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`UNITED STATES PATENT AND TRADEMARK OFFICE
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`_______________________
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`BEFORE THE PATENT TRIAL AND APPEAL BOARD
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`_______________________
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`RIMFROST AS
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`Petitioner
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`v.
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`AKER BIOMARINE ANTARCTIC AS
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`Patent Owner
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`_______________________
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`Case: IPR2020-01534
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`U.S. Patent No. 10,010,567 B2
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`_______________________
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`REPLY DECLARATION OF DR STEPHEN J. TALLON
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`RIMFROST EXHIBIT 1086 Page 0001
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`IPR2020-01534
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`U.S. Patent No. 10,010,567 B2
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`TABLE OF CONTENTS
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`TABLE OF CONTENTS ........................................................................................... 2
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`DECLARATION OF DR STEPHEN J. TALLON ................................................... 3
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`BASIS FOR OPINION .............................................................................................. 3
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`‘567 PETITION GROUNDS ................................................................................... 5
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`PO MISCHARACTERIZES BOTTINO II (EX1038) ......................................... 6
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`PO’s Attempt to Discredit Bottino II is Invalid. .................................................... 8
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`Results of Freeman and West cannot be applied to Bottino II ............................11
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`Rf values for DG, FFA and TG in various solvent systems ............................17
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`Bottino II disclosures taken as a whole ...............................................................19
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`independently describe FFA levels as the ‘unknown’ spot. ................................19
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`Reliability of Bottino II’s results .........................................................................28
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`A krill oil containing less than 3 wt% free fatty acids .........................................36
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`is obvious regardless of PO’s arguments, and is conceded by PO. .....................36
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`CONCLUDING OPINION ...................................................................................39
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`2
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`RIMFROST EXHIBIT 1086 Page 0002
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`U.S. Patent No. 10,010,567 B2
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`DECLARATION OF DR STEPHEN J. TALLON
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`I make this declaration in support of Petitioner’s Reply to Patent Owner’s (“PO’s”)
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`Response (Paper 9) to Petition in IPR2020-01534 (“POR”).
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`BASIS FOR OPINION
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`1.
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`I have reviewed the Declaration of Dr. Jacek Jaczynski, EX2001,
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`(“Jaczynski Dec.”) and accompanying exhibits, and disagree with his conclusions
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`overall and as described in detail in the discussion below.
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`2.
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`I have reviewed Patent Owner’s Response to Petition, Paper No. 09, and
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`disagree with the conclusions set forth therein and as described in detail in the
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`discussion below.
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`3.
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`I have reviewed the Deposition of Dr. Jacek Jaczynski, EX1170, from
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`IPR2020-01532 and IPR2020-01533 (“Jaczynski Dep.”). IPR2020-01532 and
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`IPR2020-01533 were brought by Petition against PO’s U.S. Patent Nos. 9,644,169
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`and 9,816,046, respectively. Like the U.S. Patent No. 10,010,567 (“the ‘567
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`patent”), they all claim priority by continuation applications to PO’s U.S. Patent
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`No. 9,375,453. See, Tallon Dec., EX1006 at ¶ 33.
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`3
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`RIMFROST EXHIBIT 1086 Page 0003
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`4.
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`Furthermore, after reviewing the foregoing, I hereby reaffirm my opinion
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`
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`U.S. Patent No. 10,010,567 B2
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`from my earlier Declaration, EX1006, including that all claims of U.S. Patent
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`10,010,567 (“the ‘567 Patent”) would have been obvious to a POSITA in view of
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`the prior art cited.
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`5.
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`In forming my opinions, I have also relied on my own education, work
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`experiences and knowledge, see my CV in my declaration, EX1006, the documents
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`referenced in Appendix E to my declaration, EX1006.
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`6.
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`PO notes (POR at 7) that I was provided with and used an older standard for
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`claim construction in which claim terms are interpreted according to their
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`“broadest reasonable construction in light of the specification”, rather than the
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`Phillips claim construction standard which I have been informed requires “that
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`claim construction begins with the ordinary and customary meanings of the terms
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`used in the claims and that the meanings of terms used in the claims should be
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`understood primarily in view of the intrinsic record, including the specification and
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`file history”. I am aware of both claim construction standards and, in my opinion,
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`my original claim construction remains the most appropriate claim construction
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`under either standard.
