The Texas Medical Center

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Cuthbert Dean
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1 ISCT Regional Meeting November 2 4, 2007 Cryopreservation - Evaluation of Different Processes Presenters: Carlos Lee & Renee Smilee

2 The Texas Medical Center

3 Center for Cell & Gene Therapy

4 The Feigin Center Past Present Future (end of 2008)

5 How to Cryopreserve Hematopoietic Cells? Principles Slow freezing procedures results in hyperosmolality & in membrane destabilization. Colligative agents (cryoprotectants) are used to prevent or mitigate damage caused by hyperosmolality. These materials, allow for easier movement of water & affect crystal growth & structure. Dimethylsulfoxide (DMSO) is a low molecular weight colligative material with high water solubility & high permeability into living cells (Lovelock & Bishop Nature 183 ( ) 1959)

6 How to Cryopreserve HSC? Principles Freezing results in various physiologic, environmental & mechanical changes. Different cells respond differently to the freezing process. A. Hubel, AABB Spring Conference 2007 N.C.Gorin, Bone Marrow & Stem Cell Processing: A Manual of Current Techniques, 1992 A slower freezing rate resulted in increased survival of bone marrow cells. CFU recovery was better with a slower freeze rate post heat of fusion.

7 Standard process: Cryopreservation Hematopoietic cells cryopreservation using programmed controlled rate freezing and dimethylsulfoxide (DMSO) as the cryoprotectant Variations: Diluents/Additives Cell Concentration Bag Source / Bag Volume DMSO Concentration / Source Process of Adding of DMSO Freezing Programs /Curves Sample Temperature measurement

8 Standard process: Cryopreservation Hematopoietic cells cryopreservation using programmed controlled rate freezing and dimethylsulfoxide (DMSO) as the cryoprotectant Variations: Diluents/Additives Cell Concentration Bag Source / Bag Volume DMSO Concentration / Source Process of Adding of DMSO Freezing Programs /Curves Sample Temperature measurement

9 Cryopreservation Diluents/Additives: RPMI Saline Plasmalyte Normosol Human Serum albumin (HSA) Autologous Plasma Plasma Protein Fraction (PPF) Purpose: Provide nutrients / protein Dilute to desired concentration Less common additives: heparin, additional ACD-A, DNAse

10 Cryopreservation RPMI Pros: Lots of nutrients/electrolytes Cons: Not approved for human use Saline Pros: Readily available, Approved for human use Cons: Nutrient poor Plasmalyte Pros: More nutrients than saline, Approved for human use Cons: May need to be ordered (institutional) Normosol Pros: More nutrients than saline, Approved for human use Cons: May need to be ordered (institutional0

11 Cryopreservation HSA Pros: For human use, defined production Cons: Relatively expensive, supply limited (allocations) Autologous Plasma Pros: autologous, relatively cheap Cons: many variables, cryoprecipitate issues PPF Pros: For human use, defined production Cons: Relatively expensive, very limited availability

12 Standard process: Cryopreservation Hematopoietic cells cryopreservation using programmed controlled rate freezing and dimethylsulfoxide (DMSO) as the cryoprotectant Variations: Diluents/Additives Cell Concentration Bag Source / Bag Volume DMSO Concentration / Source Process of Adding of DMSO Freezing Programs /Curves Sample Temperature measurement

13 Cryopreservation Cell Concentration: less than (<) 1x10 8 cells/ ml 1 5x10 8 cells/ml greater than (>) 5x10 8 cells/ml Not considered Purpose: Cell viability Product viscosity Volume of product for infusion Other variables: Processing before freezing: none, volume reduction, red cell reduction Hold times: no hold, hold overnight (ambient 4C), hold with adjusted concentration

14 Cryopreservation Less Clumping / Viscous More Volume, More DMSO, More Storage < 1x10 8 cells/ ml 1 5x10 8 cells/ml > 5x10 8 cells/ml More Clumping / Viscous Less Volume, Less DMSO, Less Storage Not considered Typically, freeze based on volume only.

15 Standard process: Cryopreservation Hematopoietic cells cryopreservation using programmed controlled rate freezing and dimethylsulfoxide (DMSO) as the cryoprotectant Variations: Diluents/Additives Cell Concentration Bag Source / Bag Volume DMSO Concentration / Source Process of Adding of DMSO Freezing Programs /Curves Sample Temperature measurement

16 Cryopreservation Bag Sources: Baxter Cryocytes American Fluroseal Kryosure Bag Volume: Range of volume for bag types Mix of different bags per freeze procedure Key Points: inventory system validated process manufacturer s specs physician s orders cell dose Other issues: over-wraps or pouches

17 Cryopreservation Site X Site Y Range of volume for bag types ml (250ml Cryobags) 20 ml (50 ml Cryobags) ml (250ml Cryobags) NA (50ml Cryobags not used) Mix of different bags per freeze procedure allowed YES NO

