IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`In re Inter Partes Reexamination of U.S. Patent No. 8,030,348
`
`Entitled:
`
`NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING
`POLYUNSATURATED FATTY ACIDS AND THEIR APPLICATIONS
`
`Issued:
`
`4 October 2011 to Sampalis
`
`DECLARATION BY DR. THOMAS GUNDERSEN IN SUPPORT OF
`
`REQUEST FOR INTER PARTES REEXAMINATION OF
`U.S. PATENT NO. 8,030,348
`
`EFS WEB Filed
`
`Mail Stop Inter Partes Reexam
`Commissioner for Patents
`P.O. Box 1450
`
`Alexandria, VA 22313-1450
`
`1, Dr. Thomas Gundersen, state as follows:
`
`1.
`
`My present position is CEO of Vitas AS - Analytical Pharma Services, Oslo, Norway. My
`
`Curriculum Vitae is attached hereto as Exhibit 1.
`
`2.
`
`I was asked by Aker Biomarine ASA to analyze lipid fractions extracted from two species of
`
`krill, Euphausia superba and Euphausia pacifica, to determine whether they contain phospholipids
`
`that have either eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) at both the sn-1 and sn-2
`
`positions of the phospholipid molecule. I did not prepare these fractions, but it is my understanding
`
`that they were obtained by the procedures described in Beaudoin I (W0 00/23 546), Beaudoin 11
`
`(Canadian Application 2,251,265) and Maruyama (Japanese Laid Open Application 2909508). The
`
`results of this analysis are attached hereto as Exhibit 2.
`
`3.
`
`As described in Exhibit 2, flactions produced according to either the Beaudoin I or Beaudoin II
`
`processes were either left unheated or heated to either 70°C for 5 minutes or 125°C for 15 minutes as
`
`described in Beaudoin I and II. The Maruyama fractions were not heated.
`
`000001
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1049
`
`000001
`
`

`
`4.
`
`As described in detail in Exhibit 2, I selected RP-HPLC, HPLC-MS/MS and Multiple Reaction
`
`Monitoring (MRM) to analyze the lipid fractions for the presence of phosphatidylcholine (PC)
`
`containing EPA at both sn-1 and sn-2 positions (PC-EPA/EPA), phosphatidylcholine with EPA at the
`
`sn-1 position and DHA at the sn-2 position or vice versa (PC-EPA/DHA, PC-DHA/EPA), and
`
`phosphatidylcholine with DHA at both of the sn-1 and sn-2 positions (PC-DHA/DHA). PC-
`
`EPA/EPA and PC-DHA/DHA (99% pure, Sigma Aldrich MO, USA) were used as reference
`
`standards. It is important to use reference standards and positive controls to verify that the analytical
`
`procedures are working and will perform their intended purpose. It is especially important to optimize
`
`mass spectrometric parameters using reference compounds.
`
`PC-EPA/EPA has a predicted molecular weight of 825.6, PC-EPA/DHA has a predicted molecular
`
`weight of 851.6, and PC-DHA/DHA has a predicted molecular weight of 877.6. The structures of the
`
`substances with molecular mass 825.6, 851.6 and 877.6 were examined by the use of RP- HPLC-
`
`MS/MS. An Agilent technologies high end linear tandem mass spectrometer (Agilent 6460 triple
`
`Quad) was interfaced with RP-HPLC using ESI ionisation in positive mode. Product ion scans of the
`
`protonated parent molecular weights (826.6 and 878.6) were then taken up for the reference standards.
`
`These data were then used as a basis for selection of multiple reaction monitoring (MRM) transitions
`
`and reconstructed ion chromatograms from the transitions described in the following were taken up.
`
`The transitions were chosen to allow us to determine whether the lipid is a phospahtidylcholine species
`
`and whether both the Sn-1 and Sn-2 positions on the phospholipid are occupied by either EPA or
`
`DHA.
`
`PL-Species
`
`
`
`
`
`
`
`félliifisé
`Reterrtioai time"‘ Parent mass Lass off
`ED
`Ufl+1]
`IEIEWEE
`IEEEENWIIIMIIIEWII
`IEIEMHE
`EMEEMEIIIIIIEMII
`Ififlfifififl
`IMIEENNEIIIIIIIEEEI
`Ififlfifififl
`flEfiEMMEIIE%IIIEEEI
`
`* HPLC-MSIMS system
`
`
`%%m%:pasIvib
`
`MRM
`
`
`
`5.
