`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`
`
`
`
`
`
`CASE IPR: Unassigned
`
`Declaration of Dr. Jeff D. Moore
`
`
`
`
`AKER BIOMARINE AS
`Petitioner
`
`v.
`
`NEPTUNE TECHNOLOGIES AND BIORESOURCES INC.
`Patent Owner
`
`
`000001
`
`
`
`I, Dr. Jeff D. Moore, state as follows:
`
`1.
`
`I am the director of Analytical Technologies for Avanti Polar Lipids,
`
`Inc. I have been retained by counsel for Petitioner Aker BioMarine AS to testify as
`
`an expert in this inter partes review.
`
`2.
`
` My report concerning analysis of total lipid content, free fatty acid
`
`content and total phospholipid content in krill extracts is attached hereto.
`
`3.
`
`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.
`
`
`
`000002
`
`
`
`%AIIanli®
`
`PEILAR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`September 13, 2013
`
`Introduction
`
`Avanti Polar Lipids, Incorporated is a Bulk Pharmaceutical Manufacturer licensed by the U.S.
`Food and Drug Administration . The analytical laboratory provides quality control testing of
`processes, raw materials and final products under current good manufacturing practices (CGMP).
`The quality control laboratory resources to include instrumentation and personnel are also
`utilized to provide fee for service laboratory testing under the Analytical Services Division. All
`instrumentation is maintained, calibrated, operationally and performance qualified (OQ/PQ). All
`personnel are professionally qualified by education and experience as well as maintain
`documented training.
`
`Samples of krill extract were provided for analysis to determine total lipid content, free fatty acid
`content and total phospholipid content. The samples were assayed by methods developed at
`Avanti. Each sample was assayed in triplicate and one sample was fortified with a relevant
`compound to determine suitability of respective methodology, i.e. precision and accuracy. The
`instrumentation, sample preparation, instrument settings, chemicals, calculations and results for
`the three assays are described herein.
`
`Samples and Receipt
`
`Samples were received by courier at Avanti Polar Lipids and unpacked for storage. All samples
`were stored at 2-8° C until time of preparation and returned to such storage between uses.
`
`Received: August 13, 2013
`Condition: no dry ice cool to touch, intact and legibly labeled
`VB 1 8/8/13 FH
`
`VB 7 8/9/13 FHE
`
`Received: August 21, 2013
`Condition: dry ice and frozen, intact and legibly labeled
`SBl BEA-P0
`
`SB5 BEA-P1
`
`SB9 BEA-P2
`
`SB 13 BEA-S0
`
`SB 17 BEA-S1
`
`SB 21 BEA-S2
`
`Received: September 10, 2013
`
`000003
`
`
`
`%Avanli®
`
`PEILAR LIFIDE, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`BEA-SUO
`
`BEA-SUI
`
`BEA—SU2
`
`Total Lipid Content
`
`The VB and SB1 thru 21 series of samples above were assayed by gravimetrical weighing upon
`extraction and drying. The total lipid content is expressed as weight % of original sample weight
`used in the assay.
`
`Principle:
`
`Lipids are fats or oils which are not soluble in water. They must be extracted and dissolved into
`non-polar solvents to form solutions. Lipid extraction techniques utilize the layering of the water
`and non-polar solvents to separate lipids from a sample. The water soluble compounds dissolve
`into the upper layer and lipids into the lower layer. Lipids extracted into the lower layer can be
`recovered by evaporating the solvent and weighing the dried residue. The weight percentage of
`lipid can be determined by the ratio of recovered lipid weight to the original sample weight. The
`Folch extraction technique (Folch et al., J Biol Chem 195 7, 226, 497) is universally accepted as a
`means to extract lipid.
`
`Reagents:
`
`Fisher Scientific HPLC grade chloroform, lot # 133740, exp. 08-08-14
`Fisher Scientific HPLC grade methanol, lot #132577, exp. 08-12-14
`Fisher Scientific HPLC grade water, lot #133911, exp. 08-12-14
`
`Instrumentation:
`
`Sartorius CP124S analytical balance (4 place), ser. #22650193.
`Calibration date: 02-27-13 / Calibration due: 02-28-14 / Suitability tested: passed 08-15-13.
`
`Sample Extraction:
`
`1. Each sample was removed from 2-8° C storage and allowed to equilibrate to ambient
`temperature.
`
`2. Each sample was weighed in triplicate into separate 12 mL, 16 X 100 mm glass test tubes with
`screw cap at 500 i 3 milligram.
`
`000004
`
`
`
`
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`oleic acid to determine method accuracy by spike recovery. One mL of 25 mg/mL oleic acid in
`chloroform, 18lWKSTK-041813, reevaluation due 10-13-13, was added to these tubes. Sample
`weights and solvent volumes are provided in table 1.
`
`Table 1: Sample wei rhts and solution volumes.
