UNITED STATES PATENT AND TRADEMARK OFFICE
`
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`
`FRESENIUS KABI USA, INC. and FRESENIUS KABI SWISSBIOSIM GmbH
`Petitioners,
`v.
`AMGEN, INC. and AMGEN MANUFACTURING LIMITED,
`Patent Owner.
`
`
`IPR2019-01183
`Patent No. 9,643,997
`Title: CAPTURE PURIFICATION PROCESSES FOR PROTEINS EXPRESSED
`IN A NON-MAMMALIAN SYSTEM
`
`
`DECLARATION OF PETER M. TESSIER, PH.D. IN SUPPORT OF
`FRESENIUS’S PETITION FOR INTER PARTES REVIEW
`OF U.S. PATENT NO. 9,643,997
`
`
`Mail Stop PATENT BOARD
`Patent Trial and Appeal Board
`United States Patent and Trademark Office
`P.O. Box 1450
`Alexandria, VA 22313-1450
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`TABLE OF CONTENTS
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`Page
`INTRODUCTION ............................................................................................... 6
` BACKGROUND AND QUALIFICATIONS ..................................................... 6
` BASES FOR OPINIONS AND MATERIALS CONSIDERED ........................ 9
` SUMMARY OF OPINIONS ............................................................................... 9
` LEGAL PRINCIPLES .......................................................................................11
`Burden of Proof ...................................................................................11
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`Claim Construction..............................................................................12
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`Prior Art ...............................................................................................12
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` Anticipation .........................................................................................13
`Obviousness .........................................................................................15
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`Person of Ordinary Skill in the Art .....................................................17
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` TECHNICAL BACKGROUND: RECOVERY AND PURIFICATION OF
`RECOMBINANT PROTEINS ................................................................................17
`Protein Structure in General ................................................................17
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`Protein Synthesis .................................................................................18
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`Recovery and Purification of Bioactive Protein..................................19
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`Step 1: Isolate and Purify the Inclusion Bodies .......................22
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`Step 2: Solubilize the Inclusion Bodies ...................................22
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`Step 3: Refold the Solubilized Protein .....................................24
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`Step 4: Capture of Refolded Protein .........................................25
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`Steps 5 and 6: Washing and Eluting .........................................28
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` PERSON OF ORDINARY SKILL IN THE ART ........................................29
` U.S. PATENT 9,643,997 AND THE CHALLENGED CLAIMS ................30
`The Specification .................................................................................31
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`The Challenged Claims .......................................................................35
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`The File History of the ’997 Patent .....................................................38
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`Claim Construction..............................................................................40
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`“applying the refold solution to a separation matrix” ...............40
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`“washing” and “eluting/elution” ...............................................43
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`“isolate/isolated” .......................................................................44
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`Expressly Defined Terms ....................................................................45
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` SCOPE AND CONTENT OF THE PRIOR ART REFERENCES ..................48
` Wang (Ex. 1003) .................................................................................48
`Reardon (Ex. 1004) .............................................................................53
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`Dietrich (Ex. 1005) ..............................................................................57
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` Komath ’944 (Ex. 1006) ......................................................................59
`Komath ’056 (Ex. 1007) ......................................................................62
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`Cutler (Ex. 1028) .................................................................................64
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` DETAILED OPINIONS ....................................................................................65
` Ground 1: The Challenged Claims Are Anticipated by Wang ...........65
`Independent Claim 9 Is Anticipated by Wang (Ex. 1003) .......65
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`The Preamble ..................................................................65
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`Solubilization Step ..........................................................66
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`Refold Step .....................................................................66
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`Capture Step ....................................................................69
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` Wash Step .......................................................................70
`Elution Step ....................................................................72
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`Claims 10, 13-21, and 23-30 Are Anticipated by Wang ..........73
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`Claim Chart ...............................................................................79
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`Ground 2: The Challenged Claims Are Obvious over Wang in View
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`of Cutler (Ex. 1028) .......................................................................................84
`Ground 3: The Challenged Claims Are Anticipated by Reardon .......85
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`Independent Claim 9 Is Anticipated by Reardon ......................86
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`The Preamble ..................................................................86
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`Solubilization Step ..........................................................86
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`Refold Step .....................................................................87
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`Capture Step ....................................................................89
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` Wash Step .......................................................................91
`Elution Step ....................................................................92
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`Claims 10, 13-21, and 23-30 Are Anticipated by Reardon ......93
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`Claim Chart ...............................................................................99
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` Ground 4: The Challenged Claims Are Anticipated by Dietrich (Ex.
