Attorney Becket N :3. T3 PN—l}€}3:"®€$TlS
`
`3} 3€§t§3—2lll 5 (Alélllvl-32174}
`
`?ATlENT
`
`EN
`
`UNl7l"‘El) STATES PATENT ANT} T"RAl}TIl\'lART{ GF T'<‘l?CTI
`
`ln Re Patent of:
`
`SAlvlPA.LlS, Fotini
`
`Confinnation l\lo.:
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`l 897
`
`Control No:
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`95/"Gt? l , 774
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`Group Art Unit:
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`399l
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`Filed:
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`FOR:
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`October l9. Zllll
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`Examiner:
`
`CAMPELL. Bruce R.
`
`TNTER PARTES REEXA.l\/T 917 US. PATENT 8,fi3{l,34l8: NATURITTL l‘«lARTNE
`SOURCTE T’E§0S§’Ti0T.flT’il}§§ CGM§’RlSll‘«lG T’CtLYUNSATURATEE)
` li‘ATTY
`
`ACTBS AND THEIR AP ?l.. lCATT{)NS
`
`Mail Stop Beelaratlon
`Connnissioncr for Patents
`
`R0. Box l4:3(3
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`Alexandria, VA 223l3~l45{}
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`DECLARATTGN BF FAUSTTNUS YEBQAH PHD. UNDER 37 C.F.R.
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`
`
`L Faustinus Yclacah, declare as follows:
`
`l.
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`2.
`
`l an: a Canadian citizen.
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`l arn the Director of KABS l_aboratories, lne., a company that offers a broad range of
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`pretluct
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`tlevelopinent services to the lbio—pl1arrnace‘utical
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`industry worlclwide. The services
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`offered include strategic planning, pre—clir1ical clevelopinent, analytical
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`testing,
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`ferrnulation
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`(leveloprnent, nranirfacturitig ef mototypes and clinical supplies, (listribution. of clinical supplies
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`to clinical sites, and chernistry, marlufacturing and controls (TCMC) aspects of regulatory affaii‘s.
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`I am also the Founcler and Principal Consul.tant ofPDlVlC Pltanna Consulting.
`
`"W
`3.
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`l olmainetl my Pl1.D., concen‘trat.ing on protein and carl)ol1yil.rate clietnistry, and my
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`M_E§c., concentrating on food chemistry, froni lVlcCsill University in Montreal, Quebec.
`
`l was a
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`post-electoral fellow at the Biotechnology Research lristitnte oftlrc National Rescarcli Council in
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`Canacla.
`
`000001
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1054
`
`000001
`
`

`
`U.S.S.N. 95./Gill ,774
`Declaration of Dr. Faustinus Yeltioali
`
`/l.
`
`l have authored twenty five papers. inany of which concern rnass spectrornetry and
`
`extraction of biomolecules, and l arn an inventor of one US. patent and six patent applications.
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`i
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`am a. Faculty Lecturer at McGill University in the Departrnent of Food Science, and I
`
`as a
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`Jounial l{eviewer' for the Jazmizal r;:fF00z.z’ Conzp0sz'ti(m and Anz.zi_;v.g*is, the Jozzrrzal (.5f.4grz'i;:'z1Itui‘e
`
`and Fem?! fihenzisnjjir, and the .Jtiw'm:z:’ of Efl‘l«’l'i"()i”tfi'l€i'£Z‘<ZZ
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`2"oxz‘c0i0g_,ii>.
`
`l also serve as a Grant
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`Reviewer for the National Science and l?.ngineering Research Council of Canada (NSERC). My
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`Curriculum Vitae is enclosed as Appendix A.
`
`l ain consideretl an expert
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`in the area. of inass spectrornetry and extraction of
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`bioniolecules.
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`6.
`
`l was engageil. by counsel l’or Neptune Technologies and Bioressources, lnc. (“Neptune”)
`
`of Quebec, Canada to analyze the Corrected Request for Reexarnination {US 95,/ll(ll,,774} filed
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`by Alger Biornarine ASA (“Ake ”}.
`
`1 am being Ltoniperlsated at my customary hourly rate for my
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`time spent on developiiig, forming, and eXpi‘essing the facts and opinions in this declaration.
`
`l
`
`have no personal interest in the ultirnate outcome. of the reexa.niination proceedings involving
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`Patent 8,{33(l,348 (“the ‘348 patent”).
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`7.
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`Specifically,
`
`l was asked to review the lfieclaration of Thornas Guhdersen, which was
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`tiled in support of the Request for Reemniiiiation filed by
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`and the
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`eclaration of Earl L
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`White, Ph.l).., which was submitted by Neptune in the prosecution of the ‘.348 patent, and to
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`opine on the Validity of the results provided therein. Further, l was aslted to express an opinion
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`on quantitati.ve aspects of the data discussed herein.
