Case 1:22-cv-00311-WCB Document 334 Filed 01/12/24 Page 1 of 46 PageID #: 20172
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`
`IN THE UNITED STATES DISTRICT COURT
`FOR THE DISTRICT OF DELAWARE
`
`IMPOSSIBLE FOODS INC.,
`
`Plaintiff,
`
`v.
`
`MOTIF FOODWORKS, INC., and
`GINKGO BIOWORKS, INC.,
`
`
`
`Defendants.
`
`
`
`
`
`Case No. 1:22-cv-00311-WCB
`
`
`
`
`
`DECLARATION OF DR. CARL BATT IN SUPPORT OF
`GINKGO BIOWORKS, INC.’S OPENING CLAIM CONSTRUCTION BRIEF
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`Case 1:22-cv-00311-WCB Document 334 Filed 01/12/24 Page 2 of 46 PageID #: 20173
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`
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`I. 
`II. 
`III. 
`IV. 
`
`V. 
`
`VI. 
`
`TABLE OF CONTENTS
`
`BACKGROUND ................................................................................................................ 1 
`QUALIFICATIONS AND EXPERTISE ........................................................................... 1 
`SCOPE OF ASSIGNMENT ............................................................................................... 5 
`LEGAL UNDERSTANDING ............................................................................................ 5 
`A. 
`Person of Ordinary Skill in the Art (POSA) ........................................................... 5 
`B. 
`Claim Construction ................................................................................................. 6 
`C. 
`Indefiniteness .......................................................................................................... 7 
`TECHNICAL BACKGROUND ......................................................................................... 9 
`A. 
`Gene Expression ..................................................................................................... 9 
`B. 
`Methylotrophic Yeast Expression Systems .......................................................... 12 
`C. 
`Promoters and transcriptional activators in P. pastoris ........................................ 13 
`THE YEAST PATENTS .................................................................................................. 14 
`A. 
`Person of Ordinary Skill in the Art for the Yeast Patents ..................................... 14 
`B. 
`The Claims and Specification of the Yeast Patents .............................................. 15 
`VII.  Opinions ............................................................................................................................ 17 
`A. 
`Term 1 - “promoter element” ................................................................................ 18 
`1. 
`A “Promoter Element” Is Not the Same as a Promoter ............................ 18 
`2. 
`The Yeast Patents Do Not Provide Any Definition of a “Promoter
`Element” ................................................................................................... 20 
`Nothing in the Specification Tells the POSA What “Something” Causes a
`Promoter Element to Modulate, Direct, or Regulate Expression .............. 22 
`Term 2a - “a Mxr1 transcriptional activator sequence” (’492 Patent, claim 1)/
`B. 
`Term 2b – “a methanol expression regulator 1 (Mxr1) transcriptional activator” (’656
`Patent, claim 1) / Term 2c - “a Mxr1 transcriptional activator sequence” (’656 Patent,
`claim 26) ........................................................................................................................... 23 
`The Specification’s Discussion of Mxr1 Transcriptional Activator
`1. 
`Sequences Is in the Context of Sequences Encoding the Mxr1 Protein ... 28 
`The Specification Contains Virtually No Discussion of Sequences to
`Which the Mxr1 Protein Binds and Contains No Disclosure of Any
`Specific Binding Sequences ...................................................................... 30 
`The Claims of the ’492 Patent Confirm to the POSA that the Phrase “a
`Mxr1 transcriptional activator sequence” in Term 2a Refers to a Coding
`Sequence ................................................................................................... 32 
`
`3. 
`
`2. 
`
`3. 
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`4. 
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`The Claims of the ’656 Patent Confirm to the POSA that the Phrase “a
`Mxr1 transcriptional activator sequence” in Term 2a Refers to a Coding
`Sequence ................................................................................................... 36 
`My Previous Opinions on the Related Terms Proposed by Motif ............ 37 
`5. 
`Term 6 - “sequence to which [the/a] Mxr1 transcriptional activator binds” ........ 38 
`
`C. 
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`ii
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`Case 1:22-cv-00311-WCB Document 334 Filed 01/12/24 Page 4 of 46 PageID #: 20175
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`I, Carl Batt, Ph.D., hereby declare:
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`I.
