UNITED STATES PATENT AND TRADEMARK OFFICE BEFORE THE PATENT TRIAL AND APPEAL BOARD RIMFROST AS Petitioner v. AKER BIOMARINE ANTARCTIC AS Patent Owner Case: IPR2017-00746 DECLARATION OF DR STEPHEN J. TALLON RIMFROST EXHIBIT 1006 page 0001
TABLE OF CONTENTS DECLARATION OF DR STEPHEN J. TALLON... 1 AGREEMENT TO PROVIDE EXPERT TESTIMONY... 8 BASES FOR OPINIONS GIVEN...10 NO FINANCIAL INTEREST IN PROCEEDING...16 QUALIFICATIONS AND RELEVANT EXPERIENCE...16 RELEVANT CONSIDERATIONS...21 LEVEL OF ORDINARY SKILL IN THE ART & THE POSITA...23 MOTIVATION TO COMBINE - OVERVIEW...24 THE 877 PATENT IS ENTITLED TO A PRIORITY...27 DATE NO EARLIER THAN JANUARY 28, 2008...27 CLAIM TERMS...28 krill oil means lipids extracted from krill...28 polar solvent means solvent or mixtures of solvents capable of extracting polar lipids comprising phospholipids...34 2 RIMFROST EXHIBIT 1006 page 0002
to denature lipases and phospholipases means to alter the conformational structure of lipases and phospholipases to reduce lipid and phospholipid decomposition...37 freshly harvested krill means recently caught krill that has not significantly degraded...41 polar entrainer means polar solvent additive to aid in extraction...43 PRIOR ART...44 Sampalis I (Exhibit 1012)...44 Budziński (Exhibit 1008)...47 Catchpole (Exhibit 1009)...52 Fricke (Exhibit 1010)...56 Fricke 1984 analysis of omega-3 fatty acids...63 Bottino (Exhibit 1007)...68 Randolph (Exhibit 1011)...71 Tanaka I (Exhibit 1014)...74 Tanaka II (Exhibit 1015)...77 3 RIMFROST EXHIBIT 1006 page 0003
Tehoharides (Exhibit 1030)...81 Sampalis II (Exhibit 1013)...82 Grantham (Exhibit 1032)...87 Yoshitomi (Exhibit 1033)...95 Bunea (Exhibit 1020)... 102 Breivik (Exhibit 1035)... 106 THE 877 PATENT (EXHIBIT 1001)... 116 The 877 Patent discloses that the prior art Neptune Krill Oil (NKO) had 75.77wt% omega-3 fatty acids attached to phospholipids as required by claims 5 and 14.... 120 THE 877 PATENT CLAIM CHART... 123 877 PETITION GROUNDS... 124 GROUND 1: CLAIMS 1-3, 6, 8-9, 11-12, 15 and 17-18... 125 [BREIVIK, CATCHPOLE AND FRICKE]... 125 Claim 1... 125 Claim 11... 125 4 RIMFROST EXHIBIT 1006 page 0004
Catching and treating krill to denature lipases and... 129 phospholipases in krill to provide a denatured krill product... 129 Catching and freshly harvesting krill... 129 Treating krill to denature lipases and phospholipases... 130 in krill to provide a denatured krill product... 130 Extracting oil from said denatured krill product with a polar solvent... 131 To provide a krill oil with from about 3% to about 10% w/w ether phospholipids; from about 27% to 50% w/w non-ether phospholipids so that the amount of total phospholipids in the krill oil is from about 30% to 60% w/w... 132 To provide a krill oil with from about 20% to 50% w/w triglycerides... 133 Claim 2... 134 Claim 12... 134 Claim 3... 136 Claim 6... 137 Claim 15... 137 Claim 8... 138 5 RIMFROST EXHIBIT 1006 page 0005
Claim 17... 138 Claim 9... 139 Claim 18... 139 GROUND 2: CLAIMS 4-5 and 13-14... 141 [BREIVIK, CATCHPOLE, FRICKE AND BOTTINO]... 141 Claim 4... 141 Claim 13... 141 Claim 5... 142 Claim 14... 142 GROUND 3: CLAIMS 7 and 16... 144 [BREIVIK, CATCHPOLE, FRICKE AND SAMPALIS I]... 144 Claim 7... 144 Claim 16... 144 GROUND 4: CLAIMS 10 and 19... 147 [BREIVIK, CATCHPOLE, FRICKE AND SAMPALIS II]... 147 Claim 10... 147 6 RIMFROST EXHIBIT 1006 page 0006
Claim 19... 147 CONCLUDING OPINION... 149 TALLON APPENDIX A... 150 877 PATENT INVALDITY GROUNDS... 150 877 PATENT CLAIM CHART... 151 7 RIMFROST EXHIBIT 1006 page 0007
I, Dr Stephen J. Tallon, do hereby make the following declaration: AGREEMENT TO PROVIDE EXPERT TESTIMONY 1. I have agreed to provide expert testimony in support of Rimfrost AS s Petition for Inter Partes Review of. My Curriculum Vitae is attached hereto as Appendix D. 2. I understand that this proceeding involves ( the 877 Patent ) entitled Bioeffective Krill Oil Compositions, (Exhibit 1001). 3. I also understand that the 877 Patent claims priority to Provisional Application No. 60/920,483, filed on March 28, 2007 ( the 483 Provisional Application, Exhibit 1005), Provisional Application No. 60/975,058, filed on September 25, 2007 ( the 058 Provisional Application, Exhibit 1004), Provisional Application No. 60/983,446, filed on October 29, 2007 ( the 446 Provisional Application, Exhibit 1003), and Provisional Application No. 61/024,072, filed on January 28, 2008 ( the 072 Provisional Application, Exhibit 1002). 4. I have reviewed each of the Provisional Applications, Exhibits 1002-1005, to determine which if any provide written support for any of the claim limitations (elements) of the 877 Patent which claim various percentages of ether 8 RIMFROST EXHIBIT 1006 page 0008
phospholipids by weight of krill oil. In my opinion, as discussed below, only the 072 Application provides any written description regarding a percentage of ether phospholipids by weight of krill oil. The other Provisional Applications, Exhibits 1003-1005, do not disclose or reference the existence of ether phospholipids. 5. I have been asked to provide my opinion regarding whether one of ordinary skill in the art at the relevant time would have understood that certain prior art references, in combination, disclose or teach each of the elements and limitations recited in the claims of the 877 Patent. I have also been asked to provide my opinion regarding whether a person of ordinary skill in the art (a POSITA ) would have been motivated or had a rationale or reason to modify or combine those certain prior art references to arrive at the elements recited in the claims of the 877 Patent. In my opinion, as discussed below, the references discussed below disclose or teach each of the elements and limitations recited in the claims of the 877 Patent and a POSITA would have had a strong rationale to combine them. 9 RIMFROST EXHIBIT 1006 page 0009
BASES FOR OPINIONS GIVEN 6. In forming my opinion, I have relied on my own education, work experiences and knowledge and my review of the following documents: a., filed September 18, 2014 (the 877 Patent) (Exhibit 1001); b. U.S. Provisional Patent Application No. 61/024,072, filed January 28, 2008 (the 072 Provisional Application) (Exhibit 1002); c. U.S. Provisional Patent Application No. 60/983,446, filed October 29, 2007 (the 446 Provisional Application) (Exhibit 1003); d. U.S. Provisional Patent Application No. 60/975,058, filed September 25, 2007 (the 058 Provisional Application) (Exhibit 1004); e. U.S. Provisional Patent Application No. 60/920,483, filed March 28, 2007 (the 483 Provisional Application) (Exhibit 1005); f. Bottino, N.R., The Fatty Acids of Antarctic Phytoplankton and Euphausiids. Fatty Acid Exchange Among Trophic Levels of the Ross Sea, Marine Biology, 27, 197-204 (1974) (Bottino) (Exhibit 1007); 10 RIMFROST EXHIBIT 1006 page 0010
g. Budziński, E., P. Bykowski and D. Dutkiewicz, 1985, Possibilities of processing and marketing of products made from Antarctic krill, FAO Fish.Tech. Pap., (268):46, (Budzinski) (Exhibit 1008); h. Catchpole and Tallon, WO 2007/123424, published November 1, 2007, Process for Separating Lipid Materials, (Catchpole) (Exhibit 1009); i. Fricke et al., Lipid, Sterol and Fatty Acid Composition of Antarctic Krill (Euphausia superba Dana), LIPIDS 19(11):821-827 (1984) (Fricke) (Exhibit 1010); j. Randolph, et al., U.S. Patent Application Publication No. US/2005/0058728 A1, Cytokine Modulators and Related Method of Use (Randolph) (Exhibit 1011); k. Sampalis [I] et al., Evaluation of the Effects of Neptune Krill Oil on the Management of Premenstrual Syndrome and Dysmenorrhea, Altern. Med. Rev. 8(2):171-179 (2003) (Sampalis I) (Exhibit 1012); l. Sampalis [II] et al.,wo 2003/011873, published February 13, 2003, Natural Marine Source Phospholipids Comprising Flavonoids, Polyunsaturated Fatty Acids and Their Applications, (Sampalis II) (Exhibit 1013); 11 RIMFROST EXHIBIT 1006 page 0011
