. § 42.104(a)) ................................................ 4 
`
`III. 
`
`Statement of the precise relief requested and the reasons therefore ............... 4 
`
`IV.  Overview .......................................................................................................... 4 
`
`POSA ..................................................................................................... 4 
`
`A. 
`
`B. 
`
`2. 
`
`Scope and content of the art before November 3, 2000 ........................ 5 
`E. coli: “the most important” host for bacterial
`1. 
`production of recombinant proteins, including growth
`factors, antibodies, and antibody fragments ............................... 6 
`Excess glucose during bacterial fermentation causes
`acetate accumulation and limits high host cell densities
`and recombinant protein production ........................................... 8 
`Glucose-limited fed-batch fermentation minimizes
`acetate accumulation and maximizes cell densities and
`recombinant protein production .................................................. 9 
`Control of recombinant protein expression used well-
`known inducible promoters, such as the phosphate-
`inducible promoter phoA ........................................................... 11 
`
`3. 
`
`4. 
`
`C. 
`
`The ’602 patent .................................................................................... 12 
`1. 
`The ’602 patent claims .............................................................. 13 
`2. 
`Summary of the prosecution of the ’602 patent ........................ 16 
`
`V. 
`
`Claim construction ......................................................................................... 20 
`
`VI. 
`
`Identification of challenge (37 C.F.R. § 42.104(b)) ...................................... 27 
`
`A.  Ground 1: Seeger anticipates claims 1, 3-4, 6, 9, 15-16, 20, 22,
`24-25, 27-28, 30, 33, and 39 ............................................................... 28 
`1. 
`Seeger anticipates claim 1 ......................................................... 28 
`
`i
`
`

`
`(a) 
`
`(b) 
`
`(c) 
`
`
`
`
`2. 
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`Seeger teaches expression of a polypeptide of
`interest in recombinant host cells regulated by an
`inducible system ............................................................. 29 
`Seeger teaches culturing the recombinant host cells
`under conditions of high metabolic and growth rate ...... 30 
`Seeger teaches reducing the metabolic rate of the
`cultured recombinant host cells at the time of
`induction of polypeptide expression ............................... 32 
`Seeger reduces the metabolic rate by reducing the
`feed rate of the carbon/energy source ............................. 36 
`Seeger’s method of reducing metabolic rate results
`in increased yield of properly-folded polypeptide ......... 36 
`(f)  A POSA would have been able to use Seeger’s
`fermentation strategy without undue
`experimentation .............................................................. 38 
`Seeger anticipates claims 3-4, 6, 9, 15-16, 20, 22, 24-25,
`27-28, 30, 33, and 39 ................................................................ 39 
`
`(d) 
`
`(e) 
`
`B. 
`
`C. 
`
`Ground 2: Claims 7-8 and 31-32 would have been obvious over
`Seeger in view of the general knowledge in the prior art ................... 43 
`
`Ground 3: Claims 10, 12, 23, 34, and 36 would have been
`obvious over Seeger and Makrides ..................................................... 46 
`
`D.  Ground 4: Claims 11, 13-14, 18, 35, and 37-38 would have
`been obvious over Seeger and Cabilly ................................................ 53 
`
`E. 
`
`Objective indicia do not support patentability .................................... 57 
`1. 
`No unexpected superior results ................................................. 59 
`2. 
`No long-felt need or failure of others ....................................... 61 
`3. 
`There is no other evidence of nonobviousness ......................... 63 
`
`VII.  Conclusion ..................................................................................................... 64 
`
`VIII.  Mandatory notices (37 C.F.R. § 42.8(a)(1)) .................................................. 64 
`
`ii
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`

`
`
`
`
`Petitioners
`Exhibit #
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`
`LIST OF EXHIBITS
`
`
`Description
`
`1001
`
`1002
`1003
`1004
`
`1005
`
`1006
`
`1007
`
`1008
`
`1009
`
`1010
`
`Anderson, D., et al., “Metabolic Rate Shifts in Fermentations
`Expressing Recombinant Proteins,” U.S. Patent No. 6,716,602 (filed
`November 1, 2001; issued on April 6, 2004)
`Declaration of Morris Z. Rosenberg, DSC.
`Curriculum Vitae of Morris Z. Rosenberg, DSC.