`
`BACKGROUND
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`1.
`
`I have been retained by Morrison & Foerster LLP on behalf of Defendant Ginkgo
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`Bioworks, Inc. (“Ginkgo”) as an independent expert in litigation brought by Impossible Foods
`
`Inc. (“Impossible”), which I understand is pending before the United States District Court for the
`
`District of Delaware (“Litigation”).
`
`2.
`
`I understand that, in this Litigation, Ginkgo is offering constructions of certain
`
`terms in claims 1, 4, 5, 7, and 14 of U.S. Patent No. 10,273,492, and claims 1, 5, 7, 8, 9, 10, 11,
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`13, 15, 16, 23, 24, 26, and 28 of U.S. Patent No. 10,689,656 (“Yeast Patents”).
`
`3.
`
`I have been asked to offer my opinion regarding the meaning of certain claim
`
`terms in the Yeast Patents, from the perspective of the person of ordinary skill in the art in the
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`patented subject matter (the “POSA”).
`
`4.
`
`I previously submitted two declarations opining on certain claim terms of these
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`same Yeast Patents, on behalf of Motif FoodWorks, Inc. (“Motif”) on June 28, 2023 and July 14,
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`2023. I understand that Impossible also accuses Motif of infringing the Yeast Patents. My
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`substantive opinions have not changed with respect to the claim terms I previously opined on,
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`but where appropriate, I have provided additional explanation and detail.
`
`II.
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`QUALIFICATIONS AND EXPERTISE
`
`5.
`
`Exhibit B-1 accompanying this declaration is a true and correct copy of my
`
`current CV. It describes in greater detail my education, publications, employment and research
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`history, and professional activities and awards.
`
`6.
`
`I have over 35 years of experience in microbiology, molecular biology, protein
`
`engineering, cell culture and food science. In 1975, I was awarded a Bachelor of Science Degree
`
`in Microbiology from Kansas State University in Manhattan, Kansas. In 1981, I was awarded a
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`1
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`Doctor of Philosophy Degree in Food Science from Rutgers University in New Brunswick, New
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`Jersey. I was a postdoctoral research associate from 1982-1984 in the Department of Nutrition
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`and Food Science and a research scientist from 1984-1985 in the Department of Applied
`
`Biological Sciences at Massachusetts Institute of Technology.
`
`7.
`
`In 1985, I joined the faculty of Cornell University as an Assistant Professor in the
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`Department of Food Science, where I was promoted to Associate Professor in 1991 and
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`Professor in 1998.
`
`8.
`
`While at Cornell University, I founded the Cornell University/Ludwig Institute
`
`for Cancer Research Partnership and served as its Director from 1999-2011. As Director, I
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`managed the design, construction and operation of a Good Manufacturing Practice (“GMP”)
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`facility on the Cornell University Ithaca campus, which resulted in the production of
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`recombinant proteins using microbial expression systems, including Pichia pastoris, that have
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`been used in clinical trials.
`
`9.
`
`I have conducted research in several areas that span the life and physical sciences.
`
`My work includes pioneering efforts in the fields of microbiology, molecular biology, and
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`nanotechnology. One area of research that I have focused on is the use of protein engineering
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`and expression systems for producing recombinant anti-cancer therapeutics and proteins in
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`general. For the past 35 years, my laboratory has been engaged in protein expression using a
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`variety of host systems including bacteria, yeast, fungi, and higher eukaryotic cells. Our work in
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`P. pastoris began in the early 1990s.
`
`10.
`
`The major area of research currently underway in my laboratory concerns
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`exploring parallel routes for the expression of therapeutic agents and constructing rational
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`approaches to scale-up of these systems. Our major focus is on the development and
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`2
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`optimization of microbial-based expression systems for tumor antigens and tumor-specific
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`antibody fragments. Our Biologics Production Facility enables us to carry out large-scale
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`production of highly-purified immunotherapeutics suitable for early-phase human clinical trials.
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`One of our products was a vaccine for schistosomiasis that was produced in P. pastoris and went
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`into a clinical trial in Brazil and Africa.
`
`11.
`
`Over the course of my career, I have authored or co-authored more than 300 peer-
`
`reviewed articles, book chapters and reviews. I have given more than 100 invited lectures
`
`worldwide. I am a named inventor on three United States patents.
