UNITED STATES PATENT AND TRADEMARK OFFICE
`
`_______________________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`_______________________________
`
`KASHIV BIOSCIENCES, LLC
`Petitioner
`
`v.
`
`AMGEN INC.
`Patent Owner
`
`____________________
`
`Case No. IPR2019-00791
`U.S. Patent No. 8,940,878
`____________________
`
`
`DECLARATION OF ANNE S. ROBINSON, Ph.D.
`IN SUPPORT OF
`PETITION FOR INTER PARTES REVIEW
`OF U.S. PATENT NO. 8,940,878
`
`
`
`
`Page 1
`
`KASHIV EXHIBIT 1002
`IPR2019-00791
`
`

`

`TABLE OF CONTENTS
`
`B. 
`
`INTRODUCTION ........................................................................................... 4 
`I. 
`QUALIFICATIONS ........................................................................................ 4 
`II. 
`III.  MATERIALS REVIEWED ............................................................................ 7 
`IV.  LEGAL STANDARDS ................................................................................... 7 
`V. 
`BACKGROUND OF THE TECHNOLOGY ................................................ 14 
`A. 
`The Basic Science of Proteins ............................................................. 14 
`1. 
`Protein Structure in General ...................................................... 14 
`2. 
`Protein Synthesis ....................................................................... 16 
`Recovering Bioactive Protein and Protein Refolding ......................... 19 
`1. 
`Isolating Inclusion Bodies ......................................................... 22 
`2. 
`Solubilizing Inclusion Bodies ................................................... 23 
`3. 
`Refolding the Solubilized Proteins ........................................... 24 
`4. 
`Using Separation Matrices to Purify Proteins........................... 26 
`5.  Washing and Eluting the Protein .............................................. 40 
`6.  Matrix Regeneration ................................................................. 41 
`VI.  STATE OF THE PRIOR ART ...................................................................... 43 
`A. 
`Ferré ..................................................................................................... 43 
`B. 
`Komath ................................................................................................ 45 
`C. 
`Rosendahl ............................................................................................ 46 
`D.  GE Handbook ...................................................................................... 47 
`VII.  DETAILED OPINIONS ................................................................................ 48 
`A.  Overview of the '878 Patent ................................................................ 48 
`B. 
`Level of Skill in the Art ....................................................................... 51 
`C. 
`Claim Construction ............................................................................. 51 
`D. 
`Claims 7, 8, 11, 12, 15, 16, 18, 19, and 21 Do Not Present Anything
`New over Ferré .................................................................................... 58 
`1. 
`Ferré discloses each and every feature of claim 7 .................... 58 
`2. 
`Ferré discloses each and every feature of claims 8, 11-12, 15-
`16, 18-19, and 21....................................................................... 66 
`
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`Page 2
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`

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`3. 
`
`Claims 7-8, 11-12, 15, and 16 Do Not Present Anything New over
`Komath ................................................................................................ 69 
`1. 
`Komath discloses each and every feature of claim 7. ............... 69 
`2. 
`Komath discloses each and every feature of claims 8, 11, 12,
`15, and 16 .................................................................................. 76 
`Claims 7-8, 11-12, 15, and 16 Are Obvious in View of Komath ....... 78 
`1. 
`Komath discloses every step of the method of claim 7 ............ 80 
`2. 
`The ordered steps of the claimed method are a well-established,
`standard procedure for purifying proteins expressed in non-
`mammalian expression systems, and no more .......................... 83 
`A POSA would have been motivated to use the steps disclosed
`in Komath in the order recited in claim 7 ................................. 86 
`A POSA would have reasonably expected success in using the
`steps of Komath in the recited order of claim 7 ........................ 91 
`Claims 8, 11-12, 15, and 16 Are Also Obvious in View of
`Komath ...................................................................................... 95 
`Claims 13 and 17 Are Obvious over Ferré or Komath in View of
`Rosendahl ............................................................................................ 97 
`Claims 18, 19, and 21 Are Obvious over Ferré or Komath in View of
`the GE Handbook ..............................................................................101 
`Objective Indicia of Nonobviousness ...............................................106 
`
`
`4. 
`
`5. 
`
`E. 
`
`F. 
`
`G. 
`
`H. 
`
`I. 
`
`
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`Page 3
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`

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`I, Anne S. Robinson, Ph.D., declare and state as follows:
`I.
