IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`
`
`AKER BIOMARINE AS
`Petitioner
`
`v.
`
`NEPTUNE TECHNOLOGIES AND BIORESOURCES INC.
`Patent Owner
`
`
`CASE IPR: Unassigned
`
`Declaration of Dr. Ivar Storrø
`
`
`
`
`
`
`
`000001
`
`
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`
`
`AKER BIOMARINE AS
`Petitioner
`
`v.
`
`NEPTUNE TECHNOLOGIES AND BIORESOURCES INC.
`Patent Owner
`
`
`CASE IPR: Unassigned
`
`Declaration of Dr. Ivar Storrø
`
`
`
`
`
`
`
`000001
`
`
`
`
`
`I, Dr. Ivar Storrø, state as follows:
`
`1. My current address is: Ullins vei 16 a, Trondheim, Norway. My present
`
`position is senior advisor at SINTEF Fisheries and Aquaculture. My Curriculum
`
`Vitae is attached hereto as Exhibit 1. I am being compensated for this analysis. I
`
`have considered the following materials in preparing this report:
`
`Exhibit
`
`Description
`
`No.
`
`1001
`
`U.S. Pat. No. 8,278,351 to Sampalis (“’351”)
`
`1002
`
`WO 00/23546 to Beaudoin (“Beaudoin I”)
`
`1003
`
`Canadian Application 2,251,265 to Beaudoin (“Beaudoin II”)
`
`1010
`
`WO97/39759 to Stoll (“Stoll”)
`
`1011
`
`Final Prospectus dated May 11, 2001 (“Final Prospectus”)
`
`1012
`
`“Neptune Technologies & Bioressources Soon to Obtain a Major
`
`Patent in Over 30 Countries” (“2011 Press Release,”)
`
`000002
`
`
`
`1031
`
`Winther et al., Elucidation of Phosphatidylcholine Composition in
`
`Krill Oil Extracted from Euphausia superba, Lipids 46(1):25-36
`
`(2011)(“Winther”)
`
`1032
`
`Eichberg, “Lecithin – It Manufacture and Use in the Fat and Oil
`
`Industry,” Oils and Soap 51-54, 1939 (“Eichberg”)
`
`1033
`
`Balassa et al., Microencapsulation in the Food Industry, Critical
`
`Reviews in Food Technology, 2:2, 245-265 (1971)(“Balassa”)
`
`1034
`
`Buchi R-220 Rotovapor® Manual
`
`1035
`
`Johnson and Lucas, Comparison of Alternative Solvents for Oils
`
`Extraction, JAOCS 60(2):229-242 (1983)
`
`1036
`
`U.S. Pat. No. 4,714,571
`
`1037
`
`Grit et al., Int. J. Pharmaceutics 50:1-6 (1989)
`
`1038
`
`Herman and Groves, Pharmaceutical Research 10(5):774-776 (1993)
`
`1049
`
`Declaration of Dr. Thomas Gundersen submitted during inter partes
`
`reexamination of parent patent U.S. 8,030,348 (“Gundersen Decl.”)
`
`000003
`
`
`
`1050
`
`Supplemental Declaration of Dr. Thomas Gundersen submitted
`
`during inter partes reexamination of parent patent U.S. 8,030,348
`
`(“Gundersen Supp. Decl.”)
`
`1051
`
`Declaration of Dr. Earl White submitted during prosecution of parent
`
`patent U.S. 8,030,348 (“2011 White Decl.”)
`
`1052
`
`Supplemental Declaration of Dr. Earl White submitted during
`
`prosecution of parent patent U.S. 8,278,351 (“White Supp. Decl.”)
`
`1053
`
`Declaration of Dr. Jacek Jaczynski from inter partes reexamination
`
`of the parent patent U.S. 8,030,348 (“Jaczynski Reexam. Decl.”)
`
`1054
`
`Declaration of Dr. Yeboah submitted during inter partes
`
`reexamination of parent patent U.S. 8,030,348 (“Yeboah Reexam
`
`Decl.”)
`
`1055
`
`Supplemental Declaration of Dr. Earl White submitted during inter
`
`partes reexamination of parent patent U.S. 8,030,348 (“White Supp.
`
`Reexam. Decl.”)
`
`1056
`
`Declaration of Dr. Shahidi submitted during inter partes
`
`000004
`
`
`
`reexamination of parent patent U.S. 8,030,348 (Shahidi Reexam.
