Spray-Freeze-Drying - sono-tek.com20Schiffter.pdf• Atmospheric freeze-drying using a cold,...

17th October 2007 IBME – Oxford University

SpraySpray--FreezeFreeze--DryingDrying

Heiko A. SchiffterHeiko A. SchiffterInstitute of Biomedical EngineeringInstitute of Biomedical EngineeringUniversity of Oxford, Great BritainUniversity of Oxford, Great Britain

in the manufacture of pharmaceuticalsin the manufacture of pharmaceuticals


The history of spray-freeze-dryingThe history of sprayThe history of spray--freezefreeze--dryingdrying19481948 Studies on the surface area of protein particles sprayStudies on the surface area of protein particles spray--frozen in the vapour over frozen in the vapour over

liquid nitrogen with subsequent freezeliquid nitrogen with subsequent freeze--drying drying [1][1]

19641964 Production of subProduction of sub--micronicmicronic powder by spraypowder by spray--freezing freezing [2][2]

19761976 SpraySpray--freezing for freezing for cryofixationcryofixation in combination with freezein combination with freeze--drying for structural drying for structural preservation as alternative to chemical fixation preservation as alternative to chemical fixation [3][3]

19781978 SpraySpray--freezing for freezing for cryopreservationcryopreservation using liquid propane using liquid propane [4][4]

19801980 Preparation of fine ceramic and alumina powder by sprayPreparation of fine ceramic and alumina powder by spray--freeze drying freeze drying [5][5]

19871987 SpraySpray--freezefreeze--drying of trehalose for diagnostic assay tablets drying of trehalose for diagnostic assay tablets [6][6]

19911991 Atmospheric sprayAtmospheric spray--freezefreeze--drying. Spraydrying. Spray--freezing at freezing at --90°C and subsequent 90°C and subsequent dehydration of frozen particles in a stream of cold, desiccant adehydration of frozen particles in a stream of cold, desiccant air ir [7][7]

Since 1999Since 1999 Various investigations of process and formulation variables in sVarious investigations of process and formulation variables in spraypray--freezefreeze--drying: drying: protein stability, microprotein stability, micro--encapsulations, etc. encapsulations, etc. [8], [9][8], [9]

19991999 Preparation of protein loaded inhalation powders by sprayPreparation of protein loaded inhalation powders by spray--freezefreeze--drying drying [10][10]

20022002 SpraySpray--freezing directly into and under the surface of cryogenic liquidfreezing directly into and under the surface of cryogenic liquids s [11][11]

20042004 Influenza vaccine reformulation for needleInfluenza vaccine reformulation for needle--free intradermal administration using free intradermal administration using sprayspray--freezefreeze--drying drying [12][12]

Since 2004Since 2004 Development of new sprayDevelopment of new spray--freezefreeze--drying equipment drying equipment [13], [14][13], [14]

Spray-Freeze-DryingSpray-Freeze-Drying



The spray-freeze-drying processThe sprayThe spray--freezefreeze--drying processdrying processThe SFD process consists of1. Atomization of liquid solutions or suspension using ultrasound, one- or two fluid nozzles

or vibrating orifice droplet generators2. Freezing of the droplets in a cryogenic liquid or cryogenic vapour3. Ice sublimation at low temperature and pressure or alternatively atmospheric freeze-

drying using a cold desiccant gas stream

SpraySpray--freezing intofreezing intovapour over liquid (SFV/L)vapour over liquid (SFV/L)

SpraySpray--freezing into freezing into liquid (SFL)liquid (SFL)

SpraySpray--freezefreeze--drying (SFD)drying (SFD)SystemsSystems

SpraySpray--freezing into freezing into vapour (SFV)vapour (SFV)

The term spray-freeze-drying is a general description for different techniques and set-ups:



Spray-freezing into vapour (SFV)(with atmospheric freeze-drying)SpraySpray--freezing into vapour (SFV)freezing into vapour (SFV)(with atmospheric freeze(with atmospheric freeze--drying)drying)

