Columbia Ex. 2076
`Illumina, Inc. v. The Trustees
`of Columbia University in the
`City of New York
`IPR2020-00988, -01065,
`-01177, -01125, -01323
`
`

`

`PROPERTY
`
`MAIWALD
`INTELLECTUAL
`
`A. Requests ...................................................................................................... 3
`
`B. Further cited documents ............................................................................ 3
`
`C. General comments and outline of arguments .......................................... 8
`
`|.
`
`General comments .................................................................................... 8
`
`||. Outline of arguments ................................................................................. 9
`
`D. Conceptual disclosure of SBS versus putting SBS into practice ........ 13
`
`|.
`
`The concept of SBS was known at the effective date of the opposed
`
`patent and plausibly established ..................................................................... 13
`
`||. The applicable case law .......................................................................... 18
`
`E.
`
`Insufficiency of disclosure ....................................................................... 20
`
`|.
`
`General comments .................................................................................. 20
`
`||. Difficulties in putting the invention into practice with a||y| and MOM as 3’-
`
`OH protective groups ...................................................................................... 22
`
`1. A||y| as 3’-OH protective group ............................................................. 24
`
`1.1
`
`1.2
`
`Finding a suitable DNA polymerase was not routine .................... 24
`
`Finding suitable cleavage conditions for a||y| was not routine ...... 30
`
`1.2.1 The cleavage conditions set forth in the patent for a||y| are not
`suitable for SBS ................................................................................... 30
`
`1.2.2 SBS-compatible cleavage conditions for a||y| were not part of the
`
`common general knowledge ................................................................ 32
`
`1.3
`
`Summary ....................................................................................... 35
`
`2. MOM as 3’-OH protective group .......................................................... 37
`
`III.
`
`Difficulties in putting the invention into practice with 3’-OH protective
`
`groups being as small as a||y| and MOM ........................................................ 39
`
`1. Requirement of fitting into the active site of a DNA polymerase .......... 4O
`
`2. Requirement of cleavability with high yields under mild conditions ...... 49
`
`3. Requirement of having chemical (electronic) properties not to be
`
`cleaved by the nucleophilic group in the active site of a DNA polymerase .52
`
`F.
`
`Inventive step ............................................................................................ 53
`
`|.
`
`D4 (Tsien) is the closest prior art and the problem underlying the claimed
`
`invention amount to providing an alternative SBS .......................................... 53
`
`||. D15 (Stemple) as closest prior art ........................................................... 59
`
`G.
`
`H.
`
`Added subject matter ............................................................................ 60
`
`Conclusion ............................................................................................. 62
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`Requests
`
`We maintain the request to revoke EP 3 034 627 B1 in its entirety on the
`
`basis of Articles 100 (a), 100 (b) and 100 (c) EPC.
`
`Our request for oral proceedings is maintained as well.
`
`Further cited documents
`
`In addition to the documents on file, we herewith file the following
`
`documents in orderto address the issues raised in the preliminary opinion
`
`in connection with the question of insufficiency of disclosure:
`
`D26:
`
`D27:
`
`D28:
`
`R. E. Ireland et al., J. Org. Chem., 1986, vol. 51, no. 5, pp. 635-
`648
`
`A. Kamal et al., Tetrahedron Letters, 1999, vol. 40, pp. 371-372
`
`H. Ruparel et al., PNAS, 2005, vol. 102, no. 17, pp. 5932-5937
`
`D29:
`
`WO 2004/018497 A2
`
`D30:
`
`D31:
`
`D32:
`
`D33:
`
`D34:
`
`D35:
`
`D36:
`
`D37:
`
`D38:
`
`D39:
`
`A. F. Gardner et al., Nucleic Acids Research, 2002, vol. 30, no. 2,
`
`pp. 605-613
`
`F. Guibé, Tetrahedron, 1998, vol. 54, pp. 2967-3042
`
`S. S. Flack, Tetrahedron Letters, 1995, vol. 36, no. 19, pp. 3409-
`3412
`
`J. Ju et al., PNAS, 2006, vol. 103, no. 52, pp. 19635-19640
`
`J. Guo et al., PNAS, 2008, vol. 105, no. 27, pp. 9145-9150
`
`Declaration by F. Romesberg dated July 1, 2020
`
`Declaration by J. Ju dated May 26, 2017
`
`Declaration by J. Kuriyan dated June 1, 2020
`
`K. Davies, The $ 1,000 Genome”, 2010, Free Press, New York,
`
`Chapter5
`
`T. W. Greene, P. G. M. Wuts, Protective Groups in Organic
`
`Synthesis, Third Edition, Chapter II, 1999, John Vlfiley & Sons, Inc.