`
`Decl.”
`
`1057
`
`Declaration of Dr. Tina Sampalis submitted during inter partes
`
`reexamination of parent patent U.S. 8,030,348 (Sampalis”)
`
`1058
`
`Declaration of Dr. Yeboah submitted during prosecution of parent
`
`patent U.S. 8,278,351 (“Yeboah ‘351 Decl.”)
`
`1059
`
`Declaration of Dr. Shahidi submitted during prosecution of parent
`
`patent U.S. 8,278,351 (Shahidi ‘351 Decl.”)
`
`1060
`
`Declaration of Dr. Jaczynski submitted during prosecution of parent
`
`patent U.S. 8,278,351 (Jaczynski ‘351 Decl.”)
`
`1061
`
`April 2, 2012 Response to Office Action, ‘351 patent
`
`1068
`
`Medina et al., J. Amer. Oil Chem. Soc. 71(5):479-82 (1994)
`
`1069
`
`U.S. Patent No. 8,030,348
`
`1079
`
`Declaration of Dr. Chong Lee submitted during inter partes
`
`reexamination of parent patent U.S. 8,030,348 (“Lee Reexam Decl.”)
`
`
`
`000005
`
`
`
`2.
`
`I was previously asked by Aker Biomarine ASA to review Declarations and
`
`references submitted by Neptune Bioresources and Technologies (Neptune) in the
`
`pending re-examination proceedings for U.S. Pat. Nos. 8,030,348 (Ex. 1069) and
`
`8,278,351 (Ex. 1001)(in which I submitted Declarations similar to this Declaration)
`
`and the very similar Declarations and references submitted by Neptune during the
`
`prosecution of the ‘351 patent. I have reviewed the ‘351 patent and the claims
`
`contained therein. It is my understanding that the ‘351 patent contains claims to
`
`krill extracts (claims 1-23); capsules, tablets, solutions, syrups or suspensions
`
`comprising the krill extracts (claims 24-46); foods, beverages or nutritional
`
`supplements comprising the krill extracts (claims 47-69); cosmetics comprising the
`
`krill extracts (claims 70-93); and Antarctic krill extracts (claim 94). The common
`
`feature of the independent claims (claims 1, 24, 47, 70 and 94) is the requirement
`
`of
`
`000006
`
`
`
`
`
`The dependent claims add limitations on other components of the composition
`
`such as omega-3 content, polyunsaturated fatty acid content, content of other lipid
`
`classes, metal content and antioxidant content.
`
`The Cold Extraction Process Described in Table 19 of Beaudoin I and Table
`
`11 of Beaudoin II Would Produce Krill Extracts with All of the Claimed
`
`Features
`
`3.
`
`Table 19 of Beaudoin I (Ex. 1002) and Table 11 of Beaudoin II (Ex. 1003)
`
`provide protocols entitled “Optimal conditions for lipid extraction from aquatic
`
`000007
`
`
`
`animal tissues.” Each of these Tables describes multistep extraction procedures
`
`that are conducted at low temperatures. Solvents are removed by evaporation under
`
`reduced pressure. It is well known in the art to remove solvents by evaporation
`
`under pressure when the product is intended for human consumption. The solvent
`
`extraction of phospholipids and oils for human consumption has been practiced for
`
`decades and solvent removal is a problem that was solved long ago.1 More
`
`recently, phospholipids have found use in the pharmaceutical industry, particularly
`
`in emulsions and liposomes. Solvent extraction of the phospholipids and use of
`
`evaporation under reduced pressure (and in particular rotary evaporation) to
`
`remove solvents in that industry is well known as well.2 Thus, when Beaudoin I
`
`and II indicate removal of solvent under reduced pressure, a person of ordinary
`
`skill in the art would immediately recognize that rotary evaporation or other
`
`evaporative procedures under reduced pressure could be used to remove solvents to
`
`a desired level that is safe for human consumption. Such techniques would be
`
`effective for removing ethanol or ethyl acetate as described in Beaudoin I and II.