m. Tanaka [I] et al., Platelet Activating Factor (PAF) Like Phospholipids Formed During Peroxidation of Phosphatidylcholines from Different Foodstuffs, Biosci. Biotech. Biochem., 59(8) 1389-1393 (1995) (Tanaka I) (Exhibit 1014); n. Tanaka (II) et al., Extraction of Phospholipids from Salmon Roe with Supercritical Carbon Dioxide and an Entrainer, Journal of Oleo Science Vol. 53 (2004) No. 9, p.17-424 (Tanaka II) (Exhibit 1015); o. Beaudoin et al., Method of Extracting Lipids From Marine and Aquatic Animal Tissues, U.S. Patent No. 6,800,299 B1 filed July 25, 2001 (Beaudoin) (Exhibit 1016); p. Folch et al., A simple method for the isolation and purification of total lipides from animal tissues, J. Biol. Chem. (1957) 226: 497-509 (Folch) (Exhibit 1017); q. Kochian et al, Agricultural Approaches to Improving Phytonutrient Content in Plants: An Overview, Nutrition Reviews, Vol. 57, No. 9, September 1999: S13-S18 (Exhibit 1018); 12 RIMFROST EXHIBIT 1006 page 0012
r. Porzio et al., Encapsulation Compositions and Processes for Preparing the Same, U.S. Patent No. 7,488,503 B1 filed March 31, 2004 ( Porzio ) (Exhibit 1019); s. Bunea, et al., Evaluation Of The Effects Of Neptune Krill Oil On The Clinical Course Of Hyperlipidemia, Altern Med Rev. 2004; 9:420 428 (Bunea) (Exhibit 1020). t. File History to, Serial No, 14/490,176 ( 877 File History) (Exhibit 1025) 1025 Part 1 - Pages 1-375 1025 Part 2 - Pages 376-724; u. File History to U.S. Patent No. 9,078,905 B2, Serial No, 14/490,221 ( 905 File History) (Exhibit 1026) 1026 Part 1 - Pages 1-450 1026 Part 2 - Pages 451-882; v. Saether et al., Lipolysis post mortem in North Atlantic krill. Comp. Biochem. Physiol. Vol. 83B, No. 1, pp. 51-55, 1986 (Saether) (Exhibit 1027); 13 RIMFROST EXHIBIT 1006 page 0013
w. Hawley s Condensed Chemical Dictionary, pp. 339, 340, 13th ed.,1997 (Hawley s) (Exhibit 1028); x. Webster s New Universal Unabridged Dictionary, 2nd ed., p. 732, 1983 (Webster s) (Exhibit 1029); y. Tehoharides, U.S. Patent Application Publication No. US/2006/0013905 A1, Anti-Inflammatory Compositions For Treating Multiple Sclerosis (Tehoharides) (Exhibit 1030); z. Grantham, G.J., The Utilization Of Krill, UNDP/FAO Southern Ocean Fisheries Survey Programme (1977) (Grantham) (Exhibit 1032); aa. Yoshitomi, U.S. Patent Application Publication No. US/2003/0113432 A1, Process For Making Dried Powdery and Granular Krill (Yoshitomi) (Exhibit 1033); bb. Breivik, U.S. Patent Application Publication No. US 2010/0143571 A1, Process for Production of Omega-3 Rich Marine Phospholipids from Krill (Breivik) (Exhibit 1035); 14 RIMFROST EXHIBIT 1006 page 0014
cc. Breivik, U.S. Provisional Patent Application No. 60/859,289, Processes for production of omega-3 rich marine phospholipids from krill, filed November 16, 2006 (Breivik 289 Provisional) (Exhibit 1036); and dd. Breivik, WO 2008/060163 A1, Process for Production of Omega-3 Rich Marine Phospholipids from Krill, International filing date November 15, 2007 (Breivik PCT) (Exhibit 1037). 15 RIMFROST EXHIBIT 1006 page 0015
NO FINANCIAL INTEREST IN PROCEEDING 7. For my work related to this Inter Partes Review, my employer, Callaghan Innovation, receives compensation for my time. I am not directly compensated by either Hoffmann and Baron, LLP or the Petitioner. I have no financial interest in this proceeding, and the potential for any future financial benefit is unaffected by the content of my testimony or the outcome of this proceeding. My compensation from my employer, Callaghan Innovation, is not in any way related to the outcome of the case. QUALIFICATIONS AND RELEVANT EXPERIENCE 8. I am currently employed as Team Manager of the Processing Team within the Integrated Bioactive Technologies Group of Callaghan Innovation, at the Gracefield Research Centre in Lower Hutt, New Zealand (an agency of the Government of New Zealand). The Integrated Bioactive Technologies group specializes in near to market research and development in the field of processing of biologically-derived raw materials to make high value nutraceuticals, food ingredients and biopharmaceuticals. My research and expertise has helped to enable a number of industries in New Zealand to make such products, including a 16 RIMFROST EXHIBIT 1006 page 0016
range of plant and marine derived extracts, including lipid extracts that are produced using a supercritical fluid extraction process. 9. Consequently, I have knowledge and expertise regarding methods of making or extracting oils from crustaceans such as krill, including lipid oils, and of their composition, including various extraction methods involving the use of supercritical fluids both with, and without, polar solvents; and the polar solvent extraction of marine biomasses to achieve the same aims. These methods are now a matter of public knowledge. See my CV attached Appendix D. 10. I began developing supercritical CO 2 + polar co-solvent extraction and fractionation processes around 2003 with my colleague Dr Catchpole, and applied them to biomasses containing phospholipids around 2004, and in particular to marine and dairy biomasses. Around this time I also investigated the use of dimethyl ether (DME) for extracting phospholipids, again mainly from marine and dairy biomasses. 11. Around this time, 2003-2004, Dr Andrew Mackenzie, also part of the Integrated Bioactive Technologies group, developed an NMR-based method for analyzing and quantifying phospholipids, based on the 31 P isotope. This analysis method was able to distinguish between very closely related phospholipids, such as 17 RIMFROST EXHIBIT 1006 page 0017
phosphatidylcholine (PC) and its ether-lipid analogue alkylacylphosphatidylcholine (AAPC); and phosphatidylethanolamine (PE) and its ether analogues phosphatidylethanolamine plasmalogen and alkylacylphosphatidylethanolamine (AAPE). 12. Prior to the use of 31 P NMR to determine the ether phospholipid content of phospholipid lipids, and though other methods for analyzing same were available, it was common industry practice to report only the total phospholipid content for the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid components. Thus, when the amount of the PC lipid component was reported, the number reported included not only PC, but its ether-lipid analogue alkylacylphosphatidylcholine (AAPC). The total then reported being the sum of the non-ether phospholipids and ether phospholipids. In a like manner, when the amount of the phosphatidylethanolamine (PE) component was reported, the number reported included not only PE, but its ether analogues phosphatidylethanolamine plasmalogen (PE plasmalogen) and alkylacylphosphatidylethanolamine (AAPE) as well. 18 RIMFROST EXHIBIT 1006 page 0018
13. When the industry began to accept the 31 P NMR standard for the first time the ether-phospholipid components started to be consistently reported as separate constituents. 14. In 2006 the Integrated Bioactive Technologies group of Callaghan Innovation (of which I am a part), began to work collaboratively with Nutrizeal Ltd (founded 1995), now operating as Pharmalink Extracts, Ltd. (Nelson, New Zealand) on the extraction of dry krill powder to produce krill lipid extracts enriched in phospholipids, and to provide a detailed compositional analysis of those extracts. 15. The work of the Integrated Bioactive Technologies group on the extraction of phospholipids using CO 2 and CO 2 + polar co-solvent led us to discover that we could separate different types of phospholipids based on their solubility. Work was performed on a variety of biomasses including krill, which demonstrated that a phospholipid-rich extract containing both ether and non-ether phospholipids was obtained. This work is described in WO 2007/123424, published November 2007, based on International Application Number PCT/NZ/2007/000087, Catchpole and Tallon, International Filing Date April 20, 2007 (Catchpole, Example 18 and Table 16, p. 24, Exhibit 1009, p. 0024). 19 RIMFROST EXHIBIT 1006 page 0019