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`D40:
`
`D41:
`
`D42:
`
`D43:
`
`D44:
`
`D45:
`
`D46:
`
`D47:
`
`D48:
`
`Declaration by S. M. Menchen dated June 1, 2020
`
`S. Lemaire-Audoire et al., Tetrahedron Letters, 1994, vol. 35, no.
`
`47, pp. 8783-8786
`
`S. Lemaire-Audoire et al., Journal of Molecular Catalysis A:
`
`Chemical 116, 1997, pp. 247-258
`
`V. M. Swamy, Synlett, 1997, pp. 513-514
`
`C.-H. Lee et al., Proc. Natl. Acad. Sci. USA, 1981, vol. 78, no. 5,
`
`pp. 2838-2842
`
`L. Levine et al., Biochemistry, 1963, vol. 2, no. 1, pp. 168-175
`
`E. J. Corey et al., Tetrahedron Letters, 1975, no. 38, pp. 3269-
`3270
`
`E. J. Corey et al., Tetrahedron Letters, 1975, no. 31, pp. 2643-
`2646
`
`Grant application by Dr. Romesberg and Dr. Ju issued in May
`2006
`
`D49:
`
`EP1 530 578 B1
`
`D50:
`
`D51:
`
`D52:
`
`D53:
`
`Submission of September 23, 2016 by Patentee
`
`during
`
`prosecution
`
`Thesis by Q. Meng, Columbia University, 2006
`
`Declaration by S. Peisayovich dated 23 March 2021
`
`Patentee’s response in IPR proceedings dated September9, 2020
`
`D54:
`
`WO 98/53300 A2
`
`D55:
`
`Bentley et al., Nature, 2008, vol. 456, no. 6, pp. 53-59
`
`D26 and D27 are named in paragraph [0007] of the contested patent as
`
`evidence of cleavage conditions for MOM and allyl which allegedly are
`
`compatible with SBS requirements. They are thus both known to the
`
`Patentee and relied upon by the Patentee fortheir sufficiency of disclosure
`
`arguments.
`
`D40 is introduced as evidence that the cleavage conditions set forth in
`
`the contested patent for allyl (i.e., D27) are not suitable for SBS. D40
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`is an expert declaration that has been submitted by the Patentee in the
`
`context of IPR proceedings relating to US patent 9,868,985, claiming
`
`priority to the same patent application as the opposed patent, and is thus
`known to the Patentee.
`
`D28 is evidence that extensive research efforts were required after the
`
`relevant date of the contested patent to identify a polymerase, namely
`
`a mutant 9°N polymerase, and cleavage conditions which would allow
`
`allyl to be used as a 3’-OH protective group for SBS. It is co-authored by
`Dr. Ju and is thus known to the Patentee. These research efforts were
`
`necessary because the polymerase and cleavage conditions in the
`
`contested patent were not functional.
`
`D29 and D30 are introduced as evidence that the only polymerase which
`
`D28 discloses to be functional with allyl, namely a particular mutant 9°N
`
`polymerase, was developed after the relevant date of the contested
`
`patent. D51 and D48 is further evidence which shows that polymerases
`
`other than the 9°N polymerase which were known at relevant date of the
`
`contested patent were not suitable for effective SBS when using allyl
`
`as a 3’-OH protective group. D51 is a PhD thesis from the group of Dr. Ju
`
`at Columbia. D48 is a grant application by Dr. Romesberg and was
`
`supported by Dr.
`
`Ju. Further, both were previously cited in US
`
`proceedings in which Columbia is participating. These documents are
`thus known to the Patentee.
