Biopreservation and Stability Considerations for Cellular Therapies – Clinical applications of HypoThermosol® and CryoStor® as ancillary or excipient reagents Aby J. Mathew, PhD Senior Vice President & Chief Technology Officer Safe Harbor Statement This presentation contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995 including, but not limited to, statements about BioLife Solutions, Inc. (the “Company”) and its future operating results, strategies, and product development plans. These forward-looking statements are based on current expectations and assumptions that are subject to risks and uncertainties. Actual results could differ materially from the results expressed or implied in these forwardlooking statements. Factors that may cause or contribute to such differences are more fully discussed, as are other factors, in Part I, Item1A. “Risk Factors” of the Company’s Form 10-K for the fiscal year ended December 31, 2011, which was filed with the SEC on March 29, 2012. In addition, any forward-looking statements represent our estimates only as of today and should not be relied upon as representing our estimates as of any subsequent date. While the Company may elect to update forward-looking statements at some point in the future, the Company specifically disclaims any obligation to do so except as may be legally necessary, even if the Company’s estimates should change. 2 BIOLIFE SOLUTIONS, INC.©2012 Biopreservation System Objectives 1. 2. Optimize commercial scale manufacturing and the broadest geographic distribution by maximizing: • Source material stability (transport time x recoverable yield of isolated cells of interest) • Manufacturing processing time (receipt of source material, cell isolation, manipulation, culturing, packaging) • Final dose stability (longest transport time that provides the highest cell viability, functional recovery, and engraftment) Minimize system costs: • 3. Minimize system risks: • 3 Direct labor and/or contracted facility time, components and supplies Process variability, component quality, supply continuity, and contamination exposure throughout the workflow process BIOLIFE SOLUTIONS, INC.©2012 Biopreservation Yield Cascading negative yield impact common to nearly all biopreservation applications • Outcomes heavily affected by lack of in-process efficiencies and optimization VIABILITY AND FUNCTION • TIME 4 BIOLIFE SOLUTIONS, INC.©2012 The Assessment Methods – Consideration to Delayed Onset Cell Death 0 Hour • • • 8 Hour Traditional survival assays do not detect the latent effects of biopreservation Need to examine samples over time to truly evaluate success (~24 hours postpreservation for recovery of viable cells; later timepoints for potency) Traditional biopreservation methods (extracellular-like media + serum + DMSO) cannot completely protect cells PBMC (Peripheral Blood Mononuclear Cells) Cryopreserved traditionally in Standard Culture Media + Serum (7%) + DMSO (10%) 5 24 Hour BIOLIFE SOLUTIONS, INC.©2012 Green = Apoptotic Cells Red = Necrotic Cells Blue = Live Cells Stability/Biopreservation Process Excipient/Ancillary Reagent Considerations 6 Ease of Use • Pre-formulated with DMSO • Sterile filtered/ USP sterility tested • Ready-to-use packaging • No end user manipulation required • Reduced contamination risk • Easily integrates into current protocols Performance • • • • • • Unique ionic balance Multiple pH Buffers Antioxidants Mitigates Apoptosis & Necrosis Osmotic support components Defined energy substrates • • • • • Improved viability Improved quality Reduced post-preservation cell death Faster/better attachment Better yields of functioning cells Quality & Regulatory • • • • • • Serum-Free and Protein-Free No animal-origin products USP components cGMP production Bioassay and Biosafety tested FDA Master Files • • • • • • Reduced contamination risk Fully defined media Lot-to-Lot consistency Quality assurance Reliable performance Support for clinical applications BIOLIFE SOLUTIONS, INC.