`
`Composition of Antarctic krill oil and methods for its
`harvesting, production and qualitative and quantitative
`analysis.
`
`Dr. Nils Hoem, Chief Scientist
`
`Aker BioMarine
`
`Newcastle Australia November 2013
`
`in this presentation material from Spectral Service , Cologne has been used for illustrative purposes
`
`0 AkerBiOMZifll if}:
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`RIMFROST EXHIBIT 1080
`
`page 0001
`
`
`
`Agenda
`
`• Introduction - a few words about krill
`• What does krill contain?
`• How do we analyze krill and krill lipids?
`- and some cumulative data.
`• Final words
`
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`RIMFROST EXHIBIT 1080 page 0002
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`
`
`Nomenclature and abbreviations
`
`FA
`EPA
`DHA
`DPA
`ASX
`NMR
`GC
`LC
`HPLC
`MS
`FID
`
`20:5 n-3
`22:6 n-3
`22:5 n-3
`
`fatty acid
`eicosapentaenoic acid
`docosahexaenoic acid
`docosapentaenoic acid
`Astaxanthin
`nuclear magnetic resonance
`Gass chromatography
`Liquid chromatography
`High pressure licuid chromatography
`Mass spectrometry
`Flame ionization detection
`
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`RIMFROST EXHIBIT 1080 page 0003
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`
`
`Antarctic Krill - Euphausia Superba
`
` Zoo plankton (crustacean)
`
` Antarctic food chain
`
` Worlds largest biomass
`
` Harvesting challenges
`
` Catch is regulated by
`CCAMLR
`
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`RIMFROST EXHIBIT 1080 page 0004
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`
`Harvesting area and ice conditions
`
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`RIMFROST EXHIBIT 1080 page 0005
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`part of Aker
`
`
`
`Agenda
`
`• Introduction - a few words about krill
`• What does krill contain?
`• How do we analyze krill and krill lipids?
`- and some cumulative data
`• Final words
`
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`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0006
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`
`
`
`What does krill contain?
`
`Shell
`
`• Chitin (poly-b-1,4-D-N-acetylglucosamine)
`
`Krill meat
`
`• Proteins
`• Lipids – high content of phospholipids
`
`Red color
`
`• Astaxanthin esters
`
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`RIMFROST EXHIBIT 1080 page 0007
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`
`
`Differences in structure and function of triglycerides and phospholipids
`
`Triglyceride (fish oils)
`
`Phospholipids (krill oil)
`
`Fatty acid
`
`Fatty acid
`
`G l y c e r o l
`
`X
`
`Phosphate
`
`Fatty acid
`
`Fatty acid
`
`Fatty acid
`
`G l y c e r o l
`
`• Non-polar
`• Hydrophobic – does NOT mix with water
`
`• Polar
`• Hydrophilic - mixes with water
`
`• Energy storage
`
`• Main structural
`component in all
`cell membranes
`
`Fat cell
`
`Membrane
`
`Structural
`form
`
`Charac-
`teristics
`
`Function
`
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`X: choline; ethanolamine; serine; inositol; glycerol
`
`RIMFROST EXHIBIT 1080 page 0008
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`
`
`What does krill contain?
`
`Main components in various krill material
`
`Total lipids
`
`Polar lipids
`
`Neutral lipid
`
`Omega-3
`
`Omega-6
`
`Proteins
`
`Water
`
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`Raw Krill (%)
`
`Krill oil (%)
`
`Krill powder (%)
`
`.
`
`5
`
`2
`
`3,7
`
`0,4
`
`0,035
`
`12
`
`80
`
`93
`
`45
`
`55
`
`25
`
`3
`
`n.a.
`
`2
`
`60
`
`28
`
`32
`
`13
`
`1,3
`
`30
`
`1
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`RIMFROST EXHIBIT 1080 page 0009
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`
`
`What does krill contain?