`
`D31 and D32 are both references which D28 refers to when explaining
`
`why the cleavage conditions disclosed in these documents were not
`
`suitable for allyl. D41 and D42 are introduced as evidence that the
`
`cleavage conditions which were used in D28 for allyl required extensive
`
`research activities and were not part of the common general knowledge.
`
`These cleavage conditions differ from those mentioned in the patent, i.e.,
`
`D27 (Kamal).
`
`D33 is another post-published publication by the group of Dr. Ju and thus
`known to the Patentee.
`It
`is
`introduced as evidence that
`the
`
`photocleavable linker as shown in the various figures of EP’627 had to be
`
`replaced by an allyl linker in order to implement efficient SBS. D50 is a
`
`submission by the Patentee during examination of the contested patent
`which comments on D33.
`
`1O
`
`D34 is another post-published publication by the group of Dr. Ju. It shows
`
`that, based on the findings of D28 (Ruparel) and D33 (Ju), only after
`
`switching to azidomethyl as a 3’-OH protective group SBS beyond 20
`
`nucleotides became possible.
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`19
`
`D35 and D37 were submitted in US Inter Partes review proceedings
`
`relating to US patents 10,407,458; 10,407,459; 10,428,380, 10,435,742;
`
`and 10,457,984. D36 was submitted on behalf of the present Patentee
`
`during examination proceedings before the USPTO relating to US
`
`15/167,917. D35-D37 are thus all known to the Patentee. They are
`
`introduced in response to D19 (Ju) to show that the application as filed
`
`fails to provide guidance for the “as small as” feature. These documents
`
`further show that different than alleged by the Patentee, azidomethyl
`
`could not be identified without an undue burden as a 3’-OH protective
`
`group which would meet the “as small as” feature.
`
`D49 is introduced as evidence establishing azidomethyl as a 3’-OH
`
`protective group for SBS methods required inventive activity.
`
`D38 is introduced to show that it was self-evident that any SBS method
`
`had to be able to sequence DNA molecules of at least 20 nucleotides.
`
`D39 is introduced as evidence of cleavage conditions for protective
`
`groups such as propargyl and methylthiomethyl (MTM) which the skilled
`
`person was aware of from organic chemistry. D43-D47 are introduced as
`
`evidence that these cleavage conditions are not suitable for SBS.
`
`D52 is introduced as evidence that other than alleged by the Patentee
`
`azidomethyl does not fit into the active site of rat DNA polymerase of
`
`Figure 1 of the contested patent.
`
`D53 is introduced as evidence that also the Patentee took the position
`
`that one cannot tell
`
`if a given protecting group such as allyl can be
`
`incorporated by any polymerase without data showing it could work. The
`
`document was submitted in US lnter Partes review proceedings relating
`
`to US patent 10,407,458.
`
`D54 is filed to show that D4 was a known reference to those working in
`
`the field of SBS. This patent was cited in the lPRs between the parties, so
`
`Columbia should be aware of this patent.
`
`D55 is a publication by the Opponent in which SBS using azidomethyl as
`
`3’-OH protective group was reported.
`
`We note that we cite declarations and statements from US proceedings
`
`such as D35-D37, D40 and D53 with respect to the technical explanations
`and factual statements made therein. We understand that the OD will
`
`need to evaluate this evidence and these statements applying the legal
`standards of the EPC.
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`Further, we herewith file Annex 1 summarizing the “Developments in DNA
`
`sequencing by synthesis (SBS) regarding the 3’-OH protective groups to
`
`achieve
`
`efficient
`
`sequencing” with
`
`special
`
`consideration
`
`of
`
`the
`
`developments before and after the effective date of the opposed patent
`
`EP’627. We refer to this Annex 1
`
`in this submission where appropriate.
`
`All of the newly cited documents are filed in direct reaction to the
`
`Response to the Opposition of April 16, 2020 and the Preliminary Opinion
`
`of the OD of August 6, 2020 in order to address the question of
`
`insufficiency of disclosure and in particular the Patentee’s allegation that
`
`the alleged invention was the first implementation of an efficient 888
`
`method and that in view of this alleged breakthrough, they would be
`
`entitled to enjoy protection not only for allyl and MOM as 3’-OH protective
`
`groups but also for 3’-OH protective groups which are “as small as” allyl
`and MOM.
