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`BEFORE THE PATENT TRIAL AND APPEAL BOARD
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`AKER BIOMARINE AS
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`Petitioner
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`v.
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`NEPTUNE TECHNOLOGIES AND BIORESOURCES INC.
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`Patent Owner
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`CASE IPR: Unassigned
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`Declaration of Mr. Bjørn Ole Haugsgjerd, MSc in Support of
`Inter Partes Review of U.S. Pat. No. 8,278,351
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`000001
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`I, Bjørn Ole Haugsgjerd, MSc, hereby declare and say:
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`1.
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`I have been retained by counsel for Petitioner Aker BioMarine AS to
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`provide an expert declaration in this action.
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`2.
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`Attached hereto are copies (Ex. 1047 and 1048) of the declarations I
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`submitted in the Inter Partes Reexamination of U.S. Patent 8,030,348. I submitted
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`a similar declaration in the Ex Parte Reexamination of U.S. Pat. No. 8,278,351.
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`The attached exhibits (Ex. 1047 and 1048) provide the details on my repeats of
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`krill oil extractions according to Beaudoin I (Ex. 1002), Beaudoin II (Ex. 1003)
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`and Maruyama (Ex. 1004). The resulting samples were sent to Dr. Richard van
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`Breemen and Dr. Thomas Gundersen for further analysis.
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`3.
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`I further declare that all statement made herein of my own knowledge
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`are true and that all statements made on information and belief are believed to be
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`true; and further that these statements were made with the knowledge that willful
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`false statements and the like so made are punishable by fine or imprisonment, or
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`both, under section 1001 of title 18 of the United States Code, and that such willful
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`false statements may jeopardize the validity of the application or any patent issued
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`thereon.
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`000002
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`000003
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`000003
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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
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`In re Inter Partes Reexamination of U.S. Patent No. 8,030,348
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`NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING
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`POLYUNSATURATED FATTY ACIDS AND THEIR APPLICATIONS
`
`4 October 2011 to Sampalis
`
`Declaration by Bjorn Ole Haugsgjerd, MSC, in Support of Reguest
`for Inter Partes Reexamination of
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`U.S. Patent NO. 8,030,348
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`EFS WEB Filed
`
`Mail Stop Inter Partes Reexam
`Commissioner for Patents
`P.O. Box 1450
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`Alexandria, VA 22313-1450
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`I, Bjorn Ole Haugsgjerd, MSc, state as follows:
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`My present position is Deputy manager at Nofima BioLab, Norway
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`At the request of Aker Biomarine ASA, I have extracted lipid fractions from Euphausia
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`superba and Euphausia pacifica by the methods described in Beaudoin I (WO 00/23546),
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`Beaudoin II (Canadian Application 2,251,265) and Maruyama (Japanese Laid Open Application
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`2909508). Following the extraction, I shipped the samples to Vitas AS, Oslo, Norway, for
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`analytical analysis. Frozen Euphausia superba was provided by Aker Biomarine ASA. Frozen
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`Euphausia pacifica was purchased from Fish and Fins Limited, East Sussex, UK.
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`000004
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`I repeated the Beaudoin I (pages 5-6 and Table I9) and Beaudoin II (page 2 and Table
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`I 1) extractions with acetone in the first step and then either ethanol or ethyl acetate in the second
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`step. The extractions were performed as described in Beaudoin I and Beaudoin II. Separate
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`extractions were conducted for Euphausia superba and Euphausia pacific. I utilized the
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`following protocol:
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`0 Grind frozen krill at 4C to reduce particle size to less than 5mm.
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`0 Extract with acetone at 4C at a samplezacetone ratio of I :6 (w/V) for 2 hours with 20
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`minutes of swirling.
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`0 Filter on organic solvent resistant filter paper under reduced pressure at 4C.
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`0 Wash solid material on filter with a samplezacetone ratio of 1:2 (w/v) with pure and cold
`acetone.
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`0 Combine filtrates, mark with Fraction I - Acetone extract E. pacifica (or superba) and
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`store at -20C.
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`0 Divide solid material on filter into two aliquots, aliquot I and aliquot 2.
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`0 Extract aliquot I with pure ethanol at samplezethanol ratio of I22 (w/v) for 30 minutes at
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`Filter on organic solvent resistant filter paper under reduced pressure at 4C.
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`0 Evaporate solvent under reduced pressure to provide Fraction Ila.
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`0 Extract aliquot 2 with pure ethyl acetate at samplezethyl acetate ratio of I :2 (w/V) for 30
`minutes at 4C.
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`Filter on organic solvent resistant filter paper under reduced pressure at 4C.
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`0 Evaporate solvent under reduced pressure to provide Fraction Ilb.
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`000005
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`0 Divide fraction Ila and llb into three aliquots and mark the samples according to the table
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`below. Flush all samples with nitrogen gas before sealing to provide an inert atmosphere.