`
`Removal of solvent under reduced pressure has the effect of allowing solvent
`
`removal at lower temperatures than at atmospheric pressure. Thus, solvent
`
`removal under reduced pressure as taught in Table 19 of Beaudoin I and Table 11
`
`1 See, e.g., Eichberg, “Lecithin – It Manufacture and Use in the Fat and Oil Industry,” Oils and Soap 51-54, 1939
`(Ex. 1022) and Johnson and Lucas, Comparison of Alternative Solvents for Oils Extraction, JAOCS 60(2):229-242
`(1983)(Ex. 1023).
`2 See, e.g., U.S. Pat. No. 4,714,571 at Column 11, lines 35-57 (Ex. 1024).
`
`000008
`
`
`
`of Beaudoin II would allow for gentle treatment of the compositions with a
`
`minimal amount of heating. In my opinion, the issues raised by Neptune in
`
`relation to heating of the samples is in fact addressed by the protocols of Table 19
`
`of Beaudoin I and Table 11 of Beaudoin II. Following these protocols would
`
`produce an undegraded krill oil.
`
`4.
`
`Beaudoin I and Beaudoin II do not teach heating for any other reason than to
`
`remove traces of solvent prior to conducting analytical procedures. The Beaudoin
`
`I extraction process is first described at pages 5-6 of Beaudoin I. Solvent is
`
`removed by evaporation under reduced pressure, with flash evaporation or spray
`
`drying being optional. Beaudoin I, p. 6, lines 7-13 and 17-19. There is no
`
`reference to removing solvent by heating to 125°C. The second place that
`
`Beaudoin I extraction process is described is in Table 19, p. 28. According to
`
`Beaudoin I, “Table 19 shows the best mode of the method in accordance with the
`
`present invention for lipid extraction of aquatic animal tissues.” Beaudoin I at p.
`
`11, lines 6-7. There is no reference to removing solvent by heating to 125°C in
`
`Table 19.
`
`
`
`5.
`
`There are two references to heating to 125°C in Beaudoin I, both related to
`
`analytical methods for studying the oil that was produced by the Beaudoin I
`
`000009
`
`
`
`process. First, on p. 7 Beaudoin I states that “To get rid of organic solvents, lipid
`
`fractions I and II are warmed to about 125°C for about 15 minutes under an inert
`
`atmosphere.” This statement occurs in the section of the specification labeled
`
`“Comparative examples” and immediately follows a description of analytical
`
`methods used to analyze the fractions including thin layer chromatography and gas
`
`liquid chromatography. Beaudoin I at p. 6, line 27 and p. 7, lines 9-20. Second, on
`
`page 10 at lines 19-30, Beaudoin I provides that: “To get rid of traces of solvents,
`
`it is important to briefly heat (to about 125°C, for about 15 min) the oil under
`
`nitrogen.” This statement also occurs in relation to analytical methods performed
`
`on Fractions I and II. The only references to heating to 125°C occur in reference to
`
`analytical procedures performed on Fractions I and II and are not part of the
`
`described production process for Fractions I and II. In any event, even if
`
`Neptune’s contentions that Beaudoin I and II do teach heating are accepted, that
`
`does not change my analysis. The heated samples would be undegraded and
`
`suitable for human consumption as explained in more detail below beginning at
`
`paragraph 14.
`
`
`
`6.
`
`Table 19 of Beaudoin I and Table 11 of Beaudoin II teach a method where
`
`all extraction steps are conducted at 4C and solvent is removed under reduced
`
`pressure. There is no step requiring heating. I have reviewed two references from
`
`0000010
`
`
`
`Neptune which further describe the Beaudoin methods: Neptune Technologies &
`
`Bioressources Inc. Final Prospectus dated May 11, 2001 (Ex. 1011) and a June 14
`
`2001 Press Release (Ex. 1012). Both of these references discuss the
`
`OceanExtractTM process which is the process described in the Beaudoin I and II
`
`publications. The May 11 2001 Prospectus states:
`
`
`
`
`
`None of the stages in the transformation process involve heating the raw
`
`material. This aspect of the Neptune OceanExtractTM process has the effect
`
`of preserving the biological activity of the krill substances, the properties of
`
`which are widely sought after by the nutraceutical, cosmetics and
`
`pharmaceutical industries.