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`‘567 PETITION GROUNDS
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`7.
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`The chart below summarizes my understanding of the grounds Petitioner is
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`asserting for the invalidity of the ‘567 patent.
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`Basis
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`35 U.S.C. §103(a)
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`Claims Challenged
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`1-5, 7-11, 15-17
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`35 U.S.C. §103(a)
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`6, 14, 20
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`35 U.S.C. §103(a)
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`12, 18
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`35 U.S.C. §103(a)
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`13, 19
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`References
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`Sampalis I (EX1012)
`Bottino II (EX1038)
`Randolph (EX1011)
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`Sampalis I (EX1012)
`Bottino II (EX1038)
`Randolph (EX1011)
`Breivik II (EX1037)
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`Sampalis I (EX1012)
`Bottino II (EX1038)
`Randolph (EX1011)
`Bottino I (EX1007)
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`Sampalis I (EX1012)
`Bottino II (EX1038)
`Randolph (EX1011)
`Fricke (EX1010)
`Yamaguchi (EX1162)
`Hardardottir (EX1164)
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`5
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`RIMFROST EXHIBIT 1086 Page 0005
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`Ground
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` 1
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`U.S. Patent No. 10,010,567 B2
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`PO MISCHARACTERIZES BOTTINO II (EX1038)
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`8.
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`PO wrongly asserts (POR at 5-6, 12-17, see also, Jaczynski Dec., EX2001 at
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`¶¶ 24-31) that “Bottino II provides no information on free fatty acid content” and
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`thus Bottino II cannot support a disclosure by it of a krill oil with less than 3% free
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`fatty acids as required by the ‘567 patent claims.
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`9.
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`As I described in my original declaration in support of the petition
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`(EX1006), Bottino II Table 2, Lipids of Antarctic krill, copied below, reports a
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`lipid fraction for E. superba krill caught at Station 11 that is labeled Unknown.
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`The ‘Unknown’ lipid fraction in Bottino II Table 2 is identified in the footnote as
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`presenting between the triglyceride and diglyceride fractions, and that the amount
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`recovered was too small to characterize further. A POSITA would have understood
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`that this fraction would include any free fatty acids that are present in the krill
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`lipids. Thus a POSITA would have understood the free fatty acid content for the
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`Station 11 Euphausia superba krill oil extract to be less than 2% - and thus the free
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`fatty acid content of the krill oil extract was necessarily less than the 3% w/w krill
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`oil claimed by the ‘567 patent. Bottino II, p. 481, EX1038, p. 0003; see also
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`Tallon Dec., EX1006, at ¶¶ 174, 459. Moreover, since Bottino II identifies 98% of
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`the lipid components of the Station 11 E. Superba, a POSITA would have
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`understood that any free fatty acids would be located in the remaining 2% labeled
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`as ‘Unknown.’ In addition, a POSITA would have known that free fatty acids
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`when analyzed by TLC will typically have an Rf value between those of
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`triglycerides and diglycerides, which as noted in the footnote to Table 2 of Bottino
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`II places the free fatty acids among the 2% ‘Unknown” components. See Bottino
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`II, EX1038 at 0003, and ¶¶ 17-29, below.
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`10. The arguments presented by PO (POR at 5-6, 12-17) in relation to the
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`disclosures of free fatty acid content by Bottino II hold no merit and do not change
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`my opinion on the obviousness of all of the ‘567 claim terms, including claims
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`related to free fatty acid content. I discuss the failings of PO’s arguments herein.
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`PO’s Attempt to Discredit Bottino II is Invalid.
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`11. PO, depending on the declaration of Jaczynski (EX2021), advances two
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`arguments related to the disclosure of free fatty acid (FFA) levels by Bottino II
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`(EX1038). Firstly that the FFA levels are not directly disclosed and that the
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`identified TLC result doesn’t represent FFA in light of results presented by
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`Freeman and West (EX2002), and secondly that the results in Bottino II are of
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`insufficient quality for a POSITA to determine FFA levels. Both arguments are
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`flawed for reasons described below.
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`12. The ‘567 patent includes two independent claims, 1 and 15, which both
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`require that the claimed krill oil comprises “less than 3% w/w free fatty acids”. As
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`Petitioner asserted in its Petition, relying on my declaration, Bottino II discloses in
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`Table 2 a krill oil containing 2±22% Unknowns which includes the free fatty acid
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`content of the krill lipid extract. Thus “the free fatty acid content of this extract
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`would be at most 2%.” Petition, Paper 2, at 22; citing Tallon Dec., EX1006 at ¶
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`174.