`1005) 102
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`Independent Claim 9 Is Anticipated by Dietrich ....................103
`The Preamble ................................................................103
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`Solubilization Step ........................................................103
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`Refold Step ...................................................................104
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`Capture Step ..................................................................105
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` Wash Step .....................................................................107
`Elution Step ..................................................................107
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`Dependent Claims 10, 13-21, and 23-30 Are Anticipated by
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`Dietrich ..............................................................................................108
`Claim Chart .............................................................................113
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`Ground 5: The Challenged Claims Are Obvious over Komath ’944 in
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`Combination with Komath ’056 ..................................................................117
`Differences between the Prior Art and the Claimed Invention
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`118
`The Challenged Claims Are Obvious over Komath ’944 and
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`Komath ’056 ......................................................................................120
`Claim 9 Is Obvious over Komath ’944 in View of Komath ’056
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`121
`The Preamble ................................................................121
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`Solubilization Step ........................................................122
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`Refold Step ...................................................................123
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`Capture Step ..................................................................125
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` Wash Step .....................................................................126
`Elution Step ..................................................................127
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`Claims 10, 13-21, and 23-30 Are Obvious over Komath ’944 in
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`View of Komath ’056 ........................................................................128
`No Secondary Considerations Support the Non-Obviousness of
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`the Challenged Claims.......................................................................135
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` CONCLUSION ...............................................................................................136
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`I, Peter M. Tessier, declare and state as follows:
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`INTRODUCTION
`I have been retained on behalf of Petitioners in connection with their
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`petition for inter partes review (“IPR”) of U.S. Patent No. 9,643,997 (“the ’997
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`patent,” Ex. 1001). I am an expert in the field of protein engineering, including the
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`commercial production of therapeutically useful proteins from non-mammalian
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`cell cultures. Counsel for Petitioners have asked me to consider the patentability
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`of claims 9-10, 13-21, and 23-30 of the ’997 patent (the “challenged claims”) in
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`light of the prior art, and to provide a tutorial on the technology described in the
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`’997 patent.
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`The opinions set forth in this declaration are my own. I am being
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`compensated at my usual rate of $600 per hour for the time spent preparing this
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`declaration, and my compensation is not contingent on the outcome of this matter
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`or on any of the opinions provided below.
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` BACKGROUND AND QUALIFICATIONS
`I have performed research in the field of protein engineering for over
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`fifteen years. My research regularly involves the purification of proteins expressed
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`from mammalian and non-mammalian cell culture systems.
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`I graduated from the University of Maine in 1998 as a Co-
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`Valedictorian with a B.S. in Chemical Engineering. I subsequently earned a Ph.D.
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`in 2003 from the University of Delaware in Chemical Engineering under the
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`supervision of Professors Lenhoff and Sandler working on protein and nanoparticle
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`interactions, solubility, crystallization, and self-assembly.
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`I have worked in academia since the early 2000s. Following my
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`Ph.D. studies, I worked as an American Cancer Society Postdoctoral Fellow at the
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`Whitehead Institute for Biomedical Research (MIT) under the supervision of
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`Professor Susan Lindquist, a specialist in molecular biology, and particularly
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`protein folding, for which she received the National Medal of Science in 2010. In
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`2007, I was appointed Assistant Professor at the Rensselaer Polytechnic Institute in
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`the Department of Chemical & Biological Engineering at the Center for
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`Biotechnology & Interdisciplinary Studies. From 2013 to 2016, I was promoted to
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`Associate Professor and then the Richard Baruch M.D. Career Development
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`(Endowed) Associate Professor, spending 2014 in Martinsried, Germany, at the
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`Max Planck Institute for Biochemistry as an Alexander von Humboldt Fellow.
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`In 2016, I was promoted to Full Professor at the Rensselaer
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`Polytechnic Institute. In 2017, I became the Albert M. Mattocks (Endowed)
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`Professor at the University of Michigan, Departments of Pharmaceutical Sciences,
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`Chemical Engineering and Biomedical Engineering, a position I continue to hold
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`today. I am also a member of the Biointerfaces Institute, a program designed to
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`promote collaboration between life and physical scientists.
