UNITED STATES PATENT AND TRADEMARK OFFICE
`
`--------------------------
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`--------------------------
`
`APOTEX INC. and APOTEX CORP.
`Petitioners,
`
`v.
`
`AMGEN INC. and AMGEN MANUFACTURING LIMITED
`Patent Owners
`
`--------------------------
`
`Case IPR2016-01542
`Patent 8,952,138
`
`--------------------------
`
`
`
`SECOND DECLARATION OF RICHARD C. WILLSON, Ph.D.
`
`Amgen Exhibit 2020
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`Page 1
`
`
`
`--------------------------
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`--------------------------
`
`APOTEX INC. and APOTEX CORP.
`Petitioners,
`
`v.
`
`AMGEN INC. and AMGEN MANUFACTURING LIMITED
`Patent Owners
`
`--------------------------
`
`Case IPR2016-01542
`Patent 8,952,138
`
`--------------------------
`
`
`
`SECOND DECLARATION OF RICHARD C. WILLSON, Ph.D.
`
`Amgen Exhibit 2020
`Apotex Inc. et al. v. Amgen Inc. et al., IPR2016-01542
`Page 1
`
`
`
`
`
`
`I.
`
`
`
`Page
`
`TABLE OF CONTENTS
`
`INTRODUCTION ....................................................................................... 4
`
`II.
`
`CLAIM CONSTRUCTION ......................................................................... 4
`
`III.
`
`INSTITUTED GROUNDS .......................................................................... 7
`
`IV.
`
`SCHLEGL AND HEVEHAN, ALONE OR IN COMBINATION, DO
`NOT RENDER CLAIMS 1-11 AND 13-24 OBVIOUS............................... 8
`
`A.
`
`B.
`
`C.
`
`Schlegl and Hevehan, Alone or in Combination, Do Not Teach
`Key Elements of Claim 1 of the ’138 Patent ...................................... 8
`
`A Person of Ordinary Skill in the Art Would Not Combine
`Schlegl And Hevehan ........................................................................19
`
`Assuming a Person of Ordinary Skill in the Art Would
`Combine Schlegl and Hevehan, Such Combination Does Not
`Render Obvious Any Claim of the ’138 Patent .................................29
`
`1. When Combined, the Teachings of Schlegl and Hevehan
`Do Not Result in the Invention Claimed in Claim 1 of the
`’138 Patent ..............................................................................29
`
`2.
`
`The Combination of Schlegl and Hevehan Does Not
`Provide a Reasonable Expectation of Success of
`Refolding Other Proteins, Including “a Protein Expressed
`in a Non-Mammalian Expression System” as Required
`by Claim 1 of the ’138 Patent..................................................34
`
`(a) Although Schlegl’s Methodology May Be
`Applicable to Refolding Bovine α-Lactalbumin, It
`Should Not Be Extrapolated to Other Proteins ..............35
`
`(b) Hevehan’s Model Should Not Be Extrapolated to
`Other Proteins ...............................................................39
`
`(c) Neither Schlegl nor Hevehan Demonstrates that
`Their Respective Refold Techniques and
`Conditions Successfully Refolded a Protein
`
`Page 2
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`
`
`
`
`Expressed in a Non-Mammalian Expression
`System ..........................................................................45
`
`D.
`
`E.
`
`Schlegl And Hevehan, Alone or in Combination, Do Not
`Render Obvious Claims 9-11 of the ’138 Patent ...............................50
`
`Schlegl and Hevehan, Alone or in Combination, Do Not Render
`Obvious Claim 18 of the ’138 Patent ................................................57
`
`V.
`
`SCHLEGL, HEVEHAN, AND HAKIM, ALONE OR IN
`COMBINATION, DO NOT RENDER OBVIOUS CLAIM 12 OF
`THE ’138 PATENT ....................................................................................61
`
`VI. CONCLUSION ..........................................................................................64
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`
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`Page 3
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`I.
`
`INTRODUCTION
`
`1.