`
`INTRODUCTION
`1.
`
`I am over the age of eighteen (18) and otherwise competent to make
`
`this declaration.
`
`2.
`
`I have been retained as an expert witness on behalf of Kashiv
`
`BioSciences, LLC for the above captioned Inter Partes Review (“IPR”). In
`
`particular, I have been asked to review U.S. Patent No. 8,940,878 (“the '878
`
`patent”) and the prior art and to offer opinions as to the state of the art as of June
`
`2009 in the field of protein purification, as well as opinions as to whether a person
`
`of ordinary skill in the art (a “POSA”) as of June 2009 would have understood
`
`claims 7-8, 11-13, 15-19, and 21 of the '878 patent as presenting anything new or
`
`non-obvious over the prior art.
`
`3.
`
`I am being compensated for my time in connection with this IPR at
`
`my standard consulting rate, which is $325 per hour or $2500 per day. My
`
`compensation is not contingent on the conclusions I reach herein or on the
`
`specifics of my testimony. I have no financial stake in the outcome of this
`
`proceeding.
`
`II. QUALIFICATIONS
`4.
`I have over twenty years’ experience in chemical and biomolecular
`
`engineering. In particular, my research interests have focused on protein folding
`
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`and refolding as well as cellular systems for optimal expression of proteins and
`
`antibodies.
`
`5.
`
`I am currently the Head of Chemical Engineering at Carnegie Mellon
`
`University, in the College of Engineering. I am also an Adjunct Professor in the
`
`Department of Chemical and Biomolecular Engineering at the University of
`
`Delaware.
`
`6.
`
`Prior to this position, I was the Catherine and Henry Boh Professor in
`
`Engineering at Tulane University, School of Science and Engineering from 2012-
`
`2018 and am now an Adjunct Professor there.
`
`7.
`
`I began my training at Johns Hopkins University, where I received an
`
`undergraduate degree in Chemical Engineering in 1988 and a Master’s degree in
`
`Chemical Engineering in 1989.
`
`8.
`
`I obtained my Ph.D. in Chemical Engineering from the University of
`
`Illinois at Urbana-Champaign in 1994.
`
`9.
`
`After obtaining my doctorate, I undertook a National Institutes of
`
`Health (“NIH”) postdoctoral fellowship in the Department of Biology at the
`
`Massachusetts Institute of Technology (“MIT”) from 1994 to 1997.
`
`10.
`
`I then served as an Assistant Professor (1997-2003), Associate
`
`Professor (2003-2008), and Full Professor (2008-2011), as well as an Associate
`
`Chair for Biochemical Engineering (2008-2011) in the Department of Chemical
`
`
`
`Page 5
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`Engineering at the University of Delaware. From 2012 until present, I have been
`
`serving as an Adjunct Professor in the Department of Chemical and Biomolecular
`
`Engineering at the University of Delaware.
`
`11.
`
`I began my training in protein-protein interactions related to
`
`chromatography and purification as an undergraduate at Johns Hopkins University,
`
`under the direction of Chris Anfinsen, Professor of Biology (and an acknowledged
`
`expert in affinity chromatography). Over the course of my career, I have produced
`
`proteins using both microbial (non-mammalian) and mammalian cell systems, and
`
`utilized various purification strategies to isolate the proteins of interest from both
`
`cells and refold mixtures, including chromatography and membrane separation
`
`strategies based on both affinity and non-affinity methods.
`
`12. Over the course of my career, I have published 90 publications in peer
`
`reviewed scientific journals, including papers relating to protein refolding and
`
`purification. Many of these papers pertain to protein refolding, protein
`
`purification, and related subjects. I am also an inventor on three patents, one of
`
`which involves protein refolding, and one which utilized chromatography for
`
`protein purification.
`
`13. A complete copy of my curriculum vitae, which further details my
`
`qualifications and experience, is attached as Exhibit 1003.
`
`
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`III. MATERIALS REVIEWED
`14.
`In forming my opinions, I have reviewed, among other things, the '878
`
`patent, papers filed in the Patent Office in connection with prosecution of this
`
`patent, which I understand to constitute the prosecution history of the patent, and
`
`the papers, publications, and other references cited herein. A full list of materials I
`
`have considered can be found in Appendix A.
