Biopreservation Considerations for GMP Bioprocessing, Clinical Development, Supply Logistics, and Patient Delivery Aby J. Mathew, PhD Senior Vice President & Chief Technology Officer
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 Conditions for the collection, processing and pre-freeze storage of cells will directly affect post-thaw quality - Acker 2012 TIME 2 BIOLIFE SOLUTIONS, INC. 2014
Effect of Storage on Marrow and Blood Kao - 2010 3 BIOLIFE SOLUTIONS, INC. 2014
Effect of Upstream Storage on Downstream Cryopreservation Transfusion epub ahead of print, 2014 4 BIOLIFE SOLUTIONS, INC. 2014
Impact of Limited Shelf Life/Stability Which sphere is your product limited to? Distribution Range Stability Constrained Potential 5 BIOLIFE SOLUTIONS, INC. 2014
Best Practices Perspective Moving Biopreservation Upstream Conventional Practice Manufactured Cell/Tissue Product Collection Transport Processing Storage Transport Application Source Material Preservation only considered for finished product Address yield issues AFTER negative system impact Recommended Best Practice Manufactured Cell/Tissue Product Biopreservation Collection Transport Processing Storage Transport Application Source Material Relevant Biopreservation Continuum Biopreservation considered from source material to delivered product Don t wait until late clinical or commercial stages to optimize! 6 BIOLIFE SOLUTIONS, INC. 2014
Cellular Response to Biopreservation Cellular responses to hypothermia and cryopreservation what happens? Metabolic activity decreases Ionic and osmotic balance is disrupted reduced membrane pump activity Free radicals accumulate Extracellular ice formation Successfully cryopreserved cells are glassy solids encased in ice Molecular stress responses are initiated Initiation of apoptotic cell death signaling pathways Secondary necrotic injury Intracellular ice formation Mechanical breakdown Membrane rupture/primary necrotic death Multiple points for preservation injury (hypothermia, thawing, ice damage) Mazur s Two-Factor Hypothesis of Freezing Injury ice damage and solute toxicity Mathew, Baust et al Hypothermic continuum as point of non-frozen cell/molecular stress Cellular priorities are survival, recovery, then functional performance 7 BIOLIFE SOLUTIONS, INC. 2014
Stability Stresses Occur at Multiple Levels Genomic Mitochondrial Proteomic Heat Shock Proteins Thermal Cycling (Transient Warming Events) Unfolded Proteins Enzymatic Caspases, Calpains, Liposomal Ionic Dysregulation Etc., etc.. 8 BIOLIFE SOLUTIONS, INC. 2014
Delayed Onset Cell Death 0 Hour 8 Hour 24 Hour Green = Apoptotic Cells Red = Necrotic Cells Blue = Live Cells Modified from Baust et al., 2005 Understand the difference between Perceived Viability and True Viability Total cell number 1.4 10 8 1.2 10 8 1 10 8 8 10 7 6 10 7 4 10 7 Population selection at work are you okay with that? -1 0 1 2 3 4 5 6 Day Modified from Stroncek,Hubel,Burger 1999 9 BIOLIFE SOLUTIONS, INC. 2014
Cryopreservation 100 VIABLE RECOVERY OF HUMAN MESENCHYMAL STEM CELLS FOLLOWING CRYOPRESERVATION IN VARIOUS FREEZE MEDIA - 1 DAY POST-THAW ASSESSED WITH ALAMARBLUE 90 80 70 PERCENT VIABILITY 60 50 40 30 20 10 0 37C CryoStor CS2 CryoStor CS5 CryoStor CS10 ProFreeze Cellvation Cell Guardian Synth-a- Freeze Recovery Sigma Freeze Media + 10% DMSO Intracellular-like, Serum-free, Protein-free Isotonic-based, Extracellular-like 10 BIOLIFE SOLUTIONS, INC. 2014
Non-Frozen Shipping/Storage Isotonic/Extracellular-like Solutions Intracellular-like Solutions Normosol-R Plasma-Lyte Viaspan HypoThermosol 3 Day Storage 1 Day Storage 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? 11 BIOLIFE SOLUTIONS, INC. 2014
GMP Considerations for Excipients/Ancillary Materials Ease of Use Performance Quality & Regulatory Pre-formulated with DMSO Sterile filtered/ USP sterility tested Ready-to-use packaging Unique ionic balance Multiple ph Buffers Antioxidants Mitigates Apoptosis & Necrosis Osmotic support components Defined energy substrates Serum-Free and Protein-Free USP components cgmp production Bioassay and Biosafety tested FDA Master Files No end user manipulation required Reduced contamination risk Easily integrates into current protocols Improved viability Improved quality Reduced post-preservation cell death Faster/better attachment Better yields of functioning cells Reduced contamination risk Fully defined media Lot-to-Lot consistency Quality assurance Reliable performance Support for clinical applications 12 BIOLIFE SOLUTIONS, INC. 2014
>100 Regenerative Medicine Clinical Applications Using Improved Intracellular-like Biopreservation Methods Stroke ALD & MS ALS UTD* AMD AMI CHF & DCM Liver Disorders GVHD Spinal Disorders HIV Lymphoma Melanoma AML Autoimmune Dis. Incontinence UTD* Cancer Wound healing Joint Repair CLI Cancer * UNABLE TO DISCLOSE 13 BIOLIFE SOLUTIONS, INC. 2014
Food for Thought Can we completely avoid DMSO in cell freezing so we can go straight from stored cells to infusion in patients? Yes, but No/Maybe: DMSO is still most effective clinical cryoprotectant. Alternatives may not be functionally effective and/or clinically appropriate (equivalent or improved Quality/Regulatory footprint). Why avoid DMSO, specifically? Myths vs. Facts. Many of our clinical partners thaw and infuse/inject. 14 BIOLIFE SOLUTIONS, INC. 2014
DMSO-Free Cryopreservation Post-thaw viability CPA1 Preliminary data on fibroblasts (TIG-20), BM MSC and red blood cells MSC 95%* TIG-20 >50%* More Details at Poster #350 CPA2 Preliminary data on fibroblasts (TIG-20), BM MSC and red blood cells MSC 80%* RBC (vitrified) 88%*** IPS (vitrified) 62%** Red Blood Cells CPA2 Control *Akron Biotech patent pending *Akron Data **Matsumura et al., Cryobiology 2011 ***El Assal, Zylberberg et al., submitted
Cold Chain Management and Supply Logistics Temperature (Degrees Centigrade) 10 0-10 -20-30 -40-50 -60-70 -80-90 Precision Thermal Packaging (CryoQ) and EPS Shipper Temperature Comparison 8⁰ -4⁰ -18⁰ -35⁰ -52⁰ -69⁰ -63⁰ -70⁰ -81⁰ -77⁰ -77⁰ -75⁰ 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 Time (Hours) CryoQ EPS Shipper 16 BIOLIFE SOLUTIONS, INC. 2014
Cold Chain Management and Supply Logistics TRAKPAD TRAKWEB 17 BIOLIFE SOLUTIONS, INC. 2014
What Do Your Cells Look Like Throughout Their LifeCycle? Intracellular-like Solution (HypoThermosol FRS) Preservation of 3 Cell Types Isotonic Solution Keratinocytes after 1 day ex vivo storage Fibroblasts after 3 days ex vivo storage Stem cells after 1 day ex vivo storage 18 BIOLIFE SOLUTIONS, INC. 2014
Contact Information Aby J. Mathew, PhD Senior Vice President and Chief Technology Officer BioLife Solutions 425-402-1400 amathew@biolifesolutions.com 19 BIOLIFE SOLUTIONS, INC. 2014