18 Standard process: Cryopreservation Hematopoietic cells cryopreservation using programmed controlled rate freezing and dimethylsulfoxide (DMSO) as the cryoprotectant Variations: Diluents/Additives Cell Concentration Bag Source / Bag Volume DMSO Concentration / Source Process of Adding of DMSO Freezing Programs /Curves Sample Temperature measurement

19 Cryopreservation DMSO Concentration: Purpose: 10% DMSO (final concentration) 5% DMSO (final concentration) Cryoprotectant DMSO toxicity DMSO related infusion reactions 5% DMSO, 6% HES (final concentration)

20 Cryopreservation 10% DMSO More DMSO volume, DMSO toxicity, DMSO related infusion reaction (standard) 5% DMSO (more recent) 5% DMSO, 6% HES (alternative) Less DMSO volume, DMSO toxicity, DMSO related infusion reaction

21 Cryopreservation DMSO Sources Cryoserv (Bioniche Pharma, formerly Edwards Life Sciences) Stemsol (Protide Pharmaceuticals) CryoSure -DMSO (WAK-Chemie Medical GmbH)

22 Standard process: Cryopreservation Hematopoietic cells cryopreservation using programmed controlled rate freezing and dimethylsulfoxide (DMSO) as the cryoprotectant Variations: Diluents/Additives Cell Concentration Bag Source / Bag Volume DMSO Concentration / Source Process of Adding of DMSO Freezing Programs /Curves Sample Temperature measurement

23 Cryopreservation DMSO Addition Process Cells DMSO Speed Chilled Soln, Chilled Fast Chilled Soln, Chilled Slow Chilled Neat, Room Temp Fast Purpose: Cryoprotectant Cell viability DMSO toxicity Ambient Soln, Room Temp Slow

24 Standard process: Cryopreservation Hematopoietic cells cryopreservation using programmed controlled rate freezing and dimethylsulfoxide (DMSO) as the cryoprotectant Variations: Diluents/Additives Cell Concentration Bag Source / Bag Volume DMSO Concentration / Source Process of Adding of DMSO Freezing Programs /Curves Sample Temperature measurement

25 Cryopreservation Freezing Program /Curves: Different Equipment End Temperature Freeze rate at different phases Purpose: Cell viability Time (duration of freeze process) Equipment functionality

26 Cryopreservation Site A Site B Site C Equipment variations Cryomed Cryomed CBS & CBS End Temperature -90 C -90 C -60 C Freeze rate at different phases see next slide

27 Example of a Freezing Curve Site A Initial cooling (-1C/min) to (-12C) Heat of fusion Post Heat of Fusion cooling (-1C/min) to (-60C) Ramp to (-18C) at (15/min) Seeding step forces nucleation Ramp to (-50C) at (-30/min) Accelerated Cooling to final temp (-5C/min) to chamber (-90C) Accelerated Cooling to final temp (-10C/min) to sample (-90C)

28 Standard process: Cryopreservation Hematopoietic cells cryopreservation using programmed controlled rate freezing and dimethylsulfoxide (DMSO) as the cryoprotectant Variations: Diluents/Additives Cell Concentration Bag Source / Bag Volume DMSO Concentration / Source Process of Adding of DMSO Freezing Programs /Curves Sample Temperature measurement

29 Cryopreservation Sample Temperature: Dummy Bag Sandwich Probe Cryovial Purpose: Indirectly measure the product s temperature without compromising the product Used in many freezing programs to determine transition from one step to another including end point

30 Cryopreservation Dummy Bag Pros: Probe has direct contact with very similar environment and materials Cons: Need a dummy bag every run; direct contact of probe with potentially biohazardous materials Sandwich Probe Pros: Probe does not contact biohazadous materials; measures temp. of product bag (s) -externally Cons: Positional & Conductivity Issues: 1) Requires very good contact with product bag (s) 2) Need to insulate probe from metal 3) may need a bag press or other arrangements to achieve good contact Cryovials Pros: Probe has direct contact with very similar materials Cons: Probe has direct contact with potentially biohazardous materials; Does not mimic the conditions of the product bags volume, area/shape, heat loss/gain

31 Cryopreservation Alternative Standard process: Hematopoietic cells cryopreservation using nonprogrammed (dump) freezing and DMSO+ Starch as the cryoprotectants Variations: Diluents/Additives Cell Concentration Bag Source / Bag Volume DMSO Source Starch Source & Concentration Dump Freezing Method