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`Guntlersen Presents Eneoni
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`
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`late and Unreliable Bate
`
`
`8.
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`l have read and reviewed the Declaration of Thomas Gundersen suhrnitted by Alzer. and
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`it is my opinion that it suffers from considerable technical deficiencies and errors which render
`
`its conclusions completely unreliable.
`
`l surnrnarize these deficiencies and errors below.
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`Gafmicrserz Preserzis Cieariy Errorzeous [Edict
`
`9.
`
`My review of the Gundersen Declaration leads rne to conclude that it contains incomplete
`
`and unreliable tlata. Gundersen erroneously presents data as distinct when it is merely an exact
`
`0007002
`
`000002
`
`

`
`UiS.S_N_ 95./(}i)l ,7’?
`Declaration of Dr. Faustihus Yeltreali
`
`cogjg of another eli1"er1iategi*arri.
`
`in my epihieii, there is clear error in tlie Guiiderseri Deelaratieh
`
`and, whether reflective ef a sloppy study marred by negligence er a fraudulent study submitted
`
`with deceitful iiite.rrt, one eaimet rely on this data at all.
`
`ll).
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`Spe.eiiieally, referring to Appendix B {if the Guhderseii Declaration, the ehremamgrahis
`
`lalieied “Sample P308-8”’ and “Sample i’3i)8~9” are identical
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`Gundersen Deelaratien,
`
`Appendix 8, pp.
`
`ll8—l9’). Similarly, the ehremategrams laltreled “Sample l)3()8—l0,”" “Sample
`
`P3i)8—l if’ and “Sarnpie P‘308—i2” are identtieai (see Gunderseii i}eeiaratien, Appendix B, pipe 19-
`
`2e).
`
`ll.
`
`Guiiderseii provided Table l, which states that “Sample P30S~8,” “Sample P3OS~9,”
`
`“Sample i’3(i8—l(i,” “Sample P3(38~ll,” and “Sample P‘308—i2” are distirietly tiii'i’ererrt samples
`
`{see page 3 of the repert appended te the Gundersen Deeiaratieti}. Belew, i i'epi'0duee, in part,
`
`Table l {eiiipliasis added, iiete the “i‘viarl<;irrg of Sample” Column):
`
`Fractieii
`)“,iE.li’)'ilJf31'
`
`Temperature
`trea.tr1'ier1t
`
`PC)
`
`.
`
`P308—8
`P3083
`
`l’3i)8-ll)
`
`:
`
`I
`
`Marle:iiig of sample
`
`.,
`
`E.superha Fraetitm Ba 7% degr 5 min
`Eauperba Fraetiei: Elia i'25 degr i5
`
`min
`
`E.saperha Fraetieh lib net heated
`
`Ehsuperha Fraetien {Eh 76} degr 5
`
`min
`
`min
`
`P3i)8~l2
`
`*
`
`Esuperha Fraetieh HE) 125 degr i5
`
`l2.
`
`i have alae attached the incomplete and urireliabie ($h,‘(OiIia1'0gT(aD'iS as eriumerated aheve
`
`as a series ef figui'es to make this point clear {see Figures l-5). By reviewing Figures 1 and 2
`
`side by side (eh pages it-i~l5 ef this i:3CCiEl1'a‘{ZiO1‘l),, it is apparent tliat the reteiitioii times, areas
`
`under the curve, and sample idehtiiieatien infermatien are exactly the same. Fer eetiveriieiiee, i
`
`have magnified the hottehi peak of each ef the elimiiiatograms labeled “Saihple l’3i)8w8” aiitl
`
`000°003
`
`000003
`
`

`
`,774
`U,S.S_N. 95./t}€)l
`Declaration of D1‘. Faustinus Yeheali
`
`“Sample l’308—9” and displayed them in Figures l and 2. The bettem eliremategrams hath have
`
`areas under the eurve ef l24384 and retention times Qf 3.758 miiiates.
`
`lt is my epihieh that it
`
`would he impossible for two distinct samples te pmvide the exact same data. Even if “Sample
`
`l’3l)8~8” and “Sample P2308-9”’ were merely repeats of eaeh other, which they are pug; according
`
`to Gtinderseifs Table 1 (above), the data would have at least some deesiiatizyn. The unreliability
`
`of this data is l’urthei' miclerseered. by the facet that the same sample l:L‘l,€Illl’l3,{$19tl.i,()l1 number appears
`
`en the ehremategrams laheled “Sample l’3(lS—8"’ and “Sample l’3(lS—9"’ and displayed in Figures
`
`l ahd. 2 (mi pages l5—l6 of this Deelaraltitm). Beth el'n'emategrarns have the fellewing sample
`
`identifier: “MSl)l 826.