`File History for U.S. Patent No. 6,716,602
`Knorre, W.A., et al., “High Cell Density Fermentation of
`Recombinant Escherichia coli with Computer-Controlled Optimal
`Growth Rate,” Annals New York Academy of Sciences 646: 300-306
`(1991)
`Jackson, D.A., et al., “Biochemical Method for Inserting New
`Genetic Information into DNA of Simian Virus 40: Circular SV40
`DNA Molecules Containing Lambda Phage Genes and the
`Galactose Operon of Escherichia coli,” Proceedings of the National
`Academy of Sciences 69(10): 2904-2909 (1972)
`Donovan, R.S., et al., “Review: Optimizing inducer and culture
`conditions for expression of foreign proteins under the control of the
`lac promoter,” Journal of Industrial Microbiology 16: 145-154
`(1996)
`Korz, D.J., et al., “Simple fed-batch technique for high cell density
`cultivation of Escherichia coli,” Journal of Biotechnology 39: 59-65
`(1995)
`Verma, R., et al., “Antibody engineering: Comparison of bacterial,
`yeast, insect, and mammalian expression systems,” Journal of
`Immunological Methods 216: 165-181 (1998)
`Seeger, A. et al., “Comparison of temperature- and isopropyl-β-D-
`thiogalacto-pyranoside-induced synthesis of basic fibroblast growth
`factor in high-cell-density cultures of recombinant Escherichia
`coli,” Enzyme and Microbial Technology 17: 947-953 (1995)
`
`iii
`
`

`
`
`
`
`Petitioners
`Exhibit #
`
`
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`Description
`
`1011
`
`1012
`
`1013
`
`1014
`
`1015
`
`1016
`
`1017
`
`1018
`
`1019
`
`Luli, G.W., et al., “Comparison of Growth, Acetate Production, and
`Acetate Inhibition of Escherichia coli Strains in Batch and Fed-
`Batch Fermentations,” Applied and Environmental Microbiology
`56(4): 1004-1011 (1990)
`Akesson, M., et al., “A simplified probing controller for glucose
`feeding in Escherichia coli cultivations,” Decision and Control 5:
`4520-4525 (2000)
`Strittmatter, W., et al., “Process for the Preparation of Recombinant
`Proteins in E. coli by High Cell Density Fermentation,” U.S. Patent
`No. 6,410,270 (International Filing Date November 28, 1996; Issued
`June 25, 2002)
`Smirnova, G.V., et al., “Influence of Acetate on the Growth of
`Escherichia coli Under Aerobic and Anaerobic Conditions,”
`Mikrobiologiya 54(2): 205-209 (1985)
`Rinas, U., et al., “Glucose as a substrate in recombinant strain
`fermentation technology,” Applied Microbiology and Biotechnology
`31: 163-167 (1989)
`CURRENT PROTOCOLS IN MOLECULAR BIOLOGY pp. 1.1.1- 1.15.8 and
`16.1-16.21 (Frederick M. Ausubel, et al., eds., Volume I,
`Supplement 3, 1995)
`Roszak, D.B., et al., “Survival Strategies of Bacteria in the Natural
`Environment,” Microbiological Reviews 51(3): 365-379 (1987)
`Akesson, M., et al., “A probing feeding strategy for Escherichia coli
`cultures,” Biotechnology Techniques 13: 523-528 (1999)
`Wangsa-Wirawan, N.D., et al., “Novel fed-batch strategy for the
`production of insulin-like growth factor 1 (IGF-1),” Biotechnology
`Letters 9(11): 1079-1082 (1997)
`
`iv
`
`

`
`
`
`
`Petitioners
`Exhibit #
`
`
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`Description
`
`1020
`
`1021
`
`1022
`
`1023
`
`1024
`
`1025
`
`1026
`
`1027
`
`1028
`
`Seeger, A., “Production of the Basic Fibroblast Growth Factor
`(bFGF) in a High Cell Density Process by means of Recombinant
`Escherichia coli,” Dissertation submitted to the Department of
`Mechanical Engineering and Electrical Engineering of the Technical
`University of Carolo-Wilhelmina (1995)
`Lin, H., “Cellular responses to the induction of recombinant genes
`in Escherichia coli fed-batch cultures,” Dissertation submitted to
`Martin-Luther-Universitat Halle-Wittenberg Faculty of Mathematics
`and Natural Sciences Department of Biochemistry and
`Biotechnology (2000)
`Sawers, G., et al., “Alternative regulation principles for the