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`Temperature
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`Fraction number
`Time (min) Marking of sample
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`treatment (°C)
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`1--
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`iTE
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`--
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`TT 0
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`Store all samples at —20C until further analysis
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`0 Heat treatments to be conducted at Vitas AS according to the table above before analysis.
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`000006
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`I repeated the Maruyama extractions with ethanol in the first step followed by washing
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`with acetone in the second step. Separate extractions were conducted for Euphausia superba and
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`Euphausia paczfica. The extractions were performed as described in Maruyama with the
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`exception that the extracts were not further purified by gel chromatography as described in
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`Maruyama. Thus, the extracts obtained were crude extracts as described by Maruyama. We
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`utilized the following protocol:
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`0 Vacuum freeze dry krill until water content is 6% or less.
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`I-lomogenize freeze-dried krill with 100% ethanol (20 parts ethanol/l part krill, w/w)
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`until total lipids are extracted.
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`0 Remove ethanol by evaporation under reduced pressure.
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`0 Re—extract with l0 parts ethanol w/w.
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`0 Remove ethanol by evaporation under reduced pressure.
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`Save a sample of the ethanol—extracted lipids for analysis. Label E. superba or E.
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`paczfica ETOH extract. Flush samples with nitrogen gas before scaling to provide an
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`inert atmosphere. Store at -20C until further analysis.
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`0 Dissolve ethanol extracted lipids in 20 parts acetone to separate into soluble and insoluble
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`fraction.
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`Separate insoluble fraction by centrifugation at 2000 rpm for 5 minutes and decant off the
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`acetone soluble fraction. Repeat wash and centrifugation with acetone twice.
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`0 Remove acetone by evaporation under reduced pressure to provide a crude phospholipid
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`extract. Save a sample of the crude phospholipid extract for analysis. Label E. superba or
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`E. pacifica crude PL extract. Flush samples with nitrogen gas before sealing to provide an
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`inert atmosphere. Store at -20C until further analysis.
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`000007
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`I further declare that all statement made herein of my own knowledge are true and that all
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`statements made on information and belief are believed to be true; and further that these
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`statements were made with the knowledge that willful false statements and the like so made are
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`punishable by fine or imprisonment, or both, under section 1001 oftitle 18 of the United States
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`Code, and that such willful false statements may jeopardize the Validity of the application or any
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`patent issued thereon.
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`Respectfully submitted,
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`l7 ’r( W;
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`ilvma;/,/Ll
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`fl» oglolw /8 ON
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`Bjorn Ole Haugsgjerd, MSC
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`Date
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`000008
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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`In re Inter Partes Reexamination of U.S. Patent No. 8,030,348
`
`NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING
`
`POLYUNSATURATED FATTY ACIDS AND THEIR APPLICATIONS
`
`4 October 2011 to Sampalis
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`Control No.:
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`95/001,774
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`October 19, 2011
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`Bruce Campbell
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`First Supplemental Declaration by Bjorn Ole Haugsgierd, MSc, in
`Support of Reguest for Inter Partes Reexamination of
`U.S. Patent N0. 8,030,348
`
`EFS WEB Filed
`
`Mail Stop Inter Partes Reexam
`Commissioner for Patents
`
`PO. Box 1450
`
`Alexandria, VA 22313-1450
`
`I, Bjnrn Ole Haugsgjerd, MSC, state as follows:
`
`My present position is Deputy Manager at Nofima BioLab, Norway
`
`At the request of Aker Biomarine ASA, I have extracted lipid fractions from Euphausia
`
`superba and Euphausia pacg’/ica by the methods described in Beaudoin I (WO 00/23546),
`
`Beaudoin II (Canadian Application 2,251,265). Following the extraction, I shipped the samples
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`to Vitas AS, Oslo, Norway, and Dr. Richard Van Breemen, of the University of Illinois for
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`analytical analysis. Frozen Euphausia superba and Euphausia paczfica were provided by Aker
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`Biomarine ASA.
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`000009
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`I repeated the Beaudoin I (pages 5-6 and Table 19) and Beaudoin II (page 2 and Table
`
`11) extractions with acetone in the first step and then either ethanol or ethyl acetate in the second
`
`step. The extractions were performed as described in Beaudoin I and Beaudoin II. Separate
`
`extractions were conducted for Euphausia superba and Euphausia pacific. I utilized the
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`following protocol:
`
`0 Grind frozen krill at 4C to reduce particle size to less than 5mm.
`
`0 Extract with acetone at 4C at a samplezacetone ratio of 1:6 (w/v) for 2 hours with 20 minutes
`of swirling.
`
`Filter on organic solvent resistant filter paper under reduced pressure at 4C.
`
`0 Wash solid material on filter with a sarnplezacetone ratio of l :2 (w/v) with pure and cold
`acetone.