`
`lil(?====825.'7.826_7 (C:\l.)1»‘~il3~‘\Al§lZl‘{ Blt:ll\/lARll\li\l’308\AE3 l l(l929‘\AB
`
`lltl929\AB ll092I9 2{}ll-09-29 ll)-35—22\(l{}r-lmlltl-()lD) ES.” By providing the exa.et same data
`
`twiee yet i'eferring to the data as erigihatiiig from two different experimental samples, Guiiclerseh
`
`himself firmly demonstrates that his data is net ereclihle. "l‘his is summarizecl belew:
`
`
`
`Retentien
`Time
`
`Area Under
`the Curve
`
`"
`
`.'.' .".L
`
`124384
`
`Masking at sample
`
`P308~8
`
`l’3()8—'9
`
`“llsuperlaa, Fraction Ila '70 degr 5
`min”
`“E.superha Fraction lla
`min”
`
`deg)‘ l5
`
`:
`
`l
`
`l3.
`
`The ehrematograms labeled “Sample l’3G8—l(l,” “Sample l’3Q8—ll.,”’ and “Sample l’3{}8~
`
`l2” alse present ideiitieal data fer allegedly distinct samples and tlierefkne provide fiirthei'
`
`iiieemplete and um‘elia'hle data. By eviewing Figui‘es 3, 4, and 5 (en pages l 6-l8 ef this
`
`Deelaratieh) side by sidet it is apparent that the retehtien times, areas under the curve, and
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`sample identification ihfermatien are exactly the
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`Fer cenvenienee, l have magnified the
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`hettem peak of each til’ the el.n'ep'iategrams labeled “Sample l)3llS~lt},” “Samiple P398-l l ,” and
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`“Sample l33{l8~l2” and displayed them in Figtires 3, 4,, and 5. The hettem ehremategrams of all
`
`three have areas und.er the curve of 5l%82l.8 and retention times {if 3.770 minutes.
`
`lt is my
`
`epihieh that it would he impossible lei‘ three distinct samples to pmvide the exact same data.
`
`Even if “Sample P3(l8~l0,” “Sample P3ll8—ll," and “Sample P3tl8—l2"’ were merely repeats et‘
`
`eaeh ether, which they are _ng_3_t according to Guhderseifs Table l (al;ieve), the data would have at
`
`least‘ some deviazioiz. The ameliahility of this data is further underscored by the appearance Of
`
`the same sample i.dentilieatieh huml;ier en the Clll‘0Tl’l3;t()_g*,1'£il'TlS labeled “Sample P308—ll},”
`
`“Sample l’308—ll,” and “Sample l’308-l2” and displayed in Figures 3-5.
`
`All
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`three
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`ehromategrams
`
`carry the
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`follewing sample
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`identifier:
`
`“l‘vlSDl
`
`826. ElC=825.7.82I6.7
`
`0004304
`
`000004
`
`

`
`U,S.S.N. 95./Gill [774
`Declaration of Dr. Fattstinus Yehoah
`
`(C:\PAD‘\Al{ER Bl0MARlN\l’308\AB ll09Z9‘\AB lll)92I9‘\AB lll)92I9 Ztll 1-09-29 ltl—3§~
`
`220G€3—lf‘>6Gl D) ES.” As with the samples discussed in Paragraph l2 ahove, hy providing the
`
`exact same data three times yet i”eferring to it as three different experiinental samples, Guridersen
`
`estahlishes that his data is not credible, This is s1irnrnai‘i:aed below:
`
` Vitas ll}
`
`Retention
`Time
`.779
`
`Area tinder the
`{Siam/‘e
`58821.8
`
`Marlelng of sample
`
`F308-10
`
`“E.superba Fraction llh
`not lieated"
`
`5
`
`P308-1 1
`
`58821.8
`3."/'.7'(l
`l “E.stiperi3a Fraction lib 70
`....................................shstégntfssssssssssssssss.ssssssssssssss
`P3G8—l2
`“E.superha Fraction llh
`3.770
`58821.8
`125 degr l5 min"
`
`
`
`l4.
`
`l note that l lirnited my review in this section to data. presented in Apperidix A ef the
`
`Giindei'sen Declaration, as the data presented in Appendix B of the Cluridersen Deelaratien is so
`
`poorly eproduced that l cannot discern most of the alleged peaks that are presented. For
`
`example, i earmnt see any data on the chrornatograms labeled “p308~4” to “p3(38~7” and can only
`
`make out faint images on the remainder of the figures presented in Appendix B ef the Gundersen
`
`Declaration.
`
`Ga‘mierserz Results are Highly Variable cznd Rafleczive of(1 Rusheci EXp8rifi’l€i2f.
`
`15.
`
`Besides the incomplete and iinreliahle data pt“6Sf31’llTt)c‘.l.,
`
`there is a pu:a:.2:liiig complete
`
`ahsenee of data in the Gnnderseri Declaration for‘ the ethyl acetate extract ME. .wper/Ba l<i'ill lipid
`
`samples ( Gnndersen Deelaratinn ‘E 5). The fact that no data was generated for these
`
`experimental sainples suggests that the experiment was not conducted carefully (see (Iluridersen
`
`Declaration, Appeiidix B, pp.