`
`Lipid classes
`
`g/100g of krill oil
`
`Triacylglycerol
`
`34,0
`
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`
`Lipid structure
`
`Lipid classes
`
`Details
`
`g/100g oil
`
`Free fatty acids
`
`7,9
`
`Other
`
`2,8
`
`44,0
`
`Phospholipids
`
`Triacylglycerol
`
`Diacylglycerol
`
`Monoacylglycerol
`
`Free fatty acids
`
`Cholesterol
`
`Cholesterol esters
`
`Phosphatidylethanolamine
`
`Phosphatidylinositol
`
`Phosphatidylserine
`
`Phosphatidylcholine
`
`Lysophosphatidylcholine
`
`Total polar lipids
`
`Total neutral lipids
`
`Total sum lipids
`
`34
`
`0,7
`
`<1
`
`7,9
`
`1,2
`
`<0,5
`
`2,6
`
`<1
`
`<1
`
`38
`
`3,4
`
`44
`
`44,7
`
`88,7
`
`RIMFROST EXHIBIT 1080 page 0010
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`
`
`What does krill contain?
`
`Fatty acid profile
`
`g/100g of krill oil
`
`Fatty acid breakdown
`
`Lipid classes
`
`Details
`
`g/100g oil
`
`7,7
`DHA
`
`EPA
`
`13,2
`
`10,7
`
`Oleic acid
`
`Palmitic acid
`
`13,5
`
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`Omega 3s
`Omega 6s
`Omega 9s
`Other
`
`w-3 (usually < 30g/100g)
`
`Eicosapentanoic acid (EPA)
`
`13,2
`
`Docosahexanoic acid (DHA)
`
`a-Linolenic acid
`
`Stearidonic acid
`
`w-6
`
`g-Linoleic acid
`
`Arachidonic
`
`w-9
`
`Oleic acid
`
`Other
`
`Palmitic acid
`
`Palmitoleic acid
`
`Stearic acid
`
`7,7
`
`1.2
`
`3
`
`0,2
`
`0,3
`
`10,7
`
`13,5
`
`2,5
`
`0,6
`
`RIMFROST EXHIBIT 1080 page 0011
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`part of Aker
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`
`
`Agenda
`
`• Introduction - a few words about krill
`• What does krill contain?
`• How do we analyze krill and krill lipids?
`- and some cumulative data
`• Final words
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0012
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`
`
`
`Analytical methods used on in the overall production of krill products 1/4
`
`Parameter
`
`Method
`
`Ph.Eur
`Ph.Eur
`
`ISO 5983-2
`/NS-EN-ISO 16634-1
`
`ISO 6496
`
`ISO 5984
`
`Bligh&Dyer
`
`AOAC 937.09
`DIN 38405-4
`AOCS Ca 2e-84
`AOAC 978.18
`
`ISO 6496
`AOAC 920.03
`
`Conway, E. I., and A. Byrne.
`Biochem. J. 27:419-429, 1933.
`Roche. Method A 23
`Roche. Method A 23
`
`Fill weight
`Disintegration
`Chemistry
`Protein
`
`Water
`
`Ash
`
`Fat (B&D)
`
`Salt
`Fluorine
`Water in oil (Karl Fischer)
`aW
`
`Dry matter
`Total volatile Nitrogen
`
`TMAO
`TMA
`Astaxanthin
`Esterified astaxanthin
`
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`RIMFROST EXHIBIT 1080 page 0013
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`
`
`Analytical methods used on in the overall production of krill products 2/4
`
`Parameter
`
`Method
`
`Ethoxyquin
`
`AOAC 996.13
`
`Naturox
`Free Fatty acids (FFA)
`Peroxide value
`Anisidin Value
`
`Acid value
`Viscosity
`Saponification value
`
`AOAC Ca 5A-40
`AOCS Cd 8b-90
`AOCS Cd 18-90
`
`ISO 660
`Brookfield 021, 1.5 rpm
`AOAC 920.160
`
`Iodine value
`
`AOCS Ja 14-91
`
`Ethanol, flash point
`Cadaverine
`Histamine
`
`GC
`Internal NOFIMA A55
`Internal NOFIMA A55
`
`Vitamin A, mg/100g
`alpha- Tocopherol, mg/100g
`Fat classes, Total phospholipids
`
`J. Sci. Agri. Vol 29 p 697-702
`
`31 P-NMR or N A881
`
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`RIMFROST EXHIBIT 1080 page 0014
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`
`
`Analytical methods used on in the overall production of krill products 3/4
`
`Parameter
`
`Method
`
`AOCS Ce 1b-89
`
`AOCS Ce 1b-89
`