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`I previously provided a declaration (EX2001) dated November 23,
`
`2016 in these proceedings in support of Patent Owner’s Preliminary Response
`
`(“First Willson Decl.”). In it, I expressed my opinion that the subject matter
`
`claimed in U.S. Patent No. 8,952,138 (“the ’138 Patent”) is novel and non-obvious,
`
`as those concepts have been explained to me, over the prior art relied upon by
`
`Apotex Inc. and Apotex Corp. (together “Apotex” or “Petitioners”).
`
`2. My professional background and qualifications, set forth in
`
`paragraphs 7-14 of the First Willson Decl. (EX2001), remain unchanged, except
`
`for my curriculum vitae (“CV”). My updated CV is attached as Exhibit 2054.
`
`3.
`
`The opinions I expressed in the First Willson Decl. (EX2001) also
`
`remain unchanged, including my views on claim construction.
`
`II. CLAIM CONSTRUCTION
`
`4.
`
`I have reviewed a copy of PTAB’s Decision Granting Institution of
`
`Inter Partes Review pursuant to 37 C.F.R. § 42.108 (“Institution Decision”). I
`
`understand that PTAB has rejected certain constructions previously adopted by the
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`United States District Court for the Southern District of Florida in a related
`
`litigation between the parties, including the constructions of two terms—“final
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`thiol-pair ratio” (“TPR”) and “redox buffer strength” (“RBS”)—on which both
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`parties to these proceedings agreed.
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`Page 4
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`5.
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`I have been informed by counsel to apply the following construction
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`for TPR: “the relationship of the reduced and oxidized species used in the refold
`
`buffer as defined by the equation [(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
`.” Institution Decision at 9 (emphasis
`
`added).
`
`6.
`
`I have also been informed by counsel to apply the following
`
`construction for RBS: “2[(cid:16)(cid:17)(cid:18)(cid:19)(cid:20)(cid:21)(cid:22)] + [(cid:24)(cid:25)(cid:19)(cid:26)(cid:27)(cid:22)(cid:20)(cid:21)(cid:22)],” wherein the oxidant and
`
`reductant concentrations are determined in the refold buffer. Institution Decision
`
`at 10.
`
`7.
`
`I understand that PTAB has interpreted “refold mixture” to mean “a
`
`mixture formed from contacting [1] the protein with [2] the refold buffer.”
`
`Institution Decision at 10. Accordingly, the protein-containing volume is separate
`
`from the refold buffer. PTAB explicitly rejected the District Court’s view that the
`
`refold mixture has a “‘high protein concentration’ . . . at or above a 1 g/L protein.”
`
`Id.
`
`8.
`
`I do not agree with the three constructions above, for reasons
`
`discussed in my declarations submitted during the claim construction phase of the
`
`related litigation (EX2007 at 10-15 and EX2008 at 11-18), and in the First Willson
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`Page 5
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`
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`
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`Decl. (EX2001) at ¶¶64-65 and 70-761. However, counsel has instructed me, for
`
`purposes of these proceedings, to apply the above constructions when considering
`
`the patentability of the subject matter claimed in the ’138 Patent in view of the art
`
`upon which this inter partes review (“IPR”) has been instituted.
`
`9.
`
`I have also been informed by counsel to apply the following
`
`constructions:
`
`• “protein” to mean “any chain of at least five naturally or non-naturally
`
`occurring amino acids linked by peptide bonds.”
`
`• “complex protein” to mean “The protein can be a complex protein,
`
`i.e., a protein that (a) is larger than 20,000 MW, or comprises greater
`
`than 250 amino acid residues, and (b) comprises two or more disulfide
`
`bonds in its native form.” Thus, a “complex protein” is either (1)
`
`larger than 20,000 MW with two or more disulfide bonds in its native
`
`form or (2) comprises greater than 250 amino acid residues with two
`
`or more disulfide bonds in its native form.
`
`1 Except for patent and patent application Exhibits, EX1002 (Robinson
`
`Declaration), EX2001 (First Willson Decl.), EX2021 (Hart Declaration), this
`
`Declaration, and EX2019 (Robinson 5/18/17 Dep. Tr.), all cites herein refer to the
`
`page numbers added by Petitioners, Amgen, or PTAB at the bottom of each
`
`Exhibit or Paper.
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`Page 6
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`
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`10.