`
`IV. LEGAL STANDARDS
`15.
`In this section, I describe my understanding of certain legal standards.
`
`I have been informed of these legal standards by Petitioner’s counsel. I am not an
`
`attorney, and I am relying only on instructions from Petitioner’s counsel for these
`
`legal standards. I have applied these understandings in my analysis as detailed
`
`below.
`
`16.
`
`I understand that in order to receive a patent an inventor must invent
`
`or discover a new and useful process, machine, manufacture, or composition of
`
`matter.
`
`17.
`
`I understand that patent protection may be granted for any new and
`
`useful process, machine, manufacture, or composition of matter, or any new and
`
`useful improvement thereof.
`
`18. With respect to the level of ordinary skill in the art at the relevant
`
`times applicable to the subject patent, I understand that factors such as the
`
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`education level of those working in the field, the sophistication of the technology,
`
`the types of problems encountered in the art, the prior art solutions to those
`
`problems, and the speed at which innovations are made may help establish the
`
`level of skill in the art. One with ordinary skill has the ability to understand the
`
`technology and make modest adaptations or advances. A person of ordinary skill
`
`in the art is also a person of ordinary creativity, not an automaton.
`
`19.
`
`In my opinion, a person of ordinary skill in the art (“POSA”) at the
`
`time of the alleged invention described in the subject patent would have had at
`
`least a Bachelor’s degree (or the equivalent) in Biochemistry or Chemical
`
`Engineering with several years’ experience in biochemical manufacturing, protein
`
`purification, and protein refolding, or alternatively, an advanced degree (Masters or
`
`Ph.D.) in Biochemistry or Chemical Engineering with emphasis in these same
`
`areas. This person may also work in collaboration with other scientists and/or
`
`clinicians who have experience in protein refolding and purification or related
`
`disciplines. A person of ordinary skill in the art relevant to the '878 patent would
`
`easily have understood the prior art references referred to herein and would have
`
`had the capacity to draw inferences from them.
`
`20.
`
`In determining the qualifications of a POSA, I considered, among
`
`other factors, the field of the alleged invention and use thereof described in the
`
`subject patent, and my experience with the educational level of practitioners in the
`
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`Page 8
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`field of protein refolding and purification or related disciplines. In addition, my
`
`opinion is based upon my background, education, and personal experience devoted
`
`to the field of protein expression, refolding, and purification.
`
`21.
`
`I consider myself to be at least a POSA of the subject patent at the
`
`time of the alleged inventions claimed therein. I have been instructed by counsel
`
`that the earliest possible “time of the invention” for the purposes of the '878 patent
`
`is June 25, 2009 (earliest filing date – provisional application). I also have been
`
`instructed that the earliest possible “critical date” is June 25, 2008 (one year prior
`
`to earliest filing date). I have not been asked to opine as to whether these dates are
`
`proper; however, in my analyses below, I have considered the ordinarily skilled
`
`artisan’s understanding as of June 25, 2009, and have confirmed that my opinions
`
`remain the same even if applying the June 25, 2008 date.
`
`22.
`
`I have been informed and understand that the first step in comparing
`
`prior art to patent claims is to properly construe the claims to determine claim
`
`scope and meaning. I understand that in Inter Partes Review proceedings, the
`
`claim terms are to be construed in accordance with the ordinary and customary
`
`meaning of such claim as understood by a POSA at the time of the invention and
`
`the prosecution history pertaining to the patent.
`
`23. Specifically, I have been informed and understand that under the
`
`standard set forth in Phillips v. AWH Corp., 425 F.3d 1303 (Fed. Cir. 2005) (en
`
`
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`banc), the claims of a patent are given their ordinary and customary meaning as
`
`would be understood by a POSA at the time of the invention. I have been
`
`informed that a Phillips construction of a claim is based on the entire record,
`
`including both intrinsic evidence (i.e., the claims, specification, and prosecution
`
`history), as well as extrinsic evidence (e.g., dictionary definitions and expert
`
`testimony).
`
`24.
`
`I understand that, once the claims of a patent have been properly
`
`construed, determining anticipation of a patent claim requires a comparison of the
`
`properly construed claim language to the prior art on a limitation-by-limitation
`
`basis.
`
`25.