Possible set-up used for spray-freezing into cryogenic vapour followed by freeze-drying under atmospheric conditions (ATMFD)

Process characteristics:• The porous flow chamber is cooled down

to the desired temperature (< -50°C) using refrigerated gases or liquid nitrogen

• Liquid feed is atomized into the cryogenic gas within the flow chamber

• Frozen droplets are collected on an exit filter at the bottom

• Atmospheric freeze-drying using a cold, desiccant gas stream

Schematic representation SFV process using liquid nitrogen gas as cryogen [13]



Spray-freezing into liquid (SFL)SpraySpray--freezing into liquid (SFL)freezing into liquid (SFL)SFL is characterized by a direct atomization of the liquid solution beneath the surface of the liquid cryogen. Possible set-up used for spray-freezing into a liquid cryogen [11] :

Process characteristics:• SFL feed solutions are atomized

beneath the surface of the liquid cryogen

• Atomization using PEEK tubing nozzles with inner diameters between 63.5 and 127µm

• Pressures up to 350bar using high pressure syringe pumps or HPLC-pumps

Schematic representation of a laboratory scale SFL process using liquid nitrogen as cryogen [11]



Spray-freezing into vapour over liquid (SFV/L)SpraySpray--freezing into vapour over liquid (SFV/L)freezing into vapour over liquid (SFV/L)SFV/L is characterized by atomization of the liquid solution or suspension into the cold gas phase of the evaporation cryogen. Possible set-ups used for spray-freezing into vapour over a liquid cryogen:

Sketch of a spray-freezing into vapour over liquid cryogen set-up using an ultrasound nozzle. Alternatively, a two-fluid nozzle with atomizing air flow can be used [15].

Picture of spray-freezing into vapour over liquid nitrogen equipment.


Benefits of spray-freeze-dryingBenefits of sprayBenefits of spray--freezefreeze--dryingdryingBenefits:• Production of a micronized pharmaceutical powder formulation for dry powder inhalation,

controlled release drug delivery systems, parenteral applications and intradermal delivery

• Cooling rates of 103K/s with liquid nitrogen achievable at a droplet size of 10µm [16]

• Rapid cooling rates support the formation of glassy water before the protein undergoes aggregation in the freeze-concentrated solution [17]

• Relaxation processes that lead to denaturation of proteins may be prevented by the formation of a glass [18]

• High process yield (>95%) compared to spray-drying (for SFV/L, SFL)• Better control of final particle size compared to spray-drying

Limitations:• Time-consuming, often discontinuous process• Inconvenient handling of some cryogenic liquids • Complicated scalability• Sterility of liquid nitrogen?




Liquid processing and jet breakupLiquid processing and jet breakupLiquid processing and jet breakupThe choice of the nozzle and the atomization conditions has a stThe choice of the nozzle and the atomization conditions has a strong influence on the rong influence on the mean and maximum droplet size of a spray and the size distributimean and maximum droplet size of a spray and the size distribution of the final powder.on of the final powder.

σρυ ⋅⋅

=d2

We

ηρυ ⋅⋅

=dRe

101 102 103 104 105 10610-3

10-2

10-1

100

101

102

AtomizationTransition

Ohn

esor

ge N

o. =

We1/

2 / R

e

Reynolds No.

Rayleigh

Atomizing flow rate 700 l/h

Liquid flow 3 ml/min

dv(50) = 21µm

OneOne--FluidFluid / / TwoTwo--FluidFluidNozzleNozzle

The liquid jet breakup is The liquid jet breakup is characterized by viscous to characterized by viscous to surface tension forces surface tension forces described using the Weber described using the Weber and Reynolds number:and Reynolds number:



Droplet size distributions (volume) of a water spray after atomization using a two-fluid nozzle (dorifice=0.7mm) with a liquid feed of 10ml/min and different atomizing air flow rate.

Droplet size distributions (volume) of a water spray after atomization using a two-fluid nozzle (dorifice=0.7mm) with a liquid feed of 10ml/min and different atomizing air flow rate.