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`13. Free fatty acids are a natural lipid component present in krill as a small
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`proportion of the total lipids in the live krill organism. Additional free fatty acids
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`may also form as a by-product of other lipids through the activity of lipases during
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`handling and processing of krill after harvesting. Consequently, the level of free
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`fatty acids present in a krill material is to some extent dependent on, and is a
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`U.S. Patent No. 10,010,567 B2
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`reflection of, the way in which the krill has been processed.
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`14. Lipase activity in krill commonly results in hydrolysis of the fatty acid
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`groups that are attached to triglycerides and phospholipids. The fatty acid is
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`cleaved to form a free fatty acid, and a residual lipid. When a single fatty acid
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`group is removed from a triglyceride the residual lipid becomes a diglyceride. If a
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`second or third fatty acid group is removed it becomes a monoglyceride or glycerol
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`respectively. Similarly for phospholipids containing fatty acyl groups, the fatty
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`acids can be cleaved to form free fatty acids and residual lyso-phospholipids.
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`Consequently, the formation of free fatty acids by lipase activity results in a
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`depletion of the levels of triglycerides and phospholipids, and an increase in the
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`levels of partial glycerides including diglycerides and lyso-phospholipids.
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`15. Bottino II used a TLC method to measure lipid class information. TLC is a
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`measurement technique in which the sample to be analysed is placed on a plate
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`coated in a silica adsorbent and exposed to a solvent or combination of solvents.
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`Different compounds travel at different rates along the plate depending on the
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`specific interaction between the compound, the adsorbent, the solvents used, the
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`composition and quantity of the sample, and other factors including time and
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`temperature. The rate of movement can be expressed as an Rf value or retardation
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`factor representing how far along the plate they travel compared to the solvent
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`front. See also the description by PO’s expert. Jaczynski Dec., EX2001 at ¶¶ 27-
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`U.S. Patent No. 10,010,567 B2
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`29. Quantitative analysis by TLC can be carried out in different ways. In the case
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`of Bottino II it was by recovering each group of compounds separately from the
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`plate and weighing them. It is a direct measurement of the weight proportion of
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`each compound that was in the initial sample.
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`16. Bottino II describes the lipid classes present in several krill samples,
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`including neutral and polar lipids classes. Results are presented for two samples of
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`E. Superba and two samples of E. Crystallorophias, collected from four different
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`locations in the Antarctic Ocean. These are all disclosed in Bottino II Table 2,
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`EX1038 at 0003. As I discussed in my previous declaration (EX1006 at ¶ 174),
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`Bottino II discloses a lipid component which is labelled as ‘Unknown’ in Table 2,
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`but is described by its position relative to other lipid components and would be
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`understood by a POSITA to represent the free fatty acid content of the samples.
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`This understanding is consistent with and supported by other disclosures by
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`Bottino II, as well as with general prior art knowledge, as discussed below.
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`Additionally, while putting forward selected arguments in an attempt to discredit
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`Bottino II, PO does not actually provide, or even attempt to provide, any
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`interpretation of Bottino II other than as a disclosure of free fatty acid content.
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`Results of Freeman and West cannot be applied to Bottino II
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`17. Dr Jaczynski argues that the spot reported as ‘Unknown’ by Bottino II, with
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`Rf between those of Triglycerides and Diglycerides, could not be free fatty acids
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`because results reported by Freeman and West (EX2002) show free fatty acids
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`with a lower Rf value:
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`Figure 1 of Freeman and West [EX2002] demonstrates
`that free fatty acids have an Rf value that is lower than that
`of diglycerides and triglycerides in the solvent systems
`utilized and therefore the free fatty acids do not travel as
`far as triglycerides or diglycerides in solvent system 1
`(panel a of Fig. 1 or the combination of solvent systems 1
`and 2 (panel c in Fig. 1).
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`As can be seen [in Freeman and West, Exhibit 2002], in
`each solvent system utilized, free fatty acids (designated
`FA in Figure 1) migrate a shorter distance than
`triglycerides (TG in Figure 1) and diglycerides (DG in
`Figure 1), and farther than phospholipids (PL in Figure 1).