16. The 31 P-NMR analysis method used in Catchpole (pp. 14 & 24, Exhibit 1009, pp. 0014 & 0024) demonstrated that ether phospholipids were an intrinsic constituent of the lipid composition of many marine organisms, including krill and krill meal. 17. We also performed lipid analysis work on existing commercial krill lipid products that were supplied to us by Nutrizeal in 2006. Upon information and belief, these products were Neptune Krill Oil (NKO), a.k.a. Krill Bill, with the krill oil in the form of gelatine capsules; and Aquasource Krill Oil, again with the krill oil in the form of gelatine capsules. 18. The encapsulated krill oils were analyzed and found to contain, among the phospholipids, both non-ether phospholipids and ether phospholipids, and high levels of omega-3 fatty acids (polyunsaturated fatty acids which have an unsaturated bond at the third carbon from the terminal end, commonly reported as as ω-3, n-3, or omega-3) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) attached to the phospholipids. 19. By 2006 and even before, EPA, DHA and phospholipids were associated with beneficial health and were recognized to be important compounds for use in the nutraceutical industry and they remain so today. 20 RIMFROST EXHIBIT 1006 page 0020
RELEVANT CONSIDERATIONS 20. I provide my opinions in this declaration as informed below. 21. I have been informed that to be entitled to an earlier priority date each claim limitation must be expressly, implicitly, or inherently supported in the originally filed disclosure, in this case in the provisional applications Exhibits 1002-1005. 22. I have been informed that a claimed invention is unpatentable if the differences between the invention and the prior art are such that the subject matter as a whole would have been obvious at the time the alleged invention was made to a person having ordinary skill in the art to which the subject matter pertains. I have also been informed that an obviousness analysis takes into account factual inquiries including the level of ordinary skill in the art, the scope and content of the prior art, and the differences between the prior art and the claimed subject matter. 23. I have been informed that there are several accepted rationales for combining references or modifying a reference to show obviousness of the claimed subject matter. Some of these rationales include the following: combining prior art elements according to known methods to yield predictable results; simple substitution of one known element for another to obtain predictable results; a 21 RIMFROST EXHIBIT 1006 page 0021
predictable use of prior art elements according to their established functions; applying a known technique to a known device to yield predictable results; choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success; and some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention. 24. I have been informed that a person of ordinary skill in the art, a POSITA, is not a specific, real individual, but rather a hypothetical individual or team of individuals working closely together, who is presumed to have known the relevant art at the time of the invention. In defining the POSITA, I have been informed to consider factors such as the educational level and years of experience not only of the person or persons who have developed the invention that is the subject of the case, but also others working in the pertinent art at the time of the invention; the types of problems encountered in the art; the teachings of the prior art; patents and publications of other persons or companies; and the sophistication of the technology. 22 RIMFROST EXHIBIT 1006 page 0022
25. I have been informed that claim terms are interpreted according to their broadest reasonable construction in light of the specification of the 877 Patent in which they appear and that the patent claim terms are also given their ordinary and customary meaning as would be understood by a POSITA in the context of the entire disclosure. LEVEL OF ORDINARY SKILL IN THE ART & THE POSITA 26. I have assessed the level of ordinary skill in the art based upon my review of the 877 Patent, the prior art, my own long standing career of working in the field of producing oils from various biomasses, including marine and other aquatic biomass, with extensive leadership roles managing projects in marine organism processing, oil extraction and compositional analysis. 27. In my opinion, as of the earliest priority date the 877 Patent is entitled to, that is January 28, 2008, see discussion below regarding Exhibit 1002, a POSITA would have held an advanced degree in marine sciences, biochemistry, organic (especially lipid) chemistry, chemical or process engineering, or associated sciences with complementary understanding, either through education or experience, of organic chemistry and in particular lipid chemistry, chemical or process engineering, marine biology, nutrition, or associated sciences; and 23 RIMFROST EXHIBIT 1006 page 0023
knowledge of or experience in the field of extraction. In addition, a POSITA would have had at least five years applied experience. MOTIVATION TO COMBINE - OVERVIEW 28. Nutraceutical products have been a growing and commercially important market for many years. An important segment of that market is based on the benefits of omega-3 fatty acids and other lipid compounds, including phospholipids, triglycerides and astaxanthin for use in providing a positive impact on health or nutrition. This was true in 2007 and before, with substantially similar new lipid products constantly being brought to the marketplace. See for example, Sampalis I (Exhibit 1012, p. 0004), and Sampalis II (Exhibit 1013). Sampalis II disclosed the many known beneficial health aspects of phospholipids (pp. 15-20, Exhibit 1013, pp. 0017-0022), specifically pointed out the benefits of phosphatidylcholine (p. 0017) and provided a laundry list of reasons why a POSITA seeking a superior source of phospholipids would have been motivated to extract them from krill. 29. The prior art disclosed fish, fish extracts, krill and krill extracts as a starting source for many of these lipid compounds. Moreover, prior art krill and krill extracts literature also disclosed that the krill extracts could be combined with 24 RIMFROST EXHIBIT 1006 page 0024
other compounds such as phytonutrients to provide additional health or nutritional benefits to the omega-3 lipid compounds. See for example, Randolph (Exhibit 1011) and Tehoharides (Exhibit 1030). 30. The health benefits of omega-3 fatty acids, particularly in connection with cardiovascular disease, were also well known. See for example Bunea, pp. 421, 426, Exhibit 1020, pp. 0002, 0007. Further, it was also well known to a POSITA that krill lipids contain high levels of phospholipids, and that a high proportion of the fatty acids are attached to these phospholipids. Further still, it was known that [k]rill oil has a unique biomolecular profile of phospholipids naturally rich in omega-3 fatty acids and diverse antioxidants significantly different from the usual profile of fish oils. The association between phospholipids and long-chain omega-3 fatty acids highly facilitates the passage of fatty acid molecules through the intestinal wall, increasing bioavailability and ultimately improving the omega- 3:omega-6 fatty acid ratio. See Bunea, p. 421, Exhibit 1020, p. 0002, col. 1-2. 31. Thus, considering the ever increasing commercial market for nutraceuticals, the POSITA would have had a strong motivation or rationale to try and combine various well known lipid processing methods to obtain nutraceutical products containing omega-3 lipids. A POSITA would have been aware of the various krill 25 RIMFROST EXHIBIT 1006 page 0025