`
`The documents submitted herewith show that the allegation underlying
`
`Patentee’s arguments, namely that by using allyl and MOM as 3’-OH
`
`protective group they provided the first efficient 888 method is wrong. The
`
`documents further establish that the skilled person could not rely on their
`
`common general knowledge to identify working 3’-OH protective groups
`
`which are “as small as” allyl and MOM without an undue burden.
`
`The documents show that new polymerases outside the common
`
`general knowledge had to be developed when using, e.g., allyl as a 3’-
`
`OH protective group. The documents further show that the cleavage
`
`conditions which are disclosed by the contested patent for allyl are not
`
`suitable for 888. They also show that suitable cleavage conditions were
`
`not available for 3’-OH protective groups such as methylthiomethyl which
`
`has been proposed by the Patentee as a 3’-OH protective group which is
`
`allegedly covered by the claims. The documents also establish that
`
`azidomethyl which has been advanced by the Patentee as another 3’-OH
`
`group which is allegedly covered by the claims does not meet the features
`of the claims.
`
`The documents are thus prima facie relevant for the assessment of the
`
`opposition ground of lack of sufficiency of disclosure.
`
`We further note that the documents filed herewith are known to the
`
`Patentee from parallel US proceedings involving the same parties.
`
`We thus respectfully request
`
`that
`
`the documents filed herewith be
`
`admitted into the proceedings.
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`27
`
`28
`
`29
`
`30
`
`31
`
`General comments and outline of arguments
`
`In this submission we will focus on the issues raised in the Response to
`
`Opposition of April 16, 2020 and the Preliminary Opinion of the OD of
`
`August 6, 2020. We emphasize that we maintain all arguments set forth
`
`with our Grounds of Opposition and reserve the right to comment on these
`
`in the oral hearing.
`
`General comments
`
`In its introduction to the technology of 888 in its Response to Opposition
`
`of April 16, 2020, the Patentee criticizes D4 (Tsien) and D13 (Dower) as
`
`being theoretical and prophetic.
`
`Yet, Patentee’s own contested patent is equally theoretical and prophetic
`
`as D4 (Tsien) and D13 (Dower) and devoid of any worked examples. The
`
`Patentee would not make 888 work for another five years after its priority
`
`date, in 2006.
`
`Critically, to finally make 888 work in 2006 required the Patentee to make
`
`several changes to its prophetic approach described in the contested
`
`patent. As just one example, discussed in more detail below, it took the
`
`Patentee years to find a polymerase that would sufficiently incorporate a
`
`3’ protected nucleotide, and the one they eventually found only became
`
`available after the claimed priority date of the Opposed patent. None of
`
`these necessary changes are reflected in the contested patent.1
`
`While Patentee criticized the prior art as showing a 100% failure rate for
`
`finding a protecting group that would work for 888, the Patentee fared no
`
`better in the years immediately following the priority date of the contested
`
`patent.
`
`It was only after the Patentee started copying the Opponent’s
`
`patented 3’ protecting group — not disclosed in the contested patent— that
`
`it achieved real success at making 888 work. However, by that time the
`
`Opponent had launched a commercial 888 product founded on that, and
`
`several of its other critical inventions that together made 888 actually
`
`work well. This achievement earned the Opponent a publication of its
`
`working method in the prestigious journal, Nature,
`
`in 2008, an unlikely
`
`honor had the patentee actually been the one to make 888 work as it has
`
`claimed in its filings.2
`
`1 We referto the discussion in section C.ll.1.1.
`
`2 See D55, p. 53, right paragraph shows that azidomethyl was used as 3’-OH
`protective group.
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`32 Patentee’s claim that it was the one that made SBS work beyond what
`
`was known in the art is disingenuous. And any suggestion that the 2001
`
`priority application contained the details necessary to do so is inconsistent
`
`with what is known about Patentee’s subsequent struggles.
`
`Outline of arguments
`
`33 We understand the Patentee to argue that the contested patent allegedly
`
`identifies for the first time the precise characteristics which modified
`
`nucleotides have to fulfil in order for SBS to efficiently work.3
`
`34 Considering the claims these characteristics apparently pertain to the
`
`properties and positioning of labels and 3’-OH protective groups in the
`
`modified nucleotides, namely:
`
`(i)
`
`The function of labels and protective groups should be split
`
`and attached to different parts of the nucleotides.