1 10 100 1000

0

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4

6

8

10

12

Vol

ume

frequ

ency

[%]

Droplet diameter [µm]

900 L/h 540 L/h 300 L/h 180 L/h

TwoTwo--FluidFluid NozzleNozzle

The droplet size distribution of a spray generated with a two-fluid nozzle depends on the nozzle geometry, the liquid properties of the formulation (surface tension, viscosity), the liquid flow rate and the atomizing air flow rate

Liquid processing and jet breakupLiquid processing and jet breakupLiquid processing and jet breakup

Atomizing gas

Liquid feed




10 100 10000

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Volu

me

frequ

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[%]


120kHz 60kHz 48kHz 25kHz

Liquid flow 2.0ml/minPower 3.0 Watt

Ultrasonic nozzles operate at a specific resonance frequency, determined primarily by the nozzle length. Atomization is achieved by sustained vibration due to a standing, sinusoidal longitudinal wave. Drop size is governed by the frequency of nozzle vibration, the surface tension and the viscosity of the atomized liquid solution

Ultrasonic nozzles operate at a specific resonance frequency, deUltrasonic nozzles operate at a specific resonance frequency, determined primarily by the termined primarily by the nozzle length. Atomization is achieved by sustained vibration dunozzle length. Atomization is achieved by sustained vibration due to a standing, sinusoidal e to a standing, sinusoidal longitudinal wave. Drop size is governed by the frequency of nozlongitudinal wave. Drop size is governed by the frequency of nozzle vibration, the surface zle vibration, the surface tension and the viscosity of the atomized liquid solutiontension and the viscosity of the atomized liquid solution

Ultrasound NozzleUltrasound Nozzle

Piezoelectric crystals

Atomizing surface

Liquid channel

Titanium amplifying section (front horn)

Ground electrode

Active electrode

Titanium rear horn

Electrical connector

(Sketch of a Sono-Tek© US-nozzle)




Monodisperse droplet generators (MDDG) / Vibrating orifice droplMonodisperse droplet generators (MDDG) / Vibrating orifice droplet generators can be used et generators can be used to optimize the droplet / particle distribution and the actual yto optimize the droplet / particle distribution and the actual yield of the process.ield of the process.

58µm water droplet from a 60µm diameter orifice MicroJet© device B5-128-5 at a dwell time of 22µs at 18 volts.

Electrical signal

Fluid inlet from a reservoir

Glass capillary connected to the fluid inlet and to an annular piezoelectric actuator

MicroFab Technologies Inc. [26]

Monodisperse droplet stream

Dispersingair inlet

Liquid inlet

Piezo crystal

Sketch of a vibrating orifice droplet generator (TSI©)




Limitations of MDDGs:• Liquid viscosity < 40.0 mPa·s• Surface tension between water and

20 mN/m• Different particle size demands

different capillary orifices or wave form conditions

• Limited production capacity• Long process times for larger amounts

of powder

Capacity: A 50µm droplet has a volume of 6.54·10 -8 ml. With a frequency of 20kHz and a solid concentration of 50mg/ml, only 3.93mg powder can be produced in one minute by one MDDG.

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Vol

ume

[%]


Orifice 40µm (Tdwell = 23µs at 11V) Orifice 60µm (Tdwell = 22µs at 18V) Orifice 60µm (Tdwell = 70µs at 30V)



Cryogenic liquids and suitabilityCryogenicCryogenic liquids and liquids and suitabilitysuitabilityPropertyProperty Liquid nitrogenLiquid nitrogen Liquid propaneLiquid propane IsopentaneIsopentane / Hexane/ Hexane ArgonArgon HFEHFE--71007100