`
`Use of single quadrupole MS showed that all samples (with the exception of the ethyl acetate
`
`extracts from E. pacifica) contained lipids that matched either the theoretical molecular weight ( PC-
`
`EPA/DHA) or both molecular and theoretical weight and the chromatographic retention time of
`
`000002
`
`000002
`
`

`
`reference standards ( PC-EPA/EPA, PC-DHA/DHA). The E. pacifica ethyl acetate fractions contained
`
`very little material. It is not unlikely that something has not worked according to the procedure during
`
`the extraction of the E. pacifca krill samples and it is doubtful that these samples were representative
`
`samples. This assumption is supported by the fact that ethanol extraction of the Same sample type gave
`
`a much higher yield and that ethyl acetate extraction of the Same type of sample but with E.Superba
`
`instead ofE. pacifica also gave a high yield. The extraction should have been repeated but there was
`
`not enough time for this. However, it is expected that if Sufficient amount of material had been used
`
`for the analysis the three phospholipid species would have been present as they were in the E. pacifica
`
`ethanol fractions and in the E.Superba ethyl acetate fractions.
`
`All samples were analyzed with MRM on the triple quadrupole, the criteria for positive identification
`
`was a positive signal at the correct retention time for the different MRMS as shown in the Table below
`
`(e.g., for PC-EPA/EPA a positive signal was required for [825+l]—>l84 and MRM1). Full results for
`
`all samples are shown in the following Table.
`
`
`
`
`
`MRM
`
`
`MRMs
`1
`
`
`
` Treatment
`[825--H]—>184
`
`Conclusion
`PC-
`
`
`°C/min
`[851+H]—>184
`
`EPA/EPA
`
`
`
`
`
`826—>524
`
`YES
`
`none
`
`YES
`
`YES
`
`Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`
`
`Vitas
`
`Frac.
`
`ID
`
`#
`
`P308-1
`
`Contains PC-DHA/DHA
` Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
`Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
`
`
` n.d. PC-EPA/EPA
`
`n.d. PC-EPA/DHA
`
`n.d. PC-DHA/DHA
`
`
`
` n.d. PC-EPA/EPA
`
`n.d. PC-EPA/DHA
`
`n.d. PC-DHA/DHA
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`NO
`
`NO
`
`NO
`
`NO
`
`NO
`
`NO
`
`NO
`
`NO
`
`000003
`
`P308—2
`
`70/5
`
`P308-3
`
`125/15
`
`none
`
`IIb
`
`70/5
`
`
`
`
`
`P308-4
`
`P308-5
`
`YES
`
`YES
`YES
`
`YES
`
`YES
`YES
`
`NO
`
`NO
`
`NO
`
`NO
`
`NO
`NO
`
`
`
`000003
`
`

`
`NO
`
`NO
`
`NO
`
`NO
`
`n.d. PC-EPA/EPA
`
`n.d. PC-EPA/DHA
`
`n.d. PC-DHA/DHA
`
`Contains PC-EPA/EPA
`
`YES
`
`YES
`
`YES
`
`YES
`
`
`
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
`Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
` Contains PC-EPA/EPA
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
`
`
` Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`
`
`Contains PC-DHA/DHA
`
` Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`P308—
`
`10
`
`P308-
`
`YES
`YES
`
`YES
`
`none
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`IIb
`
`125/ 1 5
`
`
`
`P308-6
`
`P308-7
`
`
`
`110116
`
`P208-8
`
`Ila
`
`70/5
`
`P308-9
`
`125/15
`
`NO
`
`NO
`NO
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`YES
`
`YES
`
`YES
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`
`
`
`
`Contains PC-DHA/DHA
`
`Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
` Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
`
`
` Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
`
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`YES
`YES
`
`YES
`YES
`YES
`
`YES
`YES
`
`YES
`
`YES
`YES
`
`YES
`
`YES
`
`YES
`YES
`
`YES
`
`YES
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`YES
`
`000004
`
`70/5
`
`125/ 1 5
`
`n.a.
`
`n.a.
`
`n.a.
`
`n.a.
`
`1 1
`
`1>30s—
`
`12
`
`P308-
`
`13
`
`P308-
`
`14
`
`P308-
`15
`
`P308-
`16
`
`
`
`
`
`
`
` Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
`Contains PC-EPA/EPA
`
`Contains PC-EPA/DHA
`
`Contains PC-DHA/DHA
`
`
`
`000004
`
`

`
`6.
`
`In summary, these results demonstrate that each of the lipid fractions tested (with the exception
`
`of the ethyl acetate extracts from E. pacifica) contain the following phospholipid species: PC-
`
`EPA/EPA; PC-EPA/DHA; and PC-DHA/DHA.
`
`7.
`
`I further declare that all statement made herein of my own knowledge are true and that all
`
`statements made on information and belief are believed to be true; and further that these statements
`
`were made with the knowledge that willful false statements and the like so made are punishable by
`
`fine or imprisonment, or both, under section 1001 of title 18 of the United States Code, and that such
`
`willful false statements may jeopardize the validity of the application or any patent issued thereon.