lipid constituents that had already been successfully extracted. Additionally, it was known that extracts obtained by supercritical fluid extraction processes using CO 2 were considered to be more natural than extracts produced using other solvents. Catchpole (Exhibit 1009). Additionally, a POSITA would have been further motivated to differentiate these products by combining not only the products of the various krill lipid processing methods and extracts with each other, but also to add additional beneficial compounds to the krill extracts as was disclosed in the prior art, e.g., phytonutrients, so as to be able to promote and advertise an even broader range of benefits for any final product. Finally, the POSITA already knew, and the prior art krill and krill extracts literature disclosed, capsules and encapsulation as convenient means for administering these beneficial krill extracts and additional compounds. See for example, Randolph (Exhibit 1011), Sampalis I (Exhibit 1012), Sampalis II (Exhibit 1013) and Tehoharides (Exhibit 1030). 32. Thus a POSITA would have, at most, simply be combining prior art elements according to known methods to yield predictable results to arrive at the processes and products claimed in the 877 Patent. See discussion below. 26 RIMFROST EXHIBIT 1006 page 0026
THE 877 PATENT IS ENTITLED TO A PRIORITY DATE NO EARLIER THAN JANUARY 28, 2008 33. I have read and understood the 877 Patent (Exhibit 1001), the 483 Provisional Application (Exhibit 1005), the 058 Provisional Application (Exhibit 1004), the 446 Provisional Application (Exhibit 1003), and the 072 Provisional Application (Exhibit 1002). 34. In my opinion each of the 877 Patent claims has a claim limitation requiring a certain percentage by weight of ether phospholipids and those ether phospholipid claim limitations are not expressly, implicitly, or inherently disclosed or supported in Exhibits 1003, 1004, or 1005. Exhibits 1003, 1004, and 1005 do not explicitly or inherently disclose ether phospholipids at all, or any content of ether phospholipids in krill oil. In my opinion Exhibit 1002 is the first Provisional Application that provides any written description of ether phospholipids. Thus, it is my opinion that the 877 Patent is entitled to a priority date no earlier than that of Exhibit 1002, namely January 28, 2008. 27 RIMFROST EXHIBIT 1006 page 0027
CLAIM TERMS krill oil means lipids extracted from krill 35. In the context of the 877 Patent and prior art, as discussed below, krill oil is a lipid-rich extract of krill that comprises phospholipids, as well as a lipid-rich extract of krill that comprises a blend of polar lipids (phospholipids) and neutral lipids in different proportions. The 877 Patent repeatedly refers to the krill oil composition as comprising a blend of lipid fractions. In some embodiments, krill oil composition comprises a blend of lipid fractions obtained from krill ( 877 Patent, 3:26-27, Exhibit 1001, p. 0025). In some embodiments, the blended krill oil product comprises a blend of lipid fractions obtained from Euphausia superba ( 877 Patent, 5:43-45, 6:50-52 and 7:18-20, Exhibit 1001, pp. 0027, 0028). Thus, as further discussed below, krill oil means lipids and blends of lipid fractions obtained from krill. 36. The methods of extraction are not limited. They include using, singularly and in combination, among others, polar solvents, neutral solvents, and more specifically ketone solvents, neat supercritical carbon dioxide, and supercritical carbon dioxide with a solvent modifier (co-solvent or entrainer). See, for example, 28 RIMFROST EXHIBIT 1006 page 0028
in the 877 Patent, 1:33-34, 1:59-2:2, 4:36-46, 4:59-68, 5:14-35, 5:65-6:12, 11:3-29 Exhibit 1001, pp. 0025-0027, 0030. 37. The 877 Patent discloses a well-known, two-step supercritical CO 2 in combination with ethanol extraction process, to extract two lipid fractions, a neutral lipid fraction and a polar lipid fraction. The krill oils are extracted from the krill meal in two stages, in step 1 the neutral fraction is extracted using neat supercritical CO 2 or in combination with 5% ethanol. The neutral fraction consisted mostly of triglycerides and cholesterol. In stage 2, the polar lipids (phospholipids) are extracted by adding at least 20% ethanol to the supercritical CO 2 extraction medium. 877 Patent, 9:36-43, Exhibit 1001, p. 0029. 38. The 877 Patent also discloses combinations of extraction methods. In some embodiments, krill oil is extracted from the denatured krill meal. In some embodiments, the krill oil is extracted by contacting the krill meal with ethanol. In some embodiments, krill is then extracted with a ketone solvent such as acetone. In other embodiments, the krill oil is extracted by one or two step supercritical fluid extraction. In some embodiments, the supercritical fluid extraction uses carbon dioxide and neutral krill oil is produced. In some embodiments, the supercritical fluid extraction uses carbon dioxide with the addition of a polar entrainer, such as ethanol, to produce a polar krill oil. 29 RIMFROST EXHIBIT 1006 page 0029
877 Patent, 11:3-13, Exhibit 1001, p. 0030. 39. The krill oil by this disclosure is a lipid extract produced in either a single or a two stage (step) process with solvents including ethanol, ketone solvents, and supercritical fluids with and without a polar entrainer. Two types of krill oil lipid fractions are also described, namely neutral krill oil and polar krill oil, depending on the solvent or combination of solvents used to extract them. 40. The abstract of the 877 Patent describes the actual krill oils as the oil extracted using a polar solvent after using a non-polar solvent to remove neutral lipids: The krill oils are obtained from krill meal using supercritical fluid extraction in a two stage process. Stage 1 removes the neutral lipid by extracting with neat supercritical CO 2 or CO 2 plus approximately 5% of a co-solvent. Stage 2 extracts the actual krill oils by using supercritical CO 2 in combination with approximately 20% ethanol. 877 Patent, Abstract, Exhibit 1001, p. 0001, emphasis added. 41. Furthermore, the 877 Patent states that: In some embodiments, the krill meal is first extracted with carbon dioxide followed by carbon dioxide with a polar entrainer, or vice versa. In some embodiments, the krill meal is first extracted with CO 2 supplemented with a low amount of a 30 RIMFROST EXHIBIT 1006 page 0030
polar co-solvent (e.g., from about 1% to about 10%, preferably about 5%) such a C 1 -C 3 monohydric alcohol, preferably ethanol, followed by extraction with CO 2 supplemented with a high amount of a polar co-solvent (from about 10% to about 30%, preferably about 23%) such as such a C 1 -C 3 monohydric alcohol, preferably ethanol, or vice versa. Surprisingly, it has been found that use of a low amount of polar solvent in the CO 2 as an entrainer facilitates the extraction of neutral lipid components and astaxanthin in a single step. Use of the high of polar solvent as an entrainer in the other step facilitates extraction of ether phospholipids, as well as non-ether phospholipids. See 877 Patent, 11:13-29, Exhibit 1001, p. 0030. 42. The krill oil by this disclosure is a lipid extract produced in two stages by removing some or much of the triglyceride and other neutral lipids, and then extracting the polar lipids. 43. Elsewhere, the 877 Patent also confirms that krill oil may be the blending of two or more different lipid fractions, which are then encapsulated. In particular, at 5:65-6:15 (p. 0027) the 877 Patent describes combining said polar extract and said neutral extract to provide Euphausia superba krill oil. 31 RIMFROST EXHIBIT 1006 page 0031
