1. Supercritical Carbon Dioxide- Assisted Nebulization to Produce Fine Particles of Stable Protein Formulations Robert E. Sievers* Center for Pharmaceutical Biotechnology Dept. of Chemistry and Biochemistry University of Colorado Boulder, Colorado 80309-0215 *Also affiliated with AKTIV-DRY

2. The Principle of a New CAN-BD Process: (Carbon Dioxide Assisted Nebulization with a Bubble Dryer ® ) CAN-BD mixes an aqueous solution containing the protein or drug intimately with CO 2 at 100 bar to form an emulsion. The emulsion is rapidly expanded to atmospheric pressure through flow restrictor to generate aerosols of microbubbles and microdroplets. The aerosol plume is dried at 1 to 50 ｡ C as it mixes with nitrogen or air in the drying chamber. Dry fine powders are collected.

3. Schematic of Bubble Dryer ®

4. CAN-BD Mixing Tee Drying Temperature: 1 ｡ C to 60 ｡ C

5. Aerosol spray of microbubbles and droplets generated by the CAN-BD process

6. SEM of particles from CAN-BD (with a vacuum pump) of an aqueous solution containing 10% sodium chloride, dried in only 3 seconds TEM of a particle from the same batch, confirming that the salt particles are hollow spheres of cubes ( “ Sodium- Lite ” )

7. ROOM-TEMPERATURE-STABLE ANTIBODIES, ANTIBIOTICS, AND VACCINES Strategy: Dry powders near room temperature for alveolar or nasal administration (or rapid redissolution) Studies: “Stabilization and Dehydration of Monoclonal Antibodies”- Avoid aggregation and create rapidly redissolved microparticles “Preparation of Stable Dry Powder Formulations of Live Attenuated Virus Vaccines for Nasal Delivery”- Use Bubble Dryer ｨ to process dry powders of vaccine formulations that are easier to ship, store and maintain activity. “Inhalable Antibiotics” Micronize dry powder antibiotics for delivery to alveoli

8. Primatized Anti-CD4 Antibody Size distribution (by Aerosizer) and SEM of primatized monoclonal antibody micronized by CAN-BD from a buffered solution containing saccharide and surfactant. Dried at 50 ｡ C. Mean diameter = 1.4 ｵ m; with 95% under 3.5 ｵ m 3.5% H2O (within a day of micronization) 2.0% H2O (after 6 days storage in vacuum desiccator ) Stephen P. Cape, Ph.D.

9. Primatized Anti-CD4 Antibody SEM of primatized monoclonal antibody micronized by CAN-BD. Micrograph on right is zoomed out view of the one on left. Stephen P. Cape, Ph.D.

10. Size exclusion chromatography

11. Stored Primatized Anti-CD4 Antibody ･ Stored one month at room temperature in a vacuum desiccator without apparent change. ･ TEM confirms particle morphology and indicates lower density in centers. Stephen P. Cape, Ph.D. SEM TEM

12. SEM of particles from an aqueous solution containing 0.01% tobramycin sulfate

13. SEM of particles from an aqueous solution containing 0.22% albuterol sulfate. SEM of needles generated by stirring albuterol sulfate particles in ethanol at room temperature for two hours. SEM of albuterol sulfate particles after 3 years storage over desiccant.

14. The Effects of Aqueous Solution Concentration on Particle Size

15. Some factors that determine particle size ･ Concentration Decrease in concentration decreases particle size ･ Diameter of flow restrictor (40 to 175 microns, no significant change in size) ･ Drying temperature ･ Viscosity and surface tension of soln. ･ Morphology: Solid vs. hollow particles

16. SEM of particles from 50:50 ovalbumin:trehalose (10% aq. soln.) TEM

17. SEM of particles from CAN-BD of an aqueous solution containing 66% trypsinogen and 33% trehalose (1.5% total solids). Joseph A. Villa

18. The Role of Surfactants in Protein Stabilization ･ Surfactants are attracted to the air-liquid interface ･ Likely to compete with the protein for droplet surface sites ･ The surfactant layer has the effect of reducing internal motion and surface turbulence resulting in a smoother particle surface upon dehydration. Maa, Y.-F., Costantino, H.R., Nguyen, P.-A., and Hsu, C.C. “The Effect of Operating Conditions and Formulation Variables on the Morphology of Spray-Dried Protein Particles” Pharm. Dev. Tech 2 (1997) 213-223. Porter, M.R. In “Handbook of Surfactants” 2nd ed. Blackie Academic and Professional, London, UK 1994. Chang, B.S., Kendrick, B.S., and J.F. Carpenter “Surface-Induced Denaturation of Proteins during Freezing and Its Inhibition by Surfactants” J. Pharm. Sci. 85 (1996) 1325-1330.