`
`In addition, as discussed in the First Willson Decl. (EX2001) at ¶69,
`
`the term “non-mammalian expression system” means “a system for expressing
`
`proteins in cells derived from an organism other than a mammal, including but not
`
`limited to, prokaryotes, including bacteria such as E. coli and yeast,” as defined by
`
`the patentees. EX1001, ’138 Patent at 4:63-67.
`
`11.
`
`I have applied the constructions above at ¶¶5-7 and 9-10, and for the
`
`reasons discussed herein, it is my opinion that all the claims of the ’138 Patent are
`
`not obvious in view of the cited art, Schlegl (EX1003), Hevehan (EX1004) and
`
`Hakim (sometimes referred to as “Inclonals”) (EX1006), even using the above
`
`constructions of “TPR,” “RBS,” and “refold mixture.”
`
`III.
`
`INSTITUTED GROUNDS
`
`12. The following table summarizes the claims of the ’138 Patent
`
`challenged by Apotex and the bases and references providing the grounds for
`
`instituting this IPR:
`
`Challenged Claim(s)
`
`Bases
`
`Reference(s)
`
`1-11 and 13-24
`
`12
`
`§ 103(a)
`
`§ 103(a)
`
`Schlegl and Hevehan
`
`Schlegl, Hevehan and Hakim
`
`
`Institution Decision at 34.
`
`13.
`
`It has been explained to me that “§ 103(a)” stands for the section of
`
`the patent statute pertaining to obviousness. See also First Willson Decl.
`
`Page 7
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`
`
`
`
`(EX2001) at ¶25. Thus, I understand that this IPR has not been instituted for
`
`reasons of anticipation or lack of novelty, but rather, has been instituted only on
`
`grounds of obviousness.
`
`IV. SCHLEGL AND HEVEHAN, ALONE OR IN COMBINATION, DO
`NOT RENDER CLAIMS 1-11 AND 13-24 OBVIOUS
`
`A.
`
`Schlegl and Hevehan, Alone or in Combination, Do Not Teach
`Key Elements of Claim 1 of the ’138 Patent
`
`14. PTAB stated:
`
`The question of obviousness is resolved on the basis of underlying
`
`factual determinations including: (1) the scope and content of the prior
`
`art; (2) any differences between the claimed subject matter and the
`
`prior art; (3) the level of ordinary skill in the art; and (4) objective
`
`evidence of nonobviousness.
`
`Institution Decision at 6.
`
`15.
`
`It has been explained to me that objective evidence of nonobviousness
`
`includes unexpected results and satisfaction of a long-felt need.
`
`16.
`
`It is my opinion that there are significant differences between the
`
`protein refolding method claimed in Claim 1 of the ’138 Patent and the teachings
`
`(whether combined or not) of Schlegl and Hevehan. These differences stem from
`
`the sophisticated mathematical approach taken by the inventors of the ’138 Patent
`
`for choosing optimal refold conditions to obtain maximal yields of the desired,
`
`properly-folded, protein species.
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`Page 8
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`17. As I pointed out in the First Willson Decl. (EX2001) at ¶58, I believe
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`a key teaching of the ’138 Patent, which is embodied in Claim 1 and missing from
`
`the prior art is:
`
`As described herein, the relationship between thiol buffer strength and
`
`redox thiol-pair ratio has been investigated and optimized in order to
`
`provide a reproducible method of refolding proteins at concentrations
`
`of 2.0 g/L and higher on a variety of scales. A mathematical formula
`
`was deduced to allow the precise calculation of the ratios and
`
`strengths of individual redox couple components to achieve matrices
`
`of buffer thiol-pair ratio and buffer thiol strength. Once this
`
`relationship was established, it was possible to systematically
`
`demonstrate that thiol buffer strength and the thiol-pair ratio interact
`
`to define the distribution of resulting product-related species in a
`
`refolding reaction.
`
`EX1001, ’138 Patent at 4:35-45 (emphasis added); see also id. at Figs. 1a-1f.