`
`(ii)
`
`Cleavable labels should be attached to the base of the
`
`nucleotides.
`
`(iii)
`
`Chemically cleavable protective groups should be attached to
`
`the 3’-OH group.
`
`35 Critical issues of the present proceedings clearly pertain to the properties
`
`of the 3’-OH protective groups.4 The 3’-OH protective groups must be:
`
`(i)
`
`recognized as substrates by DNA polymerases. This
`
`requirement pertains to the steric and chemical properties
`
`of the 3’-OH protective groups;
`
`(ii)
`
`cleavable under mild conditions. This requirement pertains
`
`to cleavage conditions and thus (indirectly) also reflects the
`
`chemical properties of the 3’-OH protective group;5
`
`(iii)
`
`cleavable with high yield. This requirement again pertains
`
`to cleavage conditions and thus (indirectly) also relates to
`
`the chemical properties of the 3’-OH protective group;6
`
`3 See Response to Opposition of April 16, 2020, p. 5, 4‘h paragraph.
`4 See Preliminary Opinion of August 6, 2020; items14.4 and 17.2.
`5 The requirement reflects that cleavage conditions do not negatively affect the DNA
`during the sequencing reaction.
`
`6 This requirement reflect that, unless each sequencing cycle is performed with high
`yield, it is not possible to sequence fragments having the minimal required length of
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`(iv)
`
`compatible with the nucleophilic group in the active site
`
`of DNA polymerases. This requirement also relates to the
`
`chemical properties, in particularthe electronic properties
`
`of the 3’-OH protective group.
`
`We will set forth that in section D.| that the contested patent makes no
`
`conceptual contribution over the art whatsoever in so far as the
`
`contested patent alleges
`
`to have identified the above functional
`
`characteristics that labels and 3’-OH protective groups need to fulfil.
`
`The functional properties of labels and 3’-OH protective groups had all
`
`been already advanced by prior art references such as D4 (Tsien). The
`
`conceptual requirements of cleavage conditions were known as well. We
`
`again refer to section DI and the discussion of D4 (Tsien).
`
`As noted above, the Patentee argues that the claimed invention would
`
`provide the first reduction to practice for efficient SBS by identifying
`
`the MOM and allyl group as suitable 3’-OH protective groups. According
`
`to the Patentee it would thus be justified that the claims also pertain to 3’-
`
`OH protective groups which have the same size as the MOM and allyl
`
`group and, by consequence, also provide for efficient SBS.
`
`We will address in sections E and F that by selecting these specific 3’-OH
`
`protective groups, i.e., allyl and MOM, the claimed SBS methods do not
`
`meet the requirements of Art. 83 and 56 EPC.
`
`Regarding sufficiency of disclosure,
`
`it
`
`is apparent from the functional
`
`requirements which are mentioned in above paragraph 35 that for
`
`efficient SBS it is not sufficient to know the chemical and steric properties
`
`of the 3’-OH protective group.
`
`For example, one must also know the cleavage conditions under which
`
`these groups can be deprotected and such cleavage conditions must be
`
`compatible with the requirements for SBS given that the claims pertain
`
`to such methods. Cleavability of protective group per se is not sufficient
`for SBS.
`
`As will be set forth in section E.ll.1.2, the application as filed fails to
`
`disclose chemical cleavage conditions for allyl as 3’-OH protective
`
`group which would be mild and provide a high yield.
`
`36
`
`37
`
`38
`
`39
`
`4O
`
`41
`
`42
`
`approximately 20 nucleotides. Sequenced fragments of shorter length cannot be
`
`reliably aligned to reference sequences. We point to D38, Chapter 5, p. 109, last
`sentence.
`
`10
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`43
`
`44
`
`45
`
`46
`
`47
`
`48
`
`49
`
`5O
`
`51
`
`52
`
`The cleavage conditions which are disclosed by the contested patent for
`
`allyl are not suitable for SBS. Cleavage conditions for allyl which would
`
`be compatible with 888 were also not part of the common general
`
`knowledge. Post-published evidence such as D28 (Ruparel) confirms
`
`that suitable cleavage conditions for, e.g., allyl were identified only
`
`several years after the filing date.
