Principles and Applications of Lyophilization to Biotechnology

1. Principles and Applications of Lyophilization to Biotechnology Michael J. Akers, Ph.D. Baxter BioPharma Solutions Bloomington, Indiana BIOMAN 2010 Conference Ivy Tech Community College Bloomington, Indiana July 13, 2010 Baxter Confidential

2. Organization of Presentation Baxter Confidential

3. Bloomington, Indiana - Capabilities Overview Technologies Services • Formulation development • Process development • Lyo cycle development • Analytical development • Regulatory support • Labeling and Packaging • Prefilled syringes • Lyophilization • Aseptic liquid vials • Cartridges • Diluent syringes Clinical to Commercial Baxter Confidential

4. Other Interesting Facts about Baxter BioPharma in Bloomington

5. Focus of Bloomington R&D Approximately 80% of projects in past 2 y • Formulation Development of Small Volume Injectables • Liquid • Lyophilized • Extensive experience with • Proteins • Monoclonal Antibodies • Small Molecules • Analytical Method Development • Method development and validation services available • Responsible for validation of transfer of all incoming QC methods for Bloomington facility • Expertise in cleaning validation limit calculations and method development/validation • Development stability studies • Lyophilization Process Optimization Research Programs Baxter Confidential

6. Bloomington R&D Known As Baxter Lyophilization Center of Excellence Baxter Confidential

7. Baxter BioPharma Solutions Pharmaceutical Research and Development Bloomington, IN Top: Tim Paul, Lisa Hardwick, Nathan Pease, Wendy Saffell-Clemmer, Larry Callahan Middle: Rhonda Haley,Wei Kuu, Melissa Beard, Mike Hildreth, Karen Abram, Kevin O’Bryan, Elizabeth Oslos, Michael Akers Bottom: Lindsay Stanford, Angie Kruszynski, Nathaniel Fair, Jackie Karty, Greg Sacha, Hoang Mitchell, Dan Staples, Kelly Roby, Steven Nail Baxter Confidential

8. Market segment by therapeutic area and molecule class Source: Baxter Analysis Baxter Confidential 8

9. Current/Future Facts About Biologics • Significant growth • 2005 sales: $33B • 2005 predicted 2010 sales: $82B • 1 out of 4 new drugs introduced in US/EU is a biopharm product • Monoclonal antibodies (MoAbs) • $30B of 2010 sales expected to be MoAbs • > 200 MoAbs in clinical development right now • About half of current and new biologic products require lyophilization • Many of these products will be self-administered • Obviously, there is a huge market potential for simple, cost-effective devices to be used in home health care to prepare and deliver lyophilized biologics (and other lyo products) • Especially if product is for chronic health care Baxter Confidential 9

10. Best Known (Best Selling) Lyophilized Biologics (Biopharmaceuticals) Baxter Confidential

11. Lyophilization • The terms “lyophilization” and “freeze-drying” used interchangeably • Started in the 1930s, importance grew in WWII • Lyophilization is the conversion of a liquid to a solid through the process of sublimation • A sterile solution is prepared, filled into primary containers, placed into a freeze-dryer, and frozen • The solvent in the solution is removed by directly converting it from frozen ice to water vapor. • What remains in the container are the solute components in the solid state containing very low residual moisture (typically 0.5-2.0%). Baxter Confidential

12. Schematic Overview of Processing Solution and Freeze-Dried Biopharmaceutical Dosage Forms Dispense raw materials (active and excipients) Prepare solution in appropriate mixing tank (add ingredients to Water For Injection) Thaw and pool active biopharmaceutical Wash and sterilize primary containers and closures Add active to solution, pH adjustment, final QS This is formulation bulk solution ISO 8, Grade D, Class 100,000 ISO 5, Grade A Class 100 Sterile filter formulated bulk solution Transfer to Freeze-dryers and lyophilize Aseptically fill formulated bulk solution into primary package and stopper (partial stopper if product is to be freeze-dried Fully insert stopper, remove from freeze dryer Apply aluminum overseal Storage at appropriate temperature (usually 2-8ºC) Label, sec package, storage, distribution 100% inspection Baxter Confidential

13. Preparing Product for Lyophilization • Prepare the sterile solution—compound, mix, filter • Fill into containers, typically vials • Partially insert a special designed rubber closure onto the vials • Aseptically load the vials into a freeze dry chamber • Freeze every single solution in every vial below a pre-determine critical temperature • Using appropriate application of temperature and pressure, sublime the ice from the product • Using further application of temperature and pressure, remove the necessary amount of bound water from the product • Automatically stopper the vials, neutralize the chamber • Aseptically remove the vials from the chamber and apply aluminum seals Baxter Confidential