`
`12.
`
`I have been fortunate to receive a number of distinctions during my career. In
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`1990, I was awarded the Samuel Cate Prescott Award from the Institute of Food Technologists
`
`which is given to the outstanding scientist in my field under the age of 35. In 2000, I was named
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`a Liberty Hyde Bailey Professor in the College of Agriculture and Life Sciences, a named chair
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`created to honor the most outstanding faculty in the college. It is an honor that I hold for the time
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`I am on the Cornell faculty. Today that distinction is held by only five professors among the
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`1,629 faculty at Cornell University.
`
`13.
`
`I am co-founder of the Nanobiotechnology Center (“NBTC”) at Cornell
`
`University, a National Science Foundation supported science and technology center, and was co-
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`Director of NBTC from 1999-2004. I am also co-founder of Main Street Science, a not-for-profit
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`corporation established to develop and distribute K-12 science learning materials, and the
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`founder of Nanooze, a science magazine for children.
`
`14.
`
`In 2007, I was an NSF Discovery Corps Fellow in recognition of both my
`
`extraordinary research accomplishments and my efforts to communicate advances in science and
`
`technology to the general public. In addition, I have been the recipient of a number of awards
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`3
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`from Cornell University for teaching (Teaching Excellence Award, 1990), service (Faculty
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`Fellows in Service, 2001; Kaplan Family Distinguished Faculty Fellowship in Service-Learning,
`
`2003), and diversity (CALS Kathy Berggren Diversity and Inclusion Award, 2008).
`
`15.
`
`In addition to my teaching responsibilities, I have mentored over 50 graduate
`
`students, including 45 PhD students and over 100 undergraduates in my laboratory, many of
`
`whom now hold significant positions in academia, government and the private sector, both in the
`
`United States and internationally.
`
`16. My research over the past 35 years has spanned a number of different topics
`
`including the use of recombinant protein expression systems to produce food and pharmaceutical
`
`products. My group was one of the early adopters of the Pichia expression system, and our work
`
`in the area of expression and protein secretion has been widely cited. Beginning in the mid-
`
`1990’s my group collaborated with James Cregg, an eminent researcher specializing in Pichia.
`
`Our publication on Pichia protein expression is among the most cited papers in the Pichia field
`
`in the past 10 years.
`
`17.
`
`I served as Chief Editor for Food Microbiology (1987-2000), a peer-reviewed
`
`journal. I also served on the Editorial Advisory Board of Wiley Encyclopedia of Chemical
`
`Biology (2007-2009), and as Editor (1996-1999) and Chief Editor (2d Ed., 2009-2015, 3rd Ed.
`
`2023-) for the Encyclopedia of Food Microbiology (1996-1999). I served as Editor of Elsevier’s
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`Food Microbiology, Food Science Reference Module (2015-2016). I have also been on the
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`Editorial Boards of the peer-reviewed journals: Food Biotechnology (1987-1992), Journal of
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`Food Science (2001-2008), and Protein Expression and Purification (2001-2004).
`
`18.
`
`As a champion of making scientific concepts understandable for the general
`
`public, especially young students, I lead a team that developed a series of public exhibitions
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`4
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`including “It’s a Nanoworld,” which was on a five-year tour in the United States after a six-
`
`month residency at EPCOT in Walt Disney World, and “Too Small To See,” which is also on
`
`tour after opening in EPCOT in 2007. Subsequently I designed and oversaw the construction of
`
`two additional exhibits, “Take a Nanooze Break” (2010-2020) and “Nanooze Labs” (2011-2014)
`
`that were on exhibit at Walt Disney World and Disneyland respectively.
`
`III.
`
`SCOPE OF ASSIGNMENT
`
`19.
`
` I have been asked to describe the relevant technical literature and state of the art
`
`relating to certain terms recited in some or all of the Yeast Patents and to provide my opinions
`
`regarding the meaning of such terms as they would have been understood by a POSA.
`
`20.
`
`In connection with the preparation of this declaration, I have reviewed the Yeast
`
`Patents and the prosecution history of the Yeast Patents, and various documents relevant to my
`
`opinions. A list of the documents I considered in forming my opinions are listed in Exhibit B-2.