`
`The June 14 2001 Press Release states:
`
`
`
`The industrial process for extracting Neptune OceanExtract(TM) applied to
`
`Krill, developed in close collaboration with the Université de Sherbrooke,
`
`has the distinct advantage of being a cold process that preserves the
`
`biological activity and stability of the nutritional qualities intrinsic to the
`
`most highly sought substances of Krill, such as its powerful antioxidants,
`
`phospholipids and Omega-3-6-9 fatty acids, while ensuring the microbial
`
`0000011
`
`
`
`destruction of the obtained extracts. The result is products that are healthy
`
`and safe for human consumption, with moreover an extensive lifespan for
`
`marketing purposes, without any preservatives. Neptune OceanExtract(TM) is
`
`also a simple process that allows a high extraction yield and the recycling
`
`and recovery of the extraction residues.
`
`
`
`Both of these references confirm that the processes disclosed in Beaudoin I and II
`
`do not involve heating to remove solvent and that a cold process is disclosed that
`
`preserves the biological activity of substances in the krill oil including
`
`phospholipids. The oils obtained from the process are suitable for human
`
`consumption as described in the references.
`
`7.
`
`I have further compared the protocols described in Table 19 of Beaudoin I
`
`and Table 11 of Beaudoin II with the extraction method taught in the parent ‘351
`
`patent at Column 18. As indicated in the ‘351 patent at Column 18, lines 23-27,
`
`the method is very similar to the method described in Beaudoin I and II. The table
`
`following this paragraph provides a comparison of the methods. The ‘351 patent
`
`teaches removal of solvent by flash evaporation, spray drying, or evaporation. The
`
`temperature for evaporation is not specified. Given the lack of further disclosure
`
`on solvent removal, it would be reasonable for a person of ordinary skill in the art
`
`0000012
`
`
`
`to turn to Beaudoin I or II and remove solvent under reduced pressure as described
`
`in Tables 19 and 11. I further note that both the ‘351 patent and Beaudoin I and II
`
`are consistent in conducting the extractions steps with solvent at under 5oC. Thus,
`
`if one conducted extractions from krill as described in Table 19 of Beaudoin I and
`
`Table 11 of Beaudoin II, the resulting extract would have a chemical composition
`
`that is essentially identical to the chemical compositions listed in Columns 15-19
`
`and Tables 1-7 of the ‘351 patent.
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`Patent
`
`
`
`
`
`SUMMARY OF THE
`INVENTION
` Extraction process
`
`
`
`The general extraction
`method of the present
`invention will now be
`described.
`
`Extraction of the
`phospholipid
`composition from the
`biomass is generally
`carried out by a method
`similar to the one
`described in commonly
`owned PCT publication
`
`0000013
`
`
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`
`
`Patent
`
`
`
`Fresh (or frozen)
`material (Euphausia
`pacifica and other
`species) is suspended in
`cold acetone for a given
`period of time at low
`temperature (5°C or
`lower). A ratio of krill
`acetone 1:6 (w/v) and
`an incubation time of 2
`
`The starting material
`consisting of freshly
`harvested and preferably
`finely divided marine
`and aquatic animal
`material is subjected to
`acetone extraction, for at
`about two hours and
`preferably overnight.
`
`
`
`number WO 00/23546,
`published on Apr. 27,
`2000, the disclosure of
`which is incorporated
`herein by reference.
`
`Preferably, freshly
`harvested and finely
`divided marine and
`aquatic animal material
`is subjected to acetone
`extraction, for at least
`about two hours and
`preferably overnight.
`
`0000014
`
`
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`Patent
`
`
`
`
`
`However extraction time
`is not critical to the yield
`of lipid extraction.
`
`However, extraction
`time is not critical to the
`yield of lipid extracted.
`
`
`
`h in acetone were found
`to be optimal.
` Alternatively the
`material can be kept in
`an equal volume of
`acetone at low
`temperature for long
`periods of time
`(months) under inert
`atmosphere.
`
`
`
`The size of the material To facilitate extraction, it Particle sizes of
`
`0000015
`
`
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`Patent
`
`
`
`
`
`is an important factor
`for the penetration of
`acetone. Indeed, it is
`preferable to grind
`material with
`dimensions superior to
`5 mm before getting it
`in contact with acetone.
`
`Fresh (or frozen)
`material (Euphausia
`pacifica and other
`species) is suspended in
`cold acetone for a given
`period of time at low
`temperature (5°C or
`
`
`
`is preferable to use
`particles of less than
`5mm in diameter.
`
`comminuted crustacean
`less than 5 mm are
`preferred.