`
`ll§—l7 for ehrornatograms labeled “Sainple P3084-‘lv,” “Sainple
`
`P3l}8~5,” and “Sample P3(38~6”).
`
`in fact, Gundersen himself states that the experiment “_s_ii§g_it_lgl_;
`
`
`
`have been 1‘t."£')Calt3Cl but there was not enonvh time for this” {see Gundersen Declaration fit 5;
`
`emphasis added). Fiirtiiei', on page 2 of llxhihit 2 of the Gundersen Declaration, Gundersen
`
`states that “the analysis of the samples took place between
`
`September and 4 October Ztlll.”
`
`Therefore, all within the span of just five business days, the data was acquired, the data was
`
`analyzed, a report was generated, and a declaration regarding analysis of the data was written. A
`
`oodhos
`
`000005
`
`

`
`U,S.S.N. 95./Olll ,7?‘
`Declaration of Dr. Faustinus Yehoah
`
`cornplicated expei*itnental analysis such
`
`this wenld require. far more tirne if it were conducted,
`
`properly. These facts suggest a rushed experiment and data analysis.‘
`
`Tim Gz,mde2rserz Data Sz,g,f,?'ers' Front the jleféstnoifv ii}ffZcr:‘.';
`
`lti.
`
`it
`
`is not only conventienal but also necessary, when conducting niass spectrernetry
`
`experiments, to run selvent—only (a,z’k/a “hlanlc” or “negative”? controls at the begiiniiiig of an
`
`€‘f§(’\,€‘l.”lTYl13Ilt and between runs which invelve different ex ,eriniental s ecies. Gundersen does not
`
`nevitle these crucial centrels.
`
`
`l7.
`
`"l‘hese omitted controls are crucial because they correct fer a technical difficulty in
`
`spectren'ietry known as the “tnen'ior'y e_tl’ect” or “carryover et‘t‘eet.”2 For instance, (.‘.()l'l'lEll’Ttl,lll3.l,ll.
`
`species froin a preceding experirnental run sl<:ews the subsequent preliles.
`
`This deficiency of the Gundersen Declaration is anzgziified because Gundersen ernpleys
`
`ninltiple reaction inonitoring (l\/llihvi), a highly sensitive technique used te detect very small
`
`qttamz'tié:s 0fsigm2!.3 As the detectieh is very sensitive, any cress»centarnination of species may
`
`completely hlnr actual data. Therefore, the failure et‘ Gundersen to run selvent—enly c.ontrols, in
`
`a very sensitive niass spectronietry rnethotl, casts significant doubt upon the data presented in the
`
`Declaratien.
`
`l9.
`
`Gtindersen“'s failure to contrel and cerrect fer the rneinoiy effect, in my epinion, prohahly
`
`resulted in ineerrect conclusions regarding the idetltilicatien of species. One cannot, tll(§t‘£’[f(lt'i¥,
`
`exclude with any confidence that signals froin the positive er reference standards were net
`
`carried~ever to sequential sample runs, which would result in false positive data, For instance, in
`
`the data presented in the chroniategrarns ell’ “Saniple l’3()8—l5” and “Sample l’3l38—l6” (sanie
`
`experiniental cenditions, see Gunclersen Declaratien, Appendix B, p. 22) there are considerable
`
`dittereiices in the peak intensities ohservecl where ene would expect essentially siniilar
`
`respectfully noted that some of the samples did not arrive in the laboratory Where the Gundeisen Declaration
`l lt
`expetinients Were unl"1ertal<e‘n until September
`20} l. This would leave a inaxirnuin of three business days to
`generate data, analyze data, ‘write a report,
`Write a declaration i‘egai'diti analysis oi’ these samples.
`
`”Dt3l(‘)1‘ll’ll1’lEi,lli)l] of Cai't"yo've:r and COT.\lal]1ln€tlZl0'll
`2 See‘, erg‘, Hughes e‘! alt,
`Chromatographic Assays.” F712‘ A/‘lPSJozmm/7 2007; 9
`Article 42., Appendix B.
`
`for Mass Spectremetty--Based
`
`3 See, e. Elliott .91 all, CU;-‘l‘C11’t Trends in Quantitative lJ1“0lC(31’1'1lCS. J:
`Appendix C.
`
`Spectrum,
`
`(:12): 1637/—— 1660 (2.009),
`
`oodhoe
`
`000006
`
`

`
`Ll.S.S.l\l. 95./Olll ,774
`Declaration of Dr. Faustinus Yehoah
`
`intensities. However, in the fornier, the peak intensity is in the. tens of thousands, while in the
`
`latter,
`
`the
`
`ll
`TX
`eale intensit“ is
`
`in the low hundreds
`
`l’3
`corn are,
`
`ll
`for exam _ le,
`
`the bottom
`
`chroniatograrns of both). Given this highly dirninished intensity, I cannot reasonably exclude the
`
`possibility that the higher signals bled into the suhsequent ru.n’s results Via the memory effect. A
`
`solVent—only control, which Gundersen failed to run, would have excluded the rnemory effect.