Mol. weight 28.01 g/mol 44.10 g/mol 72.15 / 86.18 g/mol 39.95 g/mol 250.0 g/mol

Characteristics Colourless, odorless, noncorrosive, nonflammable

Colourless, odorless, nontoxic

Colourless, slightly disagreeable odor

Colourless, odorless, nonirritating, nonflammable

Colourless, odorless

61°C

-135°C

1400kg/m3

1.3 kJ/kg/K

112 kJ/kg

no flash point

Toxicity testing necessary

Melting point -210.0°C -187.6°C -160°C / -95°C -189.4

Heat capacity at boiling point 2.0 kJ/kg/K 2.2 kJ/kg/K 2.3 kJ/kg/K (both) 1.1 kJ/kg/K

Limitations Asphyxiant,Leidenfrosteffect,No flash point

F+,Flash point: -104°CExplosive limit: 2.4 - 9.5%

F+ / F, Xn,Flash point: -52°C / -23°CExplosive limits: 1.4-7.6% / 1.2-7.8%

Asphyxiant,No flash point

Boiling point -195.8°C -42.1 28°C / 69°C -185.9

Liquid density at boiling point 807 kg/m3 582 kg/m3 600 kg/m3 / 654kg/m3 1394 kg/m3

Latent heat of vaporization 199 kJ/kg 425 kJ/kg 342 kJ/kg / 334 kJ/kg 161 kJ/kg



Freeze-Drying BehaviourFreezeFreeze--DryingDrying BehaviourBehaviour

0 1000 2000 3000 4000 5000-100

-80

-60

-40

-20

0

20

Secondary Drying

Drying time [min]

Tem

pera

ture

[°C

]

Primary Dyring16

18

20

22

24

26

Weight on m

icrobalance [g]

Product temperature and vial weight during freeze-drying of 3g spray-frozen trehalose droplets (50mg/ml) at a primary drying temperature of -20°C and a chamber pressure of 100mtorr

LN2-frozen solution(not atomized)

Spray-frozen product(LN2-frozen)

Empty 20ml freeze-drying vial

Shelf set temperature

Weight microbalanceSpray-frozen product



Freeze-Drying BehaviourFreezeFreeze--DryingDrying BehaviourBehaviour

0 500 1000 1500 20000

10

20

30

40

50

60

Dry

ing

Rat

e [m

g/10

min

]

Primary Drying Time [min]

Momentary freezeMomentary freeze--drying rate curve calculated from weight measurements obtained fdrying rate curve calculated from weight measurements obtained from a rom a freezefreeze--drying microbalance with 20ml standard freezedrying microbalance with 20ml standard freeze--drying vialsdrying vials

0 500 1000 1500 2000 2500 30000

5

10

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25

30

35

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45

50

Dry

ing

rate

[mg/

10m

in]

Primary Drying Time [min]

Concentration 5mg/ml Concentration 50mg/ml Concentration 350mg/ml

Comparison of spray-frozen and LN2 frozen sugar-polymer solutions with a solid content of 350mg/ml at -10°C primary drying temperature and 100mtorr pressure.

Comparison of spray-frozen sugar-polymer solutions with different solid content at -20°C primary drying temperature and 100mtorr chamber pressure

SpraySpray--freezefreeze--drieddriedsugarsugar--polymerpolymer solution solution (350mg/g)(350mg/g)

FreezeFreeze--drieddried sugarsugar--polymerpolymer solution solution (200mg/g)(200mg/g)


Particle MorphologyParticle Morphology

Particle Size and Powder MorphologyParticleParticle SizeSize and and PowderPowder MorphologyMorphology

A CB

ED

Scanning electron microscopy of spray-freeze-dried powders fromdifferent liquid solutions:

A…Insulin 5mg/ml

B…Mannitol 50mg/ml

C…Trehalose 50mg/ml

D…Maltodex./Povidon(3:4) 200mg/g

E…Sugar-Dextran (6:4) 350mg/g


Particle MorphologyParticle Morphology

Particle Size and Powder MorphologyParticleParticle SizeSize and and PowderPowder MorphologyMorphology

0.01 0.1 1 10 100 10000

5

10

15

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Num

ber f

requ

ency

[%]

Particle diameter [µm]