`Fat classes, total lipids as fatty acids
`Fatty acid profile
`Omega-3 Fatty acids
`EPA (C20:5, n-3)
`DHA (C22:6, n-3)
`DPA (C22:5, n-3)
`Omega-6 Fatty acids
`Linoleic acid (C18:2, n-6)
`Omega-9 Fatty acids
`Oleic acid (C18:1, n-9)
`Saturated fatty acids
`Monounsaturated fatty acids
`Polyunsaturated fatty acids
`Total trans-fat
`
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`RIMFROST EXHIBIT 1080 page 0015
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`
`
`Analytical methods used on in the overall production of krill products 3/4
`
`Parameter
`
`Method
`
`Metals and non-metals
`Phosphorous
`Calcium
`
`ISO 6491/Internal NOFIMA method (A37)
`ISO/CD 6869 / Internal NOFIMA method (A29)
`
`Magnesium
`
`Internal NOFIMA method (A30)
`
`Iron
`Zink
`Arcenic (in-organic)
`Arcenic (organic)
`Cadmium
`Mercury
`Lead
`Copper
`Tin
`Antimony
`PCB's
`Dioxins
`Microbiology
`TPC
`E.coli
`Coliforms
`Anaerobic Sulphitr. Clostridium
`Bacillus Cereus Pres.
`Staphylococcus Aureus
`Salmonella
`Enterobacter
`Yeast and molds
`
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`NMKL161 mod;ICP-MS/Internal NOFIMA method (A27)
`NMKL161 mod;ICP-MS
`Extraction/digestion, HPLC-ICP-MS
`Microwave assisted digestion, ICP-MS
`NMKL161 mod;ICP-MS / EN 15763:2009
`ALC 208:112 / EN 15763:2009
`NMKL161 mod;ICP-MS / EN 15763:2009
`NMKL161 mod;ICP-MS / EN ISO 11885, mod 05
`EN ISO 11885, mod 05
`EN 15763:2009
`EN 1948 modified, HRGC/HRMS
`EN 1948 modified, HRGC/HRMS
`
`NF EN ISO 4833 / NordVal Ref.no.012
`Petrifilm Select EC / NordVal Ref.no.014
`Petrifilm Select EC / NordVal Ref.no.014
`NMKL 56
`NMKL 67
`NorVal. Ref. no. 019
`AES 10/4-025/04 / NordVal Ref.no.023
`ISO 7402
`NorVal. Ref. no. 016
`
`RIMFROST EXHIBIT 1080 page 0016
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`
`
`Batch analyses performed on Superba krill oil
`
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`RIMFROST EXHIBIT 1080 page 0017
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`
`
`Compositional analysis of 184 batches of krill oil 1/4
`
`Polar lipids
`
`Neutral Lipids
`
`44,361957
`Mean
`3,3557175
`Std Dev
`0,2473868
`Std Err Mean
`Upper 95% Mean 44,850054
`Lower 95% Mean 43,873859
`Skewness
`-0,029825
`Kurtosis
`1,2234362
`
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`
`41,078804
`Mean
`4,3263125
`Std Dev
`0,31894
`Std Err Mean
`Upper 95% Mean 41,708077
`Lower 95% Mean 40,449532
`Skewness
`0,0547301
`Kurtosis
`-0,695264
`
`X: choline; ethanolamine; serine; inositol; glycerol
`
`RIMFROST EXHIBIT 1080 page 0018
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`part of Aker
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`
`
`Compositional analysis of 184 batches of krill oil 2/4
`
`Polar lipids
`
`Neutral Lipids
`
`44,361957
`Mean
`3,3557175
`Std Dev
`0,2473868
`Std Err Mean
`Upper 95% M 44,850054
`Lower 95% M 43,873859
`Skewness
`-0,029825
`Kurtosis
`1,2234362
`
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`© Aker BioMarine™
`
`41,078804
`Mean
`4,3263125
`Std Dev
`0,31894
`Std Err Mean
`Upper 95% M 41,708077
`Lower 95% M 40,449532
`Skewness
`0,0547301
`Kurtosis
`-0,695264
`
`X: choline; ethanolamine; serine; inositol; glycerol
`
`RIMFROST EXHIBIT 1080 page 0019
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`part of Aker
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`
`
`Compositional analysis of 184 batches of krill oil 3/4
`
`Phosphatidyl choline
`
`Phosphatidyl ethanolamine
`
`39,186413
`Mean
`3,1427739