`
`20.
`
`Further, another potential manifestation of the “rnernory effect” is seen in the highly
`
`variable retention tirnes presented in the Gunrlersen Declaration for what is alleged to he the
`
`same compound. For instance, selecting a random pair of chronratograrns, the hottorn traces of
`
`“Sample P308-l” and “Sarnple P‘308~7.” are purported to he the same chemical species, yet have
`
`disparate retention times of —/l.l8l and 3.747 rninutes, respectively. This is a retention time
`
`deviation of close to 26 seconds. The.refore, in one run (“Saniple P3(lS«—l”) the same compound
`
`is about ll % slower than in another {“Saniple P308-7”).
`
`ln my opinion, this should not he.
`
`Because of Gundersen’s failure to run the solvent control, I cannot exclude the. memory ellect.
`
`in fact, these data are also indicative of two dit‘feren.t chemical species incorrectly labeled by
`
`Gundersen as the same chemical species.
`
`The Gzmdersen [lard Sz{ffei".s‘ Firm: Poor Resolztttion.
`
`2l.
`
`Gundersen also presents experirnental data peaks that are not the
`
`and clearly
`
`defined speaks that are rellective of clean chroniatog;raphy. On the contrary, the peaks show a
`
`poor resolution that suggests that multiple species may be present.
`
`22.
`
`For instance, the experimental runs labeled “Sainple P3€l8~l
`
`,” “iéainple l’3l)8~2,” “Sample
`
`P3Q8—3,°’ “Sample l?‘308—l4,"’ and “Sarnple P3{}8~l6” are particularly broad and asyininetrical,
`
`which is indicative of a population of unknown species in a “peak” as opposed to a single
`
`species.
`
`ln contrast, a single species is expected to give a sharp peak in the. form of a tight
`
`Gaussi.a,n-sliaped curve.
`
`23.
`
`ln suinrnary, it is my opinion that the Gundersen l3e.claration suffers trom a nuniher of
`
`deficiencies, including incorrect and unrelia'ble data, rushed execution, and experirnental errors
`
`which render its conclusions cornpletely unreliable and not credible. As an expert l cannot give
`
`the results presented in the Gundersen declaration any weight.
`
`0007607
`
`000007
`
`

`
`U.S.S.N. 95./Gill ,7?‘
`Declaration of Dr. Fanstinus Yehoah
`
`The ‘White Declaration at Ma’ 31 2911 hresents Sound Raw Data and a Reasonable
`
`Dverall Canelnsion.
`
`l have also read the Declaration of Earl L. White,
`
`l’h.D.
`
`that was snhinitted in the
`
`W 4
`
`4.
`
`prosecution of the ‘348 patent on May 3 l, 201 l and I have heen asked to provide an independent
`
`evaluation of the correctness of its conclusions.
`
`l_1nlil<e the (Snndersen Declaration, the White
`
`Declaration provides data that is ezrpeiimentally sound and has a reliable conclusion.
`
`25.
`
`i understand Dr. White’s conclusion to he that it
`
`his “opinion that the Deatidoin Oil
`
`Fi'actiens received and tested hy {him} do not contain ?I.v‘$ [ph.ostpholipids] which have attached
`
`to them DHA and DHA, EPA and El’./%‘i, DHA and EPA, or l3l’A and DHA, at the detection limits
`
`described [LC/’l‘vl'.§_§ and MS/MS l'€(.‘.l1l'll(}l,1f.’.Sit” Theret‘ore, Dr. White could not tied any species
`
`that could. he definitively identified as those disclosed and claimed in the ‘348 patent. This is a
`
`reasonable eorielusioii based on the experimental data.
`
`26.
`
`Some cornrnentary on the White Declaration of lvlay 31, Ztlltl
`
`is helpful
`
`for
`
`understanding it.
`
`27.
`
`First, it is my expert opinion that the raw data collected by Dr. White is correct and does
`
`not suf"er from the many flaws seen in Gundersenls data.
`
`28.
`
`Second, Dr. White’s Declaration of May 3l, Ztllll does have a couple of minor
`
`itzrerpretazive errors that do _r_rg_3_§; affecct the Validity of the raw data or the overall conclusion (Le.
`
`that Dr. Wliite could not, within the limits of detection,
`
`find any species that could he
`
`definitively identified as those diselose.d and claimed in the ‘348 patent).
`
`29.
`
`A first minor error lies in Figure ll} of the Declaration of Dr. White of May 3l, Ztlltl.