44. To elaborate, the 877 Patent states that: In some embodiments, the present invention provides methods of making a Euphausia superba krill oil composition comprising: contacting Euphausia superba with a polar solvent to provide a polar extract comprising phospholipids; contacting Euphausia superba with a neutral solvent to provide a neutral extract comprising triglycerides and astaxanthin; combining said polar extract and said neutral extract to provide Euphausia superba krill oil comprising from about 30% to 60% w/w phospholipids; from about 20% to 50% triglycerides; from about 400 to about 2500 mg/kg astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage of total fatty acids in said composition, wherein from about 70% to 95% of said omega-3 fatty acids are attached to said phospholipids. In some embodiments, the methods further comprise the step of encapsulating the Euphausia superba krill oil. In some embodiments, the present invention provides a Euphausia superba krill oil produced by the methods described above. See 877 Patent, 5:65 6:15, Exhibit 1001, p. 0027. 45. The krill oil by this disclosure again includes a lipid extract produced by combining or blending two different fractions, e.g., a polar extract and a neutral extract. 32 RIMFROST EXHIBIT 1006 page 0032
46. The neutral lipids, discussed above, are mainly comprised of triglycerides, partial glycerides, free fatty acids, and sterols. The krill oil also contains astaxanthin esters and the nitrogen containing compounds trimethylamine (TMA) and trimethylamine oxide (TMAO). 47. In quality krill oil, the phospholipids are highly enriched in the omega-3 fatty acids EPA and DHA, while the neutral lipids have very low levels of these fatty acids. In an oil decomposed through the action of lipases and phospholipases, which are naturally present in the krill, the overall composition will have a high level of free fatty acids and a portion of the phospholipids will have been degraded to lyso-phospholipids (one or both fatty acids have been lysed from the molecule), such as lysophosphatidylcholine (LPC), and lysoalkylacylphosphatidylcholine (LAAPC). The free fatty acid fraction of the oil will have elevated levels of omega-3 fatty acids including EPA and DHA, as these will have been cleaved from the parent phospholipid. 48. Therefore, krill oil in accordance with the 877 Patent means lipids extracted from krill. The final composition of the krill oil is dependent upon the solvent combinations used, the extent of extraction carried out, and the ratios in which different fractions are mixed or blended together. 33 RIMFROST EXHIBIT 1006 page 0033
polar solvent means solvent or mixtures of solvents capable of extracting polar lipids comprising phospholipids 49. The claim element polar solvent is not expressly defined in the specification of the 877 Patent, but it is discussed. The 877 Patent discloses methods of making a Euphausia superba krill oil by contacting a Euphausia superba preparation, such as Euphausia superba krill meal with a polar solvent, such as ethanol to extract lipids. ( 877 Patent, Exhibit 1001, 12:24-36, p. 0030). Also disclosed is that, [i]n some embodiments, krill oil is extracted from denatured krill meal. In some embodiments, the krill oil is extracted by contacting the krill meal with ethanol. 877 Patent, 11:3-5, Exhibit 1001, p. 0030. 50. In the Background of the Invention, the 877 Patent states: In order to isolate the krill oil from the krill, solvent extraction methods have been used. See, e.g., WO 00/23546. Krill lipids have been extracted by placing the material in a ketone solvent (e.g., acetone) in order to extract the lipid soluble fraction. This method involves separating the liquid and solid contents and recovering a lipid rich fraction from the liquid fraction by evaporation. Further processing steps include extracting the recovering by evaporation the remaining soluble lipid fraction from the contents by using a solvent such as ethanol. See, e.g., WO 00/23546. 877 Patent, l:31-40, Exhibit 1001, p. 0025. 34 RIMFROST EXHIBIT 1006 page 0034
51. In the Detailed Description, the 877 Patent discloses that a polar solvent is capable of extracting a polar extract comprising phospholipids: contacting Euphausia superba with a polar solvent to provide an polar extract comprising phospholipids. 6:17-19, Exhibit 1001, p. 0027. Additionally, [i]n some embodiments, the present invention provides a method of making a Euphausia superba krill oil composition comprising contacting Euphausia superba with a polar solvent to provide an polar extract comprising phospholipids. 877 Patent, 12:12-16, Exhibit 1001, p. 0030. 52. The Detailed Description of the 877 Patent further states: In some embodiments, krill oil is extracted from the denatured krill meal. In some embodiments, the krill oil is extracted by contacting the krill meal with ethanol. In some embodiments, krill is then extracted with a ketone solvent such as acetone. In other embodiments, the krill oil is extracted by one or two step supercritical fluid extraction. In some embodiments, the supercritical fluid extraction uses carbon dioxide and neutral krill oil is produced. In some embodiments, the supercritical fluid extraction uses carbon dioxide with the addition of a polar entrainer, such as ethanol, to produce a polar krill oil. In some embodiments, the krill oil meal is first extracted with carbon dioxide followed by carbon dioxide with a polar entrainer, or vice versa. In some embodiments, the krill meal is first extracted with CO 2 35 RIMFROST EXHIBIT 1006 page 0035
supplemented with a low amount of a polar co-solvent (e.g., from about 1% to about 10%, preferably about 5%) such a C 1 -C 3 monohydric alcohol, preferably ethanol, followed by extraction with CO 2 supplemented with a high amount of a polar co-solvent (from about 10% to about 30%, preferably about 23%) such as such a C 1 -C 3 monohydric alcohol, preferably ethanol, or vice versa. Surprisingly, it has been found that use of a low amount of polar solvent in the CO 2 as an entrainer facilitates the extraction of neutral lipid components and astaxanthin in a single step. Use of the high of polar solvent as an entrainer in the other step facilitates extraction of ether phospholipids, as well as non-ether phospholipids. 877 Patent, 11:3-29, Exhibit 1001, p. 0030. Thus, the 877 Patent contemplates extraction with a polar solvent or supercritical CO 2 in the presence of a polar solvent or entrainer. 53. In practice, the polar solvent must be able to extract polar lipids, comprising phospholipids. Typical polar organic solvents (pure or mixtures) used in industrial practice that meet these criteria include alcohols (e.g., methanol, ethanol, and isopropyl alcohol), ketones (particularly acetone), and esters (e.g., ethyl acetate). 54. I therefore understand a polar solvent, in the context of the 877 Patent and prior art, to encompass both a solvent or mixtures of solvents capable of extracting polar lipids comprising phospholipids. Thus, a solvent mixture 36 RIMFROST EXHIBIT 1006 page 0036
consisting of a non-polar and polar solvent in the correct ratios, such as supercritical CO2 and above about 10% ethanol constitutes a polar solvent. to denature lipases and phospholipases means to alter the conformational structure of lipases and phospholipases to reduce lipid and phospholipid decomposition 55. The specification of the 877 Patent does not expressly define the term to denature lipases and phospholipases but does disclose and describe protein denaturation as a means to avoid decomposition of lipids in krill oil and compositions. The present invention provides methods to avoid decomposition of glycerides and phospholipids in krill oil and compositions produced by those methods. The product obtained by these new methods is virtually free of enzymatically decomposed oil constituents. The solution to the problem is to incorporate a protein denaturation step on fresh krill prior to use of any extraction technology. Denaturation can be achieved by thermal stress or by other means. After denaturation, the oil can be extracted by an optional selection of nonpolar and polar solvents including use of supercritical carbon dioxide. Krill is adapted to a very efficient nutrient digestion at very low temperatures. Therefore the enzymes are sensitive to heat and the step of applying thermal denaturation of lipases and phospholipases does not imply use of very 37 RIMFROST EXHIBIT 1006 page 0037