`
`Similar considerations apply with regard to MOM as 3’-OH protective
`group.
`
`Just naming allyl and MOM as 3’-OH protective groups was thus not
`
`sufficient to make 888 work because these 3’-OH protective are not
`
`necessarily linked to SBS-compatible cleavage conditions.
`
`At the filing date of the patent, a skilled person could thus not practice the
`
`claimed invention without an undue burden at least as far as allyl
`
`is
`
`concerned, i.e., one ofthe only two specifically disclosed 3’-OH protective
`
`groups because suitable cleavage conditions were missing.
`
`For these reasons alone, it was notjustified to extend the scope of the
`
`claims to alternatives just because these alternatives allegedly meet the
`
`size requirement of allyl and MOM.
`
`The application as filed also did not provide the skilled person with any
`
`guidance how to identify 3’-OH protective groups different than MOM or
`
`allyl which would have the above-mentioned chemical and steric
`
`properties and for which suitable cleavage conditions would be known.
`
`The skilled person could also not identify such other 3’-OH protective
`
`groups and their cleavage conditions by relying on common general
`
`knowledge. We refer to section E.l|l.
`
`The requirements of Art. 83 EPC are thus not met.
`
`Other than preliminarily found by the OD, the contribution of the claimed
`
`invention can thus not be seen in the provision ofa generally applicable
`
`functional 888 method in which the modified nucleotides can be efficiently
`
`incorporated and deprotected.7
`
`As noted above, we reject Patentee’s argument that the alleged invention
`
`would provide a new conceptual approach for SBS.
`
`To the extent that making 888 work was within the general skill of people
`
`in the art in 2001, those generally skilled artisans were equally likely to
`
`7 See Preliminary Opinion of August 6, 2020, item 17.2.
`
`11
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`get there from D4 (Tsien) or D13 (Prober) as they were from the contested
`Patent.
`
`We respectfully submit that the alleged invention does not go beyond an
`
`obvious alternative over the prior art. We refer to section F.
`
`The requirements of Art. 56 EPC are thus not met.
`
`We further respectfully submit that, when assessing the requirements of
`
`Art. 123(2) and 76(1) EPC, the OD has incorrectly limited its analysis to
`
`finding a linguistic basis for the individual features but has ignored the
`technical context in which these features are disclosed and whether the
`
`claimed feature combination pertains to technical subject matter which
`was does not reflect the technical context of this disclosure.
`
`We submit
`
`that
`
`the application as filed discloses that any 3’-OH
`
`protective group needs to have the above-mentioned chemical and
`
`steric properties.
`
`However, the “as small as” feature pertains only to the steric properties of
`
`the reference groups,
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`i.e., MOM and allyl, but omits the chemical
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`properties. The claimed subject matter is thus is based on undisclosed
`
`generalization.
`
`The requirements of Art. 123(2) and 76 (1) EPC are thus not met.
`
`53
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`54
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`55
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`56
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`57
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`58
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`12
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`PROPERTY
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`MAIWALD
`INTELLECTUAL
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`D.
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`Conceptual disclosure of SBS versus putting SBS into practice
`
`|.
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`The concept of SBS was known at the effective date of the opposed
`
`patent and plausibly established
`
`59 As explained in section C.|.4 of the Grounds of Opposition, the concept
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`of DNA sequencing by synthesis (SBS) was known in the art at the
`
`effective date of the opposed patent EP’627.