`The alterations of the Freeman and West solvent systems
`noted in Bottino II would not be expected to change this
`migration pattern. Thus, Bottino II’s “unknowns,” which
`migrated between the triglycerides and diglycerides, could
`not include free fatty acids as concluded by Dr. Tallon.
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`18.
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`Jaczynski Dec., EX2001 at ¶ 28 (detail in [] supplied). Figure 1 also
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`discloses that Freeman and West’s TLC had Rf for free cholesterol (C) higher than
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`the Rf for free fatty acids (FFA).
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`19. Dr Jaczynski’s opinion is incorrect as the Rf values described by Freeman
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`and West can’t be applied to Bottino II. Indeed, as discussed below, the alterations
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`of the Freeman and West solvent systems noted in Bottino II would be expected to
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`change, inter alia, the free fatty acid migration pattern. Bottino II describes the
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`method used in their analysis as follows:
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`For quantitative fractionation of lipid classes 20-40 mg of
`lipids were separated by the method of Freeman & West
`(1966) modified as follows: (a) Silica gel without binder
`(Adsorbosil-5, Applied Science) was used instead of silica
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`gel-G. (b) Acetic acid was eliminated from the solvent
`mixture No. 1
`to simplify
`the drying between
`developments. (c) Quantitation of the spots was done by
`gravimetry and not by colorimetry of dichromate
`reduction.
`Bottino II, EX1038 at 0002.
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`20. The method used by Bottino II, as copied above, is described as a
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`modification of the method of Freeman and West, with modifications to the solvent
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`systems used and to the type of adsorbent used (a different silica). Both of these
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`modifications are key determinants in the order of presentation of the components
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`in TLC analysis.
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`21. Though not expressly mentioned by Bottino II, but which would have been
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`known to a POSITA, other variations in operational parameters, equipment used,
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`and individual operating practices will also apply, and will further influence the
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`observed Rf values for each component ( see, e.g., Neilsen, pp. 450-451, EX1179
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`at 0004-0005) particularly for a multi-stage solvent system. These include at least
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`the development times in each solvent system, how the TLC plate is made, the size
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`and shape of the plate, how the plate is pre-conditioned, how the sample is applied
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`to the plate, the quantity and composition of the specific sample used, how the
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`plates are handled when switching between multiple solvent systems, and even on
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`atmospheric temperature and humidity. The Rf values observed in one analysis
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`cannot be directly compared to Rf values used with different solvent systems,
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`different adsorbent, different equipment, and different operating conditions. That is
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`why reference standards are used to calibrate the analysis.
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`22. Further, as also would have been known to a POSITA, many operational
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`details of the method used by Bottino II will have been adapted to meet the
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`objectives of the particular analysis being applied, and the equipment available to
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`carry out the analysis. In the case of Bottino II, the objective is characterisation of
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`the main lipid classes present in Krill, an objective that differs from that of
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`Freeman and West which is about demonstrating separation of a broad selection of
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`neutral lipid class standards. For example, Bottino II is interested in, and reports,
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`the separation of individual phospholipid classes including PC, PE, Lyso PC, and
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`PG. See Bottino II, Table 2, EX1038 at 0002. A simple comparison with the results
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`of Freeman and West, (Figure 1, EX2002 at 0002) will show that phospholipids
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`remain at the bottom of the plate and are not separated. Further, the separation and
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`quantification of free cholesterol is not of interest to Bottino II because there are
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`only trace quantities of free cholesterol in krill. See, e.g., Mimoun-Benarroch, p.
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`63, EX1099, p. 0003, Lambertsen, Table 1, p. 100, EX1101, p. 0003. The method
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`described by Freeman and West cannot be directly applied to meet the objectives
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`of the analysis in Bottino II.
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`23. The method described by Freeman and West uses two solvent systems
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`sequentially. This introduces further variables in comparing the different analysis
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`methods. In the first solvent system Bottino II specifically describes the omission
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`of acetic acid from the solvent mix. The effect of changing the solvent composition
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`is complex but acetic acid is a strongly polar compound and it would be expected
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`to influence the mobility of compounds with different polarities, as well as
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`facilitate interactions between free fatty acids and the alcohol (ethanol) present in
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`the first solvent system. These interactions are different to the interactions
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`between the solvent system and partial glycerides and would be expected to
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`influence the relative Rf values of free fatty acids compared to other components.
