UNITED STATES PATENT AND TRADEMARK OFFICE
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`AKER BIOMARINE AS
`Petitioner
`
`v.
`
`NEPTUNE TECHNOLOGIES AND BIORESSOURCES, INC.
`Patent Owner
`
`Case IPR2014-00003
`Patent 8,278,351 B1
`
`Declaration of Richard B. Van Breemen, Ph.D.
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`1, Richard B. van Breemen, Ph.D., hereby declare as follows:
`
`1.
`
`I have been retained by counsel for Petitioner Aker BioMarine AS to
`
`provide expert opinions in connection with this inter partes review.
`
`2.
`
`My background and qualifications are set forth in the declaration I
`
`submitted previously in connection with this IPR, dated September 27, 2013
`
`(Exhibit 1040).
`
`3.
`
`Attached hereto as Exhibit A are excerpts from the November 6, 2013
`
`witness statement I submitted in USITC Investigation No. 337—TA—877, In the
`
`Matter of Certain 0mega—3 Extracts from Marine or Aquatic Biomass and
`
`Products Containing the Same.
`
`4.
`
`I hereby incorporate the statements and information contained in
`
`Exhibit A into this declaration and reaffirm their truthfulness and accuracy.
`
`Dated: September l_g_, 2014
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`’@5;Ag3,Mi'gigI@ &y_,,m
`
`Richard B. van Breemen, Ph.D.
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`EXHIBIT A
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`EXHIBIT A
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`AJ(B B41109
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`RX-0579C
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`UNITED STATES INTERNATIONAL TRADE COMMISSION
`
`Washington, D.C.
`
`Before the Honorable Theodore R. Essex
`
`Administrative Law Judge
`
`In the Matter of
`
`CERTAIN OMEGA-3 EXTRACTS FROM
`MARINE OR AQUATIC BIOMASS AND
`PRODUCTS CONTAINING THE SAME
`
`Investigation No. 33'?-TA-877
`
`WITNESS STATEMENT OF RICHARD B. VAN BREEME-N, PH.D
`
`I.
`
`INTRODUCTION
`
`Q].
`
`Would you please state your name for the record?
`
`A.l.
`
`Richard Bruce van Breemen.
`
`Q.2.
`
`Where are you employed?
`
`A.2.
`
`I am a Professor of Medicinal Chemistry and Pharmacognosy at the University of
`Illinois-Chicago, or “UIC,” College of Pharmacy in Chicago, Illinois.
`
`Q.3.
`
`How long have you been with UIC?
`
`A.3.
`
`Ijoined the faculty of UIC in 1994 as an Associate Professor of Medicinal Chemistry at
`the College of Pharmacy.
`In 2000, I was promoted to the position I hold today.
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`Q.4'l.
`
`ln RDX-0504, you refer to samples you received from others and then tested. Did you
`t.est. samples in addition to the extractions you repeated‘?
`
`A.4l.
`
`I was also asked t.o test certain krill extracts that were prepared by others, including
`samples I
`received from Dr. Suzanne Budge at Dalhousie University in Halifax,
`Canada, and samples of material provided to Respondents in this litigation by Dr. Earl
`L. White, to detemline whet.her they cont.a.in the Claimed Phospholipids. All six of the
`samples I wa.s asked t.o test from Dr. Budge contained the Cla.imed Phospholipids, as
`did the three samples produced by Dr. White. While 1 do not have personal knowledge
`regarding how these nine extracts were made, it is my understanding that all of them
`were ma.de according t.o the prior art. Bea.udoin references — W0 0033546 (referred to
`as “Beaudoin I") andfor CA 2,251,265 (referred to as “Beaudoin II”).
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`IV.
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`INVALIDITY UNDER 35 U.S.C. § 102
`
`A.
`
`Claimed Phospholipids are Present in Krill Extracts Made According to
`Certain Prior Art References
`
`1.
`
`The Extracts Tested
`
`Q.46.
`
`I’d like to focus now on the repeat extractions and testing work you performed in
`reaching your opinions. Would you please identify all the extracts that you tested?
`
`A.46.
`
`Yes. RDX-0505 lists the thirteen prior art krill extracts that I tested.
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`Extracts Tested
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`Extracts I Repeated
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`-
`
`-
`
`Fujita Hexane
`
`-
`
`Fujita Once-through
`
`Fujita Hexane Ethanol
`
`- Rogozhin
`
`Extractsl Received from Others
`
`Received from Dr- Budge
`
`Produced by Dr. White
`
`Beaudoin E19
`
`Beaudoin __E{1_
`
`Beaudoin
`
`Beaudoin §L,lQ_
`
`Beaudoin SU1
`
`Beaudoin go;
`
`- White 1
`
`- White 2
`
`- White 3
`
`As I mentioned before, I repeated three different extracts by following three different
`procedures in the Fujita Reference: Fujita Hexane, Fujita Hexane Ethanol, and Fujita
`Once-through.
`I also repeated one extract by following the procedure in the Rogozhin
`patent.
`
`The nine remaining extracts that I tested were extracts that I received from others. Six
`came from Dr. Budge: Beaudoin P0, Beaudoin P1, Beaudoin P2, Beaudoin SUO,
`Beaudoin SUI, and Beaudoin SU2. The other three were provided to Respondents in
`this litigation by Dr. White: White 1, White 2, and White 3. While 1 do not have
`personal knowledge regarding how these nine extracts were made,
`it
`is my
`understanding that all of them were made according to the prior art Beaudoin
`references.