`
`Respectfully submitted,
`
`Dr. Thomas Gundersen
`
`Date: October 4, 2011
`
`000005
`
`000005
`
`

`
`EXHIBIT 1
`
`000006
`
`000006
`
`

`
`CURRICULUM VITAE OF THOMAS E GUNDERSEN
`
`Personal
`
`Name: Thomas Erik Gundersen
`
`Born: August 4th 1967
`
`Address: Lillevarmsveien 69B, 0788 Oslo
`
`Phone: +47 22 95 86 31/ +47 928 08 973
`
`E—mai1: teg@vitas.no
`
`Marital status: Married, 3 children
`
`Education
`
`1990- 1995: M.Sc. in Chemistry, majoring in Analytical Chemistry,
`University of Oslo. Title of thesis: Quantitative high performance liquid
`chromatographic determination of retinoids in human serum using on—line solid
`phase extraction and column switching. Rating 1.2 ( on a scale from 1-6 where
`1 is best)
`
`2007 Dr. Philos ( PhD) at the Medical Faculty, University of Oslo. 8
`published articles. Title: "Development of ultra—sensitive chromatographic
`tools for high—throughput analysis of retinoids in biological samples"
`
`Trial lecture: Lipidomics — Recent technological developments and
`applications
`
`Relevant work experience
`
`01/01/1998 to 12/31/2001: Recruitment Fellow of the Norwegian Cancer Society at the
`Institute for Nutrition Research, University of Oslo.
`
`01/01/2002 to 12/31/2004 Research Fellow at the Institute for Nutrition Research, University
`of Oslo.
`
`01.01 .2005-Researcher at the Institute for Nutrition Research, University of Oslo.
`
`2002 - General manager of Vitas' s
`
`2007 — General Manager Bioindex as
`
`2008 — Senior Researcher at the Medical Faculty, University of Oslo ( 20 %)
`
`000007
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`000007
`
`

`
`Courses:
`
`Course in Gas Chromatography, 1997 1 day
`
`Courses in HP- LC-Chemstation, 1998 1 day
`
`Courses Capillary electrophoresis, 1998 2 days
`
`Course LC-MS, 1999 1 day
`
`Course LC-MS, 2000, one day
`
`Courses in LC-MS/MS, 2005 3 days
`
`HSE Course for Directors, 2008 1 day
`
`GIVIP courses, one day in 2008
`
`Teaching and tutoring
`
`Supervisor for graduate students Sakhi Amrit K, Stein Joakim Thommesen and Erasmus
`exchange student Carsten Haas in the period 1998-2001.
`
`Supervisor for doctoral students Amrit K Sakhi and Olov Nordstrom in the period 2001-2003.
`
`Supervisor for doctoral students Bente Lise Halvorsen and Anette Karlsen in the period
`2002-2008.
`
`The head of the analytical "subgroup" consisting of a total of 5 persons in Rune Blomhoffs
`research group at the Institute for Nutrition Research, University of Oslo. In the period 2000-
`2006
`
`Assistant supervisor for Post-Doc Sakhi Amrit K. and E. Nasser Bastani Fellow during 2008.
`
`Invited work
`
`a) Review, Gundersen, TE & Blomhoff, R., On-line solid-phase extraction and isocratic
`separation of retinoic acid isomers in micro drilling column switching system. Methods
`Enzymol 2992430-41, 430-441 (1999).
`
`b) Review, Gundersen, TE & Blomhoff, R., Qualitative and Quantitative liquid
`chromatographic determination of natural retinoids in biological samples. J Chromatogr A.
`2001 November 1923, 935 (1-2) :13-43.
`
`c) Review, Gundersen, TE, Methods for Detecting and Identifying retinoids in tissue. J
`Neurobiol. 2006 June; 66 (7) :63]-44.
`
`Other activities
`
`000008
`
`000008
`
`

`
`Founded in 1994 AS Vitas, together with Professor Rune Blomhoff and Professor Christian A
`Drevon. AS Vitas is GMP certified analytical CRO that offers services to Academia,
`Pharmaceutical and biotech industry, hospitals and the Norwegian primary health care. The
`company is located in Oslo Innovation Centre. The analysis methods used are a direct result
`of graduate and doctoral work of Thomas Gunderson.
`
`Thomas Gundersen have from 1994 until today been central in the daily operation and
`development of business strategy and analysis in AS Vitas, and is part owner of the company.
`CEO of AS Vitas, 1 February 2002 -
`
`Founded in December 2006 the company Bioindex AS together with the technology transfer
`office of UIO. Thomas Gunderson sat on the board of Bioindex AS start-up to September
`2007.
`Is now CEO of Bioindex AS.
`
`"Expert reviewer” of fat—soluble vitamins in Finland Labquality external quality assurance
`program from September 2009 -
`
`Principal Scientist in EU projects:
`
`1. Food4Me — 2011-2015
`
`2. EPIC-CVD - 2012-2016
`
`3. Nutri Tech - 2012-2016
`
`Publications
`
`1 Gundersen, T. E., Lundanes, E. & Blomhoff, R. J Chromatogr B.Biomed.Sci.Appl. 691,
`1943-1958 (1997).
