Cryostore Validation Booklet

CRYOSTORE™ BAGVALIDATION SUMMARY

TABLE OF CONTENTS

Design Features ...........................................................................................................................................................................................1Performance Testing .....................................................................................................................................................................2 Aging Studies................................................................................................................................................................................2 Package Testing ...................................................................................................................................................................2 Gamma Sterilization Resistance ...............................................................................................2 Thermal Shock...........................................................................................................................................................................3 Long-Term Freezing Tests ...........................................................................................................................3 Multiple Freeze/Thaw Cycles ...............................................................................................................3 Bioburden...............................................................................................................................................................................................4 Sterilizer Qualification ............................................................................................................................................4 Chemical Resistance...............................................................................................................................................4 Burst Testing ...................................................................................................................................................................................5 Leak Testing ......................................................................................................................................................................................5 Production Validations .......................................................................................................................................5 Customer Validations............................................................................................................................................5Biological Evaluations ................................................................................................................................................................6 Hemolysis ...............................................................................................................................................................................................6 Acute Systemic Toxicity....................................................................................................................................6 Sub-Acute Systemic Toxicity ..............................................................................................................6 Intracutaneous Irritation ...............................................................................................................................7 Cytotoxicity .......................................................................................................................................................................................7 Maximization Sensitization .......................................................................................................................7 Pyrogenicity .....................................................................................................................................................................................8 Endotoxin Testing ..............................................................................................................................................................8 Implantation...................................................................................................................................................................................8 Physiochemical Test for Plastics ...............................................................................................8Physical Properties of OriGen EVA Film.........................................................................................9

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OVERVIEWThe OriGen CryoStore™ freezing bag is designed to freeze blood components. It has been FDA cleared and CE marked since 2003 and has been validated at dozens of institutions worldwide.

This document is intended to provide a summary of validation and performance testing already completed on the CryoStore™ freezing bag as a part of our FDA application and from experiences reported by users.

DESIGN FEATURESPRECISION FILMThe CryoStore™ Bag is made from USP Class VI precision-rolled EVA film of non-animal origin. Extrusion through tremendous rolling pressure stabilizes and orients the film polymers, producing a stronger film than a blow molded tube. The extrusion rollers can also be textured to give the film a matte finish, so the film is less likely to stick to itself and is easier to work with. The OriGen CryoStore™ bag is made from film with a matte finish on both sides, so the bag handles well yet it is easy to see the sample inside.

SPIKE PORTSTwo EVA spike ports are hermetically sealed into each bag to maintain sterility of the spike entry channel until twisted off for access by the user creating a tamper-proof design.

SAFETYThe CryoStore™ freezing bag is available with needle free access ports in the tubing set. Elimination of needles leads to a reduced risk of needlestick accidents.

SIZE RANGEThe OriGen CryoStore™ bag is available for freezing volumes of 10 to 450 mL (810 mL for CryoStore™ Conical bag).

REGULATORY COMPLIANCEOriGen products are made in compliance with ISO 13485, US FDA 21 CFR 820, MDSAP, and in conformance with Good Manufacturing Practice (GMP) standards.

CONFIGURATIONSThe product is available in a wide variety of tubing and multi-bag set configurations built to customer’s requirements.

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PERFORMANCE TESTINGAGING STUDIESThe OriGen CryoStore™ bag has been tested in accelerated aging studies to validate a four-year shelf life. Examination of the product and packaging showed no defect and no measurable functional differences between the aged product and recently sterilized products.

PACKAGE TESTINGSamples irradiated to at least 80kGy were subjected to package performance testing per ASTM D4169-16 Standard Practice for Performance Testing of Shipping Containers and Systems, using Distribution Cycle 13, followed by 4-year accelerated aging. Pouches were then subjected to visual inspections, aseptic transfer, dye penetration, and seal strength (peel testing) following label and ink compatibility testing of the packaging. All sterilized and aged packaging (including labels) maintained integrity during shipping, handling, and storage without device damage and with the sterile barrier maintained. In addition, pouch seals from each packaging run are peel tested at the start and conclusion of packaging.