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`Over the course of my career to date, I have received numerous
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`awards for my research. Most recently I was awarded the National Science
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`Foundation CAREER Award and the Alexander von Humboldt Fellowship for
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`Experienced Researchers, and I was named a Fellow of the American Institute for
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`Medical and Biological Engineering. I am a member of the American Chemical
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`Society, the American Institute of Chemical Engineers and the Society of
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`Biological Engineering.
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` My current academic research focuses on the development of next
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`generation technologies for designing, discovering, engineering, characterizing,
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`formulating and delivering biologics ranging from small affinity peptides to large
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`monoclonal antibodies for molecular imaging, diagnostic and therapeutic
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`applications. My interdisciplinary research program aims to provide new
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`fundamental insights related to protein structure and function, molecular origins of
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`protein-protein interactions, and sequence and structural determinants of key
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`protein properties, namely stability, solubility, specificity and affinity.
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`Over the course of my career, I have worked on many projects
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`investigating protein expression in both mammalian and bacterial host systems,
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`protein refolding, and chromatographic purification technology. I have published
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`60 peer-reviewed research and review papers, a book chapter on protein misfolding
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`diseases, and am an inventor on one U.S. patent.
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` A list of my publications, abstracts presented at scientific conferences,
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`appointments, affiliations, memberships in professional and research bodies and
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`selected research projects are included in my curriculum vitae, attached as Exhibit
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`1002A.
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` BASES FOR OPINIONS AND MATERIALS CONSIDERED
` The opinions in this declaration are based on my review of the ’997
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`patent, the applications and correspondence with the patent office that led to the
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`’997 patent (which I understand is called the “file history”), the “prior art” and the
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`other information cited in this declaration, as well as my extensive expertise as a
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`protein engineer working with protein expression, folding, and purification
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`processes. A list of the information I considered in preparing this declaration is
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`attached at Ex. 1002B.
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` SUMMARY OF OPINIONS
` The challenged claims (9–10, 13–21, and 23–30), are directed to the
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`purification of any protein expressed in a non-mammalian system in a limited
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`solubility form (such as inclusion bodies) by (a) solubilizing the protein in a
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`solution containing reagents that cause the proteins to disaggregate and unfold, (b)
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`forming a refold solution by diluting the solubilization solution into a buffer
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`containing reagents that allow the protein to properly refold, (c) applying the refold
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`solution to any “separation matrix” under conditions suitable for the protein to
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`associate with the matrix, (d) washing the matrix, and (e) eluting the protein. As
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`exemplified in the references I discuss below, scientists routinely used this
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`sequence of steps to purify proteins expressed in non-mammalian cells as of June
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`25, 2009, the earliest possible priority date of the ’997 patent.
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`I understand that Patent Owner (“PO”) has asserted that the claimed
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`process improved upon the prior art by eliminating the need to remove components
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`of the refold solution before applying the protein to the separation matrix.
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`However, as I discuss further below, scientists before 2009 had already purified
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`proteins expressed in a non-mammalian system without intervening steps to
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`remove components of the refold solution, and they understood that such
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`intervening steps were not necessary for the purification of all proteins using all
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`separation matrices.
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` After reviewing the prior art and applying the legal standards (set
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`forth below) provided by counsel, I have concluded that the challenged claims are
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`not patentable. In summary, the protein purification techniques described in the
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`’997 patent had been described and taught in the prior art before the earliest filing
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`date of the ’997 patent.
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` Each element of the protein purification process described in claims
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`9–10, 13–21, and 23–30 is disclosed in Wang (Ex. 1003), Reardon (Ex. 1004), and
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`Dietrich (Ex. 1005). These claims are not patentable because each of Wang,
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`Reardon, and Dietrich “anticipates” them.
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` Moreover, all of the challenged claims would have been obvious from
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`the combined disclosures of Komath ’944 (Ex. 1006) and Komath ’056 (Ex. 1007);
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`or the combined disclosure of Wang (Ex. 1003) and Cutler (Ex. 1028).
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` From these references, and from the general knowledge in the field,
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`any “person of ordinary skill in the art” would have known how to purify proteins
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`expressed in a non-native limited solubility form in a non-mammalian expression
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`system, by using the routine steps recited in the challenged claims. The claims
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`therefore are not patentable.
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` LEGAL PRINCIPLES
` To assist me in performing my assessment of patentability, counsel
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`for Petitioners have informed me about the legal standards for patentability set
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`forth below.