19. % Apparent Activity of Trypsinogen Upon Rehydration after Bubble Dryer ®

20. Key Advantages of Bubble Dryer ® ･ Microbubbles are formed ･ Reduces thermal degradation of conventional spray drying ･ Bubbles dry faster than ice cakes in freeze drying (seconds vs. hours) ･ Redissolution of dried powders is rapid (< 30 sec.) ･ Simultaneous micronization while drying

21. The best temperature window for rapid drying WITHOUT thermal degradation or exceeding the glass transition temperature is: 1 ｡ C to 50 ｡ C

22. Aktiv-Dry ™ uses a bubble dryer ® to avoid the drawbacks of traditional drying methods ･ Much faster than freeze-drying (seconds vs. hours) ･ Dries at lower temperature than conventional spray-drying (0 to 50 °C vs. much higher temperatures) ･ Enzymes, pharmaceuticals, and vaccines retain activities

23. SEM of crystalline palmitic acid particles generated by CAN-BD from an ethanolic solution containing 4% palmitic acid

24. TEM of silica particles from a suspension in water dried in the Bubble Dryer ®

25. The CAN-BD process is based on the methods invented and developed by Sievers, Carpenter, and coworkers, licensed to AKTIV-DRY ™ ･ Sievers, R.E. and Karst, U. US Patent 5639441 (1997) ･ Sievers, R.E. and Karst, U. US Patent 6095134 (2000) ･ Sievers, R.E., Sellers, S.P. and Carpenter, J.F., WO 00/75281-A2 (2001); national phase entered in US, UK, Japan, Australia, China, Italy, Spain, Germany, France, Switzerland, etc. ･ Sievers, R.E. European patent 0677332 B2, Feb.27, 2002; registered in UK, Germany, France, Switzerland. ･ Other patent applications have been filed that are divisionals of the CAN-BD patent filed April 8, 1994, and the European application.

26. AKTIV-DRY ™ Participants, Advisors and Collaborators John Carpenter, Ph.D., CU School of Pharmacy Bob Sievers, Ph.D., CU Chemistry and Biochemistry Dept. Eric Sievers, M.D., U Washington Medical School, and Fred Hutchinson Cancer Research Center Michael Routh, Ph.D., CEO, Ionics Inc. Instrument Business Group Misha Plam, Ph, D. former CEO of Sievers Instruments, Inc. (which was acquired by Ionics, Inc.) Brian Quinn, M.B.A., Ph.D., investor and start-up executive Ted Randolph, Ph.D., CU Chemical Engineering Dept. Paul Brauer, M.S., CEO of Temco Instruments, Inc. Others to be named

27. New Home of ACTIV-DRY ™

28. Bubble Dryer® Model BD-500 Manufactured by Temco Instruments

29. Conclusions A new nebulization method (CAN-BD) has been presented that can generate fine pharmaceutical particles (1-3 µm) suitable for use in a dry powder inhaler (DPI) or metered dose inhaler (MDI). Drying requires only seconds and no organic solvents are needed. The method is based on mixing CO2 and an aqueous or organic solution at room temperature and 100 bar, and then expansion of the microemulsion into a drying chamber at 0 to 70°C and below or near atmospheric pressure. Retention of enzymatic activity results when certain stabilizing sugars and surfactants are present.

30. Conclusions, cont. Since the entire nebulization and drying method is carried out at lower temperatures than in other conventional spray drying processes, it is applicable to many temperature-sensitive pharmaceuticals, vaccines, Mab and proteins. Particle formation is in thin walled drying chambers near atmospheric pressure; no high pressure autoclaves are required. The usually preferred process is continuous rather than batch; the Colorado patents claim both methods. For room temperature stable powders, dry at temperatures near room temperature.

31. Acknowledgements Financial and in-kind support of research has been provided by: –AKTIV-DRY –Bayer –Boehringer-Ingelheim –MedImmune (Aviron) –Ionics (Sievers Instrument Co.) –Temco Instruments –GlaxoSmithKline –Genentech –Aventis –Spire –Colorado Tobacco Research Fund –CU

32. Acknowledgements, cont. The data reviewed are based on the work of the students, post- docs, and associates of Bob Sievers and John Carpenter: Uwe Karst Dean Liang Tom Walsh Paul Kluetz Scott Sellers Steve Cape Ted Randolph Eric Sievers, M.D. Joseph Villa Helena Meresman Guenter Engling Janelle Kawamoto Paul Brauer Hung-yi Chang Ed Huang Graham Clark Conrad Stoldt Miranda Evans Scott Vermeer Brian Quinn and others