high temperatures. Surprisingly, it has been found that the use of mild denaturation conditions can greatly enhance the quality of krill oil. 877 Patent, 9:44-60, Exhibit 1001, p. 0029 (emphasis added). 56. Methods to carry out the denaturation, including use of chemicals, heat, or combinations thereof, are disclosed by the 877 patent: In some preferred embodiments, freshly caught krill is first subjected to a protein denaturation step. The present invention is not limited to any particular method of protein denaturation. In some embodiments, the denaturation is accomplished by application of chemicals, heat, or combinations thereof. In some embodiments, freshly caught krill is wet pressed to obtain oil and meal. In some embodiments, the meal is then heated to a temperature of about 50 C to about 100 C for about 20 minutes to about an hour, preferably about 40 minutes to denature the proteins. In some embodiments, this material is then pressed to yield a pressed cake. When this method is used on krill, only a small amount of oil is released. Most of the oil is still present in the denatured meal. 877 Patent, 10:26-40, Exhibit 1001, p. 0029 (emphasis added). Thus cooking, i.e., heating, is but one traditional method used to carry out protein denaturation. 38 RIMFROST EXHIBIT 1006 page 0038
57. Denaturation would have been, and still is, understood by a POSITA to be a process in which proteins lose the quaternary structure, tertiary structure and secondary structure which is present in their native folded state, by application of some external stress or compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), radiation or heat. 58. See for example the definition of denaturation found in Hawley s Condensed Chemical Dictionary, which defines denaturation as: denaturation. A change in the molecular structure of globular proteins that may be induced by bringing a protein solution to its boiling point or by exposing it to acids or alkalies, or to various detergents. Denaturation reduces the solubility of proteins and prevents crystallization. It involves rupture of hydrogen bonds so that the highly ordered structure of the native protein is replaced by a looser and more random structure. It is usually irreversible but in some cases is reversible, depending on the protein and the treatment involved. Hawley s, pp. 339-340, Exhibit 1028, pp. 0003-0004. 59. Proteins are like ribbons that coil to form more stable structures, for example, alpha helices and pleated sheets. The final three-dimensional structure of the protein is formed by non-covalent interactions between the 39 RIMFROST EXHIBIT 1006 page 0039
amino acids of the protein. A quaternary structure is formed when multiple three-dimensional proteins bind to form a single larger protein. 60. Thus, the looser and more random structure from denaturation causes proteins, such as enzymes, to lose their activity because the substrates can no longer bind to the active site of the enzyme. Most biological substrates lose their biological function when their native structure is altered, i.e., denatured. For example, enzymes lose their activity, because the substrates can no longer bind to the active site, and because amino acid residues involved in stabilizing substrates' transition states are no longer positioned to be able to do so. The action of lipases and phospholipases includes hydrolysis of fatty acid groups from lipids and phospholipids, respectively, to produce free fatty acids and partially or fully decomposed lipids. Active lipases and phospholipases present in krill, if not deactivated, will cause triglycerides (triacylglycerols) and glycerol-based phospholipids (phosphoglycerides) present in the krill to decompose resulting in the formation of free fatty acids. See for example, Saether, p. 51, Exhibit 1027, p. 0001; see 167, below. 40 RIMFROST EXHIBIT 1006 page 0040
61. Therefore, denaturation of the active lipases and phospholipases present in krill would have been understood to change or alter the shape or conformation of the lipases and phospholipases and inhibit their ability to decompose the krill s lipids and phospholipids. 62. Therefore, in accordance with the 877 Patent and the prior art, my understanding of the term, to denature lipases and phospholipases means to alter the conformational structure of lipases and phospholipases to reduce lipid and phospholipid decomposition. freshly harvested krill means recently caught krill that has not significantly degraded 63. The specification of the 877 Patent does not expressly include the term freshly harvested. However, the specification does refer to freshly caught krill (10:19, Exhibit 1001, p. 0029), but does not define the term or define how long the krill remains fresh after being caught. The only disclosure in the 877 Patent of the time lapse between harvesting and processing of the freshly harvested krill is found in the specification: The krill meal has been processed on board a ship in Antarctica using live krill as starting material in order to ensure 41 RIMFROST EXHIBIT 1006 page 0041
the highest possible quality of the krill meal (9:33-36, Exhibit 1001, p. 0029) and Fresh krill was pumped from the harvesting trawl directly into an indirect steam cooker, and heated to 90C (Example 6, 30:62-64, Exhibit 1001, p. 0039). 64. The 877 Patent explains that certain prior art methods used krill that had been degraded between the time it was caught until the time it was processed. The methods described above rely on the processing of frozen krill that are transported from the Southern Ocean to the processing site. This transportation is both expensive and can result in degradation of the krill starting material. Data in the literature showing a rapid decomposition of the oil in krill explains why some krill oil currently offered as an omega-3 supplement in the marketplace contains very high amounts of partly decomposed phosphatidylcholine and also partly decomposed glycerides. Saether et al., Comp. Biochem Phys. B 83B(l): 51-55 (1986) [Exhibit 1027, pp. 0001-0005]. The products offered also contain high levels of free fatty acids. 877 Patent, 2:2-13, Exhibit 1001, p. 0025 (emphasis added). 65. Webster s New Universal Unabridged Dictionary defines fresh in relevant part to mean, not spoiled, rotten, or stale; as fresh milk (p. 723, Exhibit 1029, p. 0003). 42 RIMFROST EXHIBIT 1006 page 0042
66. With regard to krill, it was well known that proteases and lipases naturally found within krill begin to digest the krill soon after catching, leading to spoilage. 67. Therefore, in accordance with the 877 Patent and my understanding of the prior art, the term, freshly harvested krill means recently caught krill that has not significantly degraded. polar entrainer means polar solvent additive to aid in extraction 68. The specification of the 877 Patent does not specifically define the term polar entrainer but it does disclose that ethanol is an example of a polar entrainer, and that it facilitates extraction (11:10-13, Exhibit 1001, p. 0030). 69. The 877 Patent (11:24-29, p. 0030) also discloses that: Surprisingly, it has been found that use of a low amount of polar solvent in the CO2 as an entrainer facilitates the extraction of neutral lipid components and astaxanthin in a single step. Use of the high [sic] of polar solvent as an entrainer in the other step facilitates extraction of ether phospholipids, as well as non-ether phospholipids. 43 RIMFROST EXHIBIT 1006 page 0043
