`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1047
`
`(cid:19)(cid:19)(cid:19)(cid:19)(cid:19)(cid:20)
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1047
`
`
`
`I repeated the Beaudoin I (pages 5-6 and Table 19) and Beaudoin II (page 2 and Table
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`11) 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 sample:acetone ratio of 1:6 (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 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
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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 sainple:ethy1 acetate 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 11b.
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`000002
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`
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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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`
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`
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`"--—
`“n—
`"--—
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`“--—
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`“-_
`
`"“—
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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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`000003
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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 Euphausz‘a superba and
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`Euphausia pacifica. 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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`0 Homogenize 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 10 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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`pacifica 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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`000004
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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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`)1/(4in/W:1“
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`V
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`q v:i0,ie//
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`270 H
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`Bjorn Ole Haugsgjerd, MSc
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`Date
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`000005
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`