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` Burden of Proof
`I understand that in order to be eligible for a patent, an invention must
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`(among other requirements) be both new and not obvious from the prior art to a
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`person of ordinary skill in the relevant technical field. In an IPR, a claim is not
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`patentable if a “preponderance of the evidence” establishes that the claim is
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`anticipated by, or would have been obvious from, the prior art. I understand “a
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`preponderance of the evidence” means “more likely than not.”
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` Claim Construction
`I understand that in an IPR, the terms in a patent claim are generally
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`given their ordinary and customary meaning as understood by a person of skill in
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`the art in the context of the entire patent disclosure and the file history (together,
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`the “intrinsic” evidence).
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`If, however, a patentee provides a specific definition for a claim term
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`in the patent disclosure, then the patentee’s definition applies. If the patentee
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`chooses to provide a definition, however, it must be done with reasonable clarity,
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`deliberateness and precision.
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`I understand that some claims are “independent” and do not refer to,
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`or incorporate, any portion of any other claim. I also understand some claims are
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`“dependent” and refer to other claims by number. The dependent claims include
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`all of the limitations of the claims to which they refer (i.e. “depend” from). A
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`“multiple dependent” claim is any dependent claim which refers to more than one
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`other claim and refers to such other claims in the alternative only.
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`Prior Art
`I have been instructed to assume for purposes of my analysis that June
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`25, 2009, is the relevant date for determining what is “prior art.” In other words, I
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`should consider as “prior art” any reference that was published prior to June 25,
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`2009.
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`I further understand that a reference is a “printed publication” if it was
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`disseminated or otherwise made available such that persons of ordinary skill in the
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`art, exercising reasonable diligence, could locate it.
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` Anticipation
`I understand that a patent claim is anticipated if a single prior art
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`document discloses every element of the claim to a person of ordinary skill in the
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`art.
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`I understand that in order to anticipate, a prior art reference must
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`generally disclose all elements of the claim arranged as in the claim. However, if
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`the reference does not expressly spell out all the limitations arranged or combined
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`as in the claim, it can still anticipate if a person of skill in the art, reading the
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`reference, would at once envisage the claimed arrangement or combination.
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`I understand that even if the prior art reference does not expressly
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`disclose a particular claim element, the prior art may still anticipate if that element
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`is “inherent” in the disclosure. A claim element is inherent if it is necessarily
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`present in, or is the natural result of, subject matter disclosed in a prior art
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`reference.
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` Regarding process claims, I understand that if a prior art process does
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`not expressly disclose a step of a claimed process but necessarily results in the
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`performance of that step, then the step is inherent in the prior art process. In
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`addition, I understand that a prior art device may be relied upon for anticipation of
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`a method claim if the method claim is directed to a function which the prior art
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`device “in its normal and usual operation,” will perform.
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`I understand that an inherent element must necessarily be present in or
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`result from what is disclosed in a prior art reference, and that inherency may not be
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`established by probabilities or possibilities.
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`I also understand that while anticipation must be based on a single
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`prior art reference, additional references can be used to make clear that a missing
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`claim element is necessarily present in the subject matter described in the
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`reference. References that are not prior art can be used to demonstrate inherency.
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`I understand that a prior art publication must be “enabling” in order to
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`anticipate a patent claim. Prior art is enabling if it describes the claimed method
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`sufficiently to allow a person of ordinary skill in the art to carry out that method.
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`I understand that a claim written in “multiple dependent form” is
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`invalid if any dependency of the claim can be shown to be invalid. For example, I
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`understand that a multiple dependent claim depending from any of claims A, B,
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`and C is invalid if the claim covers invalid subject matter when construed to
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`depend from claim A, even if the claim does not cover invalid subject matter when
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`construed to depend from claim B or claim C.
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` Obviousness
`I understand that a patent claim is obvious if the differences between
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`
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`the claim and the prior art are such that the subject matter as a whole would have
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`been obvious to a person of ordinary skill at the time the claimed invention was
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`made. For purposes of my analysis, I have been asked to assume a prior art cutoff
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`date of June 25, 2009.
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`I understand that when assessing whether a claim is obvious, I should
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`consider: (i) the scope and content of the prior art; (ii) the level of ordinary skill in
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`the art; (iii) the differences between what is claimed and the prior art; and (iv) any
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`secondary considerations that may indicate whether the claim is obvious or not.