`
`
`
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`Would you please walk us through the setup for your testing, starting with the
`equipment you used‘?
`
`UHPLC-MS analyses ofkrill oils were carried out using a high resolution Shimadzu [T-
`TOF mass spectrometer equipped with a. Shimadzu Prominence XR HPLC system.
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`Chromatographic separations were obtained using a Waters Acquity CSH C18 UHPLC
`column (2.1 mm x 150 mm; 1.7 pm).
`
`Q.161. How were your instruments configured?
`
`A.l61. The initial composition of the mobile phase was 80% methanol and 20% water
`containing 5 mM ammonium formate for 2 min followed by a 5 min gradient from 80%
`to 100% methanol. The column was re-equilibrated at 80% methanol for 3 min between
`analyses. The UHPLC mobile phase flow rate was 0.3 mL/min for the IT—TOF mass
`spectrometer and 0.4 mL.=’min for the LCMS-8040 triple quadrupole mass spectrometer.
`
`Q.l62. Did you use the standard of PC—DHA/DHA that you had purchased as part of your
`setup?
`
`A.l62. The mass spectrometers and UHPLC system were optimized for the analysis of the
`Claimed Phospholipids using a PC-DHAJDHA standard. The positive ion electrospray
`mass spectrum of standard PODHAJDHA is shown in Figure 1 on RDX—0534.
`(RX-
`0S80, Fig. 1.)
`
`RDX—0534
`
`Figure 1
`
`m
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`150
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`320
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`Figure 1. Positive ion electrospray mass spectrum of PC—DHNDHA standard
`(from SigmaiA|dn'ch).
`
`RX-0580. Fig. 1
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`The theoretical mass of PC—DHA/DHA is 878.5699. As I’ve indicated with a red box,
`the expected intact PC—DHA;'DHA ion of me: 8?8,6 was observed as the base peak of
`the mass spectrum, a result consistent with the presence of PC-DHAIDHA.
`(RX-0580,
`Fig. 1.)
`
`Q.l63. Did you do any further analysis on this ion of m/z 8'.+'8.6‘?
`
`AJ63. Yes. Turning to Figure 2 on RDX-0535, you’ll see that, using m/z 878.6] as a precursor
`ion, collision induced dissociation was used and product ion tandem mass spectrometry
`was used to obtain the tandem mass spectrum of PC-DHAJDHA.
`(RX—0580, Fig. 2.)
`
`Figure 2
`
`RDX-0535
`
`Figure 2. Positive ion electrospray product ion tandem mass spectrum of PC-
`DHNDHA ion of m/z 878.[6]_
`
`As expected, PC—DI-IAIDHA fragmented to form an abundant product ion of m.-*2 184.]
`containing the PC moiety without the fatty acids, as I’ve indicated with a red box. This
`product ion of m/Lz 184.1 is common to all PCs.
`
`RX—O580, Fig. 2
`
`I corrected a
`' As indicated with brackets in the caption of Figure 2 on RDX—0S35,
`typographical error that appeared in the caption of the same figure in my opening expert
`report (RX-0580). Unless otherwise noted, use of brackets in the captions of figures that
`appeared in RX-0580 and RX-0585 indicate such typographical error corrections.
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`Q.l64.
`
`Is there a peak corresponding to DHA in this mass spectrum?
`
`A.164. No.
`
`(2.165.
`
`ls it surprising that there are no peaks corresponding to DHA in this mass spectrum?
`
`A.l65. No.
`
`Q.l66. Why is that‘?
`
`A.166. Positive ion electrospray detects positive ions, but fatty acids like DHA form negative
`ions more readily than positive ions and would not be detected with positive ion
`electrospray.
`
`Q.l6’}'. How were you able to use the data in Figures 1 and 2 for your extract testing?
`
`A.167. Turning to Figure 3 on RDX-O53 6, you’ll see that, based on the data in Figures 1 and 2,
`an assay based on UHPLC-MSIMS was developed that utilized reversed phase UHPLC
`separation, positive ion electrospray for ionization of the phospholipids, collision-
`induced dissociation and selected reaction monitoring to record the transition from the
`intact phospholipids ions to their common product ion of m/z 184. (RX-0580, Fig. 3.)
`
`Figure 3 is a chromatogram showing this transition. This chromatogram plots the
`chromatography dimension along the X-axis and the tandem mass spectrometry
`dimension along the Y-axis. The one peak in Figure 3 indicates that the phospholipid
`was detected when it eluted at 6.9 minutes, and that it was measured at mfz 878, and as
`
`was the structurally informative ion of m/z 184, Accordingly, the phospholipid is
`identified as PC-DHAIDHA.
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`Figure 3
`
`RDX-0536
`
`— :B7B.50>'l84.‘lD(+] PC-DHNDHA
`
`Figure 3. UHPLC—MSlMS triple quadrupole analysis of a PC~DHAlDHA
`standard of 250 ngimL obtained using positive ion electrospray with collision-
`induced dissociation and selected reaction monitoring of the transition m/z 878 to
`W: 184.