`
`Sample
`_
`VB 1 8/8/13 FH__
`
`VB 1 8/8/13 FH
`
`VB 1_8_/8/13 FH
`VB 7 8/9/13 FHE
`
`VB 7 8/9/13 FHE
`
`VB 7 8/9/13 FHE
`
`VB 7 8/9/13 FHE—_SP|KE_
`
`VB 7 8/9/13 FHE-SPIKE
`
`VB 7___8/9/13 FHE-SPIKE
`BEA SB1 P0
`
`Weight (mg)
`503.0
`
`__ _
`
`CHCl3(mL)
`6.5
`
`CH30H (mL)
`3.25
`
`H2O(mL)
`2.0
`
`501.4 _
`
`499.9
`501.6
`
`499.2
`
`502.2
`
`502.0
`
`502.2
`
`499.4
`500.7
`
`501.1
`
`_
`
`_
`
`6.5
`
`6.5
`6.5
`
`6.5
`
`6.5
`
`5.5
`
`5.5
`
`5.5
`6.5
`
`6.5
`
`_
`
`__
`
`3.25
`
`3.25
`3.25
`
`3.25
`
`3.25
`
`3.25
`
`3.25
`
`3.25
`3.25
`
`3.25
`
`_
`
`_
`
`2.0
`
`2.0
`2.0
`
`2.0
`
`2.0
`
`2.0
`
`2.0
`
`2.0
`2.0
`
`2.0
`
`N
`
`_
`
`_
`
`__
`
`(mL)
`
`1.0
`
`1.0
`
`1.0
`
`BEA SB1 P0
`
`BEA SB1 P0
`
`BEA SB5 P1
`
`__
`
`_
`
`BEA sB5 P1
`
`BEA sB§J?_1
`
`BEA SB9 P2
`
`BEA SB9 P2
`
`BEA SB9 P2 _
`BEA SB13 50
`
`_BEA SB13 50
`
`BEA SB13 50
`
`_ BEA SB17 51
`BEA SB17_S_1
`BEA SB17 S1
`
`BEA SB21 52
`
`500.0
`
`499.3
`
`501.0
`
`499.8
`
`500.3
`
`499.6
`
`500.2
`500.0
`
`501.0
`
`499.3
`
`500.6
`500.6
`
`499.3
`
`6.5
`
`6.5
`
`6.5
`
`6.5
`
`6.5
`
`6.5
`6.5
`
`6.5
`
`6.5
`
`6_._5
`6.5
`6.5
`
`6.5
`
`3.25
`
`3.25
`
`2.0 _
`
`2.0
`
`3.25 _ 2.0
`
`_ A
`
`3.25
`
`3.25 __
`
`_
`
`_
`
`3.25
`
`3.25
`3.25
`
`3.25
`
`3.25
`
`3.25
`3.25
`3.25
`
`3.25
`
`_
`
`__
`
`_
`
`2.0
`
`2.0
`
`2.0
`
`2.0
`2.0
`
`2.0_
`
`2.0
`
`2.0
`2.0
`2.0
`
`__ 2.0
`
`_
`
`BEA SB21 52
`500.4
`6.5
`_
`3.25
`2.0
`3.25
`BEA SB21 S2
`.
`
`000005
`
`
`
`
`
`Avantf
`
`POLAR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`volumes of 2:1 chloroform : methanol was added to each tube, dispersed and mixed in an orbital
`shaker at room temperature for 15 minutes.
`
`5. The completely dissolved material was washed with 2.0 mL of water by vortexing and
`centrifuged at 2000 rpm to separate the layers.
`
`6. The lower layer was transferred into pre-weighed 13 X 100 mm glass test tubes with screw
`cap. The lower chloroform layer was evaporated under a gentle stream of nitrogen at ambient
`temperature under a safety hood. The remaining solvent was removed using a Genevac (EZ 2.3
`Elite Model) centrifugal evaporator for a period of 18 hours. The samples were placed in -80° C
`freezer for about 1 hour. Final drying was performed on a vacuum lyophilizer overnight.
`
`7. Each sample was weighed and the % extract / weight of sample was calculated using the
`formula:
`
`% Total Lipid = ube with extract — tube weight
`original sample weight
`
`X 100
`
`8. The accuracy of the method was evaluated by % recovery for spiked sample with oleic acid
`and calculated using the formula:
`
`% recovery = ( % wt in sample + % wt of spike) / % wt in spiked sample X 100
`
`Results:
`
`Table 2 reports the weight % of lipid from triplicate assays of each sample with calculation of
`mean and standard deviation for evaluation of precision. Table 3 reports the accuracy of the
`method from the spike recovery of oleic acid in a fortified sample.
`
`Table 2: Weight % oflipid.
`
`Sample
`
`Extract
`
`
`
`
`
`
`
`Sample
`
`VB 1 8/8/13 FH
`
`VB 1 8/8/13 FH
`
`VB 1 8/8/13 FH
`
`__\_/_B 7 8/9/13 FHE
`
`__vB 7 8/9/13 FHE
`VB 7 8/9/13 FHE
`BEA SB1 P0
`
`weight (mg) weight (mg)
`
`503.0
`
`413.2
`
`501.4
`
`499.9
`
`501.6
`
`499.2
`502.2
`500.7
`
`445.7
`
`461.2
`
`423.7
`
`408.2
`433.1
`311.2
`
`88.9
`
`92.3
`
`84.5
`
`81.8
`86.2
`62.2
`
`
`
`
`
`
`2.3
`
`I
`
`000006
`
`
`
`
`
`AVANTI POLAR LIPIDS. INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIvIsIoN
`
`
`
`62.0
`
`61.8
`
`61.6
`
`0.8
`
`
`
`BEA SB1 P0
`
`BEA SB5 P1
`
`BEA SB5 P1
`
`499.3
`
`501.0
`
`308.4
`
`290.5
`
`BEA SB5 P1
`
`BEA SB9 P2
`
`
`
`
`BEA SB9 P2
`
`BEA SB13 S0
`
`BEA SB13 S0
`
` BEA SB13 S0
`BEA SB17 S1
`
`
`
`BEA SB21 S2
`
`BEA SB21 S2
`
` BEA SB21 52
`
`
`
`Table 3: Accuracy by spike I'ecovc1'y.
