Nanoparticle and Microparticle Generation with Super- or Near-critical Carbon Dioxide

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Nanoparticle and Microparticle Generation with
Super- or Near-critical Carbon Dioxide

• E.T.S.Huang, H. Y. Chang, D. Alargov, S.P.Cape,
  ?L. Rinner, J. A. Villa, B. P. Quinn and R. E.
  SieversCenter for Pharmaceutical Biotechnology,
  ?Dept. of Chemistry and Biochemistry, and CIRES,
  ?214 UCB, University of Colorado, Boulder, CO?and
  AKTIV-DRY,?655 Northstar Ct., Boulder, CO 80301

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ROOM-TEMPERATURE-STABLE ENZYMES, ANTIBODIES, AND
PHARMACEUTICALS

• Strategy Stabilize and dry powders near room
  temperature by SF processing
• Nanoparticle and Microparticle Synthesis and
  Coating
• Inhalable Antibiotics
• Inhalable Antielastase Protein Enzyme for
  Emphysema and Cystic Fibrosis
• Micronize dry powder alpha-1-antitrypsin for
  delivery into alveoli?
• Stabilization and Dehydration of Monoclonal
  Antibodies?
• Stabilize with sugars dry and store without
  freezing or refrigeration?
• Avoid aggregation and create rapidly redissolved
  microparticles

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The Principle of a New CAN-BD Process (Carbon
Dioxide Assisted Nebulization with a Bubble
Dryer)

• In CAN-BD a solution or suspension in
  acetone,alcohol, or water is mixed 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 60 ?C as it
  mixes with nitrogen or air in the drying
  chamber.?
• Dry fine powders are collected.

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• Particles of antibiotics generated from aqueous
  solutions

0.5 solution in H2O Avg. particle size
0.97? 95 lt 1.60 µm
0.5 solution in H2O Avg. particle size
1.05? 95 lt 1.82 µm
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SOLVENTS FOR CAN-BD

• Water
• Carbon Dioxide
• Methanol
• Ethanol
• Acetone
• Acetonitrile
• Ethyl acetate
• Methylene chloride (only when required)
• Mixtures of solvents

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Size distribution of particles of
Dipalmitoylphosphatidylcholine (DPPC) from a
solution in ethanol, dried in the Bubble Dryer
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• SEM of particles generated from a 2 aqueous
  solution of lactose 2 solution of palmitic
  acid in ethanol with CO2 in a CROSS

SEM of particles generated from a 10 aqueous
solution of lactose with CO2 in a TEE
SELECTIVE LEACHING OF HETEROGENOUS PARTICLES
Particles suspended in EtOH, stirred for 0.5 hr,
and filtered Avg. aerodynamic diam.1.03µm
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• 2 Betamethasone in ETOH ?
• 2 Lactose in H2O CO2 in a CROSS

Particles suspended in ETOH, ?stirred for 0.5
hr, and filtered
Avg. Particle size 1.16 µm, 95 lt 1.96 µm
Particles suspended in H2O, stirred for 0.5 hr,
and filtered
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Two Fluid Particle?Formation (Tee)
SEM of particles generated from a?10 aqueous
solution of NaCl with?CO2
Three Fluid Particle Formation (Cross)
SEM/TEM of particles generated from a 10 aqueous
solution of NaCl 0.5 solution of PLGA in
acetone with CO2
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Two Fluid Particle Formation (Tee)
Three Fluid Particle Formation (Cross)
SEM of particles generated from a 10 aqueous
solution of NaCl with CO2
SEM of particles generated from a 10 aqueous
solution of NaCl 3 suspension of silica
nanoparticles with CO2
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Aerodynamic Size Distribution of Betamethasone
17,21-dipropionate from Ethanol

• Base Conditions
• 1200 psi
• 3 wt
• CO2 to Ethanol mass flow ratio 14.5
• Results
• Mean Diam. 0.81 µm
• 95 lt 1.34 µm

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Effect of Pressure and Flow Ratio on Particle Size
1200 psig, 3 conc. -- 14.5
1200 psig, 0.2 conc. -- 15
700 psig, 3 conc. -- 0.5
300 psig, 3 conc. -- 0.007
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COATINGPARTICLES
SEM of particles generated by CAN-BD from a
solution containing 2 ovalbumin 2 trehalose
(uncoated)
SEM of particles coated with 0.5 PLGA
SEM of particles coated with 1 PLGA
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NaCl particles generated by CAN-BD
PLA-coated NaCl particles generated by CAN-BD
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• Size distribution (by Aerosizer) and SEM of
  alpha-1-antitrypsin micronized by CAN-BD from an
  aqueous solution (2.8 AAT, 4.6 trehalose in
  0.1M sodium phosphate, 0.1 Tween 20, pH 7.0).
  Dried at 40 ?C.
• Mean diameter 2.2 ?m with 95 under 5.3
  ?m?Moisture content 7.5 H2O (initial), 1.8
  H2O (after storage in vacuum desiccator)
• Activity retained after processing 98 ? 2
• Completely redissolves in a few minutes with
  gentle swirling

Stephen P. Cape, Ph.D.
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REPRESENTATIVE SOLUTES THAT HAVE BEEN MICRONIZED
BY A LAB-SCALE CAN-BD UNIT
Antibodies Enzymes Antibiotics Water-soluble
drugs Alcohol-soluble drugs Sugars Polymers

anti-CD4, IVIG, anti-human lambda light
chain alpha-1-antitrypsin, trypsinogen,
lysozyme, lactate dehydrogenase ciprofloxcin,
moxifloxacin, tobramycin sulfate,
amoxycillin, doxycycline albuterol sulfate,
cromolyn sodium naproxen, budesonide,
betamethsone, amphotericin B, cyclosporin,
DPPC lactose, sucrose, trehalose, mannitol PLA,
PLGA, PEG
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The CAN-BD process is based on the methods
invented and developed by Sievers, Carpenter, and
coworkers, licensed exclusively 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. US Patent has
  been allowed.?
• Sievers, R.E., European Patent 0677332 B1, 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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Conclusions

• A nebulization, and drying and coating method
  (CAN-BD) has been presented that can manufacture
  dry powders of proteins, enzymes, antibodies,and
  other drugs without detectable degradation.?
• Both solutions and suspensions can be processed.?
• Coating and drying requires only seconds at near-
  ambient conditions of temperature and pressure.?
• Water and many organic solvents yield
  nanoparticles and microparticles.?
• Fluid ratios and solute concentrations determine
  size.?
• Multiple constituents can be incorporated in
  three fluids in a low volume cross to make
  heterogenous and homogenous nanoparticles and
  microparticles