`Std Dev
`0,2316884
`Std Err Mean
`Upper 95% M 39,643537
`Lower 95% M 38,729289
`Skewness
`0,2540793
`Kurtosis
`2,1289064
`
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`© Aker BioMarine™
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`2,0581522
`Mean
`0,6258012
`Std Dev
`0,0461347
`Std Err Mean
`Upper 95% M 2,1491764
`Lower 95% M 1,9671279
`Skewness
`0,5652078
`Kurtosis
`0,3084509
`
`X: choline; ethanolamine; serine; inositol; glycerol
`
`RIMFROST EXHIBIT 1080 page 0020
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`part of Aker
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`
`
`Compositional analysis of 184 batches of krill oil 4/4
`
`EPA 20:5 n-3
`
`DHA 22:6 n-3
`
`13,279348
`Mean
`1,0746318
`Std Dev
`0,0792229
`Std Err Mean
`Upper 95% M 13,435656
`Lower 95% M 13,12304
`Skewness
`-0,041249
`Kurtosis
`-0,351436
`
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`© Aker BioMarine™
`
`5,9107065
`Mean
`0,426184
`Std Dev
`0,0314187
`Std Err Mean
`Upper 95% M 5,972696
`Lower 95% M 5,848717
`Skewness
`0,0461241
`Kurtosis
`-1,045097
`
`X: choline; ethanolamine; serine; inositol; glycerol
`
`RIMFROST EXHIBIT 1080 page 0021
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`part of Aker
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`
`
`Analysis on separate lipid fractions
`
`Separation by fractionation in protic solvent(s) and/or
`flash chromatography on silica
`
`Krill
`
`Krill lipids
`
`Polar lipids
`
`Neutral lipids
`
`Free fatty acids
`
`The preferred partner
`
`© Aker BioMarine™
`
`X: choline; ethanolamine; serine; inositol; glycerol
`
`RIMFROST EXHIBIT 1080 page 0022
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`part of Aker
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`
`
`Fatty acid profile in Krill oil lipid sub fractions absolute amounts at 60% PL
`
`14.00
`
`12.00
`
`10.00
`
`8.00
`
`6.00
`
`4.00
`
`2.00
`
`0.00
`
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`© Aker BioMarine™
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`PL
`
`TG
`
`FFA
`
`RIMFROST EXHIBIT 1080 page 0023
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`part of Aker
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`
`
`Fatty acid profile in Kril oil lipid sub fractions relative amounts
`
`35.0
`
`30.0
`
`25.0
`
`20.0
`
`15.0
`
`10.0
`
`5.0
`
`0.0
`
`PL
`
`TG
`
`FFA
`
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`© Aker BioMarine™
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`RIMFROST EXHIBIT 1080 page 0024
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`part of Aker
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`
`
`Krill oil dilution in Soft gel capsules
`
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`RIMFROST EXHIBIT 1080 page 0025
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`part of Aker
`
`Krill oil
`(TG+PL mix)
`
`Volume
`=X0
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`Volume
`X1> X0
`
`=
`
`≠
`
`Triglyceride oil
`(fish oil)
`
`Volume
`=X0
`
`Fresh capsule
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`H2O
`
`Storage
`
`Volume
`X1=X0
`
`KO (hydrophilic) gain weight and
`volume because of uptake of water
`
`Found in experiments to be ≈ 10%
`
`
`
`Omega-3 Phospholipid species analysis by LC-MS/MS
`
`PC general structure
`
`The preferred partner
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`© Aker BioMarine™
`
`Winther, B., N. Hoem, et al. (2011). Lipids 46(1): 25-36.