`
`Sample Weight
`
`‘
`
` Sample
`Spike (mg)
`(mg)
`VB 7 8/9/13 FHE-SPIKE
`502.0
`445.6
`88.8
`‘
`25
`5.0
`VB 7 8/9/13 FHE—SP|KE
`502.2
`448.9
`89.4
`‘
`25
`5.0
`VB 7 8/9/13 FHE—SPlKE
`499.4
`466.4
`93.4
`‘
`25
`5.0
`Average
`90.5
`I
`
`Accuracy
`
`97.0
`97.7
`98.5
`
`000007
`
`
`
`%AvanIi®
`
`FEILAR LIPIDS, INI::.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`Free Fatty Acid Content
`
`The VB series of samples above were assayed by high performance liquid chromatography
`(HPLC) with evaporative light scattering detection (ELSD) using the extraction samples obtained
`for total lipid content. The free fatty acid content is expressed as weight % of original sample
`weight used in the Folch extraction.
`
`Principle:
`
`High performance liquid chromatography (HPLC) is a separation technique utilizing solvents
`pumped at high pressure over a column packed with uniform particulate stationary phase coated
`with materials to retain compounds of interest. A mixture of compounds can be separated
`according to their selective affinity between the coated stationary phase and the solvent
`composition flowing through the column. Evaporative light scattering detectors can detect these
`resolved compounds as they pass from the column through a light source. These molecules of
`resolved compounds scatter light proportional to their concentration, producing a peak which can
`be integrated and quantified. The concentration of the compound can be directly calculated
`through the calibration of concentration versus response curves generated from standard
`compounds. Free fatty acid (FFA) is measured in the krill lipid extracts by separation from other
`lipids on the column and quantified against a calibration curve generated for a reference standard
`of oleic acid. The weight % of FFA is the ratio of measured FFA to original sample (pre-
`extracted) weight.
`
`Reagents:
`
`Fisher Scientific HPLC grade toluene, lot # 124170, exp. 07-31-14
`Fisher Scientific HPLC grade methanol, lot #133455, exp. 08-21-14
`Fisher Scientific HPLC grade water, lot #133911, exp. 08-12-14
`Fisher Scientific HPLC grade chloroform, lot #132557, exp. 08-12-14
`System suitability standard MGDGTGFASUIT-070313 (re—eval. 10-30-13) contains the
`following in HPLC grade chloroform:
`1 mg/mL 18:1 triglyceride
`1 mg/mL 18:1 digylceride
`1 mg/mL 18:1 free fatty acid
`
`Free fatty acid calibration standard #181FFAWKSTK-041813 (re—eval. 10-30-13) at 5.1 mg/mL
`in HPLC grade chloroform.
`
`000008
`
`
`
`
`Avanlit
`
`PCILAR LIPIDE, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`Instrumentation:
`
`HPLC/ELSD System #9: Agilent model 1100 HPLC system
`PQ on 12-12-12, PM on 06-03-13, next due 12-2013.
`
`1. quaternary pump, ser. #DE43 631305
`2. auto-liquid sampler, ser. #DE33228754
`3. column oven, ser. #DE43648206
`4. vacuum degasser, ser. #JP4071 8195
`5. Sedex model 75 ELSD, ser. #0571525F
`
`Mobile Phases
`
`A = toluene
`
`B = methanol
`
`C = water
`
`Time 1min.)
`Initial
`
`10.00
`
`15.00
`
`°_/9A
`97
`
`97
`
`6.0
`
`6.0
`
`f[@ E
`3.0
`0
`
`3.0
`
`90.0
`
`90.0
`
`0
`
`4.0
`
`4.0
`
`Equilibration in initial composition for 5 min. pre-inj ection.
`Gradient flow is continuous 1 mL/min at 25° C
`
`Column = Phenomenex, Luna 4.6 X 250 mm, 5um silica, ser. #329095-3
`ELSD settings
`Nitrogen nebulizer gas pressure = 3.7 bar
`Temperature = 40° C
`Gain setting = 5
`
`System Suitability:
`
`The MGDGTGFASUIT-070313 suitability standard was diluted 1:4 in HPLC chloroform to
`contain 250 ug/mL of each component. The solution was injected 6 times at 10 IIL under the
`conditions of the method. The retention time and area of the components were evaluated for
`precision and resolution in table 4:
`
`000009
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`
`ll\lanIi®
`
`PEILAR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`Table 4: HPLC/ELSD system suitability
`18:1 TG
`
`Injection #
`
`18:1 TG (rt)
`
`(area)
`
`18:1 DG (r_t)
`
`
`
`_ 1
`
`2.699
`
`1442375
`
`3.228
`
`ANALYTICAL SERVICES DIVISION
`
`18:1 DG
`
`18:1 FFA
`
`(area)
`
`18:1 FFA (rt)
`
`(area)
`
`_
`
`1414694
`
`14218_78
`1351_769
`1428906
`1380208
`
`1425959
`
`1403902
`31082
`
`H
`
`4.380
`
`4.370
`4.383
`4.386
`4.363
`
`4.376
`
`4.376
`0.009
`
`_
`
`_
`
`1077030
`
`_1135232
`_1087389
`1093812
`1119488
`
`1124953
`
`1106317
`23363
`
`2
`3
`4
`5
`
`6
`
`mean
`
`__
`
`2.702 _
`_ 2.699
`2.706
`2.702
`
`2.706
`
`2.702
`0.003
`
`1444950
`1487072
`147_7657
`1434596
`
`1481156
`
`1461301
`23085
`
`3.225
`3.228
`3.231
`3.228
`
`3.231
`
`3.229
`0.002
`
`I
`
`_"/grsd _
`resolution
`
`
`
`0.1
`2
`0.1
`_
`_
`2
`0.2
`_
`2
`baseline
`baseline
`baseline
`
`Calibration Standard and Curve:
`
`The 181FFAWKSTK-041813 was diluted 1:5 to a concentration of 1.02 mg/mL in HPLC
`chloroform and serially diluted 1:2 to obtain 6 calibration standards ranging from 31.87 — 1020
`ug/mL. The calibration standards were injected at 10 uL. The integrated areas of the 18:1 FFA
`peak within each standard injection was plotted using a quadratic equation and forcing the origin
`through zero. Goodness of fit was calculated by regression square (r2) = 0.9988 for the
`concentrations and peak area reported in table 5.