`
`Extraction is preferably
`conducted under inert
`atmosphere and at a
`temperature in the order
`of about 5°C or less.
`
`
`The extraction is
`preferably conducted
`under an inert
`atmosphere and at a
`temperature of about 5
`degrees Celsius or less.
`
`0000016
`
`
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`
`
`lower).
` The suspension is
`swirled for a short
`period of time (about 20
`min) after acetone
`addition.
`A ratio of krillacetone
`1:6 (w/v) and an
`incubation time of 2 h in
`acetone were found to
`be optimal.
`
`Patent
`
`
`
`
`
`The mixture may be
`agitated during
`extraction and a volume
`ratio of about 6:1 of
`acetone to biomass is
`generally most
`preferred.
`
`Preferably, the beginning
`of the extraction will be
`conducted under
`agitation for about 10 to
`40 minutes, preferably
`20 minutes.
`
`Although extraction time
`is not critical, it was
`found that a 2 hour
`extraction with 6:1
`volume ratio of acetone
`to marine and aquatic
`animal material is best.
`
`0000017
`
`
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`
`
`After filtration on an
`organic solvent
`resistant filter (metal,
`glass or paper) the
`residue is washed with
`two volumes of pure
`acetone.
`
`Patent
`
`
`
`
`
`
`The solubilized lipid
`fractions are separated
`from the solid material
`by standard techniques
`including, for example,
`filtration, centrifugation
`or sedimentation.
`
`Filtration is preferably
`used.
`
`After separation by
`filtration on an organic
`solvent resistant filter
`(metal, glass or paper)
`
`The solubilized lipid
`fraction is separated
`from the solid starting
`material by known
`techniques, for example,
`by filtration,
`centrifugation or
`sedimentation.
`Filtration is preferred.
`The residue is
`optionally washed with
`acetone to recover more
`lipid and the acetone
`
`0000018
`
`
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`
`
`The combined filtrates
`are evaporated under
`reduced pressure.
`N/A
`
`The water residue
`
`Patent
`
`
`
`
`
`the residue is optionally
`washed with pure
`acetone, preferably two
`volumes (original
`volume of material) to
`recover yet more lipids.
`
`The combined filtrates
`are evaporated under
`reduced pressure.
`
`Optionally, flash
`evaporation or spray
`drying may be used.
`
`The water residue
`
`removed by flash
`evaporation or spray
`drying.
`
`Water residue is
`
`0000019
`
`
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`Patent
`
`
`
`
`
`obtained after
`evaporation is allowed
`to separate from the oil
`phase (fraction I) at low
`temperature.
`The solid residue
`collected on the fitter is
`suspended and
`extracted with two
`volumes (original
`volume of frozen
`material) of 100%
`ethanol.
`
`allowed to separate
`from the lipid extract at
`low temperature.
`The solid residue left on
`the filter from the initial
`extraction is suspended
`and extracted with 95/5
`ethyl acetate/ethanol,
`preferably two volumes
`(original volume of
`material).
`
`
`
`obtained after
`evaporation is allowed to
`separate from the oil
`phase (fraction I) at low
`temperature.
`The solid residue
`collected on the filter is
`suspended and extracted
`with alcohol, such as
`ethanol, isopropanol, t
`butanol or alternatively
`with ethyl acetate,
`preferably two volumes
`(original volume of
`material).
`
`
`0000020
`
`
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`
`
`The ethanol filtrate is
`evaporated leaving a
`second fraction of lipids
`(identified as fraction
`II).
`
`Incubation times in
`solvents may vary.
`Particle size effect the
`recovery of lipids and
`the material could be
`ground in various sizes
`of particles, depending
`on the grinder used.
`
`
`Patent
`
`
`
`
`
`
`The filtrate is evaporated
`leaving a second fraction
`of lipids (identified as
`fraction II).
`
`Although the extraction
`period is not critical, it
`was found that an
`extraction time of about
`30 minutes is sufficient
`at temperatures below
`about 5°C.
`
`
`The filtrate is
`evaporated yielding a
`second fraction of lipids.
`
`Extraction period is not
`critical although it is
`preferred to extract for
`about 30 minutes at a
`temperature below
`about 5 degrees Celsius.