`
`RIMFROST EXHIBIT 1080 page 0026
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`
`
`Omega-3 Phospholipid species
`
`Phospholipids
`
`Structure
`
`•Polar
`•Mixes with
`water
`
`Ch
`
`P
`
`FA
`
`FA
`
`Krill oil
`
` 69 choline-containing PL’s
`detected
`• 60 PL
`• 9 PLC
`
` 7 with ώ-3 FA in both SN-1
`and SN2 position
`
` 7 PC-species with an alkyl-
`acyl composition
`
`The preferred partner
`
`Major omega-3 species
`
`A
`
`B
`
`FA
`
`EPA
`
`FA
`
`DHA
`
`Ch
`
`P
`
`Ch
`
`P
`
`Minor omega-3 species
`
`C
`
`D
`
`EPA
`
`DHA
`
`EPA
`
`E
`
`EPA
`
`DHA
`
`DHA
`
`Ch
`
`P
`
`Ch
`
`P
`
`Ch
`
`P
`
`© Aker BioMarine™
`
`Winther, B., N. Hoem, et al. (2011). Lipids 46(1): 25-36.
`
`RIMFROST EXHIBIT 1080 page 0027
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`
`
`Fatty acid composition in KO species analysis by LC-MS/MS
`
`1/4
`
`KO
`
`KO-TG fraction
`
`EPA-16:0-14:0
`
`16:0-DPA-14:0
`
`16:1-16:1-14:0
`
`14:0-18:1-16:0
`
`The preferred partner
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`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0028
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`part of Aker
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`RT: 0.00 - 30.00 SM: 15B
`
`100
`
`50
`
`0
`100
`
`50
`
`0
`100
`
`50
`
`0
`100
`
`50
`
`11.15
`
`9.80
`
`1.77
`
`2.84
`
`5.63
`
`8.60
`
`6.93
`
`12.69
`
`4.08
`
`6.20
`
`7.70 8.60
`
`11.14
`
`14.46
`
`18.00
`
`19.54
`
`22.67
`
`24.32
`
`26.16
`
`27.67
`
`29.35
`
`2.99
`2.14
`
`7.58
`
`12.28
`
`12.74
`
`14.46
`
`19.41 20.18
`
`21.33 23.13 25.07
`
`26.95
`
`2.18
`
`2.97
`
`4.55
`
`6.80
`
`9.53
`
`9.55
`
`12.23
`
`14.27
`
`17.98
`
`19.37
`
`20.48 22.39
`
`24.12
`
`25.69 27.41
`
`2.98
`
`5.52
`
`8.50
`
`11.14
`
`14.38
`
`0
`100
`
`50
`
`0
`100
`
`50
`
`0
`
`0
`
`2.06
`
`6.07
`
`16.58
`
`18.23
`
`19.44
`
`21.07 22.20
`
`23.92
`
`25.74 27.58
`
`2.10
`
`2.82
`
`5.54
`
`6.59
`
`9.36
`
`6.08
`
`10.77
`
`11.18
`
`14.19
`
`15.17
`
`18.27
`
`19.54
`
`20.45
`
`24.02
`
`25.04
`
`26.63 28.36
`
`2.05
`
`2.97 5.53
`
`6.03
`
`7.76
`
`9.54
`
`14.20
`
`15.18
`
`18.67
`
`19.55
`
`20.53
`
`23.48 24.46 26.15 27.85
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`16
`14
`Time (min)
`
`18
`
`20
`
`22
`
`24
`
`26
`
`28
`
`30
`
`
`
`Fatty acid composition in KO species analysis by LC-MS/MS
`
`2/4
`
`KO?