`
`RX—O580, Fig. 3
`
`UHPLC was used to separate PC-DHA/DHA from PC-EPA/EPA and from PC-
`DHAIEPA as well as from other compounds in the krill oil extracts.
`
`Q.l68. Did you do anything between the analyses of the extracts you tested to make sure your
`equipment was getting accurate results?
`
`AJ68. Before each analysis of a krill oil sample, a blank analysis was carried out to ensure that
`there was no carryover of phospholipids from one analysis to the next.
`
`Q.169. How did you confirm that you were actually detecting each of PC—DHAfDHA, PC-
`EPAIEPA and PC-DHAIEPA in the samples you tested?
`
`A.l69.
`
`In addition to UHPLC—MSr’MS, high resolution accurate mass measurements of intact
`PC—DHAfDHA, PC—EPAJ'EPA and PC-DHAJEPA in each of the krill oil samples were
`used to confirm that their measured elemental compositions were identical
`to their
`corresponding theoretical elemental compositions. The standard practice for analyses
`such as these is that, when a high resolution measurement of an unknown molecule is
`within 10 ppm of a theoretical value, then the elemental composition of that molecule is
`confirmed. Accordingly, I used a 10 ppm window as the threshold for determining
`whether the detected ion mass matched that of the target compound.
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`Q.266.
`
`What were the results of your testing of White '1?
`
`A.266.
`
`the
`RDX-0571 through RDX-0573 include Figures 39-41 respectively, and reflect
`results of my analysis of White 1, produced in this litigation by Dr. White.
`(RX-0580,
`Figs. 39-41.) As shown in these figures, each of the three Claimed Phospholipid
`species PC-DHAIDHA, PC-EPAIEPA, and PC-DHAIEPA was detected in White 1.
`(RX-0580, Figs. 39-41.)
`
`Q.26’}'.
`
`What is shown in Figure 39?
`
`A.267.
`
`Figure 39 shows the positive ion electrospray high resolution IT TOF UI-[PLC-MS
`computer-reconstructed mass chromatograms of the White 1.
`(RX-0580, Fig. 39.) The
`top chromatogram of Figure 39 shows the detection of peaks corresponding to PC-
`EPAIEPA (mfz 826.53), PC-DHAIEPA (m..-"z 852.55) and PC-DHAIDHA (m..-"z 878.56)
`eluting at approximately 5.3, 5.5 and 5.7 minutes, respectively.
`(RX-0580, Fig. 39.)
`The bottom chromatogram of Figure 39 shows the results of an additional test where
`White 1 was spiked with a PC-DHAIDHA standard and then reanalyzed.
`(RX-0580,
`Fig. 39.)
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`Figure 39
`
`fzmeflflflil
`
`------ sass: {PC-EFNEFM
`- - - - ms: |PC-I1-lA.I’E?AI
`—-swans rrc-r.ii-wot-in:
`
`|T~TOF MS
`While1
`
`f.:.s1sssor
`}‘='
`
`PC—DI-IAIEPA
`
`Spiked with PC-DHNDHA
`standard
`
`!EI
`
`ii 13051636
`
`.. ..ss:a.ss [Po-:1-mn=.i>iu
`_. areas rvcounrnmu
`
`-—--- sass:[PC-E?:|I-|'EFA:
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`1.9
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`15
`
`::.o
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`25
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`so
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`as
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`.
`”-
`.
`Relerlionti'I‘I8ln1'ri}
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`so
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`65
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`m
`
`1'5
`
`an
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`as
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`PC-Q.li.£\JQ.l'!!3.
`with spiking
`
`Figure 39. Positive ion electrospray high resolution IT ‘_[_Q_E_UHPLC-MS computer-reconstructed mass chromatograms
`of the White 1 sample showing the detection of peaks corresponding to PC-EPNEPA(rn!z 826.53), PC-DHA/EPA(mz2
`852.55) and PCAJHNDHA (m/'2 878.58) aiming at approximately 5.3, 5.5 and 5.? minutes, respectively (top). The
`extract was spiked with a PC-‘|I_)__l-_l_.g';\_JIJ___I;I__,)'§ standard and then reanalyzed (bottom). Note that the area of the peak
`corresponding to PC-DHNDHA increased confirming the identity of PC~DHNDHA in the extract.
`
`RX—0580. Fi. 39
`
`Q.263.
`
`What do the peaks labeled in red, green, and blue indicate in the top chromatogram of
`Figure 39?
`
`A.268.
`
`The peaks correspond to the retention times of the three Claimed Phospholipids I was
`testing for. PC-EPA/EPA, indicated in red, eluted first at approximately 5.3 minutes.
`(RX—0580, Fig. 39.) PC-DHAJEPA, indicated in green, eluted next from the UHPLC-
`MS system at a retention time of approximately 5.5 minutes.
`(RX—0580, Fig. 39.) PC-
`DHAIDHA, indicated in blue, eluted last at a retention time of approximately 5.7 min.
`(RX-0580, Fig. 39,)
`
`Q.269.
`
`What is shown in Figure 40?
`
`A.269.
`
`Figure 40 shows the three high resolution accurate mass measurements of the PC—
`EPAIEPA, PC-DHA/EPA, and PC—DHA./DHA peaks respectively, previously shown in
`Figure 39. (RX-0580, Figs. 39-40.)