`
`18. More specifically, Claim 1 calls for the use of a refold buffer
`
`comprising a redox component mathematically defined as having a thiol-pair ratio
`
`ranging from 0.001 to 100 according to the equation [(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
` and a redox buffer
`
`strength of at least 2 mM according to the equation 2[(cid:16)(cid:17)(cid:18)(cid:19)(cid:20)(cid:21)(cid:22)] + [(cid:24)(cid:25)(cid:19)(cid:26)(cid:27)(cid:22)(cid:20)(cid:21)(cid:22)].
`
`Nowhere do these equations or numerical parameters appear in Schlegl or
`
`Hevehan. I have read the transcript of the deposition of Apotex’s expert, Anne S.
`
`Robinson, Ph.D., in this proceeding (EX2019), and she agrees that neither Schlegl
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`Page 9
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`nor Hevehan (nor Hakim for that matter) discloses the thiol-pair ratio and redox
`
`buffer strength equations. EX2019, Robinson 5/8/17 Dep. Tr. at 40:12-21, 67:19-
`
`68:8, 83:10-21. Nor does either reference teach the concept that the interaction of
`
`thiol-pair ratio and buffer strength will affect “the distribution of resulting product-
`
`related species in a refolding reaction.” EX1001, ’138 Patent at 4:44-45.
`
`19. A person of ordinary skill in the art (“POSITA”) would understand
`
`that the “distribution of resulting product-related species” refers to the various
`
`outcomes of a refolding reaction when the protein being refolded has enough
`
`cysteine residues such that “mis-matched” disulfide bonds can occur, resulting in
`
`mis-folded protein species. Thus, product-related species can be the correctly
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`folded protein molecule or mis-folded versions of it.
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`20. The goal of protein refolding is to maximize the properly folded
`
`protein species with all the disulfide bonds of the native protein molecule and to
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`minimize mis-folded species. By recognizing the interplay of thiol-pair ratio and
`
`buffer strength and their effect on disulfide bond formation, the inventors focused
`
`on the redox component of the refold buffer as a means to control the desired
`
`outcome. That the use of precise mathematical equations for thiol-pair ratio and
`
`buffer strength can efficiently guide the choice of optimal buffer conditions is the
`
`unique teaching of the ’138 Patent, and this teaching is reflected in the claim
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`Page 10
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`
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`language. Schlegl and Hevehan, on the other hand, are devoid of such a teaching
`
`either individually or in combination.
`
`21.
`
`In Schlegl, the disclosure about redox systems is cursory because in
`
`Schlegl’s methodology, use of redox systems is optional. EX1003, Schlegl at
`
`[0036] and [0041] (listing redox systems among optional additives, such as
`
`detergents and EDTA). I disagree with Dr. Robinson’s position, expressed at page
`
`50 of her deposition transcript (EX2019), that a skilled artisan reading Schlegl
`
`would conclude that redox systems were not “optional” with respect to disulfide
`
`bond-containing proteins and that the “optional” use of redox systems applies only
`
`to refolding proteins without disulfide bonds. EX2019, Robinson 5/18/17 Dep. Tr.
`
`at 50:4-20. Schlegl is silent on, and presents no examples of, refolding proteins
`
`without disulfide bonds. Rather than rely on redox chemicals to “reshuffle”
`
`disulfide bonds (i.e., the breaking of mismatched cysteine residues and reformation
`
`of “correct”, i.e., native, disulfide bonds), Schlegl’s method relies on extreme local
`
`dilution of unfolded protein molecules to avoid mis-folding. There is no impetus
`
`provided by Schlegl to optimize refolding through the use of a redox component in
`
`a refold buffer.
`
`22. Hevehan, who refolded proteins at concentrations higher than Schlegl,
`
`looked to chemical additives to aid disulfide bond formation and to reduce mis-
`
`folding and aggregation. Hevehan, referencing earlier studies at low protein
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`Page 11
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`
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`concentration, states that “[t]he right mixture of low molecular weight thiol
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`compounds in oxidized and reduced forms needs to be added to the renaturation
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`buffer to allow disulfide bond formation and shuffling.” EX1004, Hevehan at 5.
`
`Acknowledging that what worked at low protein concentrations “might not be
`
`appropriate when folding a protein at 1 mg/mL or higher concentrations,” Hevehan
`
`reports the use of a trial-and-error matrix approach to find appropriate conditions.