`
`KO-TG fraction
`
`FO: EPA-EPA-EPA
`
`FO: DHA-EPA-EPA
`
`FO: DPA-DHA-EPA
`
`FO: 18:1-DHA-EPA
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0029
`
`part of Aker
`
`RT: 0.00 - 30.00 SM: 15B
`
`100
`
`50
`
`0
`100
`
`50
`
`0
`100
`
`50
`
`0
`100
`
`50
`
`11.15
`
`9.80
`
`1.77
`
`2.84
`
`5.63
`
`8.60
`
`6.93
`
`12.69
`
`4.08
`
`6.20
`
`7.70 8.60
`
`11.14
`
`14.46
`
`18.00
`
`19.54
`
`22.67
`
`24.32
`
`26.16
`
`27.67
`
`29.35
`
`2.99
`2.14
`
`4.74
`
`12.28
`
`12.74
`
`14.46
`
`19.41 20.18
`
`21.33 23.13 25.07
`
`26.95
`
`0.33
`
`2.13 4.25
`
`4.83
`
`8.52
`
`8.76
`
`10.13
`
`10.73
`
`14.50
`
`15.66
`
`18.41 19.54
`
`21.56
`
`23.94 25.39
`
`27.98
`
`2.18
`
`4.43
`
`5.60
`
`6.24
`
`9.16
`
`9.89
`
`10.96
`
`17.57
`
`0
`100
`
`50
`
`0
`100
`
`50
`
`0
`
`0
`
`0.34
`
`3.57
`
`3.89
`
`2.18
`
`2.84
`
`6.23
`
`6.66
`
`6.27
`
`16.80
`
`19.76
`
`21.64 23.14
`
`25.63
`
`28.06
`
`7.97 10.40
`
`11.62
`
`12.45
`
`16.70
`
`19.16
`
`19.54
`
`22.25
`
`23.92
`
`26.66
`
`29.46
`
`0.95
`
`2.82
`
`4.53
`
`5.80
`
`7.57
`
`10.07
`
`11.96
`
`13.36
`
`16.60
`
`18.06
`
`19.46
`
`20.50
`
`24.12
`
`25.65
`
`28.12
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`16
`14
`Time (min)
`
`18
`
`20
`
`22
`
`24
`
`26
`
`28
`
`30
`
`
`
`Fatty acid composition in KO species analysis by LC-MS/MS
`
`3/4
`
`KO?
`
`KO-TG fraction
`
`EPA-EPA-EPA
`
`DHA-EPA-EPA
`
`DPA-DHA-EPA
`
`18:1-DHA-EPA
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0030
`
`part of Aker
`
`100
`
`50
`
`0
`100
`
`50
`
`0
`100
`
`50
`
`0
`100
`
`50
`
`RT: 0.00 - 30.00 SM: 15B
`4.87
`
`3.50
`
`6.26
`
`9.83 11.22
`
`1.50
`
`3.09
`
`8.62
`
`12.37
`
`15.91
`
`16.94
`
`19.35
`
`21.06
`
`21.99 23.01 25.38 28.08
`
`29.89
`
`4.87
`
`6.26
`
`2.14
`
`2.88
`
`4.77
`
`7.55 8.54
`
`9.56 10.83
`
`12.82
`
`15.08
`
`16.87
`
`19.78
`
`20.81
`
`22.43 24.31
`
`26.73
`
`29.58
`
`TG fraction
`
`TG 20:5/20:5/20:5
`
`0.38
`
`2.87
`
`4.01
`
`7.14 8.54
`
`10.92
`
`12.39
`
`14.87
`
`15.81 18.43
`
`20.48 21.40
`
`24.71
`
`26.87
`
`29.54
`
`4.85
`
`TG 22:6/20:5/20:5
`
`19.69
`
`21.82
`
`23.49
`
`26.87
`
`29.71
`
`0
`100
`
`50
`
`0
`100
`
`50
`
`0
`
`0
`
`2.11
`
`2.89
`
`6.30
`
`8.54
`
`11.08
`
`12.53
`
`15.09
`
`17.55
`
`5.18
`
`TG 22:5/22:6/20:5
`
`2.08
`
`4.03
`
`6.06
`6.31
`
`6.94 9.23
`
`12.51
`
`13.49
`
`15.20 17.44
`
`19.71
`
`21.93
`
`24.39
`
`26.75
`
`28.14
`
`0.65
`
`2.89
`
`4.79
`
`5.80
`
`8.93
`
`9.86
`
`12.01
`
`13.50 14.89
`
`19.16
`
`20.04
`
`21.54
`
`24.85
`
`26.87
`
`29.73
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`16
`14
`Time (min)
`
`18
`
`20
`
`22
`
`24
`
`26
`
`28
`
`30
`
`TG 18:1/22:6/20:5
`
`
`