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`Figure 40
`
`IT-TOF MS
`
`White 1 {Sample No. 09-1551}
`
`PC-EPAIEPA
`
`-—-i
`
`PC-D HNEPA
`
`:1:sin‘:-
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`5 i0
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`321!
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`33]
`
`H.
`1
`
`Figure 40. High resolution accurate mass measurements of the peaks corresponding to PC-EPAIEPA
`(top) eluting at a retention time of approximately 5.3 min (Figure 39), PC—EPNDHA (middle) eluting at a
`retention time of approximately 5.5 min (Figure 39), and PC-DHNDHA (bottom) eluting at a retention
`time of approximately 5.7 min (Figure 39).
`
`RX-0580, Fig. 40
`
`Q.270. What do the peaks labeled in red, green, and blue indicate in Figure 40?
`
`A.270. As indicated in red at the top of Figure 40, PC-EPAIEPA was measured at mfz 826.5379
`when it eluted.
`(RX-0580, Fig. 40.) Because the theoretical mass of PC-EPAIEPA is
`8265386 (Am = -5 ppm), the elemental composition of this phospholipid in White 1
`was determined to be identical to that of PC-EPAXEPA. As indicated in green in the
`middle of Figure 40, PC-DHAIEPA was measured at ml-"z 852.5502 when it eluted next.
`(RX-0580, Fig. 40.) Because the theoretical mass of PC—DHA;'EPA is 8525543 (Am =
`-4 ppm), the elemental composition of this phospholipid in White 1 was determined to
`be identical to that of PC-DHAIEPA. Lastly, as indicated in blue at the bottom of Figure
`40, PC-DHAJDHA was measured at m/z 878.5673 when it eluted.
`(RX-0580, Fig. 40.)
`Because the theoretical mass of PC—DHAfDHA is 878.5699 (Am = -3 ppm),
`the
`elemental compositi on was determined to be identical to that of PC—DI-IAJDHA.
`
`Q.27l. Would you please explain the results of your spiking test in Figure 39?
`
`A.27l. As shown in the bottom chromatogram of Figure 39 on RDX-0571, White 1 was spiked
`with the PC-DHAJDHA standard and reanalyzed using UT-[PLC—MS.
`(RX-0580, Fig.
`39.) The standard coeluted with one of the phospholipids in the extract — the peak for
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`PC-DHAIDHA increased and no new peak appeared — thereby identifying this
`phospholipid in White 1 as PC—DHAfDHA, indicated in blue. (RX-0580, Fig. 39.)
`
`(2.272. What is shown in Figure 41?
`
`A.272. Figure 41 shows the Positive ion electrospray UHPLC—MS/MS analysis of the Claimed
`Phospholipids in the White 1.
`(RX—0580, Fig. 41.)
`
`Figure 41
`
`—a7e.5u>1a4.1n{+j PC-DHAIDHA
`
`--—---s2a.¢o>1s4.ns[+; Pc-EPNEPA
`
`———s52.ao>1a-mom P6-DHMEPA
`
`RDX-0573
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`PC-EPNEPA
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`Figure 41. Positive ion electrospray UHPLC—MSlMS analysis of the Claimed
`Phospholipids in the White 1 sample. A triple quadrupole mass spectrometer was
`used with collision-induced dissociation and selected reaction monitoring (SRM)
`of the transitions indicated.
`
`RX-0580, Fig. 41
`
`Q.273. What do the peaks labeled in red, green, and blue indicate in Figure 41‘?
`
`A.273. Further analysis of the Claimed Phospholipids in White 1 was carried out using positive
`ion electrospray U'HPLC—l\/IS/MS with collision-induced dissociation and selected
`reaction monitoring (SRM) on a triple quadrupole tandem mass spectrometer. The SRM
`transitions that were used for analysis were m/Lz 826 to m./z 184 for PC-EPAJEPA,
`indicated in red; m./"z 852 to mxiz 184 for PODHAKEPA, indicated in green; and mxk 878
`to m./"z 184 for PC-DHAIDHA, indicated in blue.
`(RX-0580, Fig. 41.) As you can see,
`PC—EPA/EPA, PC-DHAIEPA, and PC-DHAIDHA were all detected in White 1.
`(RX-
`0S80, Fig. 41.)
`
`Q.274. What were the results of your testing of White 2 and White 3‘?
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`A.274. RDX-0574 and RDX-0575 include Figures 50-51 respectively, and reflect the results of
`my analysis of White 2 and White 3.
`(RX-0585, Attachment K, Figs. 50-51.) As
`shown in these figures, each of the three Claimed Phospholipid species PC-DHAJDHA,
`PC-EPAIEPA, and PC-DHAIEPA was detected in each of White 2 and White 3.
`(RX-
`0585, Attachment K, Figs. 50-51.)
`
`The testing I performed on White 2 and White 3 differs from the testing I performed on
`the other samples described so far.
`
`Q.275. How so?
`
`A.27S.
`
`In all of the analyses described so far, all krill oil samples were dissolved in
`chloroform,="methanol (60:40, v.="v) and then diluted with methanol. However,
`I found
`that White 2 and White 3 contained solids that did not dissolve in chlorofonnfmethanol,
`unlike the other krill oils. Therefore, I did not measure samples White 2 and White 3
`after attempting to dissolve them in chloroformfmethanol, so as not to damage my
`UPHPLC-MS-MS system.