`
`60 As evidence in this regard, reference is again made to D4 (Tsien), which
`discloses:8
`
`“In one aspect,
`
`the present invention provides a method for
`
`determining the deoxyribonucleotide sequence of a single stranded
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`DNA subject molecule. This method involves synthesizing,
`
`in the
`
`presence of a multitude of identical copies of the subject DNA, the
`
`DNA molecule which is complementary to it. This synthesis is
`
`carried out using deoxyribonucleotide triphosphates (dNTP) in a
`
`stepwise serial manner so as to simultaneously build up numerous
`
`copies of the complementary molecule, dNTP by dNTP. As each
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`dNTP is added to the growing complementary molecules,
`
`it is
`
`identified by way of an appropriate label (i.e., reporter group). By
`
`noting the identity of the bases present in this complementary
`
`molecule and using standard rules of DNA complementation, one
`
`can
`
`translate
`
`from the
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`complementary molecule
`
`to
`
`the
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`corresponding original subject molecule and thus obtain the
`
`deoxyribonucleotide sequence of the subject molecule.” (Emphasis
`
`added)
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`51 D4 (Tsien) further discloses that each dNTP is modified to contain a
`
`cleavable protective group at its 3’-OH position:9
`I
`
`‘Blocking Groups and Methods for Incorporation
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`The coupling reaction generally employs 3'hydroxyl-blocked dNTPs
`
`to prevent inadvertent extra additions.” (Emphasis in underlining
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`already in D4)
`
`62 D4 (Tsien) then discusses various aspects of the 3’-OH protective group
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`and the label on, e.g., p. 24-27. In this context, D4 considers incorporating
`
`the label into the 3’-OH protective group.10
`
`8 D4, p. 6, |. 34 — p. 7, |. 14; Claim 1.
`
`9 D4, p. 20, II. 25-27; p. 23, II. 28-31.
`10 D4, p. 21, |. 4-8.
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`13
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`MAIWALD
`INTELLECTUAL
`PROPERTY
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`63 However, D4 (Tsien) makes explicitly clearthat incorporating the label into
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`the 3’-OH protective group is only one option and that the label can as
`
`well be distinct from the 3’-OH protective group. Forthis case D4 (Tsien)
`
`points out that the label can be attached to the base of the nucleotide:11
`
`“While the above-described approaches to labeling focus on
`
`incorporating the label into the 3' -hydroxyl blocking group, there are
`
`a number of alternatives - particularly the formation of a 3'-blocked
`
`dNTP analogue containing a label such as a fluorescent group
`
`coupled to a remote position such as the base. This dNTP can be
`
`incorporated and the
`
`fluorescence measured and removed
`
`according to the methods described below.” (Emphasis added)
`
`64
`
`In fact,
`
`the first
`
`introduction to labelling in D4 (Tsien) only explicitly
`
`mentions
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`the base as
`
`a potential
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`location for
`
`the
`
`label, while
`
`acknowledging that others can exist.12
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`“When they are each tagged or labeled with different reporter
`
`groups, such as different fluorescent groups, they are represented
`
`as dA'TP, dC"TP, dG'”TP and dT'" 'TP. As will be explained in more
`
`detail below,
`
`the fact
`
`that
`
`the indication of labeling appears
`
`associated with the "nucleoside base part" of these abbreviations
`
`does not imply that this is the sole place where labeling can occur.”
`
`65 Additionally, D4 (Tsien) singles out labeling of the nucleobase portion as
`having been “reported to exhibit enzymatic competence” and “show
`
`enzymatic incorporation” of the labeled nucleotides.13
`
`66 D4 (Tsien) thus clearly teaches that the label and 3’-OH protective group
`
`can be split and attached to different positions of the nucleotide.
`
`67 D4
`
`(Tsien)
`
`also
`
`provides
`
`guidance
`
`regarding
`
`the
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`functional
`
`characteristics of the cleavable 3’-OH protective group:14
`
`“The criteria for the successful use of 3’-blocking groups include
`
`(1) the ability of a polymerase enzyme to accurately and efficiently
`
`incorporate the dNTPs carrying the 3’-blocking groups into the
`CDNA chain,
`
`11 D4, paragraph bridging p. 27-28.
`12 D4, p. 10, l. 7-14. And see Fig. 2 using that same nomenclature that had been
`defined at p. 10.
`
`13 D4, p. 28, l. 5-18.
`14 D4, p. 20, l. 28 - p. 21, l. 3.
`
`14
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`MAIWALD
`INTELLECTUAL
`PROPERTY
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`(2) the availability of mild conditions for rapid and quantitative
`
`deblocking, and
`
`(3) the ability of a polymerase enzyme to reinitiate the cDNA
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`synthesis subsequent to the deblocking stage.”