`
`0000021
`
`
`
`’265, Beaudoin II
`
`WO/23546, Beaudoin I
`
`’351 Sampalis Patent
`
`Patent
`
`Patent
`
`
`
`
`
`
`
`Temperature of the
`organic solvents, except
`tbutanol, and
`temperature of the
`sample are not critical
`parameters, but it is
`preferable to be as cold
`as possible.
`
`
`
`Temperature of the
`organic solvents and
`temperature of the
`sample are not critical
`parameters, but it is
`preferable to be as cold
`as possible.
`
`
`
`8.
`
`Formulation of phospholipid compositions into forms suitable for human
`
`consumption is also well known in the art.3 Suitable forms include tablets,
`
`capsules, solutions, syrups, suspension, powders and other known pharmaceutical
`
`3 See, e.g., WO97/39759 at p. 5, line 13-p. 6, line 2 and p. 10, line 10 - p. 9, line 12 (Ex. 1010).
`
`0000022
`
`
`
`forms.4 I further note that krill oil in itself is a solution in that it contains various
`
`dissolved components.
`
`9.
`
`I further note that Dr. Sampalis of Neptune submitted a Declaration in the
`
`re-examination of the ‘348 patent. (Ex. 1057). Dr. Sampalis states the following
`
`in her Declaration: “I discovered a krill oil extract that uniquely contained
`
`substantial amounts of phospholipids bearing Omega-3 fatty acids (such as EPA
`
`and DHA) and was suitable for human consumption.” Sampalis Decl. ¶7. “I
`
`achieved this breakthrough, in part, by avoiding the use of heat during the
`
`extraction process.” Id. ¶8. Dr. Sampalis then states that heating to 60oC, 70oC or
`
`above 100oC to remove solvents causes hydrolysis of the key phospholipids. Id.
`
`To avoid this problem and to remove sufficient of amounts of solvents, Dr.
`
`Sampalis states that “our solvent removal employed methods such as rotary
`
`evaporation and was conducted in the cold for extensive periods of time.” Id. ¶9.
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`Dr. Sampalis further states: “Therefore, I instructed that the solvent evaporation
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`experiments for the ‘348 patent be conducted using rotary evaporation at
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`temperatures under 5oC. These involved long periods of time to remove sufficient
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`solvent to render the extract suitable for human consumption.” Id. ¶10.
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`“Accordingly, among other major advances, the ‘348 patent encompasses the
`
`4 Id.
`
`0000023
`
`
`
`removal of extraction solvent at low temperatures and thus, unlike prior teaching of
`
`others, retains the structural integrity of the key components of the extract and
`
`allows the extract to be suitable for human consumption.” As I discussed above,
`
`Neptune Technologies & Bioressources Inc. Final Prospectus dated May 11, 2001
`
`(Ex. 1011) and a June 14 2001 Press Release (Ex. 1012) both state that the
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`OceanExtractTM process as disclosed in the Beaudoin publications is a cold
`
`extraction process that avoids heating and produces a product suitable for human
`
`consumption. There is no substantive difference between the Beaudoin process
`
`and the process described in the ‘351 patent. Therefore, the Beaudoin process
`
`would necessarily produce compositions containing the claimed phospholipid
`
`molecules.
`
`10. Furthermore, I have analyzed the specification of the parent ‘351 patent. At
`
`Column 18 lines 49-50, the patent provides:
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`The extraction is preferably conducted under an inert atmosphere and at a
`
`temperature of about 5 degrees Celsius or less.
`
`The extraction process is this case is the mixing of solvent and "finely divided
`
`marine and aquatic material" and the subsequent separation of these two phases: a
`
`krill extract and solid residue. There is however no example or disclosure in the
`
`‘351 patent describing removal of solvent from a krill extract via rotary
`
`0000024
`
`
`
`evaporation at less than 5oC for long periods time as described by Dr. Sampalis.
`
`Removal of solvent from krill extracts is described in the ‘351 patent (beginning at
`
`Column 18 line 54 and continuing to column 19) which provides:
`
`The solubilized lipid fraction is separated from the solid starting material by
`
`known techniques, for example, by filtration, centrifugation or
`
`sedimentation. Filtration is preferred. The residue is optionally washed with
`
`acetone to recover more lipid and the acetone removed by flash evaporation
`
`or spray drying. Water residue is allowed to separate from the lipid extract at
`
`low temperature.