`
`2. Holven, KB, Natarajan, V., Gundersen, TE., Moskaug, JO, Norum, KR & Blomhoff, R.
`Int. J Cancer 71, 654-659 (1997).
`
`3. Sakhi, AK, Gundersen, TE., Wolf, SM, Blomhoff, R. & Lundanes, E.. J Chromatogr. A
`828, 451-460 (1998).
`
`4. Christensen, EI, Moskaug, JO, Vorum, H., Jacobsen, C., Gundersen, TE., Nykjaer, A.,
`Blomhoff, R., J Am Soc Nephrol 10, 685-695 (1999).
`
`5 Gunderson, T. E. & Blomhoff, R. Methods Enzymol 299243 0-41, 430-441 (1999).
`
`6. P. Molander, TE Gundersen, C. Haas, T. Greibrokk, R. Blomhoff, E. Lundanes, Journal
`of Chromatography A 847, No. 1-2, 25 1999 59-68
`
`7. P. Molander, SJ Thommesen, IA Bruheim, R. Trones, T. Greibrokk, E. Lundanes, TE
`Gundersen, HRC Journal of High Resolution Chromatography, 22, Nr. 9, September 1999,
`pages 490-494
`
`000009
`
`000009
`
`

`
`8. Ulven SM, Gundersen TE, Weedon MS, Landaas VO, Sakhi AK, Fromm SH, Geronimo
`BA, Moskaug JO, Blomhoff R, Dev Biol 2000 in April 1915, 220 (2) :3 79-91
`
`9. SM. Ulven, TE. Gundersen, AK. Sakhi, JC. Glover and R. Blomhoff, Dev Dyn. 2001
`November; 222 (3) :341-53.
`
`10. F. Hoover, TE Gundersen, SM Ulven, J. Micha Ille, S. Blanchet, R. Blomhoff and JC
`Glover, J Comp Neurol. 2001 in July 1930, 436 (3) 1324-35
`
`11 TE Gundersen and R. Blomhoff, J . Chromatogr A. 2001 November 1923, 935 (1-2) :13-
`43
`
`12. Petrosian AM, Haroutounian JE, Gundersen TE, Blomhoff R, Fugelli K, Kanli H.
`Adv Exp With Biol. 2000; 483:453-60.
`
`13. Capillary High-Performance Liquid Chromatographic determination of lutein and
`zeaxanthin in aqueous humor from a single mouse eye. Anette Karlsen, George Alexander;
`Rune Blomhoff and Thomas E Gundersen. 1: J Chromatogr B analyte Technol BioMed
`Life Sci. 2003 in September 1925, 795 (1) :17-23.
`
`14. Identification of novel roles of the cytochrome P450 system in early embryo genesis,
`effects Wed vasculogenesis and retinoic acid homeostasis. Diana ME Otto, Colin J.
`Henderson, Diane Carrie, Megan Davey, Ralf H. Adams, Cheryl Tickle, Thomas E.
`Gundersen, Rune Blomhoff and Rolan Wolf. Mol Cell Biol. 2003 September; 23
`(17) :6103-16.
`
`15. Sakhi AK, Russnes KM, Smeland S, Blomhoff R, Gundersen TE., Simultaneous
`quantification of Reduced and oxidized glutathione in plasma needs something a two-
`dimensional chromatographic system with parallel Porous graphitized carbon columns
`coupled with fluorescence and coulometric electrochemical detection. J Chromatogr A. 2006
`February 3, 1104 (1-2) :179-89.
`
`16. Karlsen A, Blomhoff R, Gundersen TE. High-throughput analysis of vitamin C in
`human plasma with the use of HPLC with monolithic column and UV detection. J
`Chromatogr B analyte Technol BioMed Life Sci. 2005 in September 1925, 824 (1-2) :132-8.
`
`17. Drevon CA, Henriksen HB, Sanderud M, Gundersen TE, Blomhoff R. Biological
`effects of vitamin K and concentration of vitamin K in Norwegian food. Tidsskr Nor
`Laegeforen. 2004 Jun 17; 124 (12) :l650-4. Review.
`
`18. Gundersen, TE., Methods for Detecting and Identifying retinoids in tissue. J Neurobiol.
`2006 June; 66 (7) 2631-44.
`
`0000010
`
`0000010
`
`

`
`19. Sakhi AK, Blomhoff R, Gundersen TE. Simultaneous and trace determination of the
`Reduced and oxidized glutathione in minute plasma samples needs something dual mode
`fluorescence detection and column switching high performance liquid chromatography. J
`Chromatogr A. 2007 February 1923; 1142 (2) :l78-84
`
`20. Karlsen A, Blomhoff R, Gundersen TE.
