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Report of the verification of the effect of temperature and humidity to the heparin

coating

Study Number2005-061

REPORT APPROVALPrepared by Date

Jun Yamashita R&D Engineering Date

N/A N/AManufacturing DateSubhan KhanQuality and Regulatory Affairs DateJohn Toigo

Form: 811653 Rev B

6180 Jackson Road, Ann Arbor, MI 48103, U.S.A.

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1. Purpose:

The purpose of this study was to verify the effect of temperature and humidity to the heparin coating under the cyclic testing. The cyclic testing condition s followed the format established by IEC, and the temperature rate of change followed the EN45502. These worst case conditions will provide the minimal amount of heparin activity while still within this range.

2. Reference/Attachments:

2.1 The following documentation is used in conjunction with this study:　　　2.1.1 Preparation of Heparin Solution #809653 rev. E

2.1.2 Preparation of 0.1% PEI Solution #809650 rev. E 2.1.3 Preparation of 0.03% Dextran Sulfate Solution #809651 rev. E 2.1.4 Inflow conduits/ outlet pipes heparin coating process #809657 rev. D 2.1.5 Stainless Steel Disc Drawing #809684 rev. B 2.1.6 Titanium Disc Drawing #809685 rev. B 2.1.7 Thrombin Inhibition Assay (TIA) #812079 rev. A 2.1.8 THI Sterilization Criteria Procedure #811891 rev. A 2.1.9 Parts cleaning procedure# 809401 rev. D 2.1.10 LAL test products and process monitoring Procedure #811177 rev. A 2.1.11 Heparin Aging study #2004-022 2.1.12 IEC 62-2-38 Basic environmental testing procedures 2.1.13 EN45502-1: 1997 AIMD General Requirements for Safety, Marking, and Information to be provided by the manufacturer 2.2 The following equipment and tooling is used for this study:

2.2.1 Spatula, Spoon, Hemostats 2.2.2 Disposable plastic weighing trays2.2.3 Teflon coated stirring bar p/n 8112852.2.4 250 ml Beakers, Cole-Parmer p/n A-06010-552.2.5 800 ml Beakers, Cole-Parmer p/n A-06010-752.2.6 Personal protective equipment (Face shields, safety glasses, nitrile gloves, cleanroom gowns, hairnets)2.2.7 Scale, Calibration ID – 27-0026-012.2.8 Magnetic stirrer model# PC-510 27-0032-012.2.9 Magnetic stirrer model# S46415 27-0032-022.2.10 Heparin temperature controller, Calibration ID – 27-00012.2.11 Thermometer, Calibration ID – 27-0193-012.2.12 pH meter, p/n 27-0212-012.2.13 Coating process holding oven and thermometer 27-0031-012.2.14 Chemical fume hood 29-0203-022.2.15 Mixing station 27-0034-002.2.16 Heparin hood 27-0034-012.2.17 Ultrasonic cleaner model# 08892-012.2.18 LAL station model# SHKE30002.2.19 Temperature and humidity chamber PJ-349

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3. Deviations to Protocol:

The evaluation method to determine heparin activity, thrombin inhibition assay, was changed from the previous method of #78-8067-8152-8 to #812079 rev A. Therefore the acceptance criteria have changed from greater than 0.008 units/cm2 of heparin activity to the following specification. Titanium and Stainless Steel > 0.010 U/mL

THI procedures 811891A Sterilization Release Criteria and 812079A Thrombin Inhibition Assay are in a pre- released state. Protocol indicates they have already been released. This will not effect the testing performed in this protocol.

Graph 1, 2 and 3 (General cyclic test condition cycle A, B and C) is not detailed condition.The cyclic test was conducted as following condition.

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temperature (degree C) humidity (%) time (hours)

Pre condition 50 18 2450 93 5.525 80 1.550 93 6.525 93 2

0 100 325 93 2.550 93 5.525 80 1.550 93 6.525 93 8.550 93 5.525 80 1.550 93 6.525 93 2

0 100 325 93 2.550 93 5.525 80 1.550 93 6.525 93 8.550 93 5.525 80 1.550 93 6.525 93 2

0 100 325 93 2.550 93 5.525 80 1.550 93 6.525 93 8.550 93 5.525 80 1.550 93 6.525 93 2

0 100 325 93 2.550 93 5.525 80 1.550 93 6.525 93 8.550 93 5.525 80 1.550 93 6.525 93 2

0 100 325 93 2.550 93 5.525 80 1.550 93 6.525 93 8.5

9th day

10th day

5th day

6th day

7th day

8th day

1st day

2nd day

3rd day

4th day

4. Traceability:

4.1. All the materials used during the process performance qualification are listed below. Material Part number Lot numberPolyethyleneimine 801208 08503CCDextran Sulfate 801209 2324B55-100GSodium Chloride 801211 043832Citric Acid Monohydrate 801210 040033Sodium Hydroxide 801212 031355Sodium Cyanoborohydride 801214 12807Periodate Oxidized Heparin 809962 6351007252Stainless Steel Disc 809684 66159Titanium Disc 809685 80544

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Sterile 70% Isopropanol 810182 3302044Sterile water for Irrigation, USP AB-7139-09-19 16-136-JT

4.2. All equipment used during the making of the samples are listed Section 2.2.

4.3. Manufacturing records and data sheets for this study are attached.

4.4. All equipment used during the making of these samples are listed Section 2.2.

4.4.1. Sample preparationSample discs were cleaned according to parts cleaning procedure. Sample discs were strung on a wire, where each disc was separated by spacers so that the surfaces of the discs did not interfere with one another.