`
`Id. However, this approach simply varied concentrations of a reductant [DTT] and
`
`an oxidant [GSSG] in a refold mixture with a constant (and high) protein
`
`concentration (1 g/L of the model protein, lysozyme). Id. In this manner, the
`
`optimal ratios of reductant to oxidant were empirically identified. Id. at 5, 6, Fig.
`
`4.
`
`23.
`
`It should also be noted that Hevehan ultimately concluded that
`
`addition of the solubilizing agent, guanidinium chloride, to the refold buffer was
`
`more critical to optimal refolding conditions than adding redox chemicals.
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`EX1004, Hevehan at 8. In fact, Hevehan further concluded that reductant need not
`
`be added to the refold buffer at all:
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`Page 12
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`
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`Addition of GSSG’s reducing partner, GSH, to the renaturation
`
`system was not necessary due to the DTT carried over from the
`denatured [protein] solution.2
`
`EX1004, Hevehan at 3 (emphasis added). In other words, some of the carried-over
`
`DTT would reduce some of the GSSG in the refold buffer to GSH, creating a
`
`GSH/GSSG redox pair to shuffle disulfide bonds. Reliance on carry-over DTT
`
`from the solubilization solution evidences Hevehan’s lack of appreciation of the
`
`advantages of carefully controlling redox chemicals in a refold buffer. Some
`
`amount of DTT added to the solubilization solution will be consumed during
`
`solubilization and will not be available as a reductant in the refold mixture. This
`
`amount was neither measured nor calculated. Hevehan was adding an unknown,
`
`uncontrolled amount of a reductant to the refold mixture.
`
`24. Unlike the approach of the ’138 Patent, where the focus is on the
`
`redox potential of the system and refold buffers are formulated using the equation
`
`[(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
`, Hevehan’s approach reflects the state of the prior art, where
`
`concentrations of reductant and oxidant were varied, and simple ratios of
`
`[reductant] / [oxidant] were calculated, with no appreciation of the interplay of
`
`buffer strength and thiol-pair ratio, and its effect on the distribution of resulting
`
`
`2 GSH stands for reduced glutathione (sometimes also called glutathione), a
`
`reductant, and GSSG stands for oxidized glutathione, an oxidant.
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`Page 13
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`product-related species. See, e.g., EX1004, Hevehan at 5 (“. . . optimum ratios of
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`GSH/GSSG between 0.8 and 3 (DTT/GSSG between 0.3 and 0.6)”).
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`25. Combining the teachings of Hevehan about the use of redox chemicals
`
`with the Schlegl system, as Apotex, Dr. Robinson, and PTAB have done, would
`
`not make obvious to a person of ordinary skill in the art the use of the equations
`
`and numerical parameters of Claim 1 of the ’138 Patent, in particular the thiol-pair
`
`ratio expressed as [(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
` and its stated range of 0.001 to 100 in the refold
`
`buffer. Rather, the person of ordinary skill, if even inclined to use a redox system
`
`in the Schlegl methodology, would continue to formulate refold mixtures using the
`
`simple ratio of [reductant] / [oxidant] in accordance with Hevehan’s explicit
`
`teachings.
`
`26.
`
`I note that Dr. Robinson has done some calculations on the refold
`
`buffer disclosed in Schlegl (EX1002, Robinson Declaration at ¶60, fn. 3-4) and the
`
`refold mixture disclosed in Hevehan (id. at ¶68, fn. 5)3. But, in my opinion, she
`
`
`3 Although I believe Dr. Robinson incorrectly made her calculations based on the
`
`volume of the refold mixture (when they should have been based on the volume of
`
`the refold buffer, as counsel has instructed me based on PTAB’s construction of
`
`TPR), I note another error she made. For purposes of her calculations of
`
`Hevehan’s thiol-pair ratio and buffer strength, Dr. Robinson used the DTT
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`Page 14
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`
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`has taken the teachings of the ’138 Patent and applied them with hindsight to the
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`prior art. Indeed, she admitted as much at her deposition when she testified that
`
`she took values for reductant and oxidant concentrations from Schlegl and
`
`Hevehan and simply plugged them into the equations taught only in the ’138
`
`Patent. EX2019, Robinson 5/8/17 Dep. Tr. at 44:18-45:17, 68:11-69:11. The
`
`person of ordinary skill in the art, without the benefit of the disclosure of the ’138
`
`Patent, would not be able to glean from Schlegl and/or Hevehan that he or she
`
`should refold a protein with “a refold buffer comprising a redox component
`
`concentration as the reductant concentration and the GSSG concentration as the
`
`oxidant concentration. EX1002, Robinson Declaration at ¶68. As discussed in ¶23
`
`above, the redox system used to shuffle disulfide bonds in Hevehan is actually a
`
`GSH/GSSG system created when some of the DTT carried over from the
`
`solubilization step reduces some of the GSSG in the refold buffer to GSH.