GAMMA STERILIZATION RESISTANCEAll of the components of the OriGen CryoStore™ bag have been validated for gamma sterilization. Products were sterilized at 40 kGrays or more, put through an accelerated aging study, then evaluated for change or degradation in product performance. While there was some minor discoloration of PVC luer components, there was no change in physical properties. All of the components of the CryoStore™ bag are qualified for routine gamma sterilization.


THERMAL SHOCKCryoStore™ bags have been tested extensively for resistance to extreme temperature changes. Bags filled with the recommended volume of a simulated blood product were repeatedly plunged into liquid nitrogen from room temperature, then thawed. At the conclusion of the freezing cycle, the bags were pulled directly from liquid nitrogen and placed into a water bath to thaw. All bags passed the test and there was no evidence of cracks, breaks, or leaks. There was also no evidence of deformation, material loss, change of properties, or any discernible visual change of the product compared to before freezing.Note: Always follow your institution’s protocol for freezing and thawing.

LONG-TERM FREEZING TESTSSamples of CryoStore™ bags filled with a simulated blood product were put through a routine linear freezing cycle down to -80° C, then moved to the liquid phase of liquid nitrogen storage. The bags were held for 30 days, then removed and rewarmed by either gradual or rapid rewarming methods. No bag breakages occurred after either warming method.

MULTIPLE FREEZE/THAW CYCLESCryoStore™ bags filled to the recommended freeze volume with blood analog were frozen to -4° C, then moved directly to the liquid phase of the liquid nitrogen storage tank. Bags were held 24 hours and thawed in a standard water bath. The freeze/thaw cycle was repeated a total of 10 times for each bag. The bags were evaluated for any defects after each freeze/thaw cycle and no defects were found.


BIOBURDENThe manufacturing team strives to produce a product with the lowest possible number of live bacteria prior to sterilization. Bioburden testing is performed by randomly selecting product from production (prior to sterilization) and evaluating the number of live bacteria on the product. Having a low bioburden will ensure that endotoxin levels on the product are extremely low. Bioburden testing is performed throughout the production calendar year.

STERILIZER QUALIFICATIONAll CryoStore™ bags are sterilized in a gamma radiation cycle, which is qualified annually to ISO 11137. OriGen uses the VDmax method, as described in ISO 11137-2.

CHEMICAL RESISTANCEThe CryoStore™ bag has been tested for resistance to chemicals routinely used in cryopreservation and stem cell research. EVA resin suppliers have tested the chemical resistance of their resins against a wide variety of aggressive solvents and acids. Our testing has confirmed that the OriGen CryoStore™ bag is short-term resistant to 100% DMSO after 24-hour exposure (see OriGen White Paper WP1411).

Note: Resistance code 1 means the film can be treated with the substance at room temperature for several weeks without any influence on the physical, optical, andchemical properties.


Acetone

CHEMICAL

1

RESISTANCE

DMSO, 100% 1

Ethanol, Methanol, Isopropanol 1

SOURCE: DUPONT

BURST TESTINGUnsupported, sterilized and aged bags were submitted to gradually increasing air pressure (1 psi increments) until the bags burst. The acceptance criteria are achieving a minimum pressure of 5.8 psi (300 mmHg) without deformation. In all cases, failure occurred due to stretching and rupture of the film. There were no failures at the seams or ports.

LEAK TESTINGDuring routine manufacturing, single-chamber CryoStore™ bags are 100% leak tested for leaks at 10 psi with a validated test method.

PRODUCTION VALIDATIONSAll production equipment and processes used to make the CryoStore™ bag are risk assessed and validated where appropriate. Equipment is maintained and calibrated as appropriate per established operating procedures. Personnel are trained in production procedures and retrained whenever a change occurs.

CUSTOMER VALIDATIONSThe OriGen CryoStore™ bag has been independently validated at dozens of institutions via a variety of methods. Contact [email protected] for more information.


BIOLOGICAL EVALUATIONSThe OriGen CryoStore™ bag has been tested to biocompatibility compliance per ISO 10993. This data was submitted to the FDA - CBER as part of the original device approval process. In 2021, OriGen completed a new round of biocompatibility testing on the CryoStore™ bag. A summary of biocompatibility and other biological evaluation testing is below.

HEMOLYSIS (ISO 10993-4:2017 & ASTM F756)The test article was evaluated for the potential to cause hemolysis. The hemolytic index for the test article in direct contact with blood was 0.0% and the hemolytic index for the test article extract was 0.1%. Hemolytic activity of the CryoStore™ bag with rabbit blood indicates that the CryoStore™ bag was non-hemolytic.