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`24. The second solvent mixture described by Freeman and West consists of
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`diethyl-ether and hexane, a solvent mixture that typically results in elution of free
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`fatty acids with an Rf between those of triglycerides and diglycerides, consistent
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`with the position of the ‘unknown’ free fatty acid spot in Bottino II. This can be
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`seen in general prior art literature including for example Handbook of Food
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`Analysis (Second Edition): Volume 1: Physical Characterization and Nutrient
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`Analysis, Leo M.L. Nollet (2004), Chapter 9, “Fatty Acids”, Zamora and Hidalgo,
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`Table 11, p. 237 (EX1177 at 0020), copied below.
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`25.
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`In accordance with Zamora and Hidalgo, Table 11, p. 237 (EX1177 at
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`0020), the solvent system disclosed in the first column (P-E-AA) consists of
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`“petroleum ether (a mixture of hydrocarbons, commonly including or analogous to
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`hexane)-diethyl ether – acetic acid” in a ratio of 90:10:1, the solvent system
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`disclosed in the second column H-E-FA consists of “hexane-diethyl ether – formic
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`acid” in a ratio of 80:20:2. Both of these solvent systems show that Rf for free fatty
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`acids falls between the Rf s for diglycerides and triglycerides: For the P-E-AA
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`solvent system, the Rf in the first column for free fatty acids (0.18) falls between
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`the Rf for diglycerides (0.08) and the Rf for triglycerides (0.35); For the H-E-FA
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`solvent system, the Rf in the second column for free fatty acids (0.42) falls between
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`the Rf for diglycerides (0.21 and 0.24) and the Rf for triglycerides (0.63).
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`26.
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`In general, many solvent systems follow this same pattern, depending on the
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`relative polarity of the different lipids present. The Rf for free fatty acids falls
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`between those for diglycerides and triglycerides in additional solvent systems
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`including for example: the Rf in the fourth column for free fatty acids (0.62) falls
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`between the Rf for diglycerides (0.41 and 0.46) and the Rf for triglycerides (0.70);
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`the Rf in the fifth column for free fatty acids (0.39) falls between the Rf for
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`diglycerides (0.15 and 0.21) and the Rf for triglycerides (0.60); the Rf in the sixth
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`column for free fatty acids (0.50) falls between the Rf for diglycerides (0.25 and
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`0.40) and the Rf for triglycerides (0.82). I have summarized these values below.
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`Rf values for DG, FFA and TG in various solvent systems (EX1177 at 0020).
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`Compound Col. 1
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`Col. 2
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`Col. 3
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`Col. 4
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`Col. 5
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`Col. 6
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`Col. 7
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`TG
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`FFA
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`1,3 DG
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`1,2 DG
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`0.35
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`0.18
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`0.08
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`0.08
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`0.63
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`0.42
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`0.24
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`0.21
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`0.79
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`0.21
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`0.66
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`0.53
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`0.70
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`0.62
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`0.46
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`0.41
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`0.60
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`0.39
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`0.21
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`0.15
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`0.82
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`0.50
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`0.40
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`0.25
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`0.57
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`0.16
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`0.36
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`0.36
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`27. While the Rf values observed in one TLC analysis cannot be directly
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`compared to another, as supported by Zamora and Hidalgo, Table 11, p. 237
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`(EX1177 at 0020) and consistent with other prior art including that discussed
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`below, in the majority of cases Free Fatty Acids will have an Rf value between
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`those of Triglycerides and Diglycerides as seen in Bottino II.
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`28. PO’s expert is incorrect in a-priori extension of Freemen and West’s Figure
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`U.S. Patent No. 10,010,567 B2
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`1 Rf ordering (namely, from the origin of the TLC plate of: PL, MG, FAA, C, DG,
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`TG, CE) (see EX2002 at 0002) to other TLC results. Even references cited by
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`Freemen and West disclose different Rf orderings. For example, Blank et. al.,
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`Quantitative Analysts of Lipids By Thin-Layer Chromatography, EX1176, Figure
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`5 at 0003, provides the following Rf ordering: PL, C, FFA, TG, CE. That is,
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`FFA’s Rf value is greater than that for free cholesterol (C), different to that
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`disclosed in Freeman and West. See below.