`production of recombinant proteins in Escherichia coli,” Applied
`Microbiology Technology 46: 1-9 (1996)
`Makrides, S.C., “Strategies for Achieving High-Level Expression of
`Genes in Escherichia coli,” Microbiological Reviews 60(3): 512-
`538 (1996)
`Wanner, B.L., “Gene Regulation by Phosphate in Enteric Bacteria,”
`Journal of Cellular Biochemistry 51: 47-54 (1993)
` UNDERSTANDING BIOLOGY pp. 117-139 (Burton S. Guttman and
`Johns W. Hopkins III, eds., 1983)
`Carter, P., et al., “High Level Escherichia coli Expression and
`Production of a Bivalent Humanized Antibody Fragment,”
`Biotechnology 10: 163-167 (1992)
`Better, M., et al., “Escherichia coli secretion of an active chimeric
`antibody fragment,” Science 240: 1041 (1988)
`Shimuzu, N., et al., “Fed-Batch Cultures of Recombinant
`Escherichia coli with Inhibitory Substance Concentration
`Monitoring,” Journal of Fermentation Technology 66: 187-191
`(1988)
`
`v
`
`

`
`
`
`
`Petitioners
`Exhibit #
`
`
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`Description
`
`1029
`
`1030
`
`1031
`
`1032
`
`1033
`
`1034
`
`1035
`
`1036
`
`Bauer, K.A., et al., “Improved Expression of Human Interleukin-2
`in High-Cell-Density Fermentor Cultures of Escherichia coli K-12
`by a Phosphotransacetylase Mutant,” Applied and Environmental
`Microbiology 56: 1296-1302 (1990)
`Huston, J.S., et al., “Protein engineering of antibody binding sites:
`recovery of specific activity in an anti-digoxin single-chain Fv
`analogue produced in Escherichia coli,” Proceedings of the
`National Academy of Sciences 85: 5879 (1988)
`BIOPROCESS ENGINEERING PRINCIPLES pp. 257-296 (Pauline M.
`Doran, ed., 1995)
`Cabilly, S., “Growth at sub-optimal temperatures allows the
`production of functional, antigen-binding Fab fragments in
`Escherichia coli,” Gene 85: 553-557 (1989)
`Boss, M.A., et al., “Assembly of functional antibodies from
`immunoglobulin heavy and light chains synthesized in E. coli,”
`Nucleic Acids Research 12:3791-3806 (1984)
`Cabilly, S., et al., “Generation of anybody activity from
`immunoglobulin polypeptide chains produces in E. coli,”
`Proceedings of the National Academy of Sciences 81: 3273-3277
`(1984)
`Akesson, M., et al., “On-Line Detection of Acetate Formation in
`Escherichia coli Cultures Using Dissolved Oxygen Responses to
`Feed Transient,” Biotechnology and Bioengineering 64: 590-598
`(1999)
`Hoffman, F., et al., “Minimizing inclusion body formation during
`recombinant protein production in Escherichia coli at bench and
`pilot plant scale,” Enzyme and Microbial Technology 34: 235-241
`(2004)
`
`vi
`
`

`
`
`
`
`Petitioners
`Exhibit #
`
`
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`Description
`
`1037
`
`1038
`
`1039
`
`1040
`
`1041
`
`1042
`
`1043
`
`1044
`
`1045
`
`1046
`
`Jensen, E.B., et al., “Production of Recombinant Human Growth
`Hormone in Escherichia coli: Expression of Different Precursors
`and Physiological Effects of Glucose, Acetate, and Salts,”
`Biotechnology and Bioengineering 36: pp. 1-11 (1990)
`Turner, C., et al., “A Study of the Effect of Specific Growth Rate
`and Acetate on Recombinant Protein Production of Escherichia coli,
`JM107,” Biotechnology Letters 16: 891-896 (1994)
`GENES III pp. 183-218 and 732 (Benjamin Lewin ed., Third Edition,
`1987)
`Qiu, J., et al., “Expression of Active Human Tissue-Type
`Plasminogen Activator in Escherichia coli,” Applied and
`Environmental Microbiology 64: 4891-4896 (1998)
`Nan Chang, C., et al., “High-level secretion of human growth
`hormone by Escherichia coli,” Gene 55: 189-196 (1987)
`Ulrich, H.D., et al., “Expression studies of catalytic antibodies,”
`Proceedings of the National Academy of Sciences 92: 11907-11911
`(1995)
`ESSENTIAL IMMUNOLOGY pp. 31-54 (Ivan Roitt ed., Sixth Edition,
`1988)
`Rosano, G.L., et al., “Recombinant protein expression in
