Comparison of Cryopreservation Media

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An overview of various cell/tissue freeze media formulations and the CryoStor technology platform

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Comparison of Cryopreservation Media

  1. 1. Cryopreservation Media Products For Cells, Tissues, and Organs Optimizing Biopreservation Economics from Source Material to Finished ProductAugust 2010 © 2010 BioLife Solutions, Inc.
  2. 2. FORWARD LOOKING STATEMENT DISCLOSURE!This presentation contains forward-looking statements within the meaning of the PrivateSecurities Litigation Reform Act of 1995 including, but not limited to, statements aboutBioLife Solutions, Inc. (the “Company”) and its future operating results, strategies, andproduct development plans. These forward-looking statements are based on currentexpectations and assumptions that are subject to risks and uncertainties. Actual resultscould differ materially from the results expressed or implied in these forward-lookingstatements. Factors that may cause or contribute to such differences are more fullydiscussed, as are other factors, in Part I, Item1A. “Risk Factors” of the Company’s Form10-K for the fiscal year ended December 31, 2009, which is on file with the SEC andavailable at www.EDGAR.com. In addition, any forward-looking statements represent ourestimates only as of today and should not be relied upon as representing our estimates asof any subsequent date. While the Company may elect to update forward-lookingstatements at some point in the future, the Company specifically disclaims any obligationto do so except as may be legally necessary, even if the Company’s estimates shouldchange.2! OTCBB: BLFS! 2!
  3. 3. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!"#$$#%&!$()")&! We strive to be the leading provider of biopreservation tools for cells, tissues, and organs; ! to facilitate basic and applied research and commercialization of new therapies by maintaining the health and function of biologic source material! and nished products during the preservation process.!3! OTCBB: BLFS!
  4. 4. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!"#$%&(!)$*+*,! We are committed to manufacturing products in accordance with! applicable current Good Manufacturing Practices and Quality System! Regulations. Every team member in the organization is responsible for! ensuring product quality during the performance of their duties and! identifying opportunities for continuous quality improvement. ! We Will:! • Provide products and services of the highest possible standards! to satisfy our customer needs and expectations of quality, safety,! reliability and performance! • Encourage and promote a corporate culture of continuous !! quality improvement! • Maintain effective Quality Assurance Systems! • Invest to enhance the skills of all team members!4! OTCBB: BLFS!
  5. 5. PRODUCTS & SERVICES !•! Products: •! Proprietary, cGMP, serum-free, protein-free biopreservation media products for cells, tissues, and organs: •! HypoThermosol® hypothermic storage & shipping media •! CryoStor® freeze media •! BloodStor™ generic, cGMP cord blood stem cell freeze media•! Services: •! Custom biopreservation media formulation, fill, and finish into standard and custom packages •! Customers include several competitors and the US Army •! Contracted R & D to optimize biopreservation outcomes •! Biopreservation media formula development •! Verification of cell specific freezing/thawing rates and protocols 5! OTCBB: BLFS! 5!
  6. 6. REGENERATIVE MEDICINE OVERVIEW!•! Regenerative Medicine is the process of engineering living, functional cell and tissue-based therapies and administering these to patients to repair or replace tissue or organ function lost due to age, disease, damage, or congenital defects•! Target diseases include cancers, diabetes, heart disease, ALS•! Target disorders include spinal/movement, hearing loss, vision loss, neurological (i.e., stroke)•! Nearly all currently available and development stage regenerative medicine products and therapies utilize biopreservation processes in the acquisition of source material, isolation and manipulation of specific cells, and storage and shipment of a final product dose to a patient location•! Biopreservation economics greatly impact product commercialization potential •! (system costs of the shelf life & functional yield environment) 6! OTCBB: BLFS! 6!
  7. 7. REGENERATIVE MEDICINE OVERVIEW! ()*)+),-./)#0)1232+)#0-,4)5#6,7859#:!;<#•! Market activity1: •! 900 active clinical trials for new therapies !$# !"# •! 687 companies worldwide !&$# !&"#•! Market size and growth estimates2: !%$# !%"# •! Market could reach $32B by 2018 !$# !"#•! Demand for biopreservation media3 used in !" &""># #" &"%=# process development, manufacturing, and commercial product storage, transport, and freezing will mirror the end product market @)A-+1#B7,#;27C,)D),/-.7+#0)12-#:!0<# growth rate •! Increasing quality and regulatory pressure to move !$""# away from internally formulated ‘home-brew’ media !?""# !""# and reagents !&""# •! Limited risk of disruptive, competitive, !%""# technologies; BioLife products are the next !"# &""># !" #" &"%=# disruptive technology that will capture share •! Previous 9 nine quarters: BioLife product shipments to 200+ accounts 1Proteus Venture Partners, 2MedMarket Diligence, LLC, 3Management estimates 7! OTCBB: BLFS! 7!