`
`A list of any testimony I have provided in the last four years is included in Exhibit B-1.
`
`21.
`
`I am being compensated at my customary rate of $425 per hour for my work in
`
`this matter. This compensation is in no way contingent upon the outcome of this matter or upon
`
`the opinions I offer in this declaration. All of the opinions expressed in this declaration are my
`
`own.
`
`IV.
`
`LEGAL UNDERSTANDING
`
`22.
`
`I am not an attorney, but certain legal standards relevant to opinions have been
`
`explained to me by counsel.
`
`A.
`
`23.
`
`Person of Ordinary Skill in the Art (POSA)
`
`I understand that the meaning of claim terms in the patent is determined from the
`
`perspective of a POSA. I understand that a POSA is a hypothetical person who is presumed to
`
`be aware of all pertinent art, conventional wisdom in the art, and is a person of ordinary
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`5
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`creativity.
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`24.
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`In deciding the level of ordinary skill in the art, I am informed that the following
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`factors may be considered: the levels of education and experience of persons working in the
`
`field; the types of problems encountered in the field; and the sophistication of the technology.
`
`B.
`
`25.
`
`Claim Construction
`
`I understand that claim construction is a matter of law for the court to decide, but
`
`may be based on underlying facts.
`
`26.
`
`I understand that claim construction begins with the presumption that claim terms
`
`are given their ordinary and customary meaning, and that the ordinary and customary meaning of
`
`a claim term is the meaning that the term would have to a POSA at the time of the invention (i.e.,
`
`the effective filing date1), viewed in the context of the patent.
`
`27.
`
`I understand that to determine the ordinary and customary meaning of claim
`
`terms, courts may consult a variety of sources, including the claims themselves, the specification,
`
`and the prosecution history (collectively, referred to as “intrinsic evidence”), as well as relevant
`
`dictionaries and treatises, and expert testimony (collectively, referred to as “extrinsic evidence”).
`
`28.
`
`I also understand that intrinsic evidence is the most important source for
`
`determining the meaning of claim terms, but that extrinsic evidence may, in some circumstances,
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`also be useful when considered in the context of the intrinsic record. I understand that extrinsic
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`evidence cannot be used to contradict an unambiguous meaning provided in the intrinsic
`
`evidence.
`
`29.
`
`I understand that, under some circumstances, a patent applicant may clearly
`
`define a claim term in the specification or the prosecution history to mean something other than
`
`
`1 I understand that in this case, I have been asked to assume that the effective filing date is May
`11, 2015.
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`its ordinary meaning. Similarly, I understand that under some circumstances, patent applicant
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`may, in what is said during prosecution, clearly and intentionally disclaim certain claim scope.
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`30.
`
`I understand that the claim language must be considered in the context of the
`
`entire patent, including the specification and the prosecution history. Specifically, I understand
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`that the usage of a claim term within the specification and prosecution history may provide
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`guidance as to the meaning of the term and context which could assist a person of ordinary skill
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`to ascertain the plain meaning of the claim language. At the same time, I understand that the
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`specification usually cannot be used to read limitations into the claims, and that it is also
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`generally improper to read limitations out of the claims.
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`31.
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`I further understand that a patent may include two types of claims: independent
`
`claims and dependent claims. An independent claim stands alone and includes only the
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`limitations it recites. A dependent claim can depend from an independent claim or another
`
`dependent claim. I am informed that a dependent claim includes all the limitations that it recites,
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`in addition to the limitations recited in the claim or claims from which it depends.
`
`32.
`
`I understand that claim terms should usually be read and understood in a manner
`
`that gives meaning to all of the words in the claim and, further, gives each independent claim a
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`distinct meaning and scope from its dependent claims. Thus, other claims (and claim elements)
`
`of the patent in question, both asserted and unasserted, can inform the meaning of a claim term.
`
`For example, because claim terms are normally used consistently throughout the patent, the
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`usage of a term in one claim can often illuminate the meaning of the same term in other claims. I
`
`have been informed that differences among claims can also be a useful guide in understanding
`
`the meaning of particular claim terms.
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`C.
`
`33.