`Escherichia coli: advances and challenges,” Frontiers in
`Microbiology 5: pp. 1-17 (2014)
`Henry, N.G., “Effect of Decreasing Growth Temperature on Cell
`Yield of Escherichia coli,” Journal of Bacteriology 98: 232-237
`(1969)
`Kovářová, K., et al., “Temperature-Dependent Growth Kinetics of
`Escherichia coli ML 30 in Glucose-Limited Continuous Culture,”
`Journal of Bacteriology 178: 4530-4539 (1996)
`
`vii
`
`

`
`
`
`
`Petitioners
`Exhibit #
`
`
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`Description
`
`1047
`
`1048
`
`Ko, Y.-F., et al., “A Metabolic Model of Cellular Energetics and
`Carbon Flux During Aerobic Escherichia coli Fermentation,”
`Biotechnology and Bioengineering 43: 847-855 (1994)
`Skerra, A., “Use of tetracycline promoter for the tightly regulated
`production of a murine antibody fragment in Escherichia coli,”
`Gene 151: 131-135 (1994)
`
`viii
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`

`
`
`
`I.
`
`
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`Introduction
`bioeq IP AG (“Petitioner”) submits this Petition for Inter Partes Review
`
`(“IPR”) seeking cancellation of claims 1, 3-4, 6-16, 18, 20, 22-25, 27-28, and 30-
`
`39 of U.S. Patent No. 6,716,602 (“the ’602 patent”) (BEQ1001). These claims are
`
`unpatentable under 35 U.S.C. §§ 102 and 103. And the reason is simple: a § 102(b)
`
`prior art reference (Seeger, BEQ1010), neither cited nor considered by the
`
`Examiner during prosecution, describes the exact limitation Patent Owner argued
`
`was missing from the cited prior art—reducing the metabolic rate of the cultured
`
`host cells at the time of induction of polypeptide expression.
`
`To be clear, Patent Owner admitted that the primary reference relied upon by
`
`the Examiner (Knorre, BEQ1005) expressly taught reducing metabolic rate by
`
`controlling the bacterial growth rate (through a glucose feed-rate reduction), just
`
`not at the time of induction. Yet, as this Petition demonstrates, Seeger describes (or
`
`in combination with additional references renders obvious) each and every
`
`limitation of the challenged claims, including this element.
`
`The challenged claims recite methods for increasing product yield of a
`
`properly-folded polypeptide of interest produced by recombinant host cells, where
`
`expression of the polypeptide by the host cells is regulated by an inducible system.
`
`The methods comprise only two steps: (i) culturing the recombinant host cells
`
`under conditions of high metabolic and growth rate and (ii) reducing the metabolic
`
`1
`
`

`
`Petition for Inter Partes Review
`
`of U.S. Patent No. 6,716,602
`
`
`rate of the cultured host cells at the time of induction of polypeptide expression.
`
`(See, e.g., BEQ1001, 18:16-17.)1 Step (ii) is achieved by either reducing the feed
`
`rate of a carbon/energy source or reducing the amount of available oxygen, or both,
`
`as has been part of the practiced art long before the earliest effective filing date of
`
`the ’602 patent.2 (See, e.g., BEQ1001, 18:18-21.)
`
`Seeger used an inducible expression system to induce expression of a
`
`mammalian polypeptide, basic fibroblast growth factor, in a high cell density
`
`culturing system by first culturing the recombinant host cells under conditions of
`
`high metabolic and growth rate. Then, to reduce the metabolic rate of the host cells
`
`upon induction, Seeger controlled the bacterial growth rate by reducing the feed
`
`rate of a carbon/energy source—glucose. Thus, Seeger described using the exact
`
`
`1 Citations to patent literature provided as BEQ10XX, YYY:Z-Z indicate
`
`citations to column Y, at lines Z-Z. Citations to non-patent literature provided as
`
`BEQ10XX, Y:Z:Z' indicate citations to page number Y, at column number Z, and
`
`paragraph number Z'.