14. Today’s State of the Art Technology UsesAutomated Systems To Transfer ProductFrom Production Line Into the Freeze-Dryer

15. Freeze-Dryer Subsystems (What Must Be Operating Smoothly and Maintained for Lyophilization Process To Succeed?) • Heat Transfer System • Refrigeration System • Vacuum System • Stopper-in-Place System • Clean-in-Place System • Control System • Loading/Unloading System Typical Batch of a Lyophilized Biopharmaceutical Product Worth Several Million $ Baxter Pharmaceutical Solutions Baxter Confidential

16. Courtesy of Samir U. Sane, Ph.D., Genentech, Inc. Baxter Confidential

17. Product temperature lower than shelf temperature during primary drying because energy is consumed as ice sublimes to vapor. Baxter Confidential

18. Flow of Vapor from Chamber to Condenser T Vacuum Pump T P Water Vapor Flow Door Product Chamber Condenser Chamber Connecting Duct Condenser Coils Mushroom Valve(open position) Shelves with Vials Baxter Confidential

19. Schematic of Heat and Mass Transfer in the Freeze Dryer Temperature difference between chamberand condenserand pressure differentialbetween solution in vials and vacuum pumpdrives ice out of vial and onto the condenser Conversion ofsolid (ice) to vapor in chamber called sublimation Vacuum Pump Mass Transfer Condenser ΔP Heat Transfer Dry Cake Pressure gradient between sublimation front and chamber Sublimation Front Frozen Solution Thermal Fluid Shelf Thermal fluid circulates within the shelves to control temperature in chamber Baxter Confidential

20. Desired Freeze Dried Product Characteristics Intact cake Sufficient strength Uniform color Sufficiently dry Sufficiently porous Sterile Free of pyrogens Free of particulates Chemically stable—both in dry state and after reconstitution Baxter Confidential

21. Advantages of Lyophilization • Removal of water at low temperature • Ease of reconstitution • Compatible with aseptic operations • More precise fill weight control • Done properly, the freeze-dried solid has relatively high specific surface area, which promotes rapid, complete reconstitution Baxter Confidential

22. Disadvantages of Lyophilization • The drug may not be stable as a freeze-dried solid • Many biological molecules are damaged by the stresses associated with freezing, freeze-drying, or both • Not all solutes can be freeze-dried to form a pharmaceutically acceptable cake • Cost may be an issue, depending on the product Baxter Confidential

23. Common Lyophilized Products • Pharmaceuticals – large and small molecules • Bacteria • Viruses • Vaccines • Plasma • Small Zoological Specimens (Taxidermy) • Fruit • Coffee • Flowers • Water-Damaged Documents Baxter Confidential

24. Main Challenges of Lyophilization • Development of formulation that meets all the necessary critical product attribute requirements (quality appearance, potency, stability, recon time, etc) • Accurate determination of the “critical temperature” of final formulation necessary to determine conditions of lyophilization cycle (Tg’, Te, Tc) • Establishment of temperature, pressure, and time cycle settings that can achieve best product quality in shortest possible time • Transfer and scale-up of lab-developed process to production scale process • Keep all the equipment running smoothly • Special challenges in product development of lyophilized biologic formulations and cycles

25. Challenges in Lyophilization of Protein Pharmaceuticals Baxter Pharmaceutical Solutions Baxter Confidential

26. Challenges in Lyophilization of Protein Pharmaceuticals • Measurable differences in recovery of activity can be associated with differences in the thermal history of freezing • Some proteins can be subject to overdrying; that is dryer is not necessarily better • Stability of the freeze dried solid is often a concern. Most freeze dried proteins require refrigerated storage. • Solid state stability can be affected by small differences in residual moisture content. Baxter Confidential

27. Why Does Lyophilization Take So Long? • Sometimes, the properties of the formulation require that the temperature be very low, often below –30oC. This decreases the driving force • The heat required is very high, and heat transfer in freeze drying is very inefficient • Resistance to mass transfer – transport of water vapor from the sublimation front through the porous bed of partially dried solids – can be significant. • Huge batch sizes, takes time to complete the 3 stages of lyophilization

28. Final Comments • Roughly 50% of all commercial biologic (therapeutic protein products) are lyophilized. • Lyophilization technology and the expertise to use it are vital to the ability of the biopharmaceutical industry to prepare and market life-saving injectable medicines • Lyophilization is the most challenging (and most expensive) of all sterile product manufacturing unit operations Baxter Confidential

29. Acknowledgements • Thanks to all my Baxter R&D colleagues for the great collegiality we have and what makes my job so enjoyable. • Thanks to the following individuals for providing some of the slides I showed • Dr. Steven Nail • Ms. Lisa Hardwick • Ms. Wendy Saffell-Clemmer Baxter Confidential