`
`I understand that a prior art reference “anticipates” a claim, and thus
`
`renders the claim unpatentable, if all limitations of the claim are disclosed in that
`
`prior art reference, either explicitly or inherently (i.e., necessarily present or
`
`implied).
`
`26.
`
`I understand that a claim is unpatentable under 35 U.S.C. § 102(b) of
`
`the Patent Act if the invention was patented or published anywhere, or was in
`
`public use, on sale, or offered for sale in this country, more than one year prior to
`
`the filing date of the patent application. I understand that a U.S. or foreign patent
`
`qualifies as prior art under § 102(b) to a patent claim if the date of issuance of the
`
`patent is more than one year before the filing date of the patent claim. I further
`
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`understand that a printed publication, such as an article published in a magazine or
`
`trade publication or a U.S. or foreign patent application, also qualifies as prior art
`
`under § 102(b) to a patent claim if the publication occurs more than one year
`
`before the filing date of the patent.
`
`27.
`
`I have been instructed by counsel on the law regarding obviousness,
`
`and understand that even if a patent is not anticipated, it will be unpatentable if the
`
`differences between the claimed subject matter and the prior art are such that the
`
`subject matter as a whole would have been obvious at the time the invention was
`
`made to a person of ordinary skill in the pertinent art.
`
`28.
`
`I understand that a person of ordinary skill in the art provides a
`
`reference point from which the prior art and claimed invention should be viewed.
`
`This reference point prevents one from using his or her own insight or hindsight in
`
`deciding whether a claim is obvious. Thus, “hindsight reconstruction” cannot be
`
`used to combine references together to reach a conclusion of obviousness.
`
`29.
`
`I also understand that an obviousness determination includes the
`
`consideration of various factors such as (1) the scope and content of the prior art,
`
`(2) the differences between the prior art and the claims, (3) the level of ordinary
`
`skill in the pertinent art, and (4) the existence of secondary considerations of non-
`
`obviousness.
`
`
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`30.
`
`I have been informed and understand that the obviousness analysis
`
`requires a comparison of the properly construed claim language to the prior art to
`
`determine whether the claimed subject matter as a whole would have been obvious.
`
`A claimed invention can be obvious when, for example, there is some teaching,
`
`suggestion, or motivation in the prior art that would have led one of ordinary skill
`
`to modify the prior art reference or to combine prior art reference teachings to
`
`arrive at the claimed invention. In other words, even if one reference does not
`
`show the whole of the invention, if it would have been obvious to a person of
`
`ordinary skill in the art at the relevant time to add the missing pieces to the
`
`invention (for example, as a matter of standard engineering practice or application
`
`of a well-known principle in the field), then a single reference can render a claim
`
`invalid even if it does not show the whole invention. Moreover, I have been
`
`informed and understand that a combination of two or more references can render a
`
`claim invalid as obvious, whether or not there is an explicit suggestion in one of
`
`the references to combine the two references, if as a matter of engineering skill or
`
`practice in the field it would be known to do so.
`
`31. And as stated above, I understand that secondary considerations must
`
`be examined to determine whether a certain invention would have been obvious to
`
`one of ordinary skill in the art. I understand that secondary considerations of non-
`
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`obviousness are part of the obviousness inquiry under 35 U.S.C. § 103, and that
`
`some examples of secondary considerations include:
`
`(1)
`
`any long-felt and unmet need in the art that was satisfied
`
`by the invention of the patent;
`
`(2)
`
`any failure of others to achieve the results of the
`
`invention;
`
`(3)
`
`any commercial success or lack thereof of the products
`
`and processes covered by the invention;
`
`(4)
`
`any deliberate copying of the invention by others in the
`
`field;
`
`(5)
`
`(6)
`
`any taking of licenses under the patent by others;
`
`any expression of disbelief or skepticism by those skilled
`
`in the art upon learning of the invention;
`
`(7)
`
`(8)
`
`and
`
`(9)
`
`any unexpected results achieved by the invention;
`
`any praise of the invention by others skilled in the art;
`
`any lack of contemporaneous and independent invention
`
`by others.
`
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`V. BACKGROUND OF THE TECHNOLOGY
`32.