`
`2 Petitioner does not concede that the ’602 patent is entitled to a filing date
`
`of November 3, 2000. However, the ’602 patent cannot be entitled to any filing
`
`date earlier than November 3, 2000.
`
`2
`
`

`
`Petition for Inter Partes Review
`
`of U.S. Patent No. 6,716,602
`
`
`technique (reducing glucose feed-rate) to reduce metabolic rate that Patent Owner
`
`admitted was in the prior art, and Seeger did so at the time of induction.
`
`And, before November 3, 2000, a POSA running E. coli fermentations
`
`would have had a reason to apply Seeger’s reduced metabolic rate fermentation
`
`strategies to produce other recombinant proteins, such as antibodies and antibody
`
`fragments like Fab, or using well-known inducible promoter systems, such as
`
`phosphate depletion inducible systems (e.g., phoA), as recited in the dependent
`
`claims. This is so because Seeger specifically aimed to address a problem that had
`
`been identified and solved well-before the earliest priority date of the ’602 patent,
`
`namely: reduce toxic by-product accumulation, particularly acetic acid, which
`
`significantly limits host cell growth and recombinant protein production. Seeger
`
`succeeded in doing so by reducing the metabolic rate of the host cells at the time of
`
`induction of polypeptide expression.
`
`Moreover, a POSA would have successfully arrived at the limitations recited
`
`in the dependent claims identified herein with a reasonable expectation of success
`
`because recombinant E. coli fermentation strategies had long been used to produce
`
`numerous commercially-important proteins, including growth factors, antibodies,
`
`and antibody fragments. Indeed, the field had selected E. coli as “the most
`
`important” and versatile host for production of commercially-important proteins.
`
`3
`
`

`
`
`
`
`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
`
`Finally, no objective indicia of nonobviousness weigh in favor of
`
`patentability. Accordingly, Petitioner requests that the Board institute trial because
`
`Petitioner is reasonably likely to prevail with respect to at least one challenged
`
`claim based on the Grounds asserted in this Petition.
`
`II. Grounds for standing (37 C.F.R. § 42.104(a))
`Petitioner certifies that the ’602 patent is available for IPR, and Petitioner is
`
`not barred or estopped from requesting IPR of any of the challenged claims.
`
`III. Statement of the precise relief requested and the reasons therefore
`The Office should institute IPR under 35 U.S.C. §§ 311-319 and 37 C.F.R.
`
`§§ 42.1-.80 and 42.100-42.123, and cancel claims 1, 3-4, 6-16, 18, 20, 22-25, 27-
`
`28, and 30-39 of the ’602 patent as unpatentable under pre-AIA 35 U.S.C. § 102(b)
`
`and 103(a) for the reasons explained below. This petition is accompanied and
`
`supported by the Declaration of Dr. Morris Rosenberg (BEQ1002) and related
`
`materials. Petitioner’s detailed full statement of the reasons for relief requested is
`
`set forth in § VI.
`
`IV. Overview
`A.
`POSA
`A POSA is a hypothetical person who is presumed to be aware of all
`
`pertinent art, thinks along conventional wisdom in the art, and is a person of
`
`ordinary creativity. With respect to the ’602 patent, a POSA would typically have
`
`had a Ph.D. or a D.Sc. and at least two years of experience, or an M.S. and at least
`
`4
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`

`
`Petition for Inter Partes Review
`
`of U.S. Patent No. 6,716,602
`
`
`four years of experience, in recombinant protein production, specializing in
`
`biochemistry, microbiology, or chemical engineering. (BEQ1002, ¶19.) A POSA
`
`would have also typically worked as part of a multi-disciplinary team to solve a
`
`given problem, drawing upon not only his or her own skills, but also certain
`
`specialized skills of others in the team. (BEQ1002, ¶20.) For example, such a team
`
`may be comprised of a chemical engineer, microbiologist, biochemist, and/or
`
`molecular biologist. (Id.)
`
`Before November 3, 2000, the state of the art of which a POSA would have
`
`been aware included teachings provided by the references discussed in this Petition
`
`and by Dr. Rosenberg. Additionally, a POSA, based on then existing literature,
`
`would also have had general knowledge of recombinant protein production and
`
`methods of producing recombinant polypeptides. (Id.)
`
`Scope and content of the art before November 3, 2000
`
`B.
