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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
`
`Declaration by Bjorn Ole Haugsgierd2 MSc, in Support of Reguest
`for Inter Partes Reexamination of
`
`U.S. Patent NO. 8,030,348
`
`EFS WEB Filed
`
`Mail Stop Inter Partes Reexam
`Commissioner for Patents
`P.O. Box 1450
`
`Alexandria, VA 22313-1450
`
`1, Bjern 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 pacifica by the methods described in Beaudoin I (WO 00/23546),
`
`Beaudoin II (Canadian Application 2,251,265) and Maruyama (Japanese Laid Open Application
`
`2909508). Following the extraction, I shipped the samples to Vitas AS, Oslo, Norway, for
`
`analytical analysis. Frozen Euphausia superba was provided by Aker Biomarine ASA. Frozen
`
`Euphausia pacifica was purchased from Fish and Fins Limited, East Sussex, UK.
`
`000001
`
`

`

`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
`
`following protocol:
`
`0 Grind frozen krill at 4C to reduce particle size to less than 5mm.
`
`0 Extract with acetone at 4C at a sample:acetone ratio of 1:6 (w/v) for 2 hours with 20
`
`minutes of swirling.
`
`0 Filter on organic solvent resistant filter paper under reduced pressure at 4C.
`
`0 Wash solid material on filter with a samplezacetone ratio of 1:2 (w/v) with pure and cold
`acetone.
`
`0 Combine filtrates, mark with Fraction I — Acetone extract E. pacifica (or superba) and
`
`store at ~20C.
`
`0 Divide solid material on filter into two aliquots, aliquot l and aliquot 2.
`
`0 Extract aliquot l with pure ethanol at samplezethanol ratio of 1:2 (w/v) for 30 minutes at
`
`Filter on organic solvent resistant filter paper under reduced pressure at 4C.
`
`0 Evaporate solvent under reduced pressure to provide Fraction Ila.
`
`0 Extract aliquot 2 with pure ethyl acetate at sainple:ethy1 acetate ratio of 1:2 (w/v) for 30
`minutes at 4C.
`
`Filter on organic solvent resistant filter paper under reduced pressure at 4C.
`
`0 Evaporate solvent under reduced pressure to provide Fraction 11b.
`
`000002
`
`

`

`0 Divide fraction Ila and llb into three aliquots and mark the samples according to the table
`
`below. Flush all samples with nitrogen gas before sealing to provide an inert atmosphere.
`
`
`Temperature
`
`
`Fraction number
`Time (min) Marking of sample
`
`
`treatment (°C)
`
`
`
`
`
`
`"--—
`“n—
`"--—
`
`“--—
`
`“-_
`
`"“—
`
`Store all samples at —20C until further analysis
`
`0 Heat treatments to be conducted at Vitas AS according to the table above before analysis.
`
`000003
`
`

`

`I repeated the Maruyama extractions with ethanol in the first step followed by washing
`
`with acetone in the second step. Separate extractions were conducted for Euphausz‘a superba and
`
`Euphausia pacifica. The extractions were performed as described in Maruyama with the
`
`exception that the extracts were not further purified by gel chromatography as described in
`
`Maruyama. Thus, the extracts obtained were crude extracts as described by Maruyama. We
`
`utilized the following protocol:
`
`0 Vacuum freeze dry krill until water content is 6% or less.
`
`0 Homogenize freeze-dried krill with 100% ethanol (20 parts ethanol/l part krill, w/w)
`
`until total lipids are extracted.
`
`0 Remove ethanol by evaporation under reduced pressure.
`
`0 Re—extract with 10 parts ethanol w/w.
`
`0 Remove ethanol by evaporation under reduced pressure.
`
`Save a sample of the ethanol—extracted lipids for analysis. Label E. superba or E.
`
`pacifica ETOH extract. Flush samples with nitrogen gas before scaling to provide an
`
`inert atmosphere. Store at -20C until further analysis.
`
`0 Dissolve ethanol extracted lipids in 20 parts acetone to separate into soluble and insoluble
`
`fraction.
`
`Separate insoluble fraction by centrifugation at 2000 rpm for 5 minutes and decant off the
`
`acetone soluble fraction. Repeat wash and centrifugation with acetone twice.
`
`0 Remove acetone by evaporation under reduced pressure to provide a crude phospholipid
`
`extract. Save a sample of the crude phospholipid extract for analysis. Label E. superba or
`
`E. pacifica crude PL extract. Flush samples with nitrogen gas before sealing to provide an
`
`inert atmosphere. Store at -20C until further analysis.
`
`000004
`
`

`

`I further declare that all statement made herein of my own knowledge are true and that all
`
`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 oftitle 18 of the United States
`
`Code, and that such willful false statements may jeopardize the validity of the application or any
`
`patent issued thereon.
`
`Respectfully submitted,
`
`)1/(4in/W:1“
`
`V
`
`q v:i0,ie//
`
`270 H
`
`Bjorn Ole Haugsgjerd, MSc
`
`Date
`
`000005
`
`

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