`
`Q.276. When did you analyze White 2 and White 3?
`
`A.276.
`
`I had previously performed a series of preliminary qualitative analyses designed to
`identify a suitable solvent for dissolving and then diluting krill oils appropriately for
`mass spectrometric analysis. In these preliminary studies, I tried dissolving and diluting
`krill oils in methanol, The oils did not completely dissolve in methanol, so I centrifuged
`each diluted sample before mass spectrometric analysis of each supernatant. During
`this time, I analyzed White 2 and White 3 and detected PC-DHAIDHA, PC-EPAIEPA,
`and PC-DHAIEPA, as shown in Figures 50-51. (RX-0585, Attachment K, Figs. 50-51.)
`
`Q.277. What is shown in Figure 50?
`
`A.27’?. Figure 50 shows the positive ion electrospray UI-[PLC-MS-MS analysis of the Claimed
`Phospholipids in White Sample 2. (RX-0585, Attachment K, Fig. 50.)
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`Figure 50
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`—~ B78.su>1s4.1n(+)
`—~—~ a2a_4u>1a4_o5:+)
`—-—~ s52_sn>1s4.1n{+}
`
`White 2
`
`UHPLC-MS-MS triple quadrupole
`
`:1
`
`1.0
`
`...r
`
`.
`
`.
`
`....\1
`.
`5.0
`
`Figure 50. Positive ion electnospray UHPLC~MS~MS analysis of the Claimed Phospholipids in
`
`the White 2 sample. A triple guadrugole mass spectrometer was used with collision-induced
`
`dissociation and selected reaction monitoring {SRM} of the transitions indicated.
`
`RX-0585, Attachment K, Fi. 50
`
`Q.273. What do the peaks labeled in red, green, and blue indicate in Figure 50?
`
`A278. Analyses of the Claimed Phospholipids in White 2 were carried out using positive ion
`electrospray UI-[PLC—MS/MS with col1ision—induced dissociation and selected reaction
`monitoring (SRM) on a triple quadrupole tandem mass spectrometer. The SRM
`transitions that were used for analysis were mfz 826 to m/z 184 for PC—EPAJ'EPA,
`indicated in red; m/z 852 to m/z 184 for PC—DHAi'EPA, indicated in green; and my’: 878
`to m/z 184 for PC~DI-IAIDHA, indicated in blue.
`(RX—0585, Attachment K, Fig. 50.)
`As you can see, PC—EPA}'EPA, PC—DHA/EPA, and PC—DHAfDHA were all detected in
`White 2. (RX-0585, Attachment K, Fig. 50.)
`
`Q.279. What is shown in Figure 51?
`
`A.279. Figure 51 shows the positive ion electrospray UI{PLC—MS—MS analysis of the Claimed
`Phospholipids in White Sample 3. (RX-0585, Attachment K, Fig. 51.)
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`Figure 51
`
`— B?8.50>1Bd_‘I[I[+}
`~—~— B25.-1lJ>1B4.05[+)
`“H as2_an>1s4_m;+;
`
`White 3
`
`UHPLC—MS—MS triple quadrupole
`
`....-.....1-...-......,....
`2.0
`3.0
`4.0
`5.0
`
`1.0
`
`Figure 51. Positive ion electrospray L1t:LEJ,Q;MS-MS analysis of the Claimed Phospholipids in
`the White 3 sample. A triple Quadrupole mass spectrometer was used with collision-induced
`dissociation and selected reaction monitoring (SRM) of the transitions indicated.
`
`RX-0585 Attachment K Fi. 51
`
`Q.280. What do the peaks labeled in red, green, and blue indicate in Figure 51‘?
`
`A.280. Analyses of the Claimed Phospholipids in White 3 were carried out using positive ion
`electrospray UHPLC—MS/MS with collision—induced dissociation and selected reaction
`monitoring (SRM) on a triple quadrupole tandem mass spectrometer. The SRM
`transitions that were used for analysis were n:-22 826 to m/z 184 for PC—EPAJ'EPA,
`indicated in red; m/z 852 to m/z 184 for PC—DHAfEPA, indicated in green; and my’: 878
`to mfz 184 for PC~DHAJ'DHA, indicated in blue.
`(RX—0585, Attachment K, Fig. 51.)
`As you can see, PC—EPA;'EPA, PC—DHA/EPA, and PC—DHAfDHA were all detected in
`White 3. (RX-0585, Attachment K, Fig. 51.)
`
`Q.28l. Did you analyze any other krill extracts along with White 2 and White 3 during these
`preliminary qualitative analyses?
`
`A.28l. Yes, I also analyzed White 1 and the Fujita Hexane, Fujita Hexane Ethanol, Fujita
`Once—through, Rogozhin, Beaudoin P0, Beaudoin P1, and Beaudoin P2 extracts.
`
`Q.282. Did you detect the Claimed Phosphlipids in these extracts?
`
`103
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`
`A.282. Yes, I detected the Claimed Phospholipids in every sample I analyzed, every time I
`analyzed the sample.
`
`Q.283. Would you please walk us through the chromatograms associated with these analyses?