`In his Declaration, Dr. Rosenberg describes prior art teachings confirming
`
`the general knowledge of a POSA as of November 3, 2000. See In re Khan, 441
`
`F.3d 977, 988 (Fed. Cir. 2006) (stating that a person of ordinary skill possesses the
`
`“understandings and knowledge reflected in the prior art”); see also Randall Mfg.
`
`v. Rea, 733 F.3d 1355, 1362 (Fed. Cir. 2013) (“[T]he knowledge of [a person of
`
`ordinary skill in the art] is part of the store of public knowledge that must be
`
`consulted when considering whether a claimed invention would have been
`
`5
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`

`
`Petition for Inter Partes Review
`
`of U.S. Patent No. 6,716,602
`
`
`obvious.”). And Petitioner’s grounds rely on the prior art teachings, as explained
`
`below and supported by Dr. Rosenberg. (See BEQ1002, ¶¶10, 18.)
`
`1.
`
`E. coli: “the most important” host for bacterial production
`of recombinant proteins, including growth factors,
`antibodies, and antibody fragments
`
`The field of recombinant protein production advanced considerably over
`
`several decades before November 3, 2000. (See BEQ1006, 2904-2909; BEQ1007,
`
`145:1; BEQ1023, 512:1-2; BEQ1002, ¶33.) Indeed, scientists routinely used
`
`bacteria—primarily E. coli—for recombinant protein production of a wide variety
`
`of commercially-important proteins, including mammalian polypeptides, such as
`
`growth factors, antibodies, and antibody fragments. (See BEQ1007, 145:1:2;
`
`BEQ1002, ¶33.) And the field had selected E. coli as “the most important” and
`
`versatile host for recombinant protein production because, e.g., it grows at a very
`
`fast rate allowing for high-volume protein production over a short time. (See
`
`BEQ1008, 59:1:1; BEQ1002, ¶34.) Thus, a POSA had a wealth of knowledge
`
`about this versatile host and would have preferred it for recombinant protein
`
`production. (See BEQ1007, 145:1:2; BEQ1002, ¶34.)
`
`E. coli host cells also presented a POSA with an ability to express a diverse
`
`array of recombinant proteins in the cytoplasm or to target these proteins to the
`
`periplasm, using a signal sequence like the PhoA signal peptide. (See BEQ1009,
`
`170:1:3; BEQ1023, 520:2:3-4; BEQ1002, ¶¶49, 64-65.) This versatility is
`
`6
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`

`
`Petition for Inter Partes Review
`
`of U.S. Patent No. 6,716,602
`
`
`particularly important for recombinant antibodies and antibody fragments, which
`
`tend to precipitate in the reducing environment of the bacterial cytoplasm. (See
`
`BEQ1009, 170:1:3; BEQ1023, 518:1:5, 518:2:3, 520:2:2; BEQ1002, ¶¶49, 63.)
`
`Periplasmic targeting enabled production of Fab fragments (an antibody fragment)
`
`as assembled, soluble dimeric proteins that did not precipitate in the periplasm.
`
`(See BEQ 1023, 520:2:3; BEQ1042, 11909:2:3; BEQ1002, ¶¶49, 64.)
`
`Typical fermentation methods for producing recombinant proteins in E. coli
`
`involved (and still do) a growth phase, where logarithmic or exponential growth of
`
`the host cells occurs at a constant doubling rate, and a recombinant protein
`
`production phase, which can begin when the host cells are in logarithmic or
`
`exponential growth or when the host cells have reached a stationary growth phase.
`
`(See BEQ1017, 368:1:2; BEQ1016, 1.1.1:2; BEQ1002, ¶35.) The field used high
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`cell density culturing (HCDC) of the E. coli host cells as a strategy to obtain
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`“efficient recombinant protein formation.” (See BEQ1013, 1:21-23; BEQ1002,
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`¶36.) But by November 3, 2000, a POSA would have known that excess glucose
`
`feeding during either the growth phase or production phase of, e.g., an HCDC,
`
`presented a central obstacle in E. coli-based recombinant protein production
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`methods. (See BEQ1010, 947:1-1; BEQ1011, 1004, 1:1 and 1009:1:4; BEQ1014,
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`206:2 and Figure 1; BEQ1015 163:Summary; BEQ1018: 523:1:1; BEQ1028,
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`Abstract; BEQ1029, Abstract; BEQ1038, Abstract; BEQ1002, ¶¶36-38.)
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`2.