`
`EX1004, Hevehan at 3, 5. Thus, the concentrations used in the thiol-pair ratio and
`
`buffer strength calculations should have been GSSG and GSH concentrations.
`
`Hevehan does provide an expected value for [GSH]/[GSSG] in the refold mixture
`
`for the typical experiment where the refolding mixture contained 5 mM GSSG and
`
`2 mM DTT. Id. at 3. However, this expected value is not experimentally
`
`measured, and because some DTT will have been consumed during solubilization,
`
`the concentrations of GSSG and GSH would be hard to predict.
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`Page 15
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`
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`comprising a final thiol-pair ratio [defined as [(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
`] having a range of 0.001
`
`to 100 and a redox buffer strength [defined as 2[(cid:16)(cid:17)(cid:18)(cid:19)(cid:20)(cid:21)(cid:22)] + [(cid:24)(cid:25)(cid:19)(cid:26)(cid:27)(cid:22)(cid:20)(cid:21)(cid:22)]] of 2
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`mM or greater,” as called for by Claim 1 of the ’138 Patent (emphasis added).
`
`27.
`
`In connection with preparing this and my prior declaration, I have
`
`read several review articles on protein refolding that reflect the state of the art prior
`
`to the June 2009 priority date of the ’138 Patent, such as Eiberle4 (EX2030) and
`
`Lilie5 (EX2031). In none of them have I seen thiol-pair ratio expressed as
`
`[(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
`, as it is in the claims and specifications of the ’138 Patent. Rather, the
`
`state of the art at the time of the ’138 Patent invention was to express thiol-pair
`
`ratio as a simple ratio of the concentration of reductant divided by the
`
`concentration of oxidant. This is consistent with my own experience with protein
`
`refolding. Until I read the ’138 Patent, I had not seen nor heard of anyone
`
`expressing thiol-pair ratio as [(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
`.
`
`28. This state of the art is illustrated, e.g., in a review paper by Eiberle
`
`(EX2030) submitted for publication on January 4, 2010, which would encompass
`
`4 Eiberle et al., “Technical refolding of proteins: Do we have freedom to operate,”
`
`Biotechnol. J., 5, pp. 547-559 (2010).
`
`5 Lilie et al., “Advances in refolding of proteins produced in E. coli,” Current
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`Opinion in Biotechnology, 9, pp. 497-501 (1998).
`
`Page 16
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`
`
`
`
`the June 22, 2009 priority date of the ’138 Patent. In the section of the paper that
`
`addresses the use of redox systems for refolding proteins that contain cysteine
`
`residues, the authors state:
`
`Generally, a combination of a reduced and oxidized component is
`
`used, e.g., cysteine/cystine or reduced/oxidized glutathione. Suitable
`
`ratios must be found to maximize yields . . . Molar ratios of reduced to
`
`oxidized agents are recommended between 5:1 and 1:1. These ratios
`
`provide a suitable redox potential for the formation and reshuffling of
`
`disulfide bonds . . . .
`
`EX2030, Eiberle at 2 (emphasis added).
`
`29. Likewise, Lilie (EX2031) describes the renaturation/refolding buffer
`
`for proteins that contain disulfide bonds, noting that “The addition of a mixture of
`
`the reduced and oxidized forms of low molecular weight thiol reagents, such as
`
`glutathione, cysteine and cysteamine (molar ratios of reduced to oxidized
`
`compounds 1:1 to 5:1, respectively), usually provides the appropriate redox
`
`potential to allow formation and reshuffling of disulfides.” EX2031, Lilie at 3
`
`(emphasis added).