`
`Table 5: 18:1 FFA calibration curve.
`
`FFA Std #
`ug/mL
`area
`1
`1020
`3285719
`
`_ 2
`
`3
`
`4_
`
`5
`6
`
`I
`
`510
`
`255
`
`127.5
`
`63.75
`31.875
`
`1312455
`
`432470
`
`136617
`
`_
`
`36611
`6750
`
`y = l.29732X2 + 2008.87X - 103775
`
`Sample Preparation:
`The total lipid extracts performed on the VB series samples were dissolved in 5 mL of HPLC
`grade chloroform. These were further diluted 1:20 (0.1 ml in 1.9 mL) into HPLC grade
`chloroform for assay by HPLC/ELSD. Table 6 reports the weight and volumes utilized in sample
`preparation.
`
`0000010
`
`
`
`
`
`AVANTI POLAR LIPIDS, INC. — CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`Table 6: Sample preparation
` Dissolve in
`Extract
`5 mL
`1:20 dil
`
`Sample
`
`wt. (mg)
`
`(mg/mL)
`
`(mg/mL)
`
`_VB 1 8/8/13 FH
`VB 1 8/8/13 FH
`VB 1 8/8/13 FH
`VB 7 8/9/13 FHE
`vs 7 8/9/13 FHE
`VB 7 8/9/13 FHE
`VB 7 8/9/13 FHE spike
`
`__
`
`l
`
`413.2
`445.7
`461.2
`423.87
`408.2
`433.1
`445.6
`
`_
`
`|
`
`82.64
`89.14_
`92.24
`84.77
`81.64
`86.62_ __
`89.12
`
`|
`
`4.132
`4.457
`4.612 _
`4.239
`4.082
`4.331
`4.456
`
`4.489
`89.78
`448.9
`_
`_V%_/_9/__1§__F_HE spike
`93.28
`VB 7 8/9/13 FHE spike
`
`Results:
`
`Each sample was assayed in triplicate as before. Each replicate was injected once at 10 and 15
`uL. The weight % of free fatty acid in the total lipid extract was calculated using the formulas:
`
`Milligrams in extract =
`
`ug/mL X 5 mL X 20 dil. X 10/);
`1000 ug/mg
`where y = actual injection volume (10 or 15 uL).
`
`Weight% =
`
`mg in extract
`Sample weight (mg)
`
`X
`
`100
`
`The mean of 10 and 15 uL injection Values is reported for each replicate. Table 7 reports the
`sample replicate Values.
`
`Table 7: Free fally acids by HPLC/ELSD
`
`Sample
`Weight(mg)
`
`FFA/Sample
`(mg) 10uL
`
`FFA/Sample
`(mg) 15 uL
`
`FFA/Sample
`
`(mg) mean VB 1 8/8/13 FH
`
`l VB 1 8/8/13 FH
`£18/8_/13 FH
`Average
`
`Stdev
`
`VB 7 8/9/13 FHE
`
`501.6
`
`20.3
`
`23.4
`
`21.9
`
`4.4
`
`0000011
`
`
`
`%Avanli®
`
`PCILAR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`Ave rage
`
`VB 7 8/9/13 FHE
`
`Stdev
`
`Sample VB 7 8/19/13 FHE was spiked with 25 mg of oleic acid equivalent to a 5% by weight
`spike. The accuracy of the method was evaluated by calculating the spike recovery using the
`formula:
`
`% recovery = ample %FFA + spiked %FFA
`Spike sample %FFA
`
`X 100
`
`Table 8 reports the % recovery for triplicate assay of spiked samples relative to their un-spiked
`counterparts.
`
`Table 8: %Recovery of 5% oleic acid added to VB 7 8/19/13 FHE
`
`
`
`Spike Sample
`FFA/Sample
`FFA/Sample
`FFA/Sample
`FFA added
`%
`Recovery
`Wt (mg)
`(mg) 10 uL
`(mg) 15 uL
`(mg) mean
`(%)
`502.0
`41.9
`45.1
`43.5
`5.0
`107.9
`
`
`
`
`
`
`
`
`
`
`
`
`
`FFA (wt%)
`8.7
`
`
`
`
`
`
`
`5.0
`9.7
`85.0
`5.0
`9.0
`97.9
`
`96.9
`9.1
`
`
`
`_l_ __ 5_02.2___
`502.2
`
`__
`
`_I__
`
`47.3
`42.9
`
`50.6
`47.5
`
`__
`
`_
`
`48.9
`45.2
`
`Average
`
`0000012
`
`
`
`
`
`IlVanli®
`
`POLAR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`Total Phospholipid Content
`
`The samples of the BEA series were assayed by Phosphorus Nuclear Magnetic Resolution (3 ‘P-
`NMR) for total phospholipid content by weight of original sample.