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`Indefiniteness
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`I am informed that patent law requires that the claims of the patent must meet the
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`definiteness requirement. I understand that a patent is invalid for indefiniteness if its claims,
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`read in the light of the specification and prosecution history, fail to inform a POSA with
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`reasonable certainty about the scope of the invention. I further understand that even if a claim
`
`term’s definition can be reduced to words, the claim is still indefinite if a POSA cannot translate
`
`the definition into meaningfully precise claim scope, the claim is still indefinite even if the
`
`definition of the term can be reduced to words.
`
`34.
`
`I further understand that if a term in a claim is expressed as a means for
`
`performing a specified function without the recital of structure2 or material for performing that
`
`function, the claim term is construed to cover only the structure or materials disclosed in the
`
`specification (and its equivalents) for performing the claimed function. If there is no such
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`structure or materials, I understand that the term is considered indefinite.
`
`35.
`
`I understand that in determining whether a claim element is expressed as a means
`
`for performing a function, I should consider whether the claim uses the term “means for”
`
`performing a function. If it does, there is a presumption that the claim is a functional claim. If it
`
`does, there is a presumption that the claim is a functional claim. However, I also understand that
`
`merely because an element does not include the word ‘means’ does not automatically prevent
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`that element from being construed as “means” for performing a function. I understand that
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`where a term does not include the word “means,” I should consider whether the words of the
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`claim are understood by a POSA to have a sufficiently definite meaning as the name of a
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`structure. If it does not, then I should consider whether the specification provides any structure
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`for the function. If the specification does not, the claim is considered indefinite.
`
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`2 By referring to “structure” here and throughout this declaration, I do not refer to the term used
`in nucleic acid chemistry, but rather the term as it is used in patent law as it has been explained
`to me.
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`8
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`V.
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`TECHNICAL BACKGROUND
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`36.
`
`A.
`
`37.
`
`I review below certain principles that support my opinions in this declaration.
`
`Gene Expression
`
`Every organism contains a genome, which is the complete set of genetic material
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`for that organism to grow, function, and replicate. Chemically, the genome is made of
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`deoxyribonucleic acid, or DNA. DNA consists of four nucleotides or bases: “A,” “C,” “T,” and
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`“G”.3 DNA is most often found in a double-stranded form in which the two strands are
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`connected through bonds between A:T and G:C nucleotide base-pairs. DNA can, for example,
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`encode proteins, act to regulate cellular functions, or have no known function. Information
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`encoded in the DNA (a “gene”) is converted into messenger ribonucleic acid (mRNA) through a
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`process called “transcription” that is carried out by the enzyme RNA polymerase. The mRNA is
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`then “translated” into an amino acid sequence to yield a protein. It is the nucleotide sequence
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`that dictates the amino acid sequence and the structure as well as the function of the protein.
`
`38.
`
`
`As shown in the figure above, DNA provides the blueprint used to create proteins.
`
`With the advent of recombinant DNA techniques4 in the late 1970s, it became possible to
`
`
`3 These stand for adenine, cytosine, thymine, and guanosine respectively.
`4 Recombinant expression of proteins commonly involves introducing into a host cell an
`exogenous nucleic acid that contains promoter and a gene encoding the protein of interest.
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`introduce and express proteins in cells that do not naturally produce those proteins.
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`39.
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`The expression of a gene and resulting production of the protein is controlled by
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`additional DNA sequences located “upstream” of the sequence for the gene of interest.
`
`“Upstream” refers to position of the sequences that initiate transcription relative to the sequences
`
`that encode the protein. These upstream sequences are referred to as “promoters,” because they
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`help “promote” transcription of the DNA into mRNA. Every gene encoding a protein has an
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`associated promoter. As of 2015, a vast number of promoters (from different sources) had been
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`identified and studied. An example of a promoter found in the yeast Pichia pastoris is the
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`promoter for the alcohol oxidase I (AOX1) gene, which is often denoted “pAOX1.”
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`40.
`
`Promoters are categorized as “constitutive” or “inducible” promoters.
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`Constitutive promoters are “on” regardless of external stimuli (i.e., constitutively active).