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`Petition for Inter Partes Review
`of U.S. Patent No. 6,716,602
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`Excess glucose during bacterial fermentation causes acetate
`accumulation and limits high host cell densities and
`recombinant protein production
`Because E. coli grow faster on glucose than other carbon sources, media
`
`used for recombinant protein production in E. coli usually include substantial
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`concentrations of glucose to obtain high-density bacterial cultures. (See BEQ1011,
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`1004, 1:1; BEQ1002, ¶36.) However, excess glucose provided to E. coli host cells
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`grown in HCDC in the presence of oxygen (i.e., aerobic conditions), can cause the
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`formation of acidic by-products, such as acetate (or acetic acid). (Id.)
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`Acetate accumulation during HCDC E. coli fermentation presents a central
`
`obstacle to recombinant protein production because it detrimentally affects both
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`host cell growth and recombinant protein production. (See BEQ1010, 947:1-1;
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`BEQ1011, 1004, 1:1 and 1009:1:4; BEQ1014, 206:2 and Figure 1; BEQ1015
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`163:Summary; BEQ1018: 523:1:1; BEQ1028, Abstract; BEQ1029, Abstract;
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`BEQ1038, Abstract; BEQ1002, ¶¶36-38.) The acetate accumulation results from
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`an imbalance between a host cell’s glucose metabolism and respiration, which are
`
`intimately linked. (See BEQ1011, 1009:1:3; BEQ1002, ¶37.) This is because a host
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`cell uses oxygen to metabolize glucose, which means that as the glucose uptake
`
`rate (“GUR”) decreases, so does the oxygen uptake rate (“OUR”). (See BEQ1018,
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`525:Figure 1; BEQ1035, 591:Figure 1; BEQ1012, 4520:2:3 and Figure 2;
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`BEQ1002, ¶37.) Moreover, a POSA would have known that the rate at which an E.
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`Petition for Inter Partes Review
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`of U.S. Patent No. 6,716,602
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`coli host cell consumes and oxidizes a carbon source (e.g., glucose) closely
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`correlates to the metabolic rate of that host cell. (Id.)
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`3. Glucose-limited fed-batch fermentation minimizes acetate
`accumulation and maximizes cell densities and recombinant
`protein production
`
`To avoid this central obstacle (acetate accumulation) to recombinant protein
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`production methods, scientists in the field had developed methods based on a fed-
`
`batch fermentation. (See BEQ1021, 1:2(11:2); BEQ1010, 947:2:2-948:1:1;
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`BEQ1002, ¶39.) Fed-batch fermentation allowed for controlled addition of media
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`components, “to control growth conditions, such as overflow metabolism,
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`accumulation of toxic compounds and oxygen availability,” to minimize acetate
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`accumulation and increase cell mass. (Id.) Indeed, by 2000, “[f]ed-batch
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`procedures ha[d] proved to be the most effective means of maximizing cell mass
`
`concentration.” (BEQ1010, 947:2:2; BEQ1002, ¶39.) And, “controlled addition of
`
`the carbon source, e.g., by glucose limited fed-batch strategies” provided a simple
`
`means to control acetate accumulation. (See BEQ1021, 5:3(15:3)3; BEQ1010,
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`947:2:2-948:1:1; BEQ1002, ¶40.)
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`In a fed-batch process, a base media supports initial bacterial growth (“batch
`
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`3 Pincites in parenthesis for BEQ1021 refer to page numbers as indicated on
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`the label in the right-hand side in the bottom of the page.
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`of U.S. Patent No. 6,716,602
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`phase”), and a feed media is added to prevent nutrient depletion and to sustain a
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`protein production phase (“fed-batch phase”). (See BEQ1011, 1004:1:1; BEQ1002,
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`¶39.) To control acetate accumulation, researchers in the field, as of 2000,
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`routinely used high glucose amounts during batch phase and low glucose amounts
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`during fed-batch phase, to produce numerous pharmaceutically-important proteins.
`
`(See BEQ1021, 4:3(14:3); BEQ1010, 948:1:2; BEQ1020, 2:3; BEQ1002, ¶¶40-
`
`43.)