`
`Principle:
`
`Phosphorus Nuclear Magnetic Resonance (3 1P-NMR) is a selective separation method for
`compounds which contain phosphorus in their molecular structure. Phospholipids by definition
`contain phosphorus in their molecular structure. The varied phospholipids in krill have different
`molecular structures in the region of their phosphorus nucleus. These differing phosphorus nuclei
`are separated in a strong magnetic field and produce concentration proportional responses in the
`form of an integrated peak. The 3 ‘P response is calibrated within each sample by addition of an
`internal standard allowing direct measurement of the molar concentration for each phospholipid.
`The weight % of total phospholipid is a ratio of the summed measured components by the
`original sample weight.
`
`Reagents:
`
`30 mM Triphenylphosphate, TPPNMRSTD-082713, exp. 09-26-13
`1:1 CDCI3 : Methanol — 082713, exp. 10-08-13
`Dioleoyl phosphatidylcholine, lot #181PCNMRSTD-081113, exp. 08-06-17
`Dioleoyl phosphatidylethanolamine, lot #181PENMRSTD-061113, exp. 02-09-17
`Oleoyl lysophosphatidylcholine, lot #181LPC-61, exp. 06-21-15
`Oleoyl lysophosphatidylethanolamine, lot #18lLPE-49, exp. 12-15-13
`0.2 M CSEDTA (pH7), lot #073013, exp. 01-30-14
`
`Instrumentation:
`
`Bruker Avance III 400 MHZ NMR, #Z101373-1060
`
`Parameter
`Probe
`
`Abbrev.
`PROBHD
`
`Setting
`5 mm PABBO BB-
`
`Pulse Program
`Number of scans
`
`PULPROG zgig30
`NS
`512
`
`Delay (sec)
`Receiver gain
`Temperature
`Nucleus
`
`DS
`RG
`TE
`NUCl
`
`4
`2050
`290.3° K
`31 P
`
`0000013
`
`
`
`%AIIanIi®
`
`POLAR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`Sample Preparation:
`
`Sample preparation for 31P-NMR was identical for all standards, suitability samples, controls
`and samples. Each material was prepared in the following manner.
`
`1. Add 30 i 0.5 mg of test article into a 13 X 100 mm tube with screw cap.
`2. Add 100 l,lL of 3 mM TPP in chloroform to each tube.
`3. Dry to a residue under a gentle nitrogen stream at room temperature.
`4. Add 2.0 mL of 1:1 (/v) CDCI3 : methanol and gently dissolve residue.
`5. Add 1.0 mL of 0.2M Cs-EDTA to each tube and vortex mix to homogeneous suspension.
`6. Centrifuge at 2500 rpm for 10 minutes.
`7. Carefully transfer the lower layer with a glass pipet into a clean 5 mm NMR tube.
`
`System Suitability:
`
`Qualitative and quantitative assessment of instrument performance is performed using a dioleoyl
`phosphatidylcholine standard of known concentration. Quantitative accuracy should be 98 -102%
`
`181PCNMRSTD—O61113 = 6.9134 mM
`
`31P—NMR results = 6.9051
`
`% accuracy = 99.9%
`
`A mixture of phospholipid reference materials common to krill was formulated and assessed for
`acceptable accuracy and selectivity.
`
`Mix PL STD
`181PCNMRSTD—O61113
`181PENMRSTD—061113
`181LPC—61STD
`181LPE—49STD
`
`Sample Assay by 3 ‘P-NMR
`
`conc.
`
`volume
`
`final conc.
`
`shift
`
`measured
`
`_
`
`(E)
`6.9134
`7.5325
`86.6453
`58.5920
`
`(m_L)__
`1.00
`1.00
`0.13
`0.20
`
`(E)
`6.9134
`7.5325
`11.26389
`11.71839
`
`__
`
`I
`
`_
`
`I
`l
`
`(mm) __
`-0.8
`0.02
`-0.24
`0.5
`
`_
`
`m_|Vl_
`6.866
`_7.4868
`11.141
`11.9543
`
`accuracy
`99.3%
`99.4%
`98.9%
`102.0%
`
`Each sample was prepared and assayed in triplicate. The molar phosphorus content of each
`known peak was quantified from the response of the TPP internal standard added to each sample
`at a know concentration of 3.0698 mM. Conversion of mM to mg for weight % values was
`calculated by the equation:
`
`Weight % = mM phosphorus X M.W. of phospholipid X 1000 mL / L X 100
`
`0000014
`
`
`
`
`Avanlit
`
`PEILAR LIF-‘IDS, INC.
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`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`Normalized molecular weights of the krill phospholipids were pre-determined by mass
`spectrometry from the fatty acyl molecular species.
`
`Compound Q
`Lyso PE
`492.5
`770
`
`Lyso PC
`Lyso PC-2
`Ether PC
`
`other
`
`Results:
`
`534.5
`534.5
`798
`812
`812
`
`The BEA series of samples were assayed in triplicate for phospholipid content. The tables below
`report the compounds detected and their respective weight % of the original sample. Those
`reported as other are integrated signals within the 3 ‘P-NMR trace deemed as phospholipids of
`unknown identity. Their respective mM concentrations were summed and calculated using the
`molecular weight of the major PC component at 812 grams per mole.
`
`Table 9a.
`
`SB1 BEA-P0
`
`Lyso PE
`
`Lyso PC
`
`Lyso PC-2
`Ether PC
`
`Other
`
`Total wt/wt%
`
`Table 9b.
`
`
`
`0.50
`3.61
`
`19.60
`
`2.99
`
`31.02
`
`SB5 BEA-P1
`
`wt wt%
`
`Lyso PE
`
`Lyso PC
`
`Lyso PC-2
`Ether PC
`
`0.26
`2.78
`
`1.27
`
`0.40
`3.67
`
`Q
`
`0.01
`0.03
`
`0.06
`
`0.08
`0.15
`
`0.14
`
`0.06
`
`0.07
`
`s._d.