`
`30.
`
`It is clear that, as in Hevehan, the art persisted in using simple molar
`
`ratios, i.e., [reductant] / [oxidant] to identify suitable redox systems. Notably, not
`
`only is the equation of the patent for thiol-pair ratio not mentioned (i.e.
`
`[(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
`), there is nothing reported in Eiberle (EX2030) or Lilie (EX2031)
`
`Page 17
`
`
`
`
`
`about the other important teachings of the ’138 Patent, specifically that TPR and
`
`RBS are interrelated and affect distribution of product-related species such that the
`
`interrelationship also needs to be considered to achieve suitable refold conditions.
`
`Rather, as of the priority date, “for a researcher working with a novel protein,
`
`finding the most suitable conditions for expression, solubilization, and refolding of
`
`proteins a priori can be a relatively random process.” EX2032, Chow6 at 2
`
`(discussing the creation of a database of refold conditions) (emphasis added).
`
`31.
`
`I have read a copy of the Declaration of Roger A. Hart that will be
`
`submitted simultaneously with this declaration and the Patent Owner’s Response.
`
`I understand that it was his idea to use the equation for thiol-pair ratio as follows:
`
`[(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
`.
`
`32. As I mentioned above, until I read the ’138 Patent, I had not known
`
`anyone to express TPR as [(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
`, and I believe the inventors of the ’138
`
`Patent exhibited keen insight to have done so.
`
`33. Having read Dr. Hart’s declaration and studied its exhibits, this belief
`
`has been confirmed. I am persuaded that when the equation for TPR ([(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:7)](cid:11)
`
`[(cid:12)(cid:13)(cid:14)(cid:4)(cid:8)(cid:9)(cid:7)]
`
`)
`
`taught as “Equation 1” in col. 6, lines 25-28 of the ’138 Patent (EX1001) was
`
`6 Chow et al., “REFOLD: An analytical database of protein refolding methods,”
`
`Protein Expression and Purification, 46, pp. 166-171 (2006).
`
`Page 18
`
`
`
`
`
`implemented in screens for optimal refold buffers, along with the additional
`
`parameter RBS (2[(cid:16)(cid:17)(cid:18)(cid:19)(cid:20)(cid:21)(cid:22)] + [(cid:24)(cid:25)(cid:19)(cid:26)(cid:27)(cid:22)(cid:20)(cid:21)(cid:22)]) taught as “Equation 2” in col. 6,
`
`lines 35-38, it led to unexpected results and satisfied a long-felt need for a more
`
`rational, less random, design of refold buffers. Whereas results with the trial-and-
`
`error approach of varying simple ratios of reductants and oxidants produced
`
`inconsistent and random results, the use of the TPR and RBS equations led to
`
`rational results and much more easily and predictably led to suitable and optimized
`
`buffer conditions for refolding a variety of proteins.
`
`34. Thus, not only is the use of the TPR and RBS equations to design a
`
`refold buffer not obvious from Schlegl or Hevehan, alone or in combination, the
`
`unexpected results and satisfaction of a long-felt need in protein refolding could
`
`not have been predicted from the teachings of either of these references, or their
`
`combined teachings.
`
`B. A Person of Ordinary Skill in the Art Would Not Combine
`Schlegl And Hevehan
`
`35.
`
`It has been explained to me that in determining whether combined
`
`pieces of prior art render a claimed invention obvious, a threshold question needs
`
`to be answered affirmatively: Would a person of ordinary skill in the art have had
`
`reason to combine these pieces of prior art in the first place? If the answer is no,
`
`those pieces of art do not render the invention obvious.
`
`Page 19
`
`
`
`
`
`36.
`
`It has also been explained to me that the reason for combining
`
`references need not come in the form of an explicit teaching, suggestion, or
`
`motivation in a printed publication to combine the art but can also come from
`
`industry trends or simply common sense.
`
`37.
`
`I have previously explained why I do not believe a POSITA would be
`
`motivated to combine the teachings of Schlegl and Hevehan. First Willson Decl.