©2012 Stability Improvement Methods • Intracellular-like, fully-defined synthetic biopreservation formulations: – Contain key electrolytes (intracellular-like vs. isotonic/extracellular-like) – Ionic concentrations balance the intracellular state at low temperature to restrict ion and water flux • pH Buffering Capacity: – Enhanced buffering capacity specific to low temperature • Free Radical Scavengers • Osmotic stabilizing components – To reduce and control shrinking and swelling of cells during preservation • Both Permeating and Non-Permeating Cryoprotective agents (DMSO, Glycerol, Large Sugars, etc.) – reduce dependence and toxicity impact of any one agent • Optimization of cooling rate and thaw conditions during freeze/thaw • Avoidance of damaging thermal cycling in samples 7 BIOLIFE SOLUTIONS, INC.©2012 EVIDENCE SUPPORTING INTRACELLULAR-LIKE BIOPRESERVATION MEDIA AND POTENTIAL IMPROVED STABILITY 8 BIOLIFE SOLUTIONS, INC.©2012 Cryopreservation 1 DAY RECOVERY OF NORMAL HUMAN DERMAL FIBROBLASTS FOLLOWING CRYOPRESERVATION - COMMERCIAL SOLUTION COMPARISON 125 PERCENT VIABILITY 100 75 50 25 0 9 Control (37°C) CryoStor ProFreeze Aedesta Cellvation Cell Synth-a- Recovery CS5 Guardian Freeze BIOLIFE SOLUTIONS, INC.©2012 Sigma Freeze Media + 5% DMSO Non-Frozen Shipping/Storage Isotonic/Extracellular-like Solutions Plasma-Lyte Viaspan HypoThermosol 3 Day Storage 1 Day Storage Normosol-R Intracellular-like Solutions Green = Actin Cytoskeleton Red = Mitochondria Activation Blue = Nuclear Stain What is the Condition of Your Cells When They Arrive for Processing or Reach the Patient? 10 BIOLIFE SOLUTIONS, INC.©2012 EVIDENCE SUPPORTING TRANSLATION INTO CLINICAL APPLICATIONS 11 BIOLIFE SOLUTIONS, INC.©2012 HypoThermosol Case Studies – ISCT 2011 Annual Meeting 12 BIOLIFE SOLUTIONS, INC.©2012 CryoStor Case Studies – ISCT 2011 Annual Meeting 13 BIOLIFE SOLUTIONS, INC.©2012 Clinical Applications - HypoThermosol 14 BIOLIFE SOLUTIONS, INC.©2012 Clinical Applications - CryoStor 15 BIOLIFE SOLUTIONS, INC.©2012 Clinical Applications Using Improved Intracellular-like Biopreservation Methods 16 Stroke Multiple Sclerosis Amyotrophic Lateral Sclerosis Age-related Macular Degeneration Myocardial infarction Dilated Cardiomyopathy Hematologic malignancies Chronic Heart Failure Joint Repair Degenerative Joint Disease Lymphoma Melanoma Acute Myeloid Leukemia Ulcerative Colitis Urinary stress incontinence Various Cancers Wound healing Wound Repair Critical Limb Ischemia Myeloma BIOLIFE SOLUTIONS, INC.©2012 Clinically-Relevant Publications Citing Improved Intracellular-like Biopreservation Methods Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use Gulen, Maas, Julius, Warkentin, Britton, Younos, Senesac, Pirruccello, Talmadge Cryopreservation of Umbilical Cord Blood with a Novel Freezing Solution that Mimics Intracellular Ionic Composition Nicoud, Clarke, Taber, Stolowski, Roberge, Song, Mathew, Reems Evaluation of Bone Marrow-Derived Mesenchymal Stem Cells After Cryopreservation and Hypothermic Storage in Clinically Safe Medium Ginis, Grinblat, Shirvan Hyaluronan-Supplemented Buffers Preserve Adhesion Mechanisms Facilitating Cryopreservation of Human Hepatic Stem/Progenitor Cells Turner, Mendel, Wauthier, Barbier, Reid A double-blind, randomized, controlled, multicenter study to assess the safety and cardiovascular effects of skeletal myoblast implantation by catheter delivery in patients with chronic heart failure after myocardial infarction Povsic, O’Connor, Henry, Taussig, Kereiakes, Fortuin, Niederman, Schatz, Spencer, Owens, Banks, Joseph, Roberts, Alexander, Sherman Interim analysis results from the RESTORE-CLI, a randomized, double-blind multicenter phase II trial comparing expanded autologous bone marrow-derived tissue repair cells and placebo in patients with critical limb ischemia Powell, Comerota, Berceli, Guzman, Henry, Tzeng, Velazquez, Marston, Bartel, Longcore, Stern, Watling Juvenile Chondrocytes May Facilitate Disc Repair Kim, Adkisson, Wendland, Seyedin, Berven, Lotz Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs) Sarugaser, Ennis, Stanford, Davies A Phase IIa Open-Label Dose-Escalation Pilot Study Using Allogeneic Human Dermal Fibroblasts for Nasolabial Folds Lowe, Lowe, St. Clair Roberts Further details into these articles, along with links to their respective journals are listed in the current issue of BioPreservation Today 17 From Then to Now 10 years ago – improved preservation was a scientific discussion point Today – HypoThermosol® and CryoStor® are proven enabling technology within Regenerative Medicine clinical applications 18 BIOLIFE SOLUTIONS, INC.©2012 Contact Information Aby J. Mathew, PhD Senior Vice President and Chief Technology Officer BioLife Solutions 425-402-1400 [email protected] 19 BIOLIFE SOLUTIONS, INC.©2012