`I understand that the claims of the '878 patent at issue are directed to a
`
`method of purifying proteins in non-native limited solubility form expressed in
`
`non-mammalian cells. In particular, the claims recite simple and well-known steps
`
`of expressing proteins of interest in non-mammalian cells, lysing said non-
`
`mammalian cells, solubilizing proteins of interest from inclusion bodies formed in
`
`the non-mammalian cells, refolding the solubilized proteins, and purifying the
`
`refolded proteins, e.g., using chromatographic methods. See, e.g., EX1001, claim
`
`7. Each of these steps – expressing, lysing, solubilizing, refolding, and purifying
`
`proteins using a separation matrix – and implementation of these steps in the
`
`recited sequence, was well known in the art as of June 2009 and was routinely
`
`performed by a POSA to purify proteins such as inclusion body proteins from non-
`
`mammalian (e.g., bacterial) cells.
`
`33. To assist the reader in understanding the discussion of my opinions,
`
`pertinent prior art, and relevant scientific concepts, I provide the following
`
`background on the underlying technology and terminology.
`
`A. The Basic Science of Proteins
`1.
`Protein Structure in General
`34. Proteins are complex macromolecules composed of amino acid
`
`residues and have four different “levels” of structure: (i) primary structure, (ii)
`
`secondary structure, (iii) tertiary structure, and (iv) quaternary structure. EX1017,
`
`
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`Page 14
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`44-68. The three-dimensional arrangement of amino acid residues confer the
`
`protein’s biological function and activity, and it is known as the protein’s “native”
`
`structure. Id. Each of these structural levels is important. A protein’s primary
`
`structure is simply a linear chain of amino acid residues that make up the protein.
`
`Id., 20, 44. Secondary structure refers to the local structural conformation of a
`
`polypeptide chain, generally characterized by α-helices and β-sheets, as shown
`
`below, which are driven to form by intramolecular forces (i.e., hydrogen bonding).
`
`Id., 44-55. Tertiary structure refers to a three-dimensional structure of a folded
`
`polypeptide chain. Id., 55-63. Proper folding of a polypeptide chain is important
`
`as it ultimately provides a biologically active protein. Many proteins are made up
`
`of multiple polypeptide chains (also known as protein subunits), which may be the
`
`same or different. Quaternary structure refers to how protein subunits interact with
`
`each other and arrange themselves to form a larger protein complex. Id., 68-69.
`
`The diagram below illustrates the four different levels of protein structures as
`
`discussed above. Id., Figure 3.20.
`
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`Page 15
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`
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`35. Certain chemical bonds in a protein known as “disulfide bonds” are
`
`important to a protein’s native tertiary (three-dimensional) structure. See EX1017
`
`32, 58. Disulfide bonds stabilize the protein’s three-dimensional structure by
`
`forming between particular amino acids that are close in proximity – specifically,
`
`cysteine residues. Id., 31-32. When these disulfide bonds are misformed, the
`
`protein could misfold, i.e., take a structure other than its native structure. Id., 58.
`
`See infra ¶202.
`
`2.
`Protein Synthesis
`36. Generally, proteins are naturally produced by the following process.
`
`A protein’s genetic information encoded in DNA is copied to generate a messenger
`
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`RNA (mRNA) molecule, which serves as a template for the synthesis of the
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`protein. EX1017, 125-149; EX1012. Then, the genetic information stored in
`
`mRNA molecules is “read” by the ribosomes of the cell, which catalyze the
`
`assembly of amino acids into polypeptide chains. Id. This process of transcription
`
`(from DNA to RNA, e.g., mRNA) and translation (from RNA, e.g., mRNA, to a
`
`protein or polypeptide chain) is known as biosynthesis.
`
`
`
`Id.
`
`37. The protein synthesis process is not complete when the polypeptide
`
`chain is translated from a messenger RNA (mRNA) molecule. EX1012. The
`
`nascent polypeptide chain must fold into its unique three-dimensional
`
`conformation in order to become biologically active and useful to the cells. Id.
`
`Generally, the amino acid sequence of each polypeptide chain enables it to fold as
`
`soon as it emerges from a ribosome, with subsequent folding often involving the
`
`help of molecular chaperons (a class of proteins) to guide the folding of proteins to
`
`its final conformation. Id.