`
`68 The functional characteristics which the 3’-OH protective group should
`
`fulfil are further emphasized by D4 (Tsien) when commenting on
`
`deprotection of the blocked 3’-OH group,
`conditions:15
`
`i.e.,
`
`the required cleavage
`
`“Deb/ocking Methods
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`After successfully incorporating a 3'-blocked nucleotide into the
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`DNA chain, the sequencing -scheme requires the blocking group to
`
`be removed to yield a viable 3'-OH site for continued chain
`
`synthesis. The deblocking method should:
`
`(a) proceed rapidly,
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`(b) yield a viable 3'-OH function in high yield, and
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`(c) not interfere with future enzyme function or denature the
`DNA strand.
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`(d) the exact deblocking chemistry selected will, of course,
`
`depend to a large extent upon the blocking group employed.”
`
`69 This shows that D4 (Tsien) did not consider cleavability of 3’-OH
`
`protective group per se to be sufficient for SBS. Cleavage of 3’-OH
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`protective group had to be possible under conditions which are mild,
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`which are rapid, and which are quantitative. These cleavage conditions
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`reflect requirements which are inherent to the concept of 888 such as
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`that cleavage conditions must, e.g., not destroy the DNA16 and proceed
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`with a high yield.17
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`70 D4 (Tsien) does not contain any experimental data showing that the
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`claimed 888 method works. However, D4 (Tsien) relies on plausible
`
`15 D4, p. 23, l. 27 - p. 24, l. 5.
`16 As used herein “destroying” also includes denaturing of the DNA, i.e., destroying
`the double-stranded conformation of the template and the growing strand.
`17 This requirement reflects that, unless each sequencing cycle is performed with
`high yield, it is not possible to sequence fragments having the minimal required
`length of approximately 20 nucleotides. These minimal sequence lengths are
`
`required for subsequent aligning to reference sequences which underlies the
`massively parallel sequencing approach of SBS.
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`15
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`PROPERTY
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`MAIWALD
`INTELLECTUAL
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`scientific rationales, which proved valid as evidenced by later
`
`publications.
`
`71
`
`In this regard it is important to note that D4 (Tsien) not only teaches the
`
`relevance of the compatibility of a polymerase enzyme with the 3’-OH
`
`protective groups of
`
`the dNTPs, but also the requirement of mild
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`conditions for rapid and quantitative deblocking without interfering with
`
`future enzyme function or denaturing the DNA strands.18 Both aspects are
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`indeed decisive for putting 888 into practice.
`
`72 While D4 (Tsien) does not call out “small” as required criteria for a
`
`protecting group,
`
`it does provide several examples of protecting groups
`
`that are small. And in one context, refers to “lower (1-4 carbon)” groups of
`
`a certain type being better.
`
`73 The importance of the 3’-protective group being sufficiently small to
`
`allow for the incorporation of the 3’-OH blocked dNTP by a DNA
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`polymerase was described by D4 (Tsien)19 (explaining a desire to avoid
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`“large and bulky” 3’-OH substituents) and confirmed by D13 (Dower) and
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`D5 (Welch) as summarized in the herewith submitted Annex 1 under items
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`A.l.2 and A.l.3. That D4 (Tsien) disclosed small groups, and that D13
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`(Dower) and D5 (Welch) later explained the mechanisms behind the
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`choice of smaller groups (in the case of D5 (Welch), explaining it in terms
`
`of the limited space shown in the active site of polymerases by crystal
`
`structure) would have had a skilled person looking at the smaller groups
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`of D4 (Tsien) by the late 1990s. This was not a new insight provided by
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`the contested patent.
`
`74 The importance of mild conditions for
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`rapid and quantitative
`
`deblocking was confirmed by D13 (Dower) and D14 (Metzker) as
`summarized in Annex 1 under items A.ll.2 and A.ll.3.
`
`75 Moreover, D4 (Tsien) suggests allyl ethers as 3’-OH protective groups.20
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`Notably, Tsien cites to D9 (Gigg and Warren) for the allyl ether protecting
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`group and that reference specifically shows, unsurprisingly, the chemical
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`structure for the same three carbon allyl group as is used i