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`I understand that a claimed invention may be obvious when, for
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`example, there is some teaching, suggestion or motivation in the prior art that
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`would have led a person of ordinary skill in the art to modify the prior art reference
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`or to combine prior art teachings to arrive at the claimed invention, with a
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`reasonable expectation of success.
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`I also understand that a claim can be found obvious if, based on the
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`prior art, it would have been obvious for a person of ordinary skill in the art to try
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`the claimed invention by choosing from a finite number of identified, predictable
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`solutions, with a reasonable expectation of success.
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`I understand that “secondary” evidence may weigh against a finding
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`that a claimed invention would have been obvious. This secondary evidence
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`includes: (i) commercial success of a product due to the merits of what is provided
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`in the patent claim; (ii) a long-felt but unsolved need for the solution provided by
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`the patent claim; (iii) unsuccessful attempts by others to find the solution provided
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`by the patent claim; (iv) copying by others of what is claimed in the patent; (v)
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`unexpected results that arise from what is provided in the patent claim; (vi)
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`industry skepticism regarding what is provided in the patent claim; (vii) praise
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`from others in the field; and (viii) simultaneous invention by others.
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`I understand that in order to be probative of non-obviousness,
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`secondary evidence must have a “nexus” to the merits of the claimed invention. In
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`other words, the evidence must be related to the claimed invention and not what
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`was known in the prior art. For example, I understand that where alleged
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`commercial success actually results from something other than what is both
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`claimed and novel in the claim, then there is no nexus to the merits of the claimed
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`invention.
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`Person of Ordinary Skill in the Art
`I have been informed by counsel that a patentability analysis is to be
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`conducted from the perspective of a person of ordinary skill in the art to which the
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`claimed invention pertains at the time it was made. I understand that a person of
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`ordinary skill in the art is a hypothetical person that is presumed to have perfect
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`knowledge of all of the relevant prior art, and is often referred to as a “person of
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`ordinary skill in the art” or “POSA.”
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`I have been informed by counsel that in defining a POSA, the
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`following factors may be considered: (1) the educational level of the named
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`inventor; (2) the type of problems encountered in the art; (3) prior art solutions to
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`those problems; (4) rapidity with which innovations are made; and (5)
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`sophistication of the technology and educational level of active workers in the
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`field.
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` TECHNICAL BACKGROUND: RECOVERY AND PURIFICATION
`OF RECOMBINANT PROTEINS
` To assist the reader in understanding my opinions regarding the prior
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`art and claims, I have prepared a tutorial on the relevant scientific techniques and
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`principles.
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`Protein Structure in General
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` Proteins are complex molecules made up of chemical subunits known
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`as “amino acids” that are joined together by “peptide bonds” into long chains, like
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`beads on a string. ’997 patent at 6:8–11 (“the terms ‘protein’ and ‘polypeptide’ are
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`used interchangeably and mean any chain of at least five naturally or non-naturally
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`occurring amino acids linked by peptide bonds”). Proteins can perform their
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`biological function only if this “polypeptide” chain is folded into a precise three-
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`dimensional shape. Creighton 1999 (Ex. 1008) at 2020. The correct, functional
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`three-dimensional shape of a protein is often called the “native” form or structure.
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`Protein Synthesis
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` Proteins are naturally produced in both mammalian cells (e.g., mouse
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`and human cells) and non-mammalian cells (e.g., yeasts and bacteria, such as
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`Escherichia coli (commonly known as “E. coli”)). These organisms create
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`proteins by “transcribing” or duplicating information encoded in DNA to generate
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`an RNA molecule with the same encoded sequence, which then serves as a
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`template for the synthesis of a protein. Whitford (Ex. 1009) at 247; see also,
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`Horton (Ex. 1010) at 683–711. The genetic information stored in RNA is
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`“translated” or read by the ribosomes of the cell, which then use the information to
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`make protein molecules with the correct amino acid sequence. Id. This process of
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`transcription (from DNA to RNA) and translation (from RNA to a protein) is
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`known as “biosynthesis.”
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` Proteins can also be synthesized in the lab using recombinant DNA
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`technology, which has been known in the art since at least the late 1970s. Ex. 1010
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`at 719. A recombinant DNA molecule is constructed with DNA from different
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`sources, for example by inserting a segment of DNA that codes for a protein of
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`interest into another segment that facilitates insertion into the genes of a host cell.
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`Id.