70. Therefore, in accordance with the 877 Patent and my understanding of the prior art, the term, polar entrainer in accordance with the 877 Patent means a polar solvent additive to aid in extraction. PRIOR ART Sampalis I (Exhibit 1012) 71. Sampalis I, published in 2003, discloses a human clinical trial study on the use of Neptune Krill Oil (NKO ) in comparison to an 18:12 EPA:DHA fish oil (which ostensibly contains no phospholipids) to ameliorate the symptoms of PMS and dysmenorrhea. See, e.g., Sampalis I, Abstract, p. 171 (Exhibit 10012, p. 0001). 72. Sampalis I notes on page 174 (Exhibit 1012, p. 0004, left column) that Neptune Krill Oil (NKO ) is a natural health product extracted from Antarctic krill also known as Euphausia superba. Euphausia superba, a zooplankton crustacean, is rich in phospholipids and triglycerides carrying long-chain omega-3 polyunsaturated fatty acids, mainly EPA and DHA, and in various potent antioxidants... (emphasis added). This inherent composition of the krill lipid extract is consistent with the compositions variously reported for NKO with 44 RIMFROST EXHIBIT 1006 page 0044
Examples 2, 3, and 8 in the 877 Patent, see also the tables associated with these examples. 73. In further describing Neptune Krill Oil, Sampalis I describes the krill oil as being rich in potent antioxidants, including vitamins A and E, and astaxanthin, and a novel flavonoid. See Sampalis I, p. 174, left col., Exhibit 1012, p. 0004. 74. Sampalis I evaluates the effectiveness of Neptune Krill Oil, a product rich in phospholipids and triglycerides carrying long-chain omega-3 polyunsaturated fatty acids, for the management of premenstrual syndrome and dysmenorrhea (p. 174, right column, Exhibit 1012, page 0004), after first disclosing previous studies that reinforce the theory that one of the main causes of PMS is inflammation and noting the beneficial effect of omega-3 fatty acid on reducing inflammation. The balance of polyunsaturated (essential) fatty acids in the body is critical for the maintenance of healthy cell membranes and hormone regulation. During the last several decades, the average Western diet has shifted to much higher levels of omega-6 and less omega-3 fatty acid intake. Long-chain omega-6 fatty acids, such as arachidonic acid, predominating in the phospholipids of cell membranes can encourage the production of pro-inflammatory type-2 prostaglandins (PGE 2 ), while omega-3 fatty acids promote the production of anti-inflammatory prostaglandins. 45 RIMFROST EXHIBIT 1006 page 0045
Sampalis I, (p. 173, left column, Exhibit 1012, page 0003, emphasis added). 75. Sampalis I further discloses on page 174, right column (Exhibit 1012, p. 0004) that [e]ach patient was asked to take two 1-gram soft gels of either NKO or omega-3 18:12 fish oil (fish oil containing 18% EPA and 12% DHA) once daily with meals during the first month of the trial. Sampalis I concludes on page 178 (Exhibit 1012, p. 0008) that The final results of the present study suggest within a high level of confidence that Neptune Krill Oil can significantly reduce the physical and emotional symptoms related to premenstrual syndrome. Thus, an encapsulated krill oil comprising a soft gel capsule containing an effective amount of krill oil obtained from the Antarctic krill Euphausia superba is disclosed in Sampalis I. The effective amount described by Sampalis I is the equivalent of 2g (two 1-gram soft gels) of krill oil, taken daily. This is within the range of 0.2g to 10g described in the 877 Patent (see for example, 877 Patent, 7:12-14, p. 0028). 46 RIMFROST EXHIBIT 1006 page 0046
Budziński (Exhibit 1008) 76. Budziński is a review published in 1985 titled Possibilities of processing and marketing of products made from Antarctic krill. The purpose of the publication is to review current practice in krill harvesting and processing; to describe the state of the art (in 1985) of modern processing techniques; and to discuss the economics of manufacturing krill products. The publication describes stock estimates and harvesting patterns, chemical composition of the krill, challenges in processing of krill, different products that can be made from krill, technological means for producing high quality products, and processing cost estimates. Krill is of interest because it provides new sources of animal protein of marine origin.... Budziński, p. -1-, Introduction, line 1,Exhibit 1008, p. 0007. 77. The review relates primarily to Antarctic krill, which is also described as Euphausia Superba ( Euphausia superba occurs in the belt of Antarctic waters between the minimum and maximum annual range of ice cover, south of the Antarctic Convergence; its distribution is circumpolar. Budziński, p. 1, section 2.1, 1 st para., Exhibit 1008, page 0007.), and the terms are used interchangeably throughout the publication. Harvesting of krill from both the Antarctic and the Pacific is acknowledged as being well established industry practice Long-term 47 RIMFROST EXHIBIT 1006 page 0047
practice in fish harvesting on the main fishing grounds of the Atlantic and Pacific, suggest an [existing] exploitation model (Budziński, p. -28-, section 5.1.3, 1 st para., Exhibit 1008, p. 0034, ). The harvesting is carried out using ships, for example The three vessels caught a total of 8.033 tons of krill during 139 days of fishing, obtaining a daily catch rate of 57.8 tons per vessel... (Budziński, p. -4-, 2 nd para., Exhibit 1008, p. 0010). 78. One of the main processing challenges identified is the presence of active proteases The main difficulty in krill processing is caused by the very active system of proteolytic enzymes (Budziński, p. -6-, lines 1-2, Exhibit 1008, p. 0012). The presence of active lipases is also acknowledged, causing decomposition of both triglycerides and phospholipids A large role is also played by very active lipases present in the digestive tract of krill, which cause the decomposition of phospholipids and, to a lesser degree, triglycerides. A result of their activity is an increase of free fatty acids (FFA) (Budziński, p. -6-, 3 rd para., Exhibit 1008, p. 0012). The lipases described by Budziński include phospholipases because the decomposition of phospholipids is occurring. 79. The decomposition of the lipids results in high levels of free fatty acids forming, primarily from the phospholipids. Table 3 (Budziński, p. -6-, Exhibit 48 RIMFROST EXHIBIT 1006 page 0048
1008, p. 0012) shows the lipid composition of different krill samples, in which the Free Fatty Acid levels range from 2.6 to 16.1 % of total lipids. Budziński reports that The generally observed high contents of FFA (up to 31 per cent of total lipids) is, according to Ellingsen and Mohr (1981), a result of lipase and phospholipase activity. These enzymes continue to be active in raw material frozen at excessively low temperatures. According to these authors, the natural level of FFA in krill is about 4 per cent. (Budziński, p. -7-, Exhibit 1008, p. 0013). 80. To minimise the extent of enzymatic degradation, Budziński states that Storage time should be as short as possible; for krill for human consumption, it cannot exceed 3-4 hours; for krill for meal production, 8-10 hours and the vessel must be properly equipped for processing, i.e., conveyor belts to shorten the passage of the raw material and tanks (Budziński, p. -9-, para. 4, Exhibit 1008, p. 0015). 81. The importance of rapid processing is further emphasised in section 4.9 titled Location of processing facilities, which states Due to its technological properties, the raw material should be processed as soon as possible after capture. The only way to meet this requirement is to install processing facilities on-board the vessel (Budziński, p. -25-, section 4.9, lines 1-3, Exhibit 1008, p. 0031 49 RIMFROST EXHIBIT 1006 page 0049