`
`i
`
`understand that this figure was included because the Ztlll Beandoin oil did not produce product
`
`ions such that Dr. White could not identify species that might have been those disclosed and
`
`claimed in the ‘348 patent. Dr. White included Figures 9 and ll ih si.tppci't of this conclusion.
`
`However, it appears that because Dr. White did not detect characteristic product ions that are
`
`needed to definitively identit‘y the fatty acids attached to the pll0Spl'l0li,}:3i(lS at the rnolecular
`
`weights of 82.6 and 852. he included, for corhpariscn, Figure it), “from a previous Beaudoin oil,”
`
`as a “representative MS/l\2lS spectrarh for a [’pl}0Spll3.'ti(lylCll0li1'lf::l with a molecular ion at m/z
`
`ooohos
`
`000008
`
`

`
`,774
`U,S.S.N. 95./(}€)l
`Declaration of Dr. Fattstirrus Yehoah
`
`826.”
`
`White Deelaratiorr of May 31, 2611, Figure it) legend and *3 121. This figure shows
`
`what a species at the weight of a phosplioiiplti of EEiP./~‘i.r"El?"A woutlci look like if it could he
`
`tragriiehted sueh that proctuet iohs eoulti be detected. This explains why Dr. White separated this
`
`t"igutr'e trom the others in ‘El E2 of the White Declaration, which states: “lVlS/'lViS spectra for a
`
`representative Beaudoih Oil Fractiorr sample are shown in Fi tires 4-9. and il l. Fig‘ur‘e 10
`
`a
`
`E:§Q§§§§[3t‘§;§fly§__Mi_Eji{:§g1§___§}_i§f,§Q'§;{:§y;‘{}; for a {phosphamiylehehhe] with at rholeeular ion at m/is 826.5”
`
`(errrpliasis adtiedi}. This, to me,
`
`a clear irrclieatiori that Dr. Wl‘rite did not intend to present
`
`Figure lit
`
`part of the. expei'irtiehtal set upon which he was opihihg ih May, 2311. This is
`
`hoistered hy a statement in the tigttre legend of Figutr'e M), in which it is stated that the ariaiyzed
`
`sample is “from a previous Beautioiii oil.”
`
`in feet, Dr. White also iiiehtdect the date of the
`
`experiment in the upper left corners of each of the spectra irieludteci lh the figure set. Figure 10 of
`
`the White Declaration of May 31, 2011 has the iabei “09Dee07” irrt’tiea.tirrg that it is trorri a
`
`December, 2009 experirheht while all of the either figures {L9, is 1) have date labels ot7Api'ii or
`
`May, Zfii ii, iritiieatirig that they are from the experitherits commissioned for prosecution of the
`
`‘348 patent.
`
`39,
`
`1 have reviewetl an experimental report ti'oh'i the 2009 extraertiori (Appemlix B) arid
`
`conclude that, heeause of the experimehtai errors in reprochteirig Beaudoirr, the 2909 samples o
`
`»-+-,
`
`Figure l0 are rig: “Beaudoiri oil.” Those Dr. ‘vVhite’s staterrieht that Figure l0 is “from a previous
`
`Beatttftolh oil” is errorieotts. As dteser'iheci in the experirheritai report, the procedure removed the
`
`Water trorh the oil arid did riot ’<t.Cl,Cqll.i:iif3.ly heat. Therefore it wettlti he expected that the oil of
`
`Figure l0 shows enough m/z 826 species to allow protiitet ieh deteetiori (See Appendix D).
`
`31.
`
`Speeificaliy, E note that the 2009 extraction did go; replicate Beattdoin because a series of
`
`changes to the Beattdoin protoeoi were iriad ’f31‘iCI1'€ly made {see Appeittiix D):
`
`“
`
`hi the aeetorie extraetioh (“step l”), extensive evapora.tioh of the solvent and water was
`
`1.tYtCi6Y‘i.&i{e1‘E,
`
`in contrast
`
`to what was described in Beattdoin.
`
`Specifically,
`
`the
`
`e\:perirhehter* “[e.]Vapoi"ate[d]
`
`the aeetoheg” “lsleparatiedj
`
`the fat froth the water by
`
`deearitatlori after addition of 1 vohtme of aeetorieg” "‘[i']eeoVer[ed the} olig” and
`
`“ieivaporated. under Vaeuurri.”
`
`in contrast, Beautioiri’s extraction ihehtdes anti; rotary
`
`006609
`
`000009
`
`

`
`U,S.S.N. 95./Gill J74
`Declaration of Dr. Fanstinns Yehoali
`
`evaporation to reniove acetone and leaves ltl.0% moisture and Volatiles (Beautloih l,
`
`Table 13]). Such water content causes hydrolysis of the phospholipid upon heating.4
`
`5*
`
`liwrther, the experiinenterjiiilecl to heat tile arrezione e.:ttra(:t at I25°'Cfor 15 mi:/zz»::‘es, in
`
`eontradistinction f1”tJ1’11 the step taught by Beaodoin. Heat was not applied at all to
`
`Fraeti on l,
`
`E The experimenter aiidei'tool< an ethanol evaporation to yield Fraetiori ll as in Beaudoin
`
`(“step 2”} but did not hair Fmctioii 1! at 125"’C'ji)r 15 ininzzzes.