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` When recombinant DNA is inserted into a cell, it enables the cell to
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`produce the protein of interest. Id. at 734-35 In essence, recombinant DNA
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`technology turns a host cell into a “factory” to create large amounts of the desired
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`protein in an efficient manner. Id. These host cells replicate into colonies that
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`produce the desired proteins on a larger scale. Proteins that are synthesized
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`(commonly called protein “expression”) using recombinant DNA technology are
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`called “recombinant” proteins. Id.
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` While recombinant proteins can be expressed in several different host
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`systems, E. coli expression systems have been among the most widely used for
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`large-scale production of proteins because they are relatively easy to use, quick,
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`and inexpensive. Id. at 734–35.
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` Recovery and Purification of Bioactive Protein
` Following expression, recombinant proteins must be folded into their
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`precise native three-dimensional shape to function properly. Unfolded or mis-
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`folded proteins lack function and are particularly prone to aggregation, typically
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`because “hydrophobic” residues (amino acids that have lower solubility in water)
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`that are normally buried within the interior of the properly-folded protein are
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`exposed on their surfaces. Hydrophobic residues tend to stick to other
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`hydrophobic residues, which causes mis-folded protein strands to clump together
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`and form particles of aggregated protein. Stirling 2003 (Ex. 1011).
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` While bacterial host cells, such as E. coli, have the advantage of
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`efficiently synthesizing proteins, they often have difficulty producing properly
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`refolded recombinant proteins, in particular, proteins that are normally not
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`produced by the bacteria. When expressing recombinant proteins, bacteria often
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`express misfolded proteins that clump together in the cell within what are known
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`as “inclusion bodies,” as shown in the image below of a bacteria cell expressing
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`protein:
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`(Modified from Profacgen (Ex. 1012)). Inclusion bodies are “insoluble,
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`biologically inactive aggregates of partially folded protein.” Ex. 1008 at 1250.
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`20 of 137
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`In order to recover therapeutically useful, biologically active proteins
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`from these inclusion bodies, they must be “solubilized” to allow the proteins to
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`disaggregate and unfold, and then the proteins must be “refolded” into their native
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`three-dimensional shapes (these concepts are explained more fully below). Id. at
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`1251. The proteins must also be purified from components of their solubilization
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`and refolding solutions as well as remaining cell debris.
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` Techniques for recovering and purifying native, folded proteins in a
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`bioactive and stable form from inclusion bodies were well known by 2009. As
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`early as 1998, there were “over 300 reports of mammalian, plant, and microbial
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`proteins obtained and renatured from inclusion bodies formed in E. coli.”
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`Georgiou (Ex. 1014) at 48.
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` These techniques generally began with recovery of refolded proteins
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`by (1) isolating and purifying the inclusion bodies; (2) solubilizing the inclusion
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`bodies, resulting in unfolding of proteins; and (3) refolding of the solubilized
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`protein. See generally Clark 1998 (Ex. 1015) at 47–54.
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` The solution obtained after refolding usually contained undesired
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`impurities along with properly refolded proteins. Therefore, it was typical to
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`purify the refolded proteins using one or more conventional chromatography
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`methods. Following step (3), these conventional purification methods typically
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`involved (4) applying the protein solution onto a chromatography matrix under
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`conditions in which the protein will temporarily adhere to or “associate with” the
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`matrix; (5) “washing” the column using one or more solutions to remove
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`contaminants from the adhered proteins; and (6) removing or “eluting” the protein
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`from the matrix by applying one or more solutions that cause the protein to
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`dissociate from the matrix. See, e.g., Dietrich (Ex. 1005) at [008], [0072]; Cutler
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`(Ex. 1028) at 128-129. The now purified proteins are then eventually isolated from
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`the eluate. I discuss each of these steps in additional detail below.
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`Step 1: Isolate and Purify the Inclusion Bodies
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` To obtain protein synthesized from, for example, bacterial host cells,
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`the host cells were lysed, the contents of the cell were released, and the resulting
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`suspension was centrifuged to separate the lighter soluble portion (containing non-
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`target soluble proteins) from the heavier insoluble portion (containing the inclusion
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`bodies and cellular debris). Ex. 1009 at 318-22; see also Clark 2001 (Ex. 1016) at
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`202–06, Bollag 1996 (Ex. 1017) at 44–46. The inclusion bodies were then isolated
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`from the cellular debris in the insoluble fraction and washed to remove
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`contaminants.
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`Step 2: Solubilize the Inclusion