`Stability of whole blood and plasma Ascorbic acid. Eur J Clin Nutr. 2007 Feb 7
`
`21. TE Gundersen, NE Bastani, R. Blomhoff: Quantitative high-throughput determination
`of endogenous retinoids in human plasma needs something triple-stage liquid chromatography
`/ tandem mass spectrometry.Rapid commun Mass Spectrom. 2007; 21 (7) :1176-86.
`
`22. Berhe N, Halvorsen BL, Gundersen TE, Myrvang B, Gundersen SG, Blomhoff R.
`Reduced serum concentration of retinol and alpha-Tocopherol and high concentration of
`hydroperoxides are Associated with community levels of S. mansoni infection and
`schistosomal periportal fibrosis in Ethiopian school children. Am J Trop With Hyg. 2007
`May; 76 (5) :943-9.
`
`23. Noble BR, Babiuk RP, Clugston R, Underhill TM, Sun H, Kawaguchi R, Walfish PG,
`Blomhoff R, Gundersen TE, Greer JJ. Mechanisms of action of the congenital diaphragmatic
`hemia-inducing teratogen nitrofen. Am J Physiol Lung Cell Mol Physiol. 2007 August 1917
`
`24. Katy Schmidt, Catherine Hughes, JA Chudek, Sirnon Goodyear, Richard Aspden,
`Richard Talbot, Thomas E. Gundersen, Rune Blomhoff, Colin Henderson, C. Roland Wolf
`and Cheryl] Tickle. Cholesterol metabolism: the main pathway acting downstream of
`cytochrome P450 oxidoreductase in skeletal development of the limb. Molecular and cellular
`Biology, March 9 2009.
`
`24. Kristoffer Strom and Thomas E. Gundersen, Ola Hansson, Stephanie Lucas, Céline
`Fernandez, Rune Blomhoff, and Cecilia Holm. Horrnone-sensitive lipase (HSL) is Also a
`Retinyl ester hydrolase: evidence from mice lacking HSL. FASEB J. February 2009.
`
`25. Nasser Bastani, Thomas E. Gundersen and Rune Blomhoff. Determination of 8-epi
`PGF2(X1I1 human plasma, full blood, erythrocyte and urine needs something isotope dilution
`and triple-stage liquid chromatography / tandem mass spectrometry. Rapid commun Mass
`Spectrom. 2009 in August 1910, 23 (18) :2885-2890.
`
`26. Trygve Holmoy 12’ Stine Marit Moen 1’ Thomas E. Gundersen 3’ A Michael Holick
`January 4 Falch 5 Enrico Fainardi 6’ Massimiliano 6’ Ilaria Casetta 6. Vitamin D in
`cerebrospinal fluid from Patients with multiple sclerosis relapse and remission hum.
`Accepted by Multiple Sclerosis, February 2009, Published online September 2009.
`
`27. Jaensson—Gyllenbéick E, Kotarsky K, Zapata F, Persson EK, Gundersen TE, Blomhoff
`R, Agace W W. Bile retinoids imprint intestinal CD103 (+) dendritic cells with the ability two
`gene ratio gut—tropic T cells. Mucosal Immunol. Mucosal Immunol. 2011 Jul;4(4):43 8-47
`
`28. Holmcay T, Lossius A, Gundersen TE, Moen SM, Castellazzi M, Fainardi E, Casetta I.
`Intrathecal levels of vitamin D and lgG in multiple sclerosis. Acta Neurol Scand. 2011 Jul 23
`
`0000011
`
`0000011
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`

`
`Posters and oral presentations
`
`1 Gundersen, T and Blomhoff, R., Automated chromatographic determination of fat-soluble
`vitamins. Oral presentation, 13th Norwegian symposium in chromatography, 1996.
`
`2. Sakhi, AK, Gundersen TE, Ulven, SM, Blomhoff, R. & Lundanes, E Electrochemical
`detection of endogenous retinoids in mouse embryos by HPLC with on-line solid—phase
`extraction and column-switching, Posters, HPLC 9, St. Louis, USA J 998.
`
`3. Gundersen, TE, Ulven, SM, Blomhoff, R., Ultra Sensitive screening of retinoids in
`embryonic minute samples, items, retinoids 99, Strasbourg, France. 1999.
`
`4. Gundersen, TE, Dahlgren, PA, Blomhoff, R., Peak Synchronised post-column injection of
`internal standards for correction of matrix ion suppression effects in electro spray ionisation.
`Poster, 23 "1 Internatinal Symposium Wed chromatography, London, UK, 2000.
`
`5. Thomassen, A, Blomhoff, R, Gundersen, T., Transfer of chromatographic methods for
`routine determination of biological samples for capillary HPLC "plug & play"?, Oral
`presentation, 15th Norwegian symposium in chromatography, 2002.
`
`6 Sakhi, AK, Blomhoff, R, Gundersen, T., Isocratic RP-HPLC analysis of tocopherols and
`tocotrienols in plasma., Oral presentation, 15th Norwegian symposium in chromatography,
`2002.