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`Inducible promoters are turned “on” or “off” in response to external stimuli, such as temperature,
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`oxygen, or chemicals. For example, a promoter that is methanol-inducible turns on when
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`methanol is added, although it may have a basal level when there is no methanol present. pAOX1
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`is an example of a methanol-inducible promoter.
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`41. More specifically, inducible promoters are turned on, or activated, by proteins
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`(“transcriptional activators”), which typically bind to a sequence or sequences present in the
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`promoter (an “activator binding site”) and then help recruit RNA polymerase to the promoter.
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`RNA polymerase is an enzyme that carries out transcription, i.e., the generation of mRNA from
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`the DNA sequence. A simplified illustration of this process is below:
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`42. While the illustration above depicts a single activator-binding site, multiple
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`binding sites may be present and necessary to activate transcription, and the binding sites can be
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`on either DNA strand. For example, it was reported in 2016 that the AOX1 promoter includes
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`six activator binding sites for the Mxr1 transcriptional activator protein along with several other
`
`transcriptional activator protein binding sites for other proteins that help to make pAOX1
`
`methanol-inducible.5 For methanol-inducible transcription to occur, not only are the
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`transcriptional activator protein binding sites (along with protein binding) needed, the binding
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`sites need to be in proximity to other DNA sequences that are responsible for RNA polymerase
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`binding, in ways that are not completely understood to this day.
`
`43.
`
`A “consensus sequence” is a term used to describe a segment whose sequence is
`
`common across a series of other sequences that all seem to share the same function. The
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`identification of a consensus sequence doesn’t guarantee that every representative will have that
`
`function as the function of a sequence may be a matter of a wider context (i.e., the sequences
`
`surrounding the sequence of interest, and the sequence of interest’s proximal location to those
`
`other sequences, which I will refer to as “sequence, location”).
`
`44.
`
`At the time the patents at issue were filed, it was known that the Mxr1
`
`transcriptional activator protein specifically binds to sites which contains the CYCCNY
`
`consensus sequence, where “Y” is either a C or a T, and “N” represents any nucleotide. Notably,
`
`the Mxr1 “core” binding site was defined as CYCC (Kranthi 2009) but was later expanded to
`
`CYCCNY (Kranthi 2010). However the Mxr1 transcriptional activator protein may not bind to
`
`
`5 Ex. B-5, Xiaolong Wang et al., Mit1 Transcription Factor Mediates Methanol Signaling and
`Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris, 291(12) Journal of
`Biological Chemistry 6245-61 (2016) (“Wang”).
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`every CYCCNY sequence found in a promoter.6 There are ten sequences of the form CYCCNY
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`in pAOX1, but only six are putative binding sites.7 Moreover, as explained in Wang, at least two
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`other transcriptional activators (Mit1 and Prm1) work with the Mxr1 transcriptional activator to
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`positively regulate pAOX1. The presence and binding of Mit1 is essential for regulating pAOX1
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`and any resulting transcription.8 Wang also reported that Mit1 bound to different locations in
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`pAOX1 from Mxr1 (and Prm1).9
`
`B. Methylotrophic Yeast Expression Systems
`
`45.
`
`As of 2015, a variety of host cells were known to be suitable for the recombinant
`
`expression of proteins, including animal, plant, fungal (yeast) and bacterial host cells. The
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`claims of the Yeast Patents (which I discuss below) all require the use of “methylotrophic” yeast
`
`host cells, meaning yeast cells that are able to utilize methanol as a sole source of carbon and
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`energy.
`
`46. While many organisms can only metabolize sugars such as glucose to provide
`
`energy and carbon, methylotrophic yeast can utilize methanol, a simple alcohol with the formula
`
`CH3-OH, as their sole carbon source.
`
`47.
`
`The claims of the Yeast Patents also refer to specific methylotrophic yeast cells,
`
`
`6 Ex. B-6, Balla Venkata Kranthi et al., Identification of key DNA elements involved in promoter
`recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris,
`Biochimica et Biophysica Acta 1789 (2009) (“Kranthi 2009”) (identifying CYCC) as the
`consensus sequence); Ex. B-7, Kranthi et al., Identification of Mxr1p-binding sites in the
`promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic
`yeast Pichia pastoris, Yeast, 27, 705-711 (2010) (“Kranthi 2010”) (further identifying CYCCNY
`as the consensus sequence).