`
`A.283. RDX—0576 through RDX—0583 include Figures 42-49, and are the chromatograms
`associated with these analyses.
`(RX—0585, Attachment K, Figs. 50-51.) Analysis of the
`Claimed Phospholipids in each sample was carried out using positive ion electrospray
`UHPLC—MSi"MS with collision-induced dissociation and selected reaction rnonitoring
`(SRM) on a triple quadrupole tandem mass spectrometer. The SRM transitions that
`were used for analysis were 'm/z 826 to m/2 184 for PC-EPAIEPA, m/2 852 to m/z 184
`for PC—DHA,="EP.A, and
`878 to mi: 184 for PC—DHA/DI-IA.
`
`is shown in Figure 49 on RDX-0576.
`A chrornatogram for this analysis for White 1
`(RX-0585, Attachment K, Fig. 49.) As you can see, PC—EPAfEPA, PC—DHAi’EPA, and
`PC—DHAx’DHA were all detected in White 1.
`(RX-0585, Attachment K, Fig. 49.)
`
`RDX-0576
`
`Figure 49
`
`—- ars_su>1sa.1n{+}
`mm s2s_4u>1s4_u5{+;
`~—~— a52.su>1aa.1u{+;
`
`White 1
`
`UHPLC—MS-MS triple quadrupole
`
`250000:
`
`0-
`l~
`1.o'
`
`r|Ir'Il"‘|'|lI-I|l'II|'|"IIIr
`2.0
`3.9
`4.0
`so
`
`'
`
`'
`
`Figure 49. Positive ion electrospray UHPLC-MS-MS analysis of the Claimed Phospholipids in
`the White 1 sample. A triple Quadrupole mass spectrometer was used with collision-induced
`dissociation and selected reaction monitoring (SRM) of the transitions indicated.
`
`RX-0585,Attachment K. Fi . 49
`
`A chromatogram for this analysis for the Fujita Hexane extract is shown in Figure 42 on
`RDX-057?.
`(RX-0585, Attachment K, Fig. 42.) As you can see, PC—EPAi’EPA, PC-
`
`104
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`
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`
`DHAIEPA, and PC—DHAJ'DHA were all detected in the Fujita Hexane extract.
`0585, Attachment K, Fig, 42.)
`
`(RX-
`
`Figure 42
`
`j BTS_5CI>1B41D[+]
`
`-—-——- 823.-4U>‘l34_U5[+]
`-~-— a52.so>1a4.1ui+}
`
`Fujita Hexane Extraction
`UHPLC—MS—MS triple Quadrupole
`
`-KPC-.D._H£ilQ.H.&
`
`ll
`
`li
`
`0,
`
`l
`l
`
`l
`
`. .
`.111-1r'r'\r-r-1-v-r1-11":-'rr-r1-1-rt-1 r'l'lE'l"|'-"Yll"'|
`1.0
`2.0
`3.0
`4.0
`5.0
`SJ!
`7.0
`
`x
`
`mm
`
`Figure 42. Positive ion electrospray UHPLC—MS~MS analysis of the Claimed Phospholipids
`in the Fujita hexane extract. A triple Quadrupole mass spectrometer was used with collision-
`induced dissociation and selected reaction monitoring (SRM) of the transitions indicated.
`
`RX-0585, Attachment K, Fi. 42
`
`A chromatogram for this analysis for the Fujita Hexane Ethanol extract is shown in
`Figure 43 on RDX~0578.
`(RX-0585, Attachment K, Fig. 43.) As you can see, PC-
`EPAIEPA, PC-DHNEPA, and PC-DHAJDHA were all detected in the Fujita Hexane
`Ethanol extract.
`(RX—0585, Attachment K, Fig. 43.)
`
`105
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`

`
`Figure 43
`
`— 81-'S_S0>1B4_‘lD[+}
`.._.,.. g2s.4u;.1e4_u5(+]
`-—-- a52.sn>1a41n:+}
`
`Fujita Hexane."Ethanol Extraction
`UHPLC—MS—MS triple quadrupole
`
`PC-DHNEPA
`
`PC-DHAIDHA
`
`Figure 43. Positive ion electrospray UfiEi:C~MS«MS analysis of the Claimed Phospholipids in
`
`the Fujita hexanelethanol extract. A triple gggcggggfi mass spectrometer was used with
`collision-induced dissociation and selected reaction monitoring (SRM) of the transitions
`‘"°“°a*°d-
`RX-0585, Attachment K Fig. 43
`
`A chromatogram for this analysis for the Fujita Once-through extract is shown in Figure
`44 on RDX-0579.
`(RX—0S85, Attachment K, Fig. 44.) As you can see, PC—EPAfEPA_,
`PC—DHAfEPA, and PC—DHAfDHA were all detected in the Fujita Once—through extract.
`(RX—0585, Attachment K, Fig. 44.)
`
`106
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`Figure 44
`
`—- a7a.su>1a4.1o(+}
`-~— B26.4u>18d.05(+}
`--—- B52.6lJ>1B4.‘i[i(+}
`
`Fujita Once-through extraction
`U..|iEL.Q-MS~M3 triple Quemlesie
`
`°_
`,w—-r-—1r-~r--I--c——u
`10
`2.0
`
`.