`
`lnstead, Fractions l and
`
`it were merged to create a lifaction not taught by Beaadoin: a fraction having niaeh less
`
`water
`
`than the Beaiidcin fractions.
`
`This non~Beandoin oil was then treated hy
`
`“[elVaporat[ing} tinder Vacnurrf’ antl “lhleatfingl at l.’/15 “CL”
`
`As Beandoin does not teach the experimental teatures ennrneraterl above, the 2009 exzperirnent
`
`did not eor'r'ectiy replicate the lfieaucloin extractioii and thus resulted in an oil that is not BGa'U.(.lOil‘l
`
`oil.
`
`I understand that it was oil from this incorrectly~eXecnted, krill oil extraction that was sent to
`
`Dr: White lei" mass spectrcinetrie analysis. Dr. White Cll£l1‘£lClT€l‘lLZ€(l
`
`this non—Beaurloin oil in
`
`2009 and included Figure ltl froin this analysis in his Declaration of May 3 ll , Zilll l.
`
`32.
`
`For cornpleteness, l also note that there is a slight mathematical error in l’igntr'e l0, as Dr.
`
`White presentetl it in his Declaration otlwiay 3l, Ztll l. While his point was to simply show how
`
`a (leteetahle ion tragnienrtatioii locks, Di: White mistakenly ill(l6llll,i‘if::‘Ci, the two product ion peaks
`
`at m/z 524 and 542 as C205 (EPA) and Cl8:(), respectively. See White Declaration of May 3 l,
`
`Ztll 3, Figure ll}, My review of this figure shows that these two species cannot result 't’rcin the
`
`fraginentation of a peak at m/z 826. On the contrary, this fragmentation pattern is reflective of a
`
`species bearing two EPA molecules. The lragrnent at m/z 524 represents a neutral loss of the
`
`free acid of lit’/»\ front the parent phospholipiil {m/z 826), and the tragrneiit at I73/E 542 represents
`
`at neutral loss of a ketene form of EFA from the parent phospholipicl. However, as the 2009
`
`experiment was an incorrect repreduction ot‘Beandtoin,, in that water was removed and heat was,
`
`for the most part, not used, this species s',’mzi:’rZ contain nieastirahle levels of a phospholipitl with
`
`4 Seer, e. Herman and Groves “The influence of Free Fatty Acid Foiniation on the pH of Phospholipi<l~Stahili;fecl
`Triglyceride lirnulsions” Piztzrmaceuzicaf Rrzsearc/2 lU(5,}: '.7'.74~7'.7'6 (1993), Appendix E.
`
`ooddlno
`
`0000010
`
`

`
`U,S.S.N. 95./Gill ,3774
`Declaration of Dr. Fatistintis Yeheah
`
`EPA aiitl EPA. This n‘iiSl:‘dl{C., being in a. figure that does not eveii retleet Biittuilflillfi has no
`
`hearing on Dr. Whites conclusions.
`
`33.
`
`Therefore, it is my opinion that Figure it) is a failed attempt at previdiirig a COl”HpEiTElti'%’€*
`
`exaniple between the extract predtucetl in Beaudoin and the eaiiipasitieii of the ‘348 patent. This
`
`figiire, in my opinion, has no hearing an the Conclusions of Dr. White’s stutly and therefore i
`
`View the May 31,, 2011 White Declaration as experiirientally sound. despite it.
`
`34.
`
`A seeoricl minor error lies in Taliies l and 2 of Dr. Wliite’s Declaration of May 31, 203 l;
`
`particularly in Dr. White’s iclentiticatiehs of the petetitiai fatty acids that eeiilti represent
`
`differeiit m/72 signals.
`
`it
`
`appareiit that Dr. White made his calculations witheiit the kiiewletlge
`
`that ll-grill tie not possess fatty acids shorter than C145 As the m/z Values of the cempesitieri of
`
`the ‘348 patent are i.:nanil'>igu0iis, this has he hearing on Dr. White’s overall corichisien as it does
`
`net at all alter the sounrliiess efthe raw experirneiitai data. that he generated.
`
`Canirnents on
`
`antifieatian at" ‘White and Giinrierseii Data
`
`35.
`
`l have re.viewe.<l hath the Guiitiersen and White raw data and have been asked to express
`
`an epiriieri en quantitative a,spects 0f“E§}8i:}:1c
`
`The Beam:£oi.r2 Oi!" C0nmin,§‘, i§fI.4nv,
`
`(1 De fv1'z’m°mz’s' iaimozmt (:f.the PIz(2.rpii0/’ipiris' Carry.i7ng
`
`Two qfEP/i and DHA
`
`36.