`7 Ex. B-7, Kranthi 2010.
`8 Ex. B-5, Wang at 6246 & 6255 (“Mit1, Prm1, and Mxr1 are necessary for the activation of
`PAOX1. Deletion of either of these transcription factors dramatically decreases PAOX1 activity to
`<10% of that in WT [wild-type] cells and inhibits the growth of cells in the presence of
`methanol”).
`9 Ex. B-5, Wang at 6253.
`
`12
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`including Pichia pastoris or P. pastoris. A POSA would understand that the “P” refers to Pichia
`
`(a genus of yeast) and “pastoris” refers to a particular species of yeast within the Pichia genus.10
`
`There are also other species of Pichia.
`
`48. Members of the Pichia genus have several features that make them suitable for
`
`producing recombinant proteins. Among those features, they are fast growing, grow in relatively
`
`simple growth media, and grow to high densities.
`
`C.
`
`49.
`
`Promoters and transcriptional activators in P. pastoris
`
`P. pastoris natively has two alcohol oxidase genes, AOX1 and AOX2, each of
`
`which has its own promoter. At the time of the purported invention, the AOX1 promoter
`
`(pAOX1) was commonly used to drive expression of heterologous proteins in P. pastoris.11 The
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`relative strength of pAOX1’s transcription promoting function can be influenced by the binding
`
`of Mxr1 transcriptional activator proteins, along with at least two other transcriptional activator
`
`proteins, Prm1 and Mit1. These proteins positively regulate the AOX1 promoter in a cooperative
`
`fashion.12 There are also “inhibitors,” including PpNrg1, which have a negative impact on
`
`transcription.13
`
`50.
`
`A total of at least 11 binding sites have been experimentally demonstrated in the
`
`
`10 The name “Pichia” is derived from the Greek word “pikros,” meaning bitter, and it was
`initially used to describe a group of yeasts with bitter-tasting colonies. The specific epithet
`“pastoris” was added to honor the Swiss microbiologist and yeast researcher Walter Pastoris. It
`should be noted that recently P. pastoris has been renamed to Komagataella pastoris and in other
`cases Komagataella phaffi, but within this declaration, I will refer to it as P. pastoris.
`11 Ex. B-8, Thomas Vogl & Anton Glieder, Regulation of Pichia pastoris promoters and its
`consequences for protein production, New Biotechnology, 30(4) (May 2013) (“Vogl & Glieder
`2013”) (“The methylotrophic yeast Pichia pastoris is a widely used host for heterologous protein
`production . . . . P. pastoris provides a strong, methanol inducible promoter of the alcohol
`oxidase 1 (AOX1) gene.”).
`12 Ex. B-5, Wang.
`13 Ex. B-9, Figure 1, Yang, J. et al., Controlling AOX1 promoter strength in Pichia pastoris by
`manipulating poly (dA:dT) tracts, Sci Rep 8, 1401 (2018),
`https://www.nature.com/articles/s41598-018-19831-y/figures/1 (last accessed January 5, 2024).
`
`13
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`pAOX1 sequence: six specific for the Mxr1 transcriptional activator protein, three specific for
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`the Mit1 transcriptional activator protein, and two specific for the Prm1 transcriptional activator
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`protein.14 Modification of sequences within the promoter has been reported to result in
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`expression levels that vary between 6 and >160% of the wild-type[15] promoter expression
`
`levels.16
`
`VI.
`
`THE YEAST PATENTS
`
`51.
`
`I have reviewed the claims, specifications, and file histories (including
`
`proceedings at the Patent Trial and Appeal Board) of the Yeast Patents. I discuss certain aspects
`
`of these patents and the intrinsic record below.
`
`A.
`
`52.
`
`Person of Ordinary Skill in the Art for the Yeast Patents
`
`Based on my review of the ’492 and ’656 Patents (the Yeast Patents), the patents
`
`are directed to the art of recombinant protein production. In my opinion, a person of ordinary
`
`skill in this art (a POSA) for the Yeast Patents would have a Bachelor of Science degree in
`
`biochemistry, chemistry, biological sciences, or an equivalent area of study. A POSA would also
`
`ha