`
`0.01
`0.17
`
`0.02
`
`0.05
`0.10
`
`0000015
`
`
`
` ll|Ia|Ill®POLAR LIFIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`Other
`
`Total wt/wt%
`
`3.07
`
`31.51
`
`Table 9c.
`
`SB9 BEA-P2
`
`wt wt%
`
`0.39
`
`3.12
`2.67
`
`0.51
`
`4.91
`27.63
`
`5.17
`
`44.40
`
`_ L_yso PE
`
`Lyso PC
`
`__Lyso PC-2
`
`Ether gc____ _
`
`other
`
`Total wt/wt%
`
`Table 9d.
`
`SB 13 BEA-SO
`
`Table 9e.
`
`SB17 BEA-S1
`
`0.12
`
`0.12
`
`S.:d.
`
`0.02
`
`0.11
`0.07
`
`0.07
`
`0.17
`0.39
`
`0.17
`
`0.48
`
`wt wt%
`0.10
`
`0.01
`
`wt wt%
`0.15
`
`s._<'.l.
`0.01
`
`Table 9f.
`
`SB21 BEA-S2
`
`wt[wt%
`
`£46 _
`
`0.01
`
`Table 9g.
`
`ism)
`Lyso PC
`
`Lyso PC-2_ ___
`
`Ether PC
`
`other
`
`_ Wt Wt_%
`0.76
`
`0.45
`
`2.74
`23.35
`
`1.42
`
`Total wt/wt%
`
`_28.7__2 _
`
`Table 9h.
`
`_
`
`S-_d-
`0.12
`
`0.06
`
`o.22___ _
`0.40
`
`0.49
`
`0.74
`
`0000016
`
`
`
`ggmmmr
`
`POLAR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`0.95 _ _ _ 0.08
`
`Lyso PC_
`
`_ Lyso PC-2
`Ether PC
`
`I other
`Total wt/wt%
`
`Table 9i.
`BEA-SU2
`
`Lyso PC
`Lyso PC-2
`Ether PC
`
`other
`
`0.29
`2.63
`
`_ 23.66
`
`1.71
`29.24
`
`_
`wt wt%
`1.15
`
`3.0_1__
`0.41
`3.83
`
`33.78
`
`1.51
`
`0.04
`0.13
`
`0.76
`
`0.12
`0.96
`
`s._d.
`0.03
`
`__ 0.11
`0.06
`0.55
`
`0.64
`
`0.33
`
`1.50
`
`Total wt/wt%
`
`43.70
`
`Sample S1 BEA-P0 was spiked with the l8lPCNMRSTD-061113 solution to determine
`accuracy of measurement through spike recovery. Table 10 reports the accuracy for this
`experiment and the method.
`
`Table 10: Accuracy of 3lP-NMR method.
`
`Lyso PE
`
`Lyso PC
`
`Lyso PC-2
`Ether PC
`
`PC ___
`other
`
`Total
`
`Accuracy
`
`wt/wt%
`
`0.25
`5.13
`
`1.34
`
`0.52
`3.77
`
`37.43
`3.02
`
`51.47
`
`99.7%
`
`0000017
`
`
`
`%AIIanli®
`
`POLAR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`Summary
`
`The summary of test results for the 11 samples in this report is provided in table 11.
`
`Table 11: Results SLII1"]l]1£}I‘)’ in weight % as standard deviation.
`
`I Sample
`
`VB 18/8/13 FH
`
`VB 7 8/9/13 FHE
`BEA SB1 P0
`
`BEA SB5 P1
`
`BEA SB9 P2
`
`BEA SB13 50
`
`BEA SB17 S1
`
`BEA SB21 52
`
`BEA SUO
`
`BEA SU1
`
`BEA SU2
`
`Accuracy
`
`Free FA
`
`3.0i0.1
`
`3.8 i 0.5
`
`Total Lipid
`
`87.8i5.1
`
`84.2 i 2.3
`61.6 i 0.8
`
`59.6 i 1.9
`
`99.0 i 0.6
`
`94.6 i 1.6
`
`96.2 i 1.9
`
`99.6 i 0.6
`
`Total PL
`
`31.02 i 0.07
`
`31.51 i 0.12
`
`44.40 i 0.48
`
`0.10 i 0.01
`
`0.15 i 0.01
`
`0.46 i 0.01
`
`28.72 i 0.74
`
`29.24i0.96
`
`43.701 1.50
`
`98.5
`
`96.9
`
`99.7
`
`.16 ./D. Moore, Ph.D.
`
`Director, Analytical Technologies
`Avanti Polar Lipids, Inc.
`
`0000018
`
`
`
`EXHIBIT A
`
`0000019
`
`
`
`
`
`llvanlit
`
`PDLAR LIPIDS, INI::.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`September 16, 2013
`
`Introduction
`
`Avanti Polar Lipids, Incorporated is a Bulk Pharmaceutical Manufacturer licensed by the U.S.
`Food and Drug Administration . The analytical laboratory provides quality control testing of
`processes, raw materials and final products under current good manufacturing practices (CGMP).
`The quality control laboratory resources to include instrumentation and personnel are also
`utilized to provide fee for service laboratory testing under the Analytical Services Division. All
`instrumentation is maintained, calibrated, operationally and performance qualified (OQ/PQ). All
`personnel are professionally qualified by education and experience as well as maintain
`documented training.