`
`7 Gundersen, T and Blomhoff, R., Chromatographic determination of retinoids in embryonic
`tissue from mice. Oral presentation, 15th Norwegian symposium in chromatography, 2002.
`
`8. Gundersen TE, Invited plenary lecture “Kromatografidagene i Sandefjord”, January 2008.
`"High—throughput bioanalyse in routine. How to meet an increasing number of samples.
`
`9. Gundersen TE, Oral presentation MareLife March, Oslo Technopole 2009
`
`10. Gundersen TE, Oral Presentation, UCD Dublin, Human Nutrition: Where is the business?
`
`End.
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`0000012
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`EXHIBIT 2
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`0000013
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`0000013
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`Analytical Report
`
`Identification and Quantification of
`
`Specific Phospholipids in Krill Lipid
`
`Extracts by HPLC- Mass Spectrometry
`
`Sponsor: Aker Biomarine ASA
`
`Vitas project P308
`
`Sept 22-October 4”‘ - 2011
`
`By:
`
`Dr. Thomas E. Gundersen
`
`ceo
`
`Anders Dahlgren, M.S.
`
`Laboratory Manager
`
`Vitas as, Oslo Innovation Centre, Norway
`
`Vitas P308 Page 1
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`AS Vitas project: P308/ F785, F788 and 789 Protocol ID 83
`
`Please refer to this for correspondence
`
`Receipt of Samples:
`Samples were sent by Nofima, Norway and received at AS Vitas 28. September and 30. September
`and In total 16 samples were received and then stored in fridge ( 5 °C) until analysis.
`
`Date of Analysis:
`The analysis of the samples took place between 28. September and 4. October 2011 in the
`laboratories of AS Vitas.
`
`Reference to Raw Data and Journal:
`
`The raw data and results are stored at as Vitas in the P308: project archive, Perm ID 135. The work
`
`details for the actual analysis were recorded in the laboratory notebook no. 53, starting at page 1.
`
`Archiving:
`Raw data from the analysis are recorded according to Vitas archiving policy.
`The data will be archived at Vitas as for 5 years after which as Vitas will contact the customer to
`agree on transfer, retention or destruction of data.
`
`Aim of the project
`The aim of the project (Vitas P308) was to identify, if present, three specific phophatidylcholine (PC)
`structures, carrying n3—PUFA fatty acids at both the Sn—1 and Sn—2 position, in different lipid extracts
`from krill. Combinations of different chromatographic and mass spectrometric techniques were
`used to investigate this.
`
`PC—EPA/DHA; and PC-DHA/DHA.
`
`Summary of findings
`In summary, these results demonstrate that each of the lipid fractions tested (with the exception of
`the ethyl acetate extracts from E. pacifica) contain the following phospholipid species: PC—EPA/EPA;
`
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`Vitas as, Oslo Innovation Centre, Norway
`Vitas P308 Page 2
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`Introduction
`
`Vitas was asked to identify, if present, three specific phosphatidylcholine (PC) structures in
`
`different lipids extracts delivered to the Vitas laboratory in Oslo. In total 16 samples were received
`
`and registered in the Vitas database as shipment F785, F788 and 789 under Vitas project P308. An
`
`overview of the received samples is shown in table 1.
`
`Table 1 Overview of the received samples and specification of pre—ana|ytica| heat treatment
`
`Temperature treatment
`
`Marking of sample
`
`(“Cl
`
`E. pacifica Fraction Ila not heated
`
`E. pacifica Fraction Ila 70 degr 5 min
`
`E. pacifica Fraction Ila 125 degr 15
`
`E. pacifica Fraction llb not heated
`
`E. pacifica Fraction llb 70 degr 5 min
`
`E. pacifica Fraction llb 125 degr 15
`min
`
`E.Superba Fraction Ila not heated
`
`E.Superba Fraction Ila 70 degr 5 min
`
`E.Superba Fraction Ila 125 degr 15
`min
`
`E.Superba Fraction llb not heated
`
`E.Superba Fraction llb 70 degr 5
`min
`
`E.Superba Fraction llb 125 degr 15
`min
`
`Taiyo E. superba ETOH extract
`
`I--‘i-‘
`
`U1
`
`I-‘I U1
`
`l-|
`
`\I O
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`Vitas as, Oslo Innovation Centre, Norway
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`Taiyo E. pacifica ETOH extract
`
`Taiyo E. pacifica crude PL extract
`
`Taiyo E. superba crude PL extract
`
`n.a. = not applicable
`
`The phophatidylcholines to be identified and quantified were to have omega-3 poly unsaturated
`
`fatty acids (n3—PUFA) in both Sn—1 and Sn—2 position. The n3—PUFAS could be combination of the two
`n3—PUFAs A and B listed below.