`
`The solid residue left on the filter from the initial extraction is suspended
`
`and extracted with 95/5 ethyl acetate/ethanol, preferably two volumes
`
`(original volume of material). The filtrate is evaporated yielding a second
`
`fraction of lipids. Extraction period is not critical although it is preferred to
`
`extract for about 30 minutes at a temperature below about 5 degrees Celsius.
`
`There is no mention of rotary evaporation at 5oC for long periods of time for either
`
`the acetone extraction step or the ethyl acetate/ethanol extraction step. The ‘351
`
`patent indicates acetone can be removed by flash evaporation or spray drying. No
`
`conditions are specified for either method. I particularly note that spray drying of
`
`0000025
`
`
`
`oils require use of both an excipient and high temperatures.5 Inlet air temperatures
`
`generally range from 140 to 816oC. Although the temperature of the non-
`
`evaporating material is slightly lower due to the evaporation of solvent, the use of
`
`spray drying for solvent removal as taught by the ‘351 patent is clearly inconsistent
`
`with the statements by Dr. Sampalis that heat would destroy the desired
`
`phospholipids. Flash evaporation could be seen as a form of rotary evaporation,
`
`but no conditions are provided, much less the special conditions discussed by Dr.
`
`Sampalis. If it is true as stated by Dr. Sampalis that the key to obtaining a
`
`composition containing phospholipids with EPA and DHA on the phospholipid is
`
`to avoid heating during solvent removal, then the ‘351 patent contains no
`
`disclosure of how to make such phospholipid compositions because the method of
`
`rotary evaporation at less than 5oC for long periods of time is not disclosed. In
`
`fact, use of spray drying would destroy the phospholipids if Dr. Sampalis’ theory is
`
`correct.
`
`11. Dr. Sampalis states that solvent was removed by rotary evaporation at less
`
`than 5oC for long periods of time. Sampalis Decl. (Ex. 1057) ¶10. This is a very
`
`unusual use of rotary evaporation and is inconsistent with the way one of ordinary
`
`skill in the art would utilize rotary evaporation. Rotary evaporation is a process
`
`5 Balassa et al., Microencapsulation in the Food Industry, Critical Reviews in Food Technology, 2:2, 245-265
`(1971)(Ex. 1033).
`
`0000026
`
`
`
`where samples are gently heated under a vacuum while a flask containing the
`
`sample is rotated in a heat bath. The vacuum lowers the effective boiling point of
`
`the solvent allowing for removal at lower temperature. Rotation in the flask in the
`
`heat bath allows efficient heat transfer, while preventing local heating, allowing
`
`mixing and facilitating the removal of solvent from the sample. The solvent is
`
`collected in a cooled solvent receiving flask. Commercial rotary evaporation
`
`machines are designed to be operated at a sample bath temperature of 60oC with
`
`the vacuum set so that the boiling point of the solvent is at 40oC and the cooling
`
`bath set at no higher than 20oC.6 The conditions described by Dr. Sampalis are
`
`very different from these standard conditions and would require either substantial
`
`modification of commercial systems or building a special system from scratch.
`
`12. Dr. Sampalis states that the rotary evaporation conditions that are described
`
`in her Declaration (but not in the ‘351 patent) are able to produce a krill oil extract
`
`with low levels of residual solvent that is suitable for human consumption.
`
`Sampalis Decl. ¶7. Table 5.4 at p. 22 in the Buchi R-220 Manual is A Table of
`
`Solvents with vacuum in mbar required for boiling at 40oC. As described in the
`
`preceding paragraph, standard rotary evaporation systems are designed to operate
`
`by adjusting the vacuum to projected boiling point of the target solvent at 40oC. It
`
`6 Buchi R-220 Rotovapor® Manual (Ex. 1034).
`
`0000027
`
`
`
`would be very difficult, if not impossible, to establish conditions where the
`
`solvents (and associated water) used in the ‘351 patents could be effectively
`
`removed from a krill oil extract at 5oC using rotary evaporation. Since the ‘351
`
`patent does not describe the conditions and equipment used, it impossible to
`
`evaluate whether or not solvent would be effectively removed in the manner
`
`claimed. I further note that the Sampalis Declaration does not describe the
`
`equipment, how it was modified, or how long the rotary evaporation needs to be
`
`conducted. “Long periods of time” is ambiguous.