`
`The eoiielusieii of the White Declaration, that Dr. White could not find any species of
`
`phespholipids carrying two of EPA and DHA Within the detectiori
`
`limits of the experiinerit
`
`conciueted, is sound. The species detectecl at m/z Values at 826 and 852 represeiit amounts in a
`
`range en the order of only (ll ta 2. % of the ;)hr).s;:9i2r)ii';2ids Q)" the 0116
`
`i understand that
`
`phosphelipids represent aheut 409/?) of the total lipids in krill eiil and therefore, the raw data of
`
`Tables l arid 2 efttie White i:§©Cl£i1”Ei'E’.‘iGIl shows that the atneurit of phespiielipids carryitig two ef
`
`tag. Winther er ./,zZ., Elucidation of Phosphatiidylclioline Composition in Krill Oil Extra<;:te<i from ,Eupimzzsia
`See,
`.rz/perba LzTpz'iz’s 46 (if):
`(2.0l Ll), Apgieiitiix F.
`
`This is based on the rev» ' data presented in Tables 1 and 2 of the White Declaration efl\/lay 31, 2011.
`
`7 See, Winther at 511., Elucidation oi‘ Phosphatidylcholiiie Ceniposition in Krill Oil Extracted from Ezyzpizamia
`superrba Lipids 46 (1): 25-36, page 29 (Agspenrlix F, an Ahei‘ paper that stated: “the PtclClio content (if the undilutecl
`hrill oil was determined to he 34 i 5% (W/W)"); See (rim Beaudein I, Table 14; ‘348 patent, Column 15, lines 3235.
`
`oodolon
`
`0000011
`
`

`
`[774
`U,S.S.N. 95./(}€)l
`Declaration of Dr. Faustirnts Yehoali
`
`EPA and DHA the total Beaurioin oii is only about 9.05 to l.l%. This is a tie minimis amount
`
`ofphospholipids carrying two ot‘E.PA or DE-ISA.
`
`3'7.
`
`Uiifortariately, clue to the rnyriad experirhental errors of the Guhtierseri Declaration,
`
`l
`
`cannot quantity its data. Given this,
`
`it
`
`my opinion that even if there is any of the
`
`phospliolipids carrying two of EPA or DEA, it is likely a very sinall arnount. Speeitieally, it is
`
`noted that (Iluntlersen needed to turn to l_.,C-l\’lRlVt detection to allegedly find the species. LC»
`
`l\/ERM is a taiidein rnass speetrornetrie technique that is sevens] 0m’ez"s of iizrzgnitafre more
`
`sensz'tive than standard, t_,ti3—MS or LC~l‘\/lS‘”‘.8 LC—l‘vlRt\/it is usually used when the detection and
`
`quantification of extremely low or trace levels of analyte is desired.
`
`38.
`
`Having established that there is, at rnost, a tie mzinimzls amount of phospholipids carrying
`
`two of il3l’A or DEA in Beaadoin,
`
`l have also ascertained What percentage of inzrzcz
`
`;)h0.sp}20lz'pid3:,
`
`i"e,g'ardZess of l'dz£’i'£fi'fj? of the fatzjv at:-ta’ citzains, in the Beauclein phospholipid
`
`sample is intact, Based on Tables l and 2 of the White Declaration ofl‘v’lay 31, 2011, and the
`
`fact that a Cl-4./Cl 4 phospholipitl is the lowest intact molecular weight species possible in lorill, l
`
`ealeulate this Value to he at about 35% of the }:)h0S‘pl10lilpitlS.9 Therefore, only about l5‘?/2; of the
`
`total Beandoin oil, regardless of the identity of the fatty acid chains, is intact.
`
`[remainder of page left inteiitioiially blank]
`
`8 ,See Elliott ei c:.7., “Current Trends in Quantitative Proteornics.” J Mass. Speci.r.:2r;«z., 44
`Appendix C.
`
`l63'.7'——l660 (2009),
`
`the fact that l-trill does not
`lii inaldng my assignments, l ‘.l[lliZ€(l
`calculation.
`9 l used Dr. Whi’te"s raw data to do
`contain fatty acids longer than C14 and therefore, a pliospliatidylcholiiie bearing two C14 moieties would he the
`
`lowest molecular weight intaet phosphatidyleholine possible. Such a
`would he expected to h
`a molecular
`Weight of about 677' (this is tlie stun oi‘ two C14 chains, glycerol, and choline). Tlierefore, any 721/: Value of 677 or
`higher was classified as intact. The same would apply ifphosphatidylethanolaniine is considered (inolecular Weight
`cutoff would be about 635- the sum of two Clfi: chains, glycerol, and ethanolarnine).
`
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