`
`38. Proteins can also be made in the laboratory using recombinant DNA
`
`technology, which has been known in the art since at least the 1970s. For example,
`
`the use of recombinant DNA technology in producing biologically functional
`
`proteins was patented by Cohen and Boyer in 1974, and the first commercial
`
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`Page 17
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`production was human insulin by Eli Lilly in 1981. See EX1013; EX1014;
`
`EX1015; EX1016. Recombinant DNA is formed by combining two or more pieces
`
`of DNA, often from different sources. The recombinant DNA is then inserted into
`
`a host cell, which undergoes a similar biosynthesis process as discussed above, to
`
`produce a desired protein that the cell typically does not synthesize. EX1017, 182-
`
`183. In essence, recombinant DNA technology turns the host cell into a “factory”
`
`that creates a large amount of the desired protein in a highly efficient manner. See
`
`EX1016, 5. Proteins that are expressed using recombinant DNA technology are
`
`called recombinant proteins. EX1017, 182-183.
`
`39. Recombinant DNA technology can be used in both mammalian and
`
`non-mammalian cells (referred to as “expression systems”) to produce proteins.
`
`While the mammalian expression systems typically produce recombinant proteins
`
`that are biologically active, the low yield and challenges associated with using
`
`mammalian expression systems, such as long cell cultivation times and
`
`requirements for expensive bioreactor runs and medium components, significantly
`
`increase production costs. EX1011, 1. In contrast, non-mammalian expression
`
`systems (such as bacterial expression systems) can provide higher yields for a
`
`lower cost, because non-mammalian cells can grow quickly and to a high cell
`
`concentration. Id. Scientists thus generally turned to very-high-yield bacterial
`
`expression systems to express recombinant proteins. Id. One well-established host
`
`
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`organism for use in producing recombinant proteins is Escherichia coli, commonly
`
`referred to as E. coli. The biochemistry and genetics of E. coli are very well
`
`known, and E. coli is easily grown; thus, it is typically the organism of choice for
`
`many researchers to produce a high yield of desired proteins. EX1018, 1; EX1017,
`
`182-183; EX1023, 1.
`
`B. Recovering Bioactive Protein and Protein Refolding
`40. As discussed above, a protein must be properly folded into its native,
`
`three-dimensional structure in order to perform its biological function. EX1017,
`
`44-68. Generally, recombinant proteins produced in expression systems (e.g., non-
`
`mammalian expression systems) can exist as (1) soluble proteins in their bioactive,
`
`native structure; or (2) insoluble proteins in their non-native forms – that is, having
`
`a structure other than the protein’s bioactive, native three-dimensional structure.
`
`For example, these insoluble proteins can accumulate in host cells as intracellular
`
`protein aggregates, forming “inclusion bodies.” The figure below (reproduced
`
`from EX1065, Figure 2) depicts a bacteria cell expressing a misfolded protein that
`
`then accumulates in inclusion bodies. See also EX1011, 1; EX1023, 1; EX1021.
`
`
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`
`
`EX1065, 3; see also EX1018, 3-4.
`
`41.
`
`Inclusion bodies have been known for several decades to contain
`
`between 35-95% of the overexpressed recombinant protein of interest, as well as
`
`DNA, ribosomal RNA, lipids, other proteins, and water. EX1020, 2, 4; EX1018, 2;
`
`EX1021, 9.
`
`42. Scientists generally believed that inclusion bodies are the result of
`
`using non-mammalian expression systems to express heterologous proteins (i.e.,
`
`proteins that are not naturally synthesized by the cells). For example, recombinant
`
`proteins tend to aggregate inside bacterial cells such as E. coli because of the
`
`conditions used to produce a high protein expression level in the bacterial host
`
`cells (e.g., culture media, growth temperature, and how the protein is expressed in
`
`bacterial cells). EX1010, 4, 9; EX1018, 1. Bacterial host cells provide for a more
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`rapid intracellular production of recombinant proteins than the natural protein
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`generation process in mammalian cells. As a result, the bacterial host cells have
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`trouble “keeping up” with this rapid rate of recombinant protein production and the
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`nascently-produced proteins misfold; for example, resulting in misfolded proteins
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`that have hydrophobic surfaces exposed to an aqueous environment surrounding
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`the protein. These misfolded proteins may preferentially interact with other
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`molecules via non-specific hydrophobic interactions to form inclusion bodies.
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`EX1018, Abstract.
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`43.