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`29. As demonstrated above, the particular solvent systems used in TLC
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`significantly influences the Rf value for, inter alia, FFAs, triglycerides and
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`18
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`diglycerides and, as discussed above, Freeman and West and Bottino II used
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`different solvent systems, as well as other functional and operational differences,
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`thus the results reported in the Freeman and West chromatograph and the results
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`reported in Table 2 Bottino II for, at least, FFAs are not comparable.
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`Bottino II disclosures taken as a whole
`independently describe FFA levels as the ‘unknown’ spot.
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`30. To understand Bottino II it is necessary to examine what is disclosed in
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`Bottino II itself as well as other prior art knowledge that supports the
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`interpretation.
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`31. Firstly, it is well known that free fatty acids would have been present in the
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`krill lipids analysed by Bottino II. A POSITA would have understood this, and
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`PO’s expert agrees that this is the case. “Given the content of diglycerides,
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`lysophosphatidylcholine, and phosphatidylglycerol reported in Table 2, it would be
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`reasonable to assume that free fatty acids were present in the sample but not
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`reported.” Jacyznski Dec., EX2001 at ¶ 29 (emphasis supplied).
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`32. The presence of free fatty acids in krill lipids was well reported in other
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`prior art. For example Fricke, EX1010 at 0003, discloses a free fatty acid content
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`in freshly cooked krill “ranging from 1% to 3% of total lipids” and that this “low
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`FFA content of freshly caught krill also was confirmed by Ellingsen (11)” and
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`Phleger, EX1173, Table 1 at 0004, discloses free fatty acids content for total lipid
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`extracts from E. Superba krill ranging from 1.1% to 1.8%.
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`33. Fricke also describes higher levels of free fatty acids in samples which have
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`been subjected to lipase activity (“In the 1977 sample the FFA content [16.1%] is
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`about twice that of the 1981 sample [8.5%]. The high value could be caused by the
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`longer storage time of the 1977 sample. A residual lipolytic activity against
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`phospholipids exists even at temperatures of -30 C and below” (EX1010 at 0002).
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`Thus free fatty acids are expected to be present in Bottino II’s analysis at levels of
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`1% to 3% representing fresh krill, or higher levels if lipase activity has occurred,
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`and their presence would have been expected to be included in the analysis
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`reported by Bottino II.
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`34. Dr Jaczynski concedes that these free fatty acids were present, but is unable
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`to offer an alternative explanation to free fatty acids being the ‘unknown’ spot,
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`other than to “speculate that the lack of data on free fatty acid content is due to
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`poor resolution of spots representing lipids on the TLC plates …”. Jaczynski Dec.
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`at ¶ 29. However, Bottino reports a number of other components of the krill oil
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`which constitute only 1% of the total oil (see Table 2, Exhibit 1020, page 0003, for
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`example Lyso-PC, and PG content for Station 11 are recorded as 1%) and Bottino
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`II is able to quantify these. Thus free fatty acids would not have been missed by
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`Bottino II due to poor resolution, as alleged by Dr Jaczynski, unless they were
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`present at levels lower than at least 1%.
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`35. Secondly, as discussed above at ¶¶ 24-26 and further discussed below, the
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`presence of free fatty acids with a Rf factor between those of triglycerides and
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`diglycerides, where the ‘unknown’ spot is located in Bottino II, is common in other
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`prior art using TLC analysis with similar low polarity elution solvents designed for
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`neutral lipid separation.
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`36. Yamaguchi discloses analysis of krill oil extracts from two types of krill
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`powder using TLC analysis. Figure 2 from Yamaguchi (EX1162 at 0003) is
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`reproduced below. The plates labelled I and II show spots produced using a solvent
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`mixture of diethyl-ether petroleum-ether and acetic acid where free fatty acids
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`(identified as spot #4) lies between those of triglycerides (spot #3) and diglycerides
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`(spot #5), in the same location as the ‘unknown’ spot in Bottino II.
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`Figure 2 shows TLC patterns of the oils extracted with SC-
`CO2 and the residual lipids. By co-TLC with standard
`reagents, the main component of the extracted oils was
`found to be triglycerides (spot 3) and the minor
`components were
`identified as hydrocarbon
`(1),
`cholesteryl ester (2), free fatty acids (4), diglycerides (5),
`cholesterol (7), monoglycerides (8), and carotenoids (6
`and 9), as illustrated in chromatogram I.
`***
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`Yamaguchi, EX1162 at 0002-0003 (emphasis supplied).