`
`(EX2001) ¶¶111-113, which state:
`
`111. A POSITA would not combine Schlegl and Hevehan because
`
`they are fundamentally different and incompatible approaches to
`
`protein refolding. On the one hand, as discussed above, Schlegl is a
`
`mechanical approach to achieve protein refolding at extremely dilute
`
`concentrations of unfolded protein. On the other hand, Hevehan is a
`
`chemical approach (focused on the addition of denaturant and oxidant,
`
`but not reductant, in the refold buffer) to achieve protein refolding at
`
`high protein concentrations.
`
`
`
`112. A POSITA understands that refolding protein at dilute
`
`concentrations and high concentrations are diametric opposites. The
`
`approach of refolding protein at dilute concentrations (like Schlegl) is
`
`meant to avoid the problem of aggregation by means of physical
`
`separation between the protein molecules. Refolding proteins at high
`
`concentrations (like Hevehan) necessarily reduces or eliminates the
`
`physical separation between the protein molecules, and other (e.g.,
`
`chemical) means are needed to avoid aggregation and achieve proper
`
`Page 20
`
`
`
`
`
`refolding. EX1004, Hevehan at 1 (“. . . low recovery of correctly
`
`folded protein is often due to aggregation . . . The most direct means
`
`of minimizing aggregation is by decreasing protein concentration.”);
`
`EX1003, Schlegl at [0008] (“The higher the protein concentration, the
`
`higher the risk of intermolecular misfolding, and vice versa.”).
`
`Hevehan primarily addresses the problem of protein aggregation by
`
`controlling the amount of denaturant (GdmCl) in the refold buffer.
`
`EX1004, Hevehan at 2 (“In particular, addition of solubilizing agents
`
`[denaturant] in nondenaturing concentrations to the renaturation
`
`buffer seemed to be most effective at decelerating the rate of
`
`aggregation.”) A POSITA, in my opinion, would not view the
`
`teachings of Hevehan as providing a benefit, much less an
`
`improvement, to Schlegl’s system where the problem of aggregation is
`
`being addressed by means of extreme dilution. See supra at ¶¶97-98.
`
`
`
`113. A POSITA understands that adding Hevehan’s denaturant and
`
`oxidant chemicals to Schlegl’s dilute refolding method would have
`
`been viewed as making Schlegl’s process more costly and
`
`complicated. The volume of the “refolding buffer solution” in Schlegl
`
`is large. See, e.g., EX1003, Schlegl at [0040] (“In its simplest
`
`embodiment, the method of the invention is a batch process that
`
`comprises, as its essential step, the above-defined mixing operation, in
`
`which a feed stream having a high concentration of unfolded protein
`
`and a low flow rate is combined with a refolding buffer solution
`
`having a high flow rate.”) (Emphasis added). Adding denaturant and
`
`oxidant chemicals to the refolding buffer solution stream would
`
`increase the already-considerable cost of preparing it. These
`
`Page 21
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`
`
`
`
`chemicals also can be corrosive, and their use entails substantial
`
`disposal costs. See, e.g., EX2017, EP0657466 at 1:34-37 (prior
`
`processes “produce a dilute urea waste solution which results in a
`
`difficult and expensive disposal problem.”).
`
`I stand by my previous opinions. In my view, it defies common sense that a
`
`POSITA would apply Hevehan’s conditions for refolding proteins at high
`
`concentrations, which employ costly redox chemicals, with a technique
`
`(Schlegl) seeking to avoid or minimize the use of chemicals by refolding
`
`proteins at ultra-low concentrations of unfolded protein, where the sheer
`
`physical isolation of each unfolded protein molecule limits aggregation and
`
`allows the molecule to fold properly on itself.
`
`38.
`
`I have read the PTAB’s Institution Decision at pages 13-15 and
`
`understand it to have been persuaded, at least preliminarily, by Dr. Robinson’s
`
`view that a POSITA “would look to Hevehan to solve the problem of refolding
`
`proteins at higher concentrations, and would have known the methods of Hevehan
`
`could apply to the dilution refolding methods of Schlegl.” Institution Decision at
`
`13 (citation om
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