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`In addition, the bacterial expression systems may not provide an
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`optimum condition for eukaryotic proteins to properly fold into their native
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`structure. Specifically, the intracellular environment of bacterial cells, is often
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`termed as a “reducing redox environment,” which does not promote the formation
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`of disulfide bonds. EX1010, 6. Thus, eukaryotic proteins that contain disulfide
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`bonds in their native state are not able to properly form those bonds within
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`bacterial cells, thereby facilitating aggregation and inclusion body formation. Id.
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`44. Recombinant proteins expressed in E. coli were known to have the
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`specific problem of forming inclusion bodies. EX1022; EX1018, 2. To remedy
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`this problem, various methods for recovering proteins successfully in a bioactive
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`and stable form from inclusion bodies were developed. Indeed, Gergiou and Valax
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`reported that “[a]s of 1998, there have been over 300 reports of mammalian, plant,
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`and microbial proteins obtained and renatured from inclusion bodies formed in E.
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`coli.” EX1020, 1. The general approach for recovering proteins from inclusion
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`bodies follows a three-step process: (1) isolating inclusion bodies from host cells;
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`(2) solubilizing inclusion bodies; and (3) refolding the solubilized protein. See
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`EX1023, 1. See also EX1052, 2 ; EX1020. During this three-step process, the host
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`cells (e.g., bacterial cells) were typically broken open (“lysed”) to isolate the
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`inclusion bodies. Id. The inclusion bodies were subsequently solubilized, thereby
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`forming a solution containing denatured proteins, including the protein of interest.
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`Id. The solubilization solution containing the protein of interest in a denatured
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`state was then combined with a “refold” solution to cause the denatured protein
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`(i.e., the protein in a non-native form) to restore its native bioactive conformation
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`(“refolding”). Id. See also EX1022, 1-4; EX1023, 1-3.
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`1.
`Isolating Inclusion Bodies
`45. To isolate inclusion bodies, bacterial host cells (e.g., E. coli)
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`containing the inclusion bodies undergo disruption of their cell membrane, for
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`example, through high-pressure homogenization or a combination of mechanical
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`(e.g., sonication) and enzymatic (e.g., lysozyme) methods (known as “lysing” the
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`cells). EX1017, 187-188; see also EX1022, 1. Once the host cells are lysed, the
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`contents of the cells are released, and the resulting suspension is processed (e.g., by
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`centrifugation) to separate the lighter soluble portion (containing the soluble
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`proteins) from the heavier insoluble portion (containing the inclusion bodies and
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`cellular debris). EX1017, 189-192; EX1022, 1.
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`2.
`Solubilizing Inclusion Bodies
`46. After the inclusion bodies are isolated from the insoluble fraction, the
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`inclusion bodies are washed to remove nonspecifically surface-absorbed materials
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`and other contaminants such as membrane-associated proteins. EX1022, 1. The
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`isolated inclusion bodies are typically washed, e.g., using components such as
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`ethylenediaminetetraacetic acid (EDTA) and low concentrations of denaturants
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`and/or weak detergents such as Triton X-100. Id.
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`47. The washed inclusion bodies are then solubilized with chemicals that
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`disrupt the interactions between protein molecules of the inclusion bodies (e.g.,
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`decrease non-covalent interactions between protein molecules, and/or reduce
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`undesirable inter- and/or intra-molecular disulfide bonds). This solubilization step
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`is used to “denature” the protein into an unfolded state. Id., 2.
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`48. As of June 2009, there were a variety of methods that could be used to
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`solubilize inclusion body proteins. Inclusion body proteins were commonly
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`solubilized with denaturants. See, e.g., id., 2-3. Common denaturants include urea
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`and guanidine chloride. See, e.g., id.; EX1017, 217; EX1023, 5. While high
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`concentrations (e.g., 6-8M) of urea were typically used to solubilize inclusion
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`bodies, low concentrations of urea (e.g., 2M urea) could be used at an alkaline pH
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`(e.g., pH 11.0 to 12.5). EX1005, 10. See also EX1022, 2.
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`49. Surfactants (short for “surface active agent”) and/or reductants (short
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`for “reducing agents”) were also known to solubilize inclusion bodies. EX1022, 2;
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`EX1050, 16. Certain surfactants (e.g., detergents) may offer the advantage that the
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`solubilized protein may already display biological activity, thus avoiding the need
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`for a refold step. EX1022, 2. Common detergents include sodium dodecyl sulfate
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`(SDS) and n-cetyl trim