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`37. Further examples include TLC analysis of krill oil by Tsuyuki (EX1172),
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`which shows the presentation of Free Fatty acids between triglycerides and
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`diglycerides using a solvent system consisting of petroleum ether, diethyl ether,
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`acetic acid.
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`Tsuyuki, EX1172, Figure 1 at 0004.
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`38. Thirdly, Bottino II describes several krill compositions in Table 2 in addition
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`to the one for Station 11, and the only consistent explanation of the combined
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`results is if the unknown spot is free fatty acids. See Table 2 from Bottino II
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`(EX1038 at 0003) below:
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`Bottino II describes results for E. Superba harvested from two stations, identified
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`in Table 2 as Station 8 and Station 11. For Station 11, a triglyceride content of 36%
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`is reported, a diglyceride content of 4%, a complex lipid content of 58%, and an
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`‘unknown’ content of 2%. In contrast, Station 8 is recorded as having a decreased
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`triglyceride content of 8% and a decreased complex lipid content of 54%, an
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`increased diglyceride content of 17% and ‘unknown’ content of 21%. The results
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`for station 11 are consistent with the known lipid composition of fresh krill. The
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`results for station 8 are consistent with a krill oil which has experienced lipase
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`activity – i.e., the triglyceride level is reduced due to the removal of a fatty acid
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`group by the enzymes, and are converted into diglycerides and free fatty acids. The
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`24
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`RIMFROST EXHIBIT 1086 Page 0024
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`diglyceride content for station 8 increased significantly, from 4% at Station 11 to
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`17% at Station 8, therefore a proportional increase in free fatty acids is expected.
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`The ‘unknown’ spot in Bottino II’s analysis for Station 8 meets this expectation,
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`and the only reasonable interpretation for the results of Bottino II is for the
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`‘unknown’ spot to represent free fatty acid content. Similarly, the reduced level of
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`total complex lipid in Station 8 indicates a degree of lipase hydrolysis converting
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`phospholipids into smaller (lyso) phospholipids and adding to the free fatty acid
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`content.1 All of these effects were well known to a POSITA. As noted above, Dr
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`Jaczynski concedes that the presence of diglycerides and lysophospholipids gives
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`the expectation of a corresponding presence of free fatty acids. Jaczynski Dec.,
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`EX2001 at ¶ 29.
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`39.
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`In summary, all of these points serve to identify the ‘unknown’ spot in
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`Bottino II as representing the free fatty acid content: The presence of free fatty
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`acids is expected at a level that would have been able to be measured by Bottino II
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`1 Results given by Bottino II for E. Crystallorophias, stations 13 and 16 in Table II
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`also demonstrate the same pattern, with reduced triglyceride and phospholipid
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`content at station 16 corresponding with elevated levels of diglycerides and free
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`fatty acid (‘unknown’) levels.
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`and would be expected to have been reported; the presentation of free fatty acids
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`with a Rf factor between those of triglycerides and diglycerides, as observed by
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`Bottino II, is common (see discussion above); and comparison of the results
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`reported for Stations 8 and 11 (and 13 and 16) by Bottino II demonstrates that the
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`‘unknown’ spot must be free fatty acids for a consistent interpretation of the
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`results. In contrast, PO has not demonstrated that the results of Freeman and West
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`using a different TLC method changes this interpretation; has not proposed an
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`explanation for why free fatty acids are not reported by Bottino II (as alleged by
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`PO) other than ‘speculation’; and has not provided any interpretation of what the
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`‘unknown’ spot in Bottino II might be if not free fatty acids.
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`40. Finally, PO’s expert has even recently co-authored an article disclosing FFA
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`falling between DG and TG in a TLC analysis of krill. The analysis of krill oil and
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`krill oil fractions by TLC is described by Showman (EX1174, published in 2020),
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`of which PO’s expert Jaczynski is an author. Figure 1 shows Free Fatty acids
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`between triglycerides and diglycerides in a solvent system including hexane,
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`diethyl ether, acetic acid.
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`26
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`RIMFROST EXHIBIT 1086 Page 0026
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`Showman, EX1174, Figure 1 at 0004.
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`Reliability of Bottino II’s results
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`41. Dr Jaczynski discusses the reliability of the data reported by Bottino II and
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`argues that “the data is not reliable and would be disregar
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