  8. 8. BIOPRESERVATION WORKFLOW! Enabling end to end biopreservation workflow optimization with newly integrated solutions; from source material to final product! <%(/.1&1.=+()*>(.?@(A*%)*B161)1.+=1*C1,%"%)1* 4)5/.("1&&*&-(.%)6* 9-(.+61*+),* !"#$%&%()*+),* (.*0.117%)6* 8$3-$.%)6*$/*-(*+* &:%/21)-*(0*;)+3*-.+)&/(.-+()*(0*&($."1* %&(3+-1,*(.* "3%)%"+3*,(&1* "133*/.(,$"-*,(&1* 2+-1.%+3* 2+)%/$3+-1,*"133&* -(*/+1)-* !""#$%&()*+,%#,-.$)/(".%*,(,0.&1./%&$(23,-$#/*++(23,4,5*6(7*$8,9&""68,:;.(+, Pre-filled biologic packaging products BioLife biopreservation media products from partners 8! OTCBB: BLFS! 8!
  9. 9. ISSUES WITH HOME-BREW BIOPRESERVATION MEDIA!•! Internally formulated biopreservation media products: •! Based on historical, non-optimized formulas •! Made daily/weekly and subject to lot-lot variability •! Often not made with USP grade components •! Often not made under current GMP •! Subjected to less stringent (non-lot specific) release testing for sterility, endotoxin, mycoplasma, etc. !"#$%&()*"+,--( !"#$%&$()*#+,-.) ""*,*(?@/5306( J3-0"*3+/5(I"*8@5/:"#( A#,B+3,#0(C"*DE"C( 73830,9()*,-,*2/:"#( F53#3+/5(,G,+0-( ./0+1(2/*3/435306( 7"C(-+/5/435306( !"0("):83H,9(I"*(4,-0(( )*/+:+,-( 73830,9(*,5,/-,(0,-:#;( <=>(;*/9,(+"8)"#,#0-( 9! OTCBB: BLFS! 9!
  10. 10. BIOLIFE RESEARCH DISCOVERIES!•! Preservation induced cell damage and death •! Metabolic activity decreases •! Ionic and osmotic balances are disrupted •! Free radicals accumulate •! Molecular stress responses are initiated•! Two injury mechanisms •! Necrosis – mechanical destruction of cell membrane •! Apoptosis – enzyme based, programmed cell death•! Two injury exposure periods •! During hypothermic storage and freezing •! During re-warming/thawing•! Delayed preservation induced cell injury – not apparent or appreciated •! Immediate post-thaw assays do not accurately indicate cell viability •! Perceived Viability ! True Viability•! Traditional preservation media formulations do not adequately protect cells from preservation induced stress•! Innovation opportunities to re-formulate preservation media to mitigate underlying causes of preservation related cell damage and death 10! OTCBB: BLFS!
  11. 11. BIOPRESERVATION ECONOMICS! !"#$%&.&,-)*&/(01+"2&•! Overall system performance is constrained by limited stability, yield loss, and processing costs !"#$%&()*)!•! Incremental and cumulative optimization of biopreservation !"#$%& processes can maximize the yield- cost relationship•! System optimization should strive to move up and to the left ()*#+&,-)*& !"#$%&()*+,-./"01.(2+3145#*+6125#+ 11! OTCBB: BLFS!
  12. 12. IMPACT OF LIMITED SHELF LIFE! )&%+%2#&(3%,"-%$41*+(5#+6.(12! OTCBB: BLFS! !"#$%&%"()*+,"-#%+./(0*".+1#&(
  13. 13. FORMULATION INNOVATION!•! Intracellular-like media: •! Ionic concentrations are tuned to balance the intracellular state at low temperature to reduce gradient formation and water flux•! pH buffering capacity: •! Organic and inorganic buffers include bicarbonate and HEPES •! Effective from 15°C to 2°C•! Free radical scavengers•! Osmotic stabilizing components •! To reduce and control shrinking and swelling of cells during preservation process temperature changes•! Serum-free energy substrates•! Pre-formulated with cryoprotective agents (2%, 5%, or 10% DMSO) •! No user mixing required•! Fully-defined components •! USP/multi-compendial or highest available grade 13! OTCBB: BLFS!