`
`A. Eicosapentaenoic acid (EPA), 20:5(n-3), with IUPAC name: (5Z,8Z,11Z,14Z,17Z)—eicosa-5,8,11,14,17-
`
`pentenoic acid and a structure:
`
`
`
`Figure 1 EPA structure
`
`B. Docosahexaenoic acid (DHA), 22:6(n—3), with UIPAC name:
`
`(4Z,7Z,10Z,13Z,16Z,19Z)—docosa—
`
`4,7,10,13,16,19—hexaenoic acid and the structure;
`
`it
`
`
`
`Figure 2 DHA structure
`
`Substituting the fatty acids A and B with the R in the generic phophatidylcholine structure below will
`
`give four possible structures. Three of these are listed in table 1. The forth structure is an isomeric
`structure of PC-EPA/DHA where EPA and DHA have changed place (PC—DHA/EPA). These two isomeric
`
`structures cannot easily be distinguished and nor is it relevant for the purpose of this examination to
`
`achieve this separation. Thus these isoform are treated as one in this study. PC—EPA/EPA
`
`and PC—DHA/DHA are available from commercial sources and pure reference substances of these
`
`were purchased and used in this study, PC-EPA/DHA are to our knowledge not commercially
`available.
`
`C. Generic structure of a phosphatidylcholine structure
`
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`Vitas as, Oslo Innovation Centre, Norway
`Vitas P308 Page 4
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`

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`«CH3 NJ
`
`i ‘xfixgf
`ii
`
`‘EH2
`
`: t
`
`it’
`\CH3
`
`Figure 3 Generic structure of a phosphatidylcholine molecule
`
`Table 2 Possible combination of DHA and EPA on a phophatidylcholine backbone, theoretical exact
`
`molecular weight (MW), observed MW of authentic reference standards and retention time in the
`
`described reversed phase chromatographic system. Molecular structures are shown in figure 4 and 5.
`
`
`
`*E|utes in between PC—EPA/EPA and PC—EPA/DHA
`
`O
`LFH3
`H=c /\/‘~ 9
`O
`O—l?“O\/«—hIl‘—CH3
`O
`O
`0
`0:43
`
`H3C——"‘"'_‘
`
`Figure 4 Molecular structure of PC—EPA/EPA (1,2—Dieicosapentaenoyl-sn—g|ycero—3—phosphocholine)
`
`
`
`Figure 5 Molecular structure of PC—DHA/DHA (1,2—Didocosahexaenoyl—sn—g|ycero-3—phosphocho|ine)
`
`Scientific approach
`
`The extracts to be examined contain a very complex mixture of lipids. Reversed phase liquid
`
`chromatography will be used to separate phospholipids from triacylglycerols, free fatty acids,
`
`Cholesterol esters and other lipids. The chromatographic step will also coarsely separate different
`
`species of phophatidylcholine to increase the sensitivity of the assay and reduce the risk of false
`
`positives.
`
`This chromatographic step will then be combined with different types of on—line mass spectrometry
`as described below.
`
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`Vitas as, Oslo Innovation Centre, Norway
`Vitas P308 Page 5
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`Mass spectrometry (MS) and Tandem mass spectrometry (MS/MS). HPLC—MS will be used to develop
`
`the chromatographic method and to investigate the mass of the intact phospholipids and match
`
`these with reference standards. MS/MS, in the form of a triple quadrupole, will be used to perform
`
`product ion scans and multi reaction monitoring (MRM) on the intact phospholipids to further
`
`confirm the structure and identify the acyl groups attached to the lipid.
`
`Materials
`
`99 % pure PC—EPA/EPA (1,2—Dieicosapentaenoy|—sn—g|ycero—3—phosphocholine) and 99 % pure PC-
`
`DHA/DHA (1,2—Didocosahexaenoyl—sn-glycero—3—phosphocholine) were purchased from Sigma—Aldrich
`
`MO , USA. Dichloromethane, hexane, formic acid, ammonium formate, isopropanol and methanol
`
`were obtained from VWR Norway.
`
`Instrumentation
`
`LC—MS and MS/MS were delivered by Agilent technologies, Palo Alto, California, USA.
`
`Pre analytical heat treatment
`
`Heat treatment of samples were performed at 70 °C and 125 °C. Both treatments were performed by
`
`placing a suitable heat block inside the oven of a gas chromatograph, set to the either 70°C or
`
`125°C.
`
`for at least one hour. The sample vials were then heated for the required time, allowed
`
`cooling to room temperature standing on the laboratory bench, before analysis by HPLC—Mass
`spectrometry.
`
`Sample pre treatment
`
`For Vitas ID P308 1-3 and 7-9, both heat treated or non heat treated samples were dissolved in
`
`chloroform/methanol (2:1 v/v) concentration of approximately 30 mg/ml. For Vitas ID P308 4-6 and
`
`10-16, both heat treated or non heat treated samples were dissolved in chloroform/hexane (6:4 v/v)
`
`concentration of approximately 30 mg/ml. Samples were loaded into the automatic autosampler for
`
`lF l|IIl l
`
`HPLC—MS analysis.