`
`Samples of krill extract were provided for analysis to determine total lipid content, free fatty acid
`content and total phospholipid content. The samples were assayed by methods developed at
`Avanti. Each sample was assayed in triplicate and one sample was fortified with a relevant
`compound to determine suitability of respective methodology, i.e. precision and accuracy. The
`instrumentation, sample preparation, instrument settings, chemicals, calculations and results for
`the three assays are described herein.
`
`Samples and Receipt
`
`Samples were received by courier at Avanti Polar Lipids and unpacked for storage. All samples
`were stored at 2-8° C until time of preparation and returned to such storage between uses.
`
`Received: August 13, 2013
`Condition: no dry ice cool to touch, intact and legibly labeled
`VB 1 8/8/13 FH
`VB 7 8/9/13 Fl-IE
`
`Received: August 21, 2013
`Condition: dry ice and frozen, intact and legibly labeled
`SBI BEA-P0
`SB5 BEA—Pl
`SB9 BEA-P2
`
`SB 13 BEA-S0
`SB 17 BEA-S1
`SB 21 BEA-S2
`
`Received: September 10, 2013
`Condition: dry ice and frozen. intact and legibly labeled
`SB25 BEA-SUO
`
`0000020
`
`
`
`
`
`Avanlit
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
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`
`ANALYTICAL SERVICES DIVISION
`
`SB29 BEA-SU I
`SB33 BEA-SU2
`
`Total Lipid Content
`
`The VB and SB] thru 21 series of samples above were assayed by gravimetrical weighing upon
`extraction and drying. The total lipid content is expressed as weight % of original sample weight
`used in the assay.
`
`Principle:
`
`Lipids are fats or oils which are not soluble in water. They must be extracted and dissolved into
`non-polar solvents to form solutions. Lipid extraction techniques utilize the layering of the water
`and non-polar solvents to separate lipids from a sample. The water soluble compounds dissolve
`into the upper layer and lipids into the lower layer. Lipids extracted into the lower layer can be
`recovered by evaporating the solvent and weighing the dried residue. The weight percentage of
`lipid can be determined by the ratio of recovered lipid weight to the original sample weight. The
`Folch extraction technique (Folch et a1., JBiol Chem 1957, 226, 497) is universally accepted as a
`means to extract lipid.
`
`Reagents:
`
`Fisher Scientific HPLC grade chloroform, lot # l33740, exp. 08-08- l4
`Fisher Scientific HPLC grade methanol, lot #l32577, exp. 08-I2-I4
`Fisher Scientific HPLC grade water, lot #1339] 1, exp. 08- 12-14
`Free fatty acid calibration standard #l8lFFAWKSTK-041813 (re-eval. 10-30-13) at 5.1 mg/mL
`in HPLC grade chloroform.
`
`I nstrumentation:
`
`Sartorius CPl24S analytical balance (4 place), ser. #22650 I93.
`Calibration date: 02-27-13 / Calibration due: O2-28-I4 / Suitability tested: passed 08-I5-I3.
`
`Sample Extraction:
`
`l. Each sample was removed from 2-8° C storage and allowed to equilibrate to ambient
`temperature.
`
`2. Each sample was weighed in triplicate into separate I2 mL, I6 X 100 mm glass test tubes with
`screw cap at 500 i 3 milligram.
`
`0000021
`
`
`
` .
`
`__ POLAR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. — CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`3. Sample VB 7 8/9/13 FHE was weighed and transferred again in triplicate for fortification with
`oleic acid to determine method accuracy by spike recovery. One mL of25 mg/mL oleic acid in
`chloroform, 181 WKSTK-041813, reevaluation due 10-13-13, was added to these tubes. Sample
`weights and solvent volumes are provided in table 1.
`
`
`
`
`CH3OH(_mL)
`Hzg(mL)
`_(mL)
`CHC|3(mL)
`_____ix‘.......
`_
`.2.-9.
`_.
`..___§:5_5...
`
`3.25
`2.0
`6.5
`
`3.25
`2.0
`6.5
`I _W__
`3.25
`2.0
`6.5
`..........._-3.-§_
`_____i‘?.......................... __
`5.-5
`6.5 W
`3.25
`_
`2.0
`5.5
`3.25
`_ W 2.0
`__.I/3 25
`I.[_.__......2-0
`_
`3.25
`2.0
`3.25
`2.0
`W
`3.25
`2.0
`3.25
`2.0 W
`__
`3.25
`2.0
`__ _
`. _. 9-25..-
`........ 2-0
`. ._.........2.-_0
`2.0
`__ ___2__._0_
`2._0
`2.0_
`2.0
`
`_______
`
`__
`
`
`
`__ 1.0
`__
`
`1.0
`
`
`
`
`
`
`
`
`
`____..
`
`
`
`
`_
`
`
`
`_
`_
`
`_.
`
`_
`
`Table 1: Sam rile wI:iB,|its and solution volumes.
`Sample
`_ Weight (mg)
`_
`Sample
`VB 18Z§[1_3_F_”__......_ 5°_3_-‘L _
`VB 1 8/8/13 FH
`501.4
`VB 1 8/8X13 FH
`499.9
`vB 7 8/9/13 FHE
`501.6__
`‘(B 7 8/9/13 FHE
`499-2
`___VB 7 3/9/13 FH_E
`502._2
`VB78/9/13 FHE-SPIKE
`502.0
`V3 7._9../_9/13 EHE'5P'KE._____
`502-2”
`VB 7 B/9g13 FHE-SPlKE_l_
`499.4
`BEA SB1 PO
`500.7
`BEA SB1 P0
`501.1
`BEA SB1 PO
`_ 500.0
`M BEA sB5 P1
`499.3
`3.51.1595 I.’_1
`504-0
`BEA$B_§.._.F’1_.