`Fatty acid composition in KO species analysis by LC-MS/MS
`
`4/4
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0031
`
`part of Aker
`
`RT: 0.00 - 30.00 SM: 15B
`4.90
`
`3.53
`
`6.29
`
`9.86 11.25
`
`AKB33483-1
`
`1.53
`
`3.12
`
`8.65
`
`12.40
`
`12.85
`
`17.90
`
`19.38
`
`21.09
`
`22.02 23.04 25.41 28.11
`
`29.92
`
`1.28
`
`2.91
`
`5.71
`
`7.01
`
`11.25
`
`9.86
`
`8.65
`
`12.81
`
`9.86
`
`11.19
`
`14.52
`
`17.06
`
`18.19
`
`20.60
`
`21.44
`
`24.49
`
`26.45
`
`28.12
`
`29.11
`
`AKB33483-2
`
`2.87
`
`3.51
`
`6.97
`
`6.38
`
`8.59
`
`12.25 12.72
`
`9.86
`
`11.23
`
`AKB33483-3
`
`16.84
`
`18.12
`
`20.48
`
`22.91
`
`24.57
`
`25.71
`
`28.15
`
`29.53
`
`3.56
`
`2.88
`
`8.67
`
`7.00
`
`12.32
`
`12.76
`
`17.01
`
`18.30
`
`21.16
`
`22.16
`
`25.40
`
`27.38
`
`29.48
`
`AKB33483-4
`
`100
`
`50
`
`100
`
`50
`
`100
`
`50
`
`100
`
`50
`
`100
`
`50
`
`100
`
`50
`
`100
`
`50
`
`1.07
`
`4.46
`
`9.86
`
`11.22
`
`AKB33483-5
`
`3.16
`
`3.55
`
`6.63
`
`7.00
`
`8.68
`
`12.33 12.77
`
`15.53 17.03
`
`20.96
`
`22.91
`
`24.50
`
`26.46
`
`28.08
`
`29.53
`
`3.15
`
`3.55
`
`6.99
`
`5.74
`
`2.43
`
`2.90
`
`5.69
`
`6.99
`
`11.20
`
`9.86
`
`8.66
`
`12.76
`
`11.21
`
`9.86
`
`8.66
`
`12.75
`
`AKB33483-6
`
`14.49
`
`15.28
`
`18.37 20.83
`
`22.78
`
`24.49
`
`26.06
`
`28.25
`
`29.33
`
`14.52
`
`18.06
`
`19.60
`
`22.73
`
`24.38
`
`26.58
`
`27.73
`
`29.41
`
`AKB33483-7
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`
`12
`
`16
`14
`Time (min)
`
`18
`
`20
`
`22
`
`24
`
`26
`
`28
`
`30
`
`
`
`Analysis of KO by NMR
`
`•
`
`•
`
`31P-NMR: has become standard method for phospholipid classification and quantitation
`(AKBM)
`• Validated method
`• Relatively large Round Robin Test (21 different labs with some variation in
`instrumentation)
`
`1H-NMR and 13C-NMR increasingly used for
`Total omega-3
`•
`Total number of double bounds
`•
`EPA and DHA and their sn1, sn2 positioning
`•
`Ether-phospholipids
`•
`• Other lipids
`• Other impurities (TMNO)
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0032
`
`part of Aker
`
`
`
`Analysis of KO by NMR - phospholipids
`
`1/2
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0033
`
`part of Aker
`
`For the quantification two measurements were performed for each sample: one with 1 whole capsule
`
`and one with only the filling of 1 whole capsule. Each test item and 20 mg of the internal standard
`
`(TPP) were exactly weighed (see tables “Calculation of content”, Chapter 6 - NMR Spectroscopy),
`dissolved in 10 ml CDCl3 / MeOH / Cs-EDTA, aq. (2/4/4) and measured. The integrated signals of
`test item and internal standard were used for calculation.