SFD - Insulin 5mg/g SFD - Sugar-Polymer 350mg/g

Comparison of final particle size distributions of spray-freeze-dried powders with different initial solid concentration, both atomized with a 25kHz US-nozzle and a flow rate of 0.5ml/min:

Particle size distribution (number) after spray-freeze-dryingand stirring at 3000rpm in Cyclohexane for 5 minutes

A

E

d(n,0.5) = 0.089µm

d(n,0.5) = 59.35µm

SpecificSpecific SurfaceSurface AreaArea = 39.3 = 39.3 –– 56.7m56.7m22/g/g

Tap Tap DensityDensity = 0.01g/ml= 0.01g/ml

Tap Tap DensityDensity = 0.84g/ml= 0.84g/ml


Protein StabilityProtein Stability

Influence of atomization / droplet formation and cryogenic freezing on product stabilityInfluence of atomization / droplet formation Influence of atomization / droplet formation and cryogenic freezing on product stabilityand cryogenic freezing on product stability

Residual enzyme activity of lysozyme (50mg/g) and α-chymotrypsin (5mg/g) after the different unit operations of the spray-freeze-drying procedure with a primary drying temperature of -20°C and a chamber pressure of 100mtorr.

LysozymeLysozyme αα--ChymotrypsinChymotrypsin

USUS--nozzle (60kHz) nozzle (60kHz) into large staininto large stain--less less steal bowlssteal bowls

USUS--nozzle (60kHz) nozzle (60kHz) using 20ml freezeusing 20ml freeze--drying vialsdrying vials

USUS--nozzle (60kHz) using 20ml nozzle (60kHz) using 20ml freezefreeze--drying vialsdrying vials

96.9 ± 1.9 % 93.3 ± 2.2 %

Liquid atomization (LA) 85.9 ± 1.4 % 91.2 ± 2.3 %

Liquid atomization and freezing in LN2

95.2 ± 1.9 % 96.0 ± 0.7 %

Formulated with trehalose and PS 80 97.2 ± 1.6 %

Liquid atomization and freeze-drying 91.0 ± 4.3 % 91.1 ± 2.0 % 78.3 ± 4.3 %




1740 1720 1700 1680 1660 1640 1620 1600-0.05

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0.05

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0.25

Nor

mal

ized

abs

orpt

ion

units

Wavenumber [cm-1]

Untreated Pure SFD α-chymotrypsin Subtraction spectrum Formulated α-chymotrypsin Subtraction spectrum

1720 1700 1680 1660 1640 1620 1600-0.05

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Untreated Pure SFD α-chymotrypsin Subtraction spectrum Heated α-chymotrypsin (95°C) Subtraction spectrum

Influence of spray-freeze-drying process steps on the secondary structure of α-chymotrypsindetermined using Fourier-Transform Infrared Spectroscopy:




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Emission wavelength [nm]

Untreated chymotrypsin Heated chymotrypsin (95°C)

Influence of the spray-freeze-drying process steps on the tertiary structure of α-chymotrypsindetermined using fluorescence spectroscopy with an excitation wavelength of 295nm:

SFDSFD--procedureprocedure Peak Peak emissionemissionwavelengthwavelength [nm][nm]

UntreatedUntreated 341.98341.98

Liquid Liquid atomizationatomization 342.92342.92

HeatHeat treatedtreated (95°C)(95°C) 347.92347.92

341.96341.96

343.69343.69

FormulatedFormulated withwithtrehalose and PS80trehalose and PS80 342.30342.30

Liquid Liquid atomizationatomizationand and freezingfreezing

Liquid Liquid atomizationatomizationand and freezefreeze--dryingdrying


SFD ApplicationSFD Application

Needle-free Ballistic Powder InjectionNeedleNeedle--free Ballistic Powder Injectionfree Ballistic Powder InjectionMechanism and benefits of needle-free powder injection / immunization [23] :

• Proven safety and efficiency [24]