ACUTE SYSTEMIC TOXICITY (ISO 10993-11:2017)Systemic injection compares the potential toxic effects of CryoStore™ bags extracted in various solutions to unexposed solutions by injection into mice. The test article was extracted in sodium chloride USP solution and sesame oil, NF. There was no difference between the extracts and the unexposed solutions when injected into mice; the test article did not show greater biological reactivity compared to the controls. There was no mortality or evidence of systemic toxicity observed.

SUB-ACUTE SYSTEMIC TOXICITY (ISO 10993-11:2017)The test article was extracted in 0.9% sodium chloride USP solution and sesame oil, NF. These extracts were evaluated for systemic toxicity in rats following repeated intravenous and intraperitoneal injections over four weeks. Parenteral administration of the CryoStore™ bag extract did not produce systemic toxicity in rats.


INTRACUTANEOUS IRRITATION (ISO 10993-10:2010)The test article is extracted 0.9% sodium chloride USP solution and sesame oil, NF and is injected into rabbits. There was no erythema or edema from the sodium chloride test extract. There was no erythema or edema to very slight erythema and edema from the sesame oil test extract. The difference between the test and the control mean scores was less than 1.0. The requirements of the test were met.

*This was tested to MHLW, Japan Part 5 and ISO is the reference standard.

CYTOTOXICITY (ISO 10993-5:2009)The test article was evaluated to determine the potential for cytotoxicity. The assay evaluated the potential for leaching substances from the article to produce cytotoxicity by measuring the effect on colony formation of Chinese hamster lung cells. The CryoStore™ bag did not indicate cytotoxicity per ISO 10993-5.

MAXIMIZATION SENSITIZATION (ISO 10993-10:2010)The test article was evaluated for the potential to cause delayed dermal contact sensitization in the guinea pig maximization test. The test article was extracted in 0.9% sodium chloride USP and sesame oil, NF. Each extract was intradermally injected and occlusively patched to guinea pigs. The CryoStore™ Bag extracts showed no evidence of causing delayed dermal contact sensitization in the guinea pig and was not considered a sensitizer in the guinea pig maximization test based on the criteria set forth in ISO 10993-10.


PYROGENICITY (USP <151> AND ISO 10993-11:2017)The purpose of this study was to determine whether an extract of the test article induced a pyrogenic response following intravenous injection in rabbits.

The total rise of rabbit temperatures during the 3-hour observation period was within acceptable USP requirements. The CryoStore™ bag met the requirements for the absence of pyrogens.

ENDOTOXIN TESTING (USP <85> AND USP <161>) Endotoxin testing is performed throughout the production calendar on the CryoStore™ product line. Results have passed the acceptance criteria of having less than 20 USP Endotoxin Units (EU) per device.

IMPLANTATION (ISO 10993-6)Samples of the CryoStore™ bag film were implanted in rabbit muscle for seven days and compared to negative control implant sites. Macroscopic and microscopic evaluation of the test article and the implant sites showed no toxic effects of the sample.

PHYSIOCHEMICAL TEST FOR PLASTICS (USP <661>)This test determines the physical and chemical properties of the water for injection extract of the test article, and the CryoStore™ film met the USP 25 <661> physiochemical requirements for plastics.


PHYSICAL PROPERTIES OF ORIGEN EVA FILM


*(GRAMS PER 100 IN2 PER 24 HRS)**(CC PER 100 IN2 PER 24 HRS)

Note: The values in this table have not been validated by OriGen and should be used for reference only.

COMPLIANCEUSP Class VI and ISO 10993

Durometer Hardness

PHYSICAL CHARACTERISTICS

76A D-2240

TEST, ASTMRESULT

Specific Gravity 0.91 to 0.96 -

Tensile Strength, psi 4530 - 4810 -

Elongation @ break 850.00% -

Melting Point >73°C -

Tear Resistance, lb/in 366 -

Glass Transition <-32°C D-1790

WVTR* 0.785 E-96-80

Oxygen transmission** D-3985

CO2 Transmission 813 D-1434

Optical Transmission 92% -

84.6

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Documents

Cryostore Validation Booklet