(emphasis added)). Rapid processing of krill shortly after harvesting while it is still fresh is thus described, for which the only practical solution is to carry out the processing on-board the vessel. 82. Further examples of processing equipment on-board vessels are also given, including The Polish machine was improved during subsequent cruises (Budziński, p. -14-, Exhibit 1008, p. 0020), and Still, it is possible to install 6-8 peelers with auxiliary equipment on a typical factory trawler (Budziński, pp. -14- through -16-, Exhibit 1008, pp. 0020-0022). 83. Budziński proposed immediate processing of the caught krill. The exploitation option assumes that krill will be processed on the vessel immediately after each haul. Other options have also been considered, especially catching krill by medium-sized trawlers and transporting fresh for processing on land or transshiping onto floating processing plants. However, none of these options seems feasible because raw krill spoil too rapidly (Budziński, p. -27-, section 5.1.1(k), Exhibit 1008, p. 0033). 84. Budziński discloses that heat treatment is known to cause denaturation of proteins, including enzymes, thus providing for a denatured krill product. Many examples of using heat treatment to preserve the quality of the krill and krill lipids 50 RIMFROST EXHIBIT 1006 page 0050
are given. For example In Japan, large krill with eggs are considered a delicacy. They must be boiled for approximately 5 minutes at 90 C before freezing so that no blackening and autolysis occur after defrosting. (Budziński, p. -10-, Exhibit 1008, p. 0016); Larger quantities of raw material for production could be stored because precooking permits longer storage. (Budziński, p. -12-, Exhibit 1008, p. 0018); and In industrial practice, a traditional method of krill-meal production is being used: cooking and pressing or centrifuging and drying.... On the basis of model-based and commercial trials, the optimum temperature is believed to be 80-85 C. Budziński, p. -20-, Exhibit 1008, p. 0026, emphasis added, references omitted). 85. Further benefits of the thermal processing are described, including reduced fluoride levels by stopping the migration of fluoride from the shell to the muscle in frozen krill (Budziński, p. -18-, Exhibit 1008, p. 0014). 86. Extraction of krill oil using organic solvents is described, as is the potential value of the krill oil - Krill oil was only obtained by extraction with the help of various organic solvents. Because of its high content of phospholipids and fatty acids, krill oil could be a valuable raw material for oil, cosmetic and pharmaceutical industries. Budziński, p. -24-, Exhibit 1008, p. 0030. 51 RIMFROST EXHIBIT 1006 page 0051
Phospholipids are included in the description of the krill oil that has been extracted, teaching the successful use of a solvent with suitable solvency to extract polar lipids. Catchpole (Exhibit 1009) 87. Catchpole and Tallon filed a PCT in April 2007 on the extraction and fractionation of phospholipids from various feedstocks using supercritical CO2 with a polar co-solvent. The polar co-solvent is a polar entrainer. It was noted that supercritical fluid extraction processes using CO 2 were becoming increasingly popular because of processing and consumer benefits. For example, CO 2 can be easily removed from the final product by reducing the pressure, whereupon CO 2 reverts to a gaseous state giving a completely solvent free product. The extract is considered to be more natural than extracts produced using other solvents. See Catchpole, p. 2, lines 18-22, Exhibit 1009, p. 0002. 88. Catchpole also discloses that it is an object of the invention described therein to provide a process for producing a product that contains desirable levels of particular phospholipids. See Catchpole, p. 3, lines 27-29, Exhibit 1009, p. 0003. 52 RIMFROST EXHIBIT 1006 page 0052
89. Catchpole further discloses that the described compositions and methods may be employed in a number of applications including infant formulas, brain health, sports nutrition and dermatological compositions. See Catchpole, p. 25, lines 9-13, Exhibit 1009, p. 0025. 90. Furthermore, Catchpole notes that phospholipids have been implicated in conferring a number of health benefits including brain health, skin health, eczema treatment, anti-infection, wound healing, gut microbiota modifications, anti-cancer activity, alleviation of arthritis, improvement of cardiovascular health, and treatment of metabolic syndromes. Catchpole, p. 1, line 11 to p. 0002, line 2, see also p. 25, lines 9-13, Exhibit 1009, pp. 0001, 0002, 0025. 91. The polar co-solvent comprises at least 10 % of the CO 2 by mass and preferably consists of a C1 C3 mono-hydric alcohol (most preferably ethanol) optionally containing a small amount of water (0 40 %) (see, for example, Catchpole, p. 4, lines 11-15, Exhibit 1009, p. 0004). The solvent mixture of CO2 and polar co-solvent (polar entrainer) constitutes a polar solvent according to the preceding definition described here. The feedstocks containing phospholipids included dairy and marine biomasses, and krill. See Catchpole, p. 7, lines 11-14, Exhibit 1009, p. 0007. 53 RIMFROST EXHIBIT 1006 page 0053
92. The invention of Catchpole is the description of means for fractionating a mixture of phospholipids, based on their differential solubility in supercritical CO2 + polar co-solvent. The most soluble phospholipids are extracted into the mixed solvent, whilst the least soluble phospholipids are concentrated in the residue. Dried krill powder in Example 18: Fractionation of Krill Lipids of Catchpole (p. 24, Exhibit 1009, p. 0024) was extracted with supercritical CO 2 to firstly extract neutral lipids (mainly triglycerides) and then with a CO 2 + ethanol mixture, the residual powder was then extracted with CO 2 and absolute ethanol, using a mass ratio of ethanol to CO 2 of 11 %, to produce a second extract labelled Extract 2, and a residual un-extracted fraction labelled Residue. Catchpole, p. 24, lines 3-19, Exhibit 1009, p. 0024. 93. The krill powder was a freeze-dried powder (Catchpole, p. 24, lines 1-4, Exhibit 1009, p. 0024). 94. Extract and residue fractions were analyzed for phospholipid content and profiled by 31 P-NMR. Phospholipids that were able to be measured using this technique included phosphatidylchoine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), plasmalogens (PL), phosphonolipids (PP), alkylacylphospholipids (ALP), sphingomyelin (SM), ceramide 54 RIMFROST EXHIBIT 1006 page 0054
aminoethylphosphonate (CAEP), phosphatidyl-serine (PS), and cardiolipin (CL). Catchpole, p. 14, lines 7-11, Exhibit 1009, p. 0014. 95. The composition of the resulting krill oil from the second extraction using CO 2 + ethanol mixture (Extract 2) is disclosed in Table 16 (Exhibit 1009, p. 24) as containing 4.6% AAPC (an ether phospholipid), 0.2% AAPE (an ether phospholipid), 39.8% PC, 0.3% PE and 0.2% CL giving a total phospholipid content of 45.1%, a phosphatidylcholine (PC) level of 39.8%, and an ether phospholipid (AAPC+AAPE) content of 4.8%. Catchpole, p. 24, Exhibit 1009, p. 0024. 96. Thus, Catchpole discloses (a) a krill oil obtained by polar solvent extraction, (b) the krill oil having an ether phospholipid content between about 3% to about 10% (4.8%) by weight of the krill oil, (c) the krill oil containing from about 30% to 60% (45.1%) total phospholipids by weight of krill oil, (d) the krill oil containing at least 30% (39.8%) phosphatidylcholine by weight of krill oil, and (e) the krill oil 55 RIMFROST EXHIBIT 1006 page 0055