4.4.2. Heparin coatingSample discs were coated according to the designated procedure under the nominal process parameter.

4.1.3 LAL Extraction Sample discs were submerged in USP sterile water for irrigation for one hour and thoroughly dried to simulate the LAL test.

4.1.4 Sterilization Samples were packed in Tyvek pouches and sent for sterilization.

　　　 4.1.5 Cyclic testing

Samples were sent to Shonan R&D center in Japan, and were processed temperature and humidity cyclic test. Samples operating record is attached.

5. Results: Table 1: Summary of results, values for heparin activity are in U/mL unless otherwise noted.

Titanium disc Stainless Steel discNon- Cyclic tested (n=10)

Cyclic tested (n=10)

Non- Cyclic tested (n=10)

Cyclic tested (n=10)

Average (U/mL) 0.031485 0.019255 0.029004 0.014490Standard Deviation 0.001790 0.002988 0.002079 0.001670Maximum 0.033463 0.022690 0.030666 0.016935Minimum 0.027280 0.013450 0.023496 0.011829Acceptance criteria Greater than 0.01 U/mL Greater than 0.01 U/mL

Figure 1: Effect of cyclic testing (temperature and humidity changes) on heparin activity for

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Titanium substrate

Titanium disc result (Average)

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Ti Non- cyclic tested Ti Cyclic tested

(U/mL)

Figure 2: Effect of cyclic testing (temperature and humidity changes) on heparin activity for Stainless Steel substrate

Stainless Steel disc result (Average)

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

SS Non- cyclic tested SS Cyclic tested

(U/mL)

Figure 3: Heparin activity summary and data distribution for titanium Non- Cyclic tested

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Figure 4: Heparin activity summary and data distribution for titanium Cyclic tested

Figure 5: Heparin activity summary and data distribution for Stainless Steel Non-Cyclic tested

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Figure 6: Heparin activity summary and data distribution for Stainless Steel Cyclic tested

6. Conclusions:

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Based on the results detailed in section 5 it can be seen from figures1 and figure 2 that temperature and humidity changes have effect on heparin activity degradation.

It can also be seen for each substrate, that heparin activity exhibits a normal distribution (α=.05) except for Stainless Steel Non- Cyclic tested. One Stainless Steel sample of Non- Cyclic tested is lower than the others (nine data), however the heparin activity was still 130% higher than the acceptance criteria.

Review of the results detailed in section 5 indicate that for both Non - Cyclic tested and Cyclic tested samples the acceptance criteria of 0.01 U/mL for heparin activity is exceeded in all cases for both substrates.

Therefore it can be concluded that under the cyclic test, the existing heparin coating process is able to deliver heparin activity levels that can be sustained for Pump Product Requirement Specification already defined. ( Temperature 0-50 degree C, Humidity 10-95%RH)

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Appendix

Raw Data

Description Heparin activity (U/mL)Ti Cyclic tested 0.01570157Ti Cyclic tested 0.013450259Ti Cyclic tested 0.022690417Ti Cyclic tested 0.020401569Ti Cyclic tested 0.02198518Ti Cyclic tested 0.022130982Ti Cyclic tested 0.019782409Ti Cyclic tested 0.018009169Ti Cyclic tested 0.017907359Ti Cyclic tested 0.020488999

Ti Non- cyclic tested 0.03119723 Ti Non- cyclic tested 0.032153489 Ti Non- cyclic tested 0.027279883 Ti Non- cyclic tested 0.030050063 Ti Non- cyclic tested 0.032016683 Ti Non- cyclic tested 0.031305612 Ti Non- cyclic tested 0.033463128 Ti Non- cyclic tested 0.033364324 Ti Non- cyclic tested 0.032360844 Ti Non- cyclic tested 0.031657313

SS Cyclic tested 0.016458968SS Cyclic tested 0.016934723SS Cyclic tested 0.011829397SS Cyclic tested 0.014267233SS Cyclic tested 0.012012158SS Cyclic tested 0.015156427SS Cyclic tested 0.015095411SS Cyclic tested 0.013884363SS Cyclic tested 0.015248436SS Cyclic tested 0.014010853

SS Non- cyclic tested 0.028532044SS Non- cyclic tested 0.028697968SS Non- cyclic tested 0.029929655SS Non- cyclic tested 0.023496048SS Non- cyclic tested 0.030050063SS Non- cyclic tested 0.029148951SS Non- cyclic tested 0.028865615SS Non- cyclic tested 0.030396127SS Non- cyclic tested 0.030665802SS Non- cyclic tested 0.03025274

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Ti Non- cyclic tested Ti Cyclic tested

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

SS Non- cyclic tested SS Cyclic tested

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Ti Non- cyclictested

Ti Cyclic tested SS Non- cyclictested

SS Cyclictested

Series1