  14. 14. PROPRIETARY PRODUCTS! HypoThermosol® ! CryoStor®! Storage & Shipping Media ! Freeze Media!!! Formulary improvements to organ !! Based on HypoThermosol formula preservation media !! For cryopreservation of biologics at!! Serum-free and protein-free - 80°C to -196°C!! For 2 - 8°C short term storage and !! Pre-formulated with 2%, 5%, or 10% shipment of biologics DMSO!! Enables functional restart upon return !! Serum-free and protein-free to normothermic temperature !! Outperforms other commercial!! Extends stability of specimens, products and home-brew freeze pancreata, liver sections, tumor cocktails biopsies, tissue samples, bone marrow 14! OTCBB: BLFS!
  15. 15. PRODUCT VALUE PROPOSITION! @A%+31B3!CD%%(@1;EFA%AFGD+1;B(:A91D(EF;9H!+%( 123)$4/-(5$".4067$2( !"#$%&$"!( )#*$+,-".$/$0( 89:%;<%-"4.<:-=>6?("#$%&#()*+,$-!.$#)(%&#$$!"#,$/0%&#$$!1."23/45$6,!7)+*/,8!9:;"!;+0)<+9,)#$-!&=>!;+6,$#!&/*$!.,$#/*/,8?!@0-,A/0?!B!C$**%D+6$-!E$*$+6$!F$6G04! 15! OTCBB: BLFS!
  16. 16. REGULATORY STATUS!•! Preservation solutions are not devices or drugs; considered excipient orancillary material in clinical applications •! Ancillary material: used in manufacturing cell, gene, or tissue-engineered products, but not intended to be in final product (washed or removed) •! Excipient: component intended to be part of the final product at clinical application•! We help customers comply with Good Tissue Practices (GTP – 1271.210) •! Highest quality reagents must be utilized in the process of developing Human Cellular, Tissue, and Tissue-Based Products (HCT/Ps) •! “all reagents must be verified to meet specifications designed to prevent circumstances that increase the risk of the introduction, transmission, or spread of the introduction, transmission, or spread of communicable diseases”•! Type II FDA Master Files regularly updated for both HypoThermosol® and CryoStor® product platforms 16! OTCBB: BLFS! 16!
  17. 17. QUALITY SYSTEMS!•! Certified to: •! ISO 13485:2003 medical device quality systems management•! Adherence to: •! 21 CFR part 820 Quality Systems Regulation of Good Manufacturing Practices for medical devices (GMP) •! 21 CFR parts 210 and 211 covering GMP for Aseptic Production •! ISO 13408 manufacturing processes for aseptically-processed health care products •! Volume 4, EU GMP Guidelines, Annex 1: Manufacture of Sterile Medicinal Products •! ISO 14644 Clean Rooms and Associated Controlled Environments•! Product Release Criteria: •! Sterility Tested USP <71> •! Endotoxin Tested USP <85> •! Cell based preservation efficacy assay •! pH •! Appearance 17! OTCBB: BLFS! 17!
  18. 18. PRODUCT ADOPTION!•! Our biopreservation media products are incorporatedinto numerous development stage and commercialbiologic products •! cell therapies •! umbilical cord blood units •! cells and cell lines used in drug screening•! Critical supplier status with leading regenerativemedicine and life science tools companies 18! OTCBB: BLFS! 18!
  19. 19. CRYOPRESERVATION MEDIA PRODUCTS – 5% DMSO! Recovery of Human Mesenchymal Stem Cells Following Cryopreservation With Serum-Free CryoStor® Comparison to Serum Containing Home-Brew Media 125 100% Recovery of Viable Cells 75 50 25 0 37C CryoStor 95% Serum FBM FGM Normosol Normosol * Plasma-lyte Plasma-lyte CS5 * Medium Medium * * 5% DMSO 19! alamarBlue® stain for metabolic activity * Contains serum 19!
  20. 20. CRYOPRESERVATION MEDIA PRODUCTS – 5% DMSO! Recovery of Normal Human Dermal Fibroblasts Following Cryopreservation With Serum-Free CryoStor® Commercial Solution Comparison: Serum and Serum-Free 125 100% Recovery of Viable Cells 75 50 25 0 Control CryoStor ProFreeze Aedesta Cellvation Cell Synth-a- Recovery * Sigma FGM + 5% (37°C) CS5 Guardian Freeze Freeze * DMSO * 20! alamarBlue® stain for metabolic activity * Contains serum 20!