`
`One of many examples is Seeger, which describes a fed-batch, HCDC
`
`fermentation process to produce a recombinant human growth factor, basic
`
`fibroblast growth factor (“bFGF”), in E. coli. (BEQ1010, 947, Abstract, 948:1:3;
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`BEQ1002, ¶43.) Seeger avoided “accumulation of toxic levels of acetic acid” by
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`using a three phase fed-batch process comprising a batch phase, characterized by
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`unlimited E. coli growth (µmax = 0.51 h-1), followed by two fed-batch phases of
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`successively-reduced growth rates (µset = 0.12 h-1 to 0.08 h-1). (BEQ1010, 925:1:1-
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`2:1, 950:2 Figure 3, 952:2:1; BEQ1002, ¶¶ 43, 55.) At the time of induction of
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`bFGF polypeptide expression, Seeger shifted the growth rate from 0.12 h-1 to 0.08
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`h-1 which “was sufficient to prevent accumulation of acetic acid during fed-batch
`
`phase 2 and to allow expression of bFGF.” (BEQ1010, 952:2:1; BEQ1002, ¶¶43,
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`57.) Thus, Seeger provided one of several examples of production of “more total
`
`and more soluble” recombinant protein by limiting glucose availability in HCDC
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`of U.S. Patent No. 6,716,602
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`fermentation at the time of recombinant protein induction to limit growth rate.
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`(BEQ1010 953:1:3; BEQ1002, ¶43.)
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`4.
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`Control of recombinant protein expression used well-known
`inducible promoters, such as the phosphate-inducible
`promoter phoA
`
`As of November 3, 2000, researchers in the field achieved high yields of
`
`recombinant proteins using inducible promoter systems. (BEQ1007, 145:2:1;
`
`BEQ1002, ¶45.) Inducible promoters, as compared to constitutive promoters, allow
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`for separation of cell growth from the recombinant protein production phase and
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`generally avoid the metabolic burden associated with coordinated cell growth and
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`recombinant protein production. (BEQ1007, 145:1:3 and 145:2:1; BEQ1016,
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`16.1.1:1:4; BEQ1002, ¶45.) This facilitates increased cell mass accumulation
`
`before recombinant protein production and thus, higher total recombinant protein
`
`yields. (BEQ1007, 145:2:1; BEQ1002, ¶45.)
`
`And researchers had available a number of inducible promoters (or inducible
`
`expression systems) suitable for E. coli. (BEQ1022, Table 4; BEQ1023 1996,
`
`Table 1; BEQ1002, ¶46.) For example, Makrides disclosed a list of twenty-nine
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`inducible promoters, including phoA, a phosphate-depletion inducible promoter.
`
`(BEQ1023, Table 1; BEQ1002, ¶62.) The art provided guidance for choosing a
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`promoter: 1) the promoter must be strong (e.g., “resulting in the accumulation of
`
`protein making up to 10-30% or more of the total cellular protein”); 2) “exhibit a
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`Petition for Inter Partes Review
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`of U.S. Patent No. 6,716,602
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`minimal level of basal transcriptional activity”; and 3) induction should be simple
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`and cost-effective. (BEQ1023, 513:2:2-514:1:2; BEQ1002, ¶¶46, 62.) As
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`confirmed by Dr. Rosenberg, a POSA aware of this guidance would have easily
`
`selected a suitable promoter from Makrides’ twenty-nine commonly-used
`
`promoters. (BEQ1002, ¶¶46, 62, 129.)
`
`For example, a POSA knew that the phoA promoter satisfied several of these
`
`criteria. (BEQ1022, Table 4; BEQ1023 1996, Table 1; BEQ1024, 48:1:Table I;
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`BEQ1026, 163:2:4; BEQ1042, 11900:1:2 and Abstract; BEQ1002, ¶¶46, 62, 129.)
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`Not only does the phoA promoter induce protein expression at “more than 1000-
`
`fold,” it is “essentially silent,” exhibiting minimal basal transcriptional activity.
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`(BEQ1022, 5:2:2; BEQ1002, ¶47.) And because induction is simple, requiring only
`
`limiting the phosphate concentration, the promoter had been used routinely to
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`produce several recombinant proteins in HCDC. (See BEQ1022, 5:2:2; BEQ 1023,
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`513:2:2-514:1:2; BEQ1040, Abstract and 4892:2:2; BEQ1041, Abstract;
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`BEQ1042, 11900:1:2 and Abstract; and BEQ1026, Abstract; BEQ1002, ¶¶47, 62.)
`
`C. The ’602 patent
`Against this background in which the prior art described well-established
`
`methods for avoiding acetate accumulation to inc