Supercritical Carbon Dioxide-?Assisted Nebulization to?Produce Fine Particles of?Stable Protein Formulations

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

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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 CO2 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.

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Schematic of Bubble Dryer
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CAN-BD Mixing Tee?Drying Temperature 1?C to 60?C
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• Aerosol spray of microbubbles and droplets
  generated by the CAN-BD process

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

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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 ?m with 95 under 3.5 ?m?
• 3.5 H2O (within a day of micronization)?
• 2.0 H2O (after 6 days storage in vacuum
  desiccator)

Stephen P. Cape, Ph.D.
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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.
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Size exclusion chromatography
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Stored Primatized Anti-CD4 Antibody
TEM
SEM

• ?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.
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• SEM of particles from an aqueous solution
  ?containing 0.01 tobramycin sulfate

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SEM of needles generated by stirring albuterol
sulfate particles in ethanol at room temperature
for two hours.

• SEM of particles from an aqueous solution
  containing 0.22 albuterol sulfate.

SEM of albuterol sulfate particles after 3 years
storage over desiccant.
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The Effects of Aqueous Solution Concentration on
Particle Size
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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

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SEM of particles from 5050 ovalbumintrehalose
(10 aq. soln.)
TEM
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• SEM of particles from CAN-BD of an aqueous
  solution containing 66 trypsinogen and 33
  trehalose (1.5 total solids).

Joseph A. Villa
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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.

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Apparent Activity of Trypsinogen Upon
Rehydration after Bubble Dryer
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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 (lt 30
  sec.)?
• ?Simultaneous micronization while drying

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• The best temperature?
• window for rapid drying?
• WITHOUT thermal?
• degradation or exceeding?
• the glass transition?
• temperature is
• 1?C to 50?C

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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

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

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

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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

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New Home of ACTIV-DRY
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• Bubble Dryer
• Model BD-500
• Manufactured
• by Temco
• Instruments

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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 70C and below or near
  atmospheric pressure.?
• Retention of enzymatic activity results when
  certain stabilizing sugars and surfactants are
  present.

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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.

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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

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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