`
`Reversed phase chromatography (RP-HPLC)
`
`RP—HPLC was performed with a Agilent technologies 1200 HPLC system using A: water
`
`/methanol/formic acid/ammonium formate ( 500:500:1:1 v/v/v/w). B lsopropanol /formic
`
`acid/ammoniumformate(1000:1:1 v/v/w). Flow 0.6 ml/min. The separating column was a
`
`Phenomex Kinetic XB C-18, 150 mm x 4.6 mm i.d. ( 2.6 pm particles). The temperature on the
`
`column was 60 °C. A full description of this method is given in Appendix C.
`
`A representative chromatogram of a krill oil sample separated with this system is shown in Figure 6
`trace A and B.
`
` "mm"ii'BiiJiiwH*¢531:s\.:'‘a‘11o9:29xaé’uoqa9~:.a'"bfidééiifi-né-§9'fi3sE7i2v36bLdi;oi.bfEs§i5G"*"" ""7 "
`?
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`9
`
`min 1
`
`.
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`4 34
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`
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`Vitas as, Oslo Innovation Centre, Norway
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`0000019
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`
`Page 1
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`Both traces are obtained using ESI ionisation in positive mode. Trace B shows the total ion
`
`chromatogram between m/z = 700-900. This will cover masses for PC—14:O—P16:1 to PC—DHA/DHA .
`Trace A shows the reconstructed ion chromatogram of m/Z = 184 from analysis of a krill oil sample.
`
`This ion is present in all phosphatidylcholines and illustrates that the chromatographic window contains the
`
`class of substances in question.
`
`‘" 'iisb1s2aiféiE§éI7zT21§T7'(c':'\Iv}\b\Ar:'Raiouammr-‘sums’" ’1T0e2T 1'io0imAaéE02ia2o1:—ui2§ 1n?sT2m2’"EEi'1Iij Es "
`
`
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`
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`
`St. PC—EPA/EPA m/z = 826
`
`
`MSD1 S78, EIE=fi'a‘7.7378.7 (C:\PAD'\AKER BlDIARlN\F'.£3'-AB 11OQI&\AE 11El929\AB 0910292011-U3-E 1335-.‘.72lClD1—Ui0LDj ES
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`
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`
`Figure 7
`
`St. PC—DHA/DHA m/z = 878
`
`u
`
`9
`
`10
`
`12
`
`14
`
`10
`
`Figure 7 shows selected ion monitoring (SIM) traces for the reference standard PC—EPA/EPA and PC-
`
`DHA/DHA, showing the retention time for the specific phospholipids and that these also are within
`
`the chromatographic window. The retention times can be found in table 3. A full description of the
`
`single quadrupole mass spectrometric conditions is given in Appendix C.
`
`
`
`PL—Species
`Theoretical MW Mwstandard Retentiontime Quantifier
`
`
`
`
`
`218
`
`PC—EPA/EPA
`PC-EPA/DHA
`851.6 3- 852-6
`
`PC—DHA/DHA
`877.6
`877.5
`878.6
`219
`
`HPLC -Mass Spectrometry (MS)
`
`From the total ion chromatogram, masses for the three phospholipid combinations shown in table 3,
`
`were digitally extracted. The reconstructed ion chromatograms (RIC) are shown in figure 8.
`
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`V00020 7
`Vitas P308 Page
`itas as, Oslo Innovation Centre, Norway
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`0000020
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`

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`Trace A represents the TIC, trace B represents PC-DHA/DHA and trace C represents PC—DHA/EPA
`
`while trace D represents PC—EPA/EPA. It should be noted that there are only one narrow and
`
`symmetric peak in the entire chromatographic window for each trace. This indicates that there is
`
`probably only one phospholipid with this molecular weight in the sample. We also see that the
`
`retention time ofthese peaks matches perfectly the retention time of the reference standards (table
`
`1 and figure 7). For PC—EPA/DHA we do not have a reference standard but the sample contains a
`
`molecule that matches the theoretical molecular weight and that elutes in between the two others
`
`as anticipated.
`
`HPLC-MS/MS
`
`The structure of the substances with molecular mass 825.6, 851.6 and 877.6 were then further
`
`examined by the use of RP— HPLC-MS/MS. An Agilent Technologies high end linear tandem mass
`
`spectrometer (Agilent 6460 triple Quad) was interfaced with RP—HPLC using ESI ionisation in positive
`
`mode. Product ion scans of the protonated parent molecular weights (826.6 and 878.6) were then
`
`taken up using reference standards. Product ion scan for PC—EPA/EPA and PC-DHA/DHA (B) is shown
`
`in figure 9A and 9B.
`
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`
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`
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`0 7" 200 225 250 275'cuums vs. Mass»lo—Chavge (m/2)
`
`Figure9A Product ion scan of PC—EPA/EPA
`
`Vitas as, Oslo Innovation Centre, Norway
`
`V1tas P