• Genetic vaccines can prevent as well as treat infectious diseases

• Less vaccine is required for immunization• Minimization of cost and side-effect due to

dose reduction• No needle stick injuries• Stable powders do not require a

refrigeration chain• No particular skill needed for injection• Does not cause bleeding• Pain free (subjective)→ Successful Phase I Clinical Trials with

reformulated and spray-freeze-driedflu vaccine (Fluvirin©)


SFD-powders for needle-free powder injectionSFDSFD--powderspowders forfor needleneedle--freefree powderpowder injectioninjection

Newer devices employing powder aspiration:• Device relies on the “Venturi” effect to suck powder

into the gas stream• High speed gas creates a low pressure region and

lets the powder flow from the cassette into the high speed gas

• A small amount of swirl in the gas distributes the powder on the target

Pressurised micro-cylinder

Break-off tip

Bursting membranepowder cassette

Silencer / filter

Nozzle

PowderMed/Pfizer© Device Particle Therapeutics© Device



Particle requirements for needle-free ballisticdelivery• Uniform particle size (40-60µm)• High density ( > 800kg/m3 )• Sufficient mechanical particle strength to

withstand acceleration and impact on the skin• Excipients for parenteral use and to stabilize

biomolecules

SFD-powders for needle-free powder injectionSFDSFD--powders for needlepowders for needle--free powder injectionfree powder injection

417 µm417 µm417 µm

424 µm424 µm424 µm 395 µm395 µm395 µm

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Particle diameter [µm]

Sugar-polymer mixture Sugar-polymer mixture

after injection at 60 bar

Particle stability upon acceleration and injection into an attrition chamber determined via laser light diffraction.

Particle penetration into agar test-beds after injection with 60bar helium.



SummarySummarySummary

• Spray-freeze-drying (SFD) in general is a feasible method to produce peptide and protein loaded powders for pulmonary and parenteral applications;

• Different SFD method have been developed for powder production;• The right choice of the atomization and liquid delivery system as well as the right

atomization conditions have a strong impact on the final product quality;• Spray-frozen solution behave different to regular frozen solutions and need under the

same primary drying condition a longer drying time;• Peptides and proteins suffer stress during atomization that can be reduced by addition of

suitable excipients to the formulation, like sugars or interfacial active substances;• SFV/L has shown to be a suitable method to produce powders for ballistic protein and

vaccine delivery.

Further SFD projects in the Institute of Biomedical Engineering in Oxfordand Particle Therapeutics Ltd:• Development of automated SFV/L laboratory and pilot plant equipment;• Pre-clinical trials of SFD and Powder Injection in treatment of diabetes.


Dr. Heiko A. SchiffterDrug Delivery Research

Institute of Biomedical EngineeringDepartment of Engineering ScienceUniversity of Oxford

43 Banbury RoadOxford, OX2 6PEGreat Britain

Mail: [email protected]: http://www.eng.ox.ac.uk

Contact detailsContact detailsContact details

Dr. Heiko A. SchiffterHead of Peptide and Protein Formulation

Particle Therapeutics LtdUnit 5, Begbroke Science Park

Sandy LaneYarton, OX5 1PFGreat Britain

Mail: [email protected]: http://www.particletherapeutics.com


[1] Benson, S.W.; Ellis, D.A. (1948): Surface areas of proteins. I. Surface areas and heat of adsorption. Journal of the American Chemical Society. Vol. 70; pages 3563-3569.

[2] Werly, E.F.; Baumann, E.K. (1964): Production of submicronized powder by spray-freezing.Archives of environmental health. Vol. 9; pages 567-571

[3] Pfaller, W. et al. (1976): A comparison of the ultrastructure of spray-frozen and freeze-etched or freeze-dried bull and boar spermatozoa with that after chemical fixation. Journal of Reproduction and Fertility. Vol. 48(2); pages 285-290.

[4] Lang, R.D.; Bronk, J.R. (1978): A study of rapid mitochondrial structural changes in vitro by spray-freeze-etching. Journal of Cell Biology. Vol. 77(1); pages 134-147.