`49.9-.8
`__BEA sB9___I32
`500.3
`BEA sB9 P2_
`499.6
`BEA sB9 P2
`500.2
`BEA sB13 so _
`500.0
`_
`BEA sB13 so
`501.0”
`.
`BE/*....$B1_3
`2.0 __
`3.25 ____ml_
`6.5
`I
`500.6
`WBEA SB17 s1
`3-25 _ _ ......._ _LO__ .1 _
`._5._-..-5...
`500-5_______________
`BEA 5317 51
`9..-___2._-‘I..-
`.
`.
`.. 2-0
`.._
`499-5 . . 5-5 __
`BE’§_..5B17_51
`. 3-2.5
`.
`. 2.0
`_ -
`. 499-3
`..
`6-5.
`....
`BEAsB21s2
`...3:.25_......
`_..2.-0
`.
`._
`-
`___5_0°-4
`__
`5-—‘z.......
`.9EA-992152-
`
`
`BEA SB21 s2
`. , 6.5 3.25 2.0
`
`
`
`.____
`
`_
`
`I
`
`_
`
`..
`_
`
`..
`
`_
`
`h
`
`_
`
`I__
`__
`
`_______________
`
`5.5
`6.5
`6.5
`6.5
`6.5
`
`____
`1915..
`6.5
`6.5
`6.5
`6.5 ___
`_ 6.5
`.
`
`__
`
`_
`
`_
`
`_
`
`__
`
`3.25
`3.25
`3 25
`3.25 _
`§v._2w5__
`
`_
`
`_
`
`.
`
`________..
`I .
`
`_
`1
`
`............
`
`I
`
`I
`
`.........-
`
`.
`
`__
`.
`
`_
`
`......-
`
`__
`
`I
`
`.
`
`..... _
`.......................
`
`4. The samples were extracted using a Folch extraction technique whereby approximately 20
`
`0000022
`
`
`
`
`
`Avalllli
`
`POLAR‘ Lu=u:>s, IND.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAI SERVICES DIVISION
`
`volumes of2:l chloroform : methanol was added to each tube, dispersed and mixed in an orbital
`shaker at room temperature for IS minutes.
`
`5. The completely dissolved material was washed with 2.0 mL of water by vortexing and
`centrifuged at 2000 rpm for 10 minutes to separate the layers.
`
`6. The lower layer was transferred into pre-weighed 13 X 100 mm glass test tubes with screw
`cap. The lower chloroform layer was evaporated under a gentle stream of nitrogen at ambient
`temperature under a safety hood. The remaining solvent was removed using a Genevac (EZ 2.3
`Elite Model) centrifugal evaporator for a period of 18 hours. The samples were placed in -80° C
`freezer for about 1 hour. Final drying was performed on a vacuum lyophilizer overnight.
`
`7. Each sample was weighed and the % extract/ weight of sample was calculated using the
`formula:
`
`% Total Lipid = tube with extract — tube weight
`original sample weight
`
`X 100
`
`8. The accuracy of the method was evaluated by % recovery for spiked sample with oleic acid
`and calculated using the formula:
`
`% recovery = (% wt in sample + % wt ofspike) / % wt in spiked sample X 100
`
`Results:
`
`Table 2 reports the weight % oflipid from triplicate assays of each sample with calculation of
`mean and standard deviation for evaluation of precision. Table 3 reports the accuracy ofthe
`method from the spike recovery ofoleic acid in a fortified sample.
`
`Table 2: Wciulil % ofll aid.
`
`Sample
`Extract
`
`
`
`
`
`
`
`
`weight (mg) weight (mg)
`Sample
`413.2_ __
`__ 503.0___
`VB 1 8/8/13 FH
`501.4
`445.7
`VB 1 8/8/13: FH
`499.9 T.
`461.2
`VB 1 8/8/13 FH
`5(_)_1_.6
`_\_/B__7 8/9/13 FH?_l
`4"}.-.2......_.
`_\(B_75fi/,13__F_'1E .__ /I912_______t.
`VB 7 8/9/13 FHE E
`502._2__ QM 433_._1
`
`
`BEA SB1 PO __
`500.7
`_
`311.2
`.___'§_§5_$fl'°9_.._. _.,_...l..----5%_1____......203 7
`
`% Extract
`82.1
`88.9
`92.3
`
`_
`
`_
`
`_
`
`0000023
`
`
`
`
`
`IIIIEIIIIT
`
`._ F'I:n_AR LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANALYTICAL SERVICES DIVISION
`
`BEA SB1 PD
`
`BEA 535 PE__
`
`BEA 535 R1
`BEA 535 P1
`
`BEA 539 P2
`
`BEA SB9 P2
`
`BEA 539 P2
`
`BEA SB13 S0
`
`BEA SB13 SO
`
`BEA SB13 S0
`
`BEA SB17 S1
`
`BEA 5817 S1
`
`BEA SB21 S2
`
`BEA SB21 S2
`
`BEA SB21 52
`
`Sample
`VB78/9/13 FHE-SPIKE
`VB 7 3/9/13 FHE—SP|KE
`
`.
`.
`
`'
`
`,
`.
`
`_
`
`_
`
`,
`.
`
`Accuracy
`__ 100.8
`97.9
`
`.
`.
`
`__
`
`0000024
`
`
`
`
`llvanlf
`
`F'I:iL..cu:I LIPIDS, INC.
`
`AVANTI POLAR LIPIDS, INC. - CONFIDENTIAL BUSINESS INFORMATION, SUBJECT
`TO PROTECTIVE ORDER.
`
`ANA