`
`
`
`Analysis of KO by NMR - phospholipids
`
`2/2
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0034
`
`part of Aker
`
`TPP
`Internal Standard
`Test item
`Excel 14.0
`
`PC
`1-LPC
`2-LPC
`PI
`LPI
`PS
`LPS
`SPH
`PE
`LPE
`APE
`PG
`DPG
`PA
`LPA
`Rest
`Sum
`Phosphorus
`
`Integral Molecular weight mMol
`24.82
`326.29
`0.0623
`
`Integral Molecular weight mMol
`47.46
`790.0
`0.1191
`2.00
`534.5
`0.0050
`15.98
`534.5
`0.0401
`0.00
`907.0
`0.0000
`0.00
`629.5
`0.0000
`0.00
`833.0
`0.0000
`0.00
`555.5
`0.0000
`0.00
`812.0
`0.0000
`4.07
`770.0
`0.0102
`1.26
`492.5
`0.0032
`3.03
`1032.0
`0.0076
`0.00
`820.0
`0.0000
`0.00
`774.0
`0.0000
`0.00
`746.0
`0.0000
`0.00
`468.5
`0.0000
`1.38
`812.0
`0.0035
`75.18
`
`Initial weight [mg] Content [%] Number P
`20.34
`99.9
`1
`442.66
`1
`Content [mg]
`94.0616
`2.6819
`21.4280
`0.0000
`0.0000
`0.0000
`0.0000
`0.0000
`7.8622
`1.5568
`7.8448
`0.0000
`0.0000
`0.0000
`0.0000
`2.8112
`
`Weight-%
`21.25
`0.61
`4.84
`---*)
`---*)
`---*)
`---*)
`---*)
`1.78
`0.35
`1.77
`---*)
`---*)
`---*)
`---*)
`0.64
`31.23
`1.32
`
`Mol-%
`63.13
`2.66
`21.26
`---*)
`---*)
`---*)
`---*)
`---*)
`5.41
`1.68
`4.03
`---*)
`---*)
`---*)
`---*)
`1.84
`100.00
`
`
`
`Analysis of KO by NMR – proton NMR for total n-3
`
`1/4
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0035
`
`part of Aker
`
`Fig. 83
`
`1H-NMR spectrum of test item AKB33483-6 (CDCl3/DMSO-d6)
`
`
`
`Analysis of KO by NMR – proton NMR for total n-3
`
`2/4
`
`RIMFROST EXHIBIT 1080 page 0036
`
`part of Aker
`
`Product
`
`A B C D E F G
`
`The preferred partner
`
`© Aker BioMarine™
`
`
`
`Analysis of KO by NMR – carbon NMR for fatty acids in
`PL and TG fractions
`
`3/4
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0037
`
`part of Aker
`
`Fig. 85
`
`13C-NMR spectrum of test item AKB33483-6 (CDCl3/DMSO-d6)
`
`
`
`Analysis of KO by NMR – carbon NMR for fatty acids in
`PL and TG fractions
`
`4/4
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0038
`
`part of Aker
`
`
`
`Analysis of KO by NMR ether PL
`
`1/2
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0039
`
`part of Aker
`
`
`
`Agenda
`
`• Introduction - a few words about krill
`• What does krill contain?
`• How do we analyze krill and krill lipids?
`- and some cumulative data
`• Final words
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0040
`
`part of Aker
`
`
`
`Analysis of KO by NMR
`
`•
`•
`
`Krill oil is a complex mixture of lipids
`Krill oil can not be directly compared with regular Triglyceride oils
`
`• Analytical methods needs to be adapted specifically for the KO matrix
`
`• Regular methods for determination of oxidative status of the lipid mixture is not
`sensitive nor specific enough to be more than marginally useful
`(e.g. Anisidine or TBARS methods)
`
`•
`
`Krill oils will always have more free fatty acids as well as water content than TG,
`thus are more prone to hydrolysis than TG-oils
`
`• Methods that can distinguish between TG from KO and FO is desirable
`• Methods for direct (and sensitive enough) measurement of molar amount of
`double bounds could be useful to investigate oxidative status and development
`
`•
`
`Krill oil standards, both PL and TG are needed
`
`The preferred partner
`
`© Aker BioMarine™
`
`RIMFROST EXHIBIT 1080 page 0041
`
`part of Aker
`
`
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