`rwr--1-r--vr--I---1-u——ur—-|--i—r—i"r-1-—u--u—-1-r
`3.0
`4.6
`5.0
`6.0
`
`.
`
`u——r-—iu
`
`an
`
`Figure 44. Positive ion eiectrospray Ui;iPi:C-MS-MS analysis of the Claimed Phosphoiipids in
`the Fujita once-through extract. Atriple guadruggle mass spectrometer was used with collision-
`induced dissociation and selected reaction monitoring (§fi_M_) of the transitions indicated.
`
`RX-0585, Attachment K. Fig. 44
`
`A chrornatograrn for this analysis for the Rogozhin extract is shown in Figure 45 on
`RDX-0580.
`(RX-0585, Attachment K, Fig. 45.) As you can see, PC-EPA/EPA, PC—
`DHAXEPA, and PC—DHA/DHA were all detected in the Rogozhin extract.
`(RX-0585,
`Attachment K, Fig. 45,)
`
`107
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`RDX-0580
`
`Figure 45
`
`*: 8?8.5U>‘lBa'l-.1U[+}
`*"*-"'" 326.Ilfl>‘l84.D5[+}
`-“* 352.BE}>‘lB-l».1U[+]
`
`Rogozhin (Canada)
`UHPLC-MS-MS triple quadrupole
`
`1.0. ‘I
`
`zfol
`
`I
`
`I
`
`‘3'.u'
`
`’
`
`'
`
`'4:a'
`
`I
`
`I
`
`'53:"
`
`Figure 45. Positive ion electrospray UHPLC-MS—MS analysis of the Claimed Phospholipids
`in the Rogozhin extract. A triple guadruggle mass spectrometer was used with collision-
`induced dissociation and selected reaction monitoring (SRM) of the transitions indicated.
`
`RX-«U585, Attachment K. Fig. 45
`
`A chromatogram for this analysis for the Beaudoin Extract P0 is shown in Figure 46 on
`RDX-0581.
`(RX—0585, Attachment K, Fig. 46.) As you can see, PC-EPAIEPA, PC-
`DHAIEPA, and PC—DHAfDHA were all detected in Beaudoin Extract P0.
`(RX—0585,
`Attachment K, Fig. 46.)
`
`108
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`

`
`RDX-0531
`
`Figure 46
`
`i sra.5u>134.1o(+}
`—— a25.4c>1a4_n5(+;
`——-— s52.so>1aa_1o[+;
`
`Beaudoin Extract P0
`
`UHPLC-MS-MS triple quadrupole
`
`I’
`
`l
`
`2.0
`
`Figure 46. Positive ion electrospray UHPLC—M$—MS anaiysis of the Ciaimed Phospholipids in
`
`the Beaudoin extract PO. A triple guadrugole mass spectrometer was used with collision-
`induced dissociation and selected reaction monitoring {SRMJ of the transitions indicated.
`
`RX-0585, Attachment K, Fig. 46
`
`A chromatogram for this analysis for the Beaudoin Extract P1 is shown in Figure 47 on
`RDX—0582.
`(RX-0585, Attachment K, Fig. 47.) As you can see, PC-EPA/EPA, PC—
`DHAXEPA, and PODHAJDHA were all detected in Beaudoin Extract P1.
`(RX-0585,
`Attachment K, Fig. 47.)
`
`109
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`Figure 47
`
`RDX-0582
`
`T‘ BT3.5|]>1El4_1fl{+}
`'"*"*—' B2E_4l]>184_fl5(+}
`“‘“ B52.G0>1B4.1U(+)
`
`159cc?
`
`125cc:
`
`Beaudoin Extract P1
`
`UHPLC-MS—MS triple quadrupole
`
`I--rr-‘TI
`2.0
`
`I
`
`‘I
`
`l
`
`.
`
`'1‘-1" I
`4.0
`
`I
`
`r
`
`-r-
`
`r
`5.0
`
`-1-
`
`.
`
`Figure 47. Positive ion electroepray UHPLC—MS—MS analysis of the Claimed Phospholipids in
`the Beaudoin extract P1. A triple Quadrupole mass spectrometer was used with collision-
`induced dissociation and selected reaction monitoring (SRM) of the transitions indicated.
`
`RX—0585, Attachment K. Fig. 47
`
`A chromatcgram for this analysis for the Beaudoin Extract P2 is shown in Figure 48 on
`RDX—0583.
`(RX—058S, Attachment K, Fig. 48.) As you can see, PC-EPA/EPA, PC—
`DHAXEPA, and PC~DHAJ’DHA were all detected in Beaudoin Extract P2.
`(RX-0585,
`Attachment K, Fig. 48,)
`
`110
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`
`Figure 48
`
`209$. T s?3.5n>1s4 1o(+}
`f
`——— s2e_4a>1s4.n5(+)
`1759“:
`———— a52.5u>13x11u(+;
`
`Beaudoin Extract P2
`UHPLC-MS-MS triple quadrupole
`
`15°°°'£
`__
`12500-
`
`1oooo='
`
`7500
`
`soon-
`
`25uc_
`
`D.
`1|IIII|III-|I\lI§IIll|l|Il|IIll|IIll
`1.0
`2.0
`3.0
`4.0
`5.0
`6.0
`?.0
`
`‘Pa’!