`
`13. Dr. Sampalis states that “I discovered a krill oil extract that uniquely
`
`contained substantial amounts of phospholipids bearing Omega-3 fatty acids (such
`
`as EPA and DHA) and was suitable for human consumption.” Sampalis Decl. (Ex.
`
`1057) ¶7. There is no data in the ‘351 patent that support the statement that the
`
`extract contains “substantial amounts” of phospholipids bearing Omega-3 fatty
`
`acids. No quantitative data of the relative amounts of particular phospholipid
`
`species having EPA at the sn-1 or -2 positions is provided in the ‘351 patent.
`
`Figures 1-3 in the ‘351 patent are chromatograms of the product produced in
`
`Example 1. ‘351 Patent, Column 22, lines 14-22. Example 1 in turn describes the
`
`isolation of phospholipids by Thin Layer Chromatography (TLC), HPLC and
`
`subsequent analysis by Gas Chromatography (GC) and mass spectometry. ‘351
`
`0000028
`
`
`
`Patent, Column 22, line 35 – Column 24, line 45. Tables 8-10 present a summary
`
`of the results. The data and methods used provide no information on the presence
`
`or amount of phospholipids with EPA or DHA at the sn-1 and-2 positions of the
`
`phospholipid. In fact, the data indicate phospholipids with a mixture of fatty acids,
`
`including palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) in addition
`
`to EPA and DHA. Thus, from this data, it is impossible to determine of any one
`
`phospholipid species contained DHA or EPA at both sn-1 and -2 positions.
`
`Neptune’s Theory of Degradation of Phospholipids is not Supported by the
`
`Cited Evidence
`
`14.
`
`In its response dated April 2, 2102, in the parent ‘351 patent (Ex. 1061),
`
`Neptune made repeated arguments that heating would destroy the claimed
`
`phospholipid molecules. See April 2, 2012 Response at 30-35. Neptune states that
`
`“hydrolysis of phospholipids is known to occur with heat, as is well established in
`
`the literature.” As described above, there is no reason to believe that heating was a
`
`part of the Beaudoin process. Nevertheless, the references cited by Neptune in
`
`support of its theory of hydrolysis of phospholipids by heating are not applicable or
`
`relevant to the mild heating conditions of 125C for 15 minutes which Neptune
`
`alleges is taught by Beaudoin.
`
`0000029
`
`
`
`15. Neptune has no provided no experimental evidence supporting its theory of
`
`degradation of phospholipids by hydrolysis under the heating conditions described
`
`in the Beaudoin references. Such an experiment would be relatively simple and
`
`involve comparing a non-heated krill extract sample containing phospholipids with
`
`DHA or EPA at the sn-1 and -2 position with a sample of the krill extract heated as
`
`described in the Beaudoin references.
`
`16.
`
`Instead of addressing this issue experimentally, Neptune relied on the
`
`Declaration of Chong Lee (Ex. 1079) and several literature references: Medina et
`
`al., J. Amer. Oil Chem. Soc. 71(5):479-82 (1994)(Ex. 1068); Grit et al., Int. J.
`
`Pharmaceutics 50:1-6 (1989)(Ex. 1037); and Herman and Groves Pharmaceutical
`
`Research 10(5):774-776 (1993)(Ex. 1038). None of these references are relevant
`
`to whether phospholipids would have been hydrolyzed during the alleged
`
`Beaudoin heating step of 125C for 15 minutes.
`
`17. Medina et al. does not provide an analysis of phospholipids, instead Medina
`
`et al. analyzes degradation of trigylcerides. Table 1 of Medina et al. does list
`
`PC/PE, but contains no information on hydrolysis. Figure 1 of Medina et al.
`
`contains no discernible information on phospholipids. The rest of the paper
`
`discusses only hydrolysis of triglycerides.
`
`0000030
`
`
`
`18. Grit et al. analyzes liposomes, a dispersion of phospholipids in water. This
`
`solution has a high water content, and water is the continuous phase, as opposed to
`
`extracted krill oil where the oil is the continuous phase. A calculation based on an
`
`average molecular weight of phospholipid on 750, gives a concentration of
`
`phospholipid in Grits work of 2.2 % and water 97.8 %. As the rate of hydrolysis
`
`increases with the concentration of water (moisture), hydrolysis in liposomes can
`
`be expected to very different from hyd
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