[5] Green, D.J.; Hutchinson, S. (1980): Solution spray-dried and freeze-dried sodium beta-alumina powders: preparation and hot pressing. Materials Science Monograph.

[6] Bollin, E.Jr.; Fletcher, M.G. (1987): Trehalose as excipient and stabilizer for diagnostic assay tablets. Patent: US 4762857.

[7] Mumenthaler, M.; Leuenberger, H. (1991): Atmospheric spray-freeze drying: a suitable alternative in freeze-drying technology. International Journal of Pharmaceutics. Vol. 72(2), pages 97-110.

[8] Costantino, H.R. et al. (1999): Protein spray-freeze-drying. Effect of atomization conditions on particle size and stability. Pharmaceutical Research. Vol. 17(11), pages 1374-1383.

[9] Costantino, H.R. et al. (2002): Protein spray-freeze-drying. Effect of formulation variables on particle size and stability. Journal of Pharmaceutical Sciences. Vol. 91(2), pages 388-395.

LiteratureLiteratureLiterature


[10] Maa, Y.F. et al. (1999): Protein inhalation powders: spray-drying vs spray-freeze-drying.Pharmaceutical research. Vol. 16(2), pages 249-254.

[11] Yu, Z. et al. (2002): Preparation and characterization of microparticles containing peptide produced by a novel process: spray freezing into liquid. European Journal of Pharmaceutics and Biopharmaceutics. Vol. 54(2), pages 221-228.

[12] Maa, Y.F. et al. (2004): Influenza vaccine powder formulation development: spray-freeze-drying and stability evaluation. Journal of Pharmaceutical Sciences. Vol. 93(7), 1912-1923

[13] Finlay, W., Wang, Z. (2005): Powder formulation by atmospheric spray-freeze-drying. Patent WO 2005061088.

[14] Leuenberger, H. et al. (2006): Spray-freeze-drying in a fluidized bed at normal and low pressure. Drying Technology. Vol. 24(6), pages 711-719.

[15] Sonner et al. (2002): Spray-freeze-drying for protein powder preparation. Journal of Pharmaceutical Science. Vol. 91(10), pages 2122-2139

[16] Franks, F. (1982): The properties of aqueous solutions at subzero temperatures. In Franks, F. (Editor), Water and aqueous solutions at subzero temperatures. Plenum Press, New York.

[17] Zhongshui et al. (2006): Spray-freezing into liquid versus spray-freeze-drying. Influence of atomization on protein aggregation and biological activity. European Journal of Pharmaceutical Science. Vol. 27, pages 9-18.

[18] Heller et. al (1999): Protein formulation and lyophilization cycle design. Biotechnology and Bioengineering. Vol. 63(2), pages 166-174.




[19] Webb, S. et al. (2002): Surface adsorption of recombinant human interferon-γ in lyophilized and spray-lyophilized formulations. Journal of Pharmaceutical Science. Vol. 91(6), pages 1474-1487

[20] Sono-Tek Inc. (2006): http://www.sono-tek.com[21] Gieseler, H. (2004): Product morphology and drying behaviour delineated by a freeze-drying

microbalance. Ph.D. thesis. University of Erlangen, Germany.[22] Sigma-Aldrich (2006): http://www.sigma-aldrich.com[23] Dean et al. (2003): Powder and particle-mediated approaches for delivery of DNA and protein

vaccines into epidermis. Comparative Immunology, Microbiology and Infectious Deseases. Vol. 26, pages 373-388.

[24] Dean, H.J., Chen, D. (2004): Epidermal powder immunization against influenza. Vaccines. Vol. 23, pages 681-686.

[25] Jumah, N. A. (2004): The development of non-metallic powders for biolistic DNA delivery and the analysis of biolistic delivery by two-photon microscopy. Ph.D. thesis. University of Oxford. Institute for Biomedical Engineering.

[26] Microfab Technologies Inc. (2006): http://www.microfab.com

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