32 Cryopreservation of HSC - Current Practices DMSO DMSO & Hydroxyethyl Starch (HES) Final: 10% (5%) DMSO Final: 5% DMSO 6% HES Programmed controlled rate freezing Nonprogrammed /noncontrolled rate (dump) freezing Key cooling rate of 1 0 C/min Key cooling rate ~ 3 0 C/min Storage in Liquid Nitrogen bank Ping Law & Harold Meryman, Chapter 22, Bone Marrow Processing & Purging A Practical Guide, 1991 Storage in -80 o C Freezer up to 2 yrs (Or transfer to LN bank) Patrick Stiff, Chapter 23, Bone Marrow Processing & Purging A Practical Guide, 1991

33 Cryopreservation Associated Processes: Storage Thawing Post Thaw Assessments Liquid Nitrogen Liquid Phase Vapor Phase Mechanical (-135) (- 80) With or without LN back up Bedside In the Lab Transferred Washed Other No Assessment Assess Surrogate Sample cryovial Segment Assess Product Diluent & ratio

34 Evaluation of Methodology Things to Consider / Decisions to Make: Types (sources) of HSC products DMSO only versus DMSO/HES Programmed versus non-programmed Preparatory procedures and resultant volumes Expected duration of storage Identify (select) vendors of equipment, supplies, reagents Location for the equipment, procedure Search for (identify & select) existing procedures from references, colleagues

35 Remarks There are many variations in the process of cryopreserving cells. Why fix what is not broken In many centers, the procedure was developed years ago with good clinical outcomes and they are reluctant to change Evaluation and implementation of a new or replacement method is typically costly in time and money: Equipment, Inventory systems, Validation & Qualification costs, Re-training, Retro-fitting, Monitoring

36 Things to Do: Evaluation of Methodology Break the whole process into components parts Establish expectations from the component parts Consider the expectations when purchasing the equipment, supplies, reagents Develop qualification and validation plans / procedures Evaluate the whole Develop training modules

37 Things to Do: Evaluation of Methodology Establish expectations from the component parts : Examples: Cell recovery / viability post volume reduction: >90% & > 95%, respectively Cell recovery / viability post freeze & thaw: >50% & >80%, respectively Storage temperature does not exceed -150oC with static hold time of 10 days Programmable freezer will consistent perform the specified program and the freeze curve will be printable Cryobags contents will be sterile when used as per SOP and can hold 70 ml of product and fit inside the cassettes. Post freeze, 95% of the bags will pass our drop protocol DMSO is sterile and will pass our toxicity protocol

38 Things to Do: Evaluation of Methodology Develop qualification & validation plans/procedures: Examples: SOPs for process validation, equipment qualification these should guide the user: what needs to be qualified / validated, how to proceed, what & how to document, who should perform the testing and what approvals are necessary Develop a qualification and/or validation plan for the equipment / procedures Perform the qualification/validation procedures and documentation Evaluate the whole: Examples: Put all the component parts together Develop a validation plan for the whole process.

installed in the right environment Operational Qualification Show that the instrument functions according to specification in

39 Evaluation of Methodology Equipment Qualification 4 Stages: Design Qualification define functional & operational specifications decision to purchase this particular model Installation Qualification document that expected instrument was received, properly installed in the right environment Operational Qualification Show that the instrument functions according to specification in the selected environment Performance Qualification Show that the instrument performs according to specification during routine use

40 Evaluation of Methodology Equipment Qualification Documentation Form Design Qualification Description Indicate function Indicate specs to be met: Key performance characteristics Sample volume Operational range Health, safety & environmental concerns Reason for selecting that model Installation Qualification Date of Delivery Order complete? Location for instrument? Does location meet spec? Installer Date of Install If applicable, did it pass selfcheck,? Calibration/service established? Comments/Issues

41 Evaluation of Methodology Equipment Qualification Documentation Form Operational Qualification Must be completed before placed into use Prospective validation Validation plan Calibrated? SOP written? Performance Qualification Provide evidence that equipment is performing as expected over time: table of results over time retrospective validation calibration/service record Re-Qualification Must be performed for critical equipment if: Moved? Damaged / Repaired? Modified / Upgraded? Intend to use for different purpose?

Retrospective a procedure that has been employed without formal validation.

42 Evaluation of Methodology Procedure Validation Prospective new procedure, equipment or whenever there is a significant change to a validated procedure. Retrospective a procedure that has been employed without formal validation. Process validation SOP directs user in the process but does not specify the methods or expectations Expectations are to be determined by the user to be outlined in the Validation Plan.

43 Procedure Validation Sample Process SOP to be validated Type of validation List of Expectations (endpoints) Methods or procedures and # of experiments Anticipated completion date Signatures / approval

44 Final Remarks If you are considering adopting a new procedure expect a lengthy evaluation and implementation process Patience and perseverance

CryoStor CS2, CS5 and CS10 FREEZE MEDIA

CryoStor CS2, CS5 and CS10 FREEZE MEDIA Pre-Formulated Serum-Free Protein-Free cgmp Manufactured FDA Master File Sterility, Endotoxin, and Cell-Based Release Testing CryoStor, a series of cell-specific,