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`0 Combine filtrates and evaporate solvent under reduced pressure.
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`0 Allow water residue obtained after evaporation to separate from oil phase (Fraction I) at 4C.
`Store Fraction I at 4C.
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`0 Divide solid material on filter into two aliquots, aliquot l and aliquot 2.
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`0 Extract aliquot l with pure ethanol at samplezethanol ratio of 1:2 (w/v) for 30 minutes at 4C.
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`Filter on organic solvent resistant filter paper under reduced pressure at 4C.
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`0 Evaporate solvent under reduced pressure to provide Fraction Ila.
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`0 Extract aliquot 2 with pure ethyl acetate at sample:ethyl acetate ratio of l :2 (w/v) for 30
`minutes at 4C.
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`Filter on organic solvent resistant filter paper under reduced pressure at 4C.
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`0 Evaporate solvent under reduced pressure to provide Fraction IIb.
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`0000010
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`0 Divide Fraction I into three aliquots. In an oil bath, heat one aliquot to 60C for 5 minutes and
`another aliquot to 125C for 15 minutes under an inert atmosphere, label as E. pacifica (or
`superba) Fraction I heat treated 60C or 125C. Store at —20C until further analysis. Label the
`third aliquot as E. pacifica (or superba) Fraction I not heated. Store at -20C until further
`analysis.
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`0 Divide Fraction Ila into three aliquots. In an oil bath, heat one aliquot to 70C for 5 minutes
`and another aliquot to 125C for 15 minutes under an inert atmosphere, label as E. pacifica (or
`superba) Fraction Ila/ethanol heat treated 70C or 125C. Store at -20C until further analysis.
`Label the third aliquot as E. pacifica (or superba) Fraction Ila/ethanol not heated. Store at -
`20C until further analysis.
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`0 Divide Fraction IIb into three aliquots. In an oil bath, heat one aliquot to 70C for 5 minutes
`and another aliquot to 125C for 15 minutes under an inert atmosphere, label as E. pacifica (or
`superba) Fraction IIb/ethyl acetate heat treated 70C or 125C. Store at -20C until further
`analysis. Label the third aliquot as E. pacifica (or superba) Fraction IIb/ethyl acetate not
`heated. Store at -20C until further analysis.
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`0 The samples were marked as follows:
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`Fraction number
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`Temperature
`treatment (°C)
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`Time (min)
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`Marking of sample
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`E. superba I — E. superba 1/ Acetone Not heated
`E. superbal
`E. superba I / Acetone 60C
`E. superbal
`E. superba I / Acetone 125C
`E. superba Ila — E. superba Ha / Etanol Not heated
`E. superba Ha
`E. superba IIa/ Etanol 70C
`E.superbana
`E. superba IIa/ Etanol l25C
`Not heated _ E. superba Ilb / Ethyl acetate Not heated
`E. superba IIb
`70
`E. superba Ilb — E. superba IIb / Ethyl acetate 70C
`125
`E. superba Hb —
`E. superba Hb / Ethyl acetate 125C
`apacmcai
`—
`E. pacifica I
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`E. pacifica I / Acetone Not heated
`E. pacifica I / Acetone 60C
`E. pacifica I/ Acetone 125C
`125
`Not heated — E. pacifica Ila / Etanol Not heated
`70
`E. pacifica Ila / Etanol 70C
`125
`E. pacifica Ila / Etanol 125C
`Not heated — E. pacifica llb / Ethyl acetate Not heated
`E. pacifica IIb / Ethyl acetate 70C
`E. pacifica Hb / Ethyl acetate 125C
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`E. pacifica I
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`E. pacifica Ila
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`E. pacifica Ila
`E. pacifica Ila
`E. pacifica Ilb
`E. pacifica IIb
`E. pacifica Ilb
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`0000011
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`It is my understanding that Neptune’s experts have criticized the heating procedure used
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`in the first set of repeats.
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`I understand that Dr. Gundersen heated the samples in a heat block that
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`had been placed in an oven, which is a reasonable heating method that one of skill in the art
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`would use to heat samples prior to analysis as described in Beaudoin.
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`I disagree with these
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`criticisms, but used an oil bath in this set of extractions to remove that criticism.
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`I further declare that all statement made herein of my own knowledge are true and that all
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`statements made on information and belief are believed to be true; and further that these
`
`statements were made with the knowledge that willful false statements and the like so made are
`
`punishable by fine or imprisonment, or both, under section 1001 of title 18 of the United States
`
`Code, and that such willful false statements mayjeopardize the validity of the application or any
`
`patent issued thereon.
`
`Respectfully submitted,
`
`_‘
`V’
`;~,)m4 yo %;-mwi. c J
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`”./
`
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`
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`/n/ / (5,
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`
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`,
`K L /#3
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`Bjorn Ole Haugsgjerd, MSc
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`Date
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`0000012