  21. 21. CRYOPRESERVATION MEDIA PRODUCTS – 5% DMSO! Representative Fluorescent Micrographs Illustrating Post Cryopreservation Morphology of NHDF Stored in Serum-Free CryoStor®CryoStor® CS5 ProFreeze® Aedesta® Cellvation®Cell Guardian® Synth-a-Freeze® Recovery Sigma !"##$!! %#&!! ()#!! "#$%!&()*+,-,()%! 81()#/)%2910!"#$:0$)%! =>#-,09!?(01%! 21! ./0--)121%!3456&7! 81()690#+,9;!<,2! @),#/*(!
  22. 22. CRYOPRESERVATION MEDIA PRODUCTS – NO NEED FOR SERUM! Recovery of Human Mesenchymal Stem Cells Following Cryopreservation With Serum-Free CryoStor® Comparison to Serum Containing Home-Brew Media 125 100 % Recovery of Viable Cells 75 50 25 0 Control CS2 CS5 CS10 FGM + 2% FGM + 5% FGM + 10% DMSO * DMSO * DMSO * CryoStor Culture Media 22! alamarBlue® stain for metabolic activity * Contains serum 22!
  23. 23. CRYOPRESERVATION MEDIA PRODUCTS – NO NEED FOR SERUM! Representative Fluorescent Micrographs Illustrating Post Cryopreservation Morphology of hMSC Stored in Serum-Free CryoStor® CryoStor® CS2 CryoStor® CS5 CryoStor® CS10 FGM + 2% DMSO FGM + 5% DMSO FGM + 10% DMSO !"##$!! %#&!! ()#!! "#$%!&()*+,-,()%! 81()#/)%2910!"#$:0$)%! =>#-,09!?(01%! 23! ./0--)121%!3456&7! 81()690#+,9;!<,2! @),#/*(! 23!
  24. 24. CRYOPRESERVATION MEDIA PRODUCTS – NO NEED FOR SERUM! Recovery of Human Mesenchymal Stem Cells Following Cryopreservation With Serum-Free CryoStor® Comparison to Serum Containing Home-Brew Media 125 100 % Recovery of Viable Cells 75 50 25 0 37C CryoStor 90% Serum * MSCGM MSCGM Normosol Normosol * Plasma-lyte Plasma-lyte CS10 (Medium) (Medium) * * (no serum) 10% DMSO 24! alamarBlue® stain for metabolic activity * Contains serum 24!
  25. 25. POST PRESERVATION DIFFERENTIATION CAPACITY! Representative Fluorescent Micrographs Illustrating Post Cryopreservation Differentiation Capacity and Morphology of hMSC Stored in CryoStor® hMSC (Undifferentiated) hMSC Osteoblasts !"##$!! %#&!! ()#!! "#$%!&()*+,-,()%! 81()#/)%2910!"#$:0$)%! =>#-,09!?(01%!25! ./0--)121%!3456&7! 81()690#+,9;!<,2! @),#/*(! 25!
  26. 26. CRYOPRESERVATION MEDIA PRODUCTS – NO NEED FOR SERUM! Recovery of hMSC Differentiated Osteoblast Cells Following Cryopreservation With Serum-Free CryoStor® Comparison With Serum Containing Home-Brew Media 125 100 % Recovery of Viable Cells 75 50 25 0 Control CS2 CS5 CS10 MSCGM + 2% MSCGM + 5% MSCGM + 10% DMSO * DMSO * DMSO * 26! alamarBlue® stain for metabolic activity * Contains serum 26!
  27. 27. CRYOPRESERVATION MEDIA PRODUCTS – NO NEED FOR SERUM! Recovery of Human Dental Pulp Stem Cells Following Cryopreservation with Serum-Free CryoStor Comparison to Serum Containing Home-Brew Media 125 100 % Recovery of Viable Cells 75 50 25 0 37C CryoStor 90% Serum * RPMI RPMI Normosol Normosol * Plasma-lyte Plasma-lyte CS10 (Medium) (Medium) * * (no serum) 10% DMSO 27! alamarBlue® stain for metabolic activity * Contains serum 27!
  28. 28. TECHNOLOGY SUMMARY!•! Scientific validation across a broad set of biologics, offering significantperformance and quality advantages•! Proven ability to extend stability and improve post-preservation viability,recovery, and function of specimens, source material, and isolated cells•! Viewed as a critical manufacturing reagent supplier by numerous celltherapy companies and cell suppliers•! Demand for our proprietary products will closely follow pace ofincreased regulatory and quality oversight of reagents used in clinicalproduct development, processing, storage, transportation, and infusion 28! OTCBB: BLFS! 28!

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