`
`Figure 48. Positive ion electrospray gfiflisgfi-MS-M8 analysis of the Claimed Phospholipids in
`
`the Beaudoin extract P2. A triple guadrup_gLe_ mass spectrometer was used with collision-
`induced dissociation and selected reaction monitoring (SRM} of the transitions indicated.
`
`RX-0585, Attachment K, Fig. 48
`
`Ill
`
`AKBM 1109
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`AKBM 1109
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`RX-0579C.0111
`
`

`
`Q.3l8. Have you reviewed any analyses Neptune and its retained experts have relied on
`previously with respect to testing for the Claimed Phospholipids?
`
`A.3l8.
`
`l have.
`
`Q.3l9. How does your analysis compare to those Neptune analyses?
`
`A.3l9. My analysis relating to the presence of the Claimed Phospholipids in the prior art krill
`extracts I tested is more reliable and comprehensive than similar analyses Neptune and
`its retained experts have relied on previously.
`In other words, my analysis presents
`even more evidence that the Claimed Phospholipids are present than the reports and
`studies Neptune and its experts commissioned and relied upon.
`
`Q.320. Would you please give an example of why that is the case?
`
`l’ve shown on RDX-0589C, according to pages WHITEITC-00000419 and
`A.320. As
`WHITEITC-00000421 of Dr. White’s 2009 report, Dr. White analyzed a “Beaudoin Oil
`Sample” for Neptune for identity and relative concentrations of “phospholipids
`containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the sn-1
`and sn-2 positions of the glycerol backbone.”
`“The PLs of interest
`included
`phosphatidylcholine
`(PC),
`phosphophatidylethanolamine
`[sic]
`(PE)
`and
`phosphatidylinositol (PI).”
`
`12]
`
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`
`Dr. White Analyzed a Beaudoin Oil Sample in 2009 with Tandem Mass Spectrometry.
`Finding Molecules having the Same Molecular Weights as the Theoretical Molecular
`Weights for Intact PC-EPAIEPA (826). PC-DHA.-‘EPA {B52}. and PC-DHAIDHA {B78}
`
`RDX—0589C_
`
`bottle to MD)! Biofiinalytical Laboratory
`
`in a brown opaque 250 mL
`
`believed to be present at 85%, 7% and 8% for PC, PE and PL. respectively.
`
`These PL classes were
`
`Tanle I
`
`Phgzfimq
`
`c1a:oxc :05
`
`C110-'C?°'5''
`'5” 0"” ""
`cn-5.-'<::a a"
`
`
`
`121:.-n,'<'m.>"r??.:3.rc1o 4;"
`
`toaniancniinn ma ouuntmum.-n ol
`mm anlwllplda in Baauduin on
`No. Lu; as-1551
`smmmm
`
`"°”‘:'|':n::::l':;'f”' '"“
`-v-1*
`-‘M
`
`mu
`
`fiemvax
`
`1
`
`Phospholipids Detected In Beaudoin Oil Sample
`
`HT
`
`FlElan
`
`:-4:
`
`‘'5 W1’? 9"‘
`F‘:
`5':
`"mu.
`""
`
`9r: we I».=5 mmu
`
`Pl":-."l'P.\
`Pl. v.'.i'E 9.‘.
`r-zu w.r|.Jl-in
`ucmnmx
`
`l’.i'UI5.I'C1iI'0
`no 5;-:13-1' '
`(3 am: 15. 1"
`(.22-c.rc15:o"
`t'1l:l:lJ.-‘L ::i::*-'-
`-ti
`x-.'-cu
`'
`v
`r. ax-i'..'r IS: 1"
`— — ' - i
`
`_
`
`RX—0503C WH|TElTC—00000419, 421
`
`As shown in Table 1 of Dr. White’s 2009 report on RDX-0503C, Dr. White concluded
`that he detected the following Claimed Phospholipids in the “Beaudoin Oil Sample” he
`analyzed: PC-EPAIEPA, PC-DHA/EPA, and PC-DHAIDHA. He claims, however,
`that he could not differentiate PC-DHA/EPA.
`
`Q.321. What did Dr. White base his conclusions on?
`
`A.321. Dr. White based his conclusions on his findings, through tandem mass spectrometry, of
`molecules having the same molecular weights as the theoretical molecular weights for
`intact PC-EPAIEPA (826), PC-DHAJEPA (852), and PC-DHAJDHA (878).
`
`Q.322. How is your analysis more reliable or comprehensive than Dr. White’s?
`
`A.322. My analysis achieves an even higher level of confidence than Dr. White’s, at least for
`the reasons that I used an authentic standard as a positive control, and each of my
`spectra shows not only the parent (826, 852, and 878) ions associated with the species
`of interest but also shows product ions of m/z 184 containing the PC moiety without the
`fatty acids. I also confirmed that the compounds I detected contained the exact same
`elemental composition as the target species.
`
`122
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`
`(2.331. Are there any other examples showing your analyses to be more reliable and
`comprehensive than Neptune’s?
`
`A.33l. As previously mentioned, Neptune submitted a May 31, 2011 declaration by Dr. White
`attaching his May 29, 2011 “Final Report” to the USPTO in connection with the
`reexamination of the ‘348 Patent. As shown on RDX-0591, Dr. White stated on page
`NEPSTFITC-00003353 of his declaration that he detected compou