Formation of stable submicron protein particles by thin film freezing.
OBJECTIVE
Highly stable, submicron lactate dehydrogenase (LDH) and lysozyme particles may be produced by thin film freezing (TFF) of aqueous solutions followed by lyophilization.


METHODS
The LDH activity was determined by measuring the decrease in absorbance of NADH over time for the reaction of pyruvate to lactate. For lysozyme the particle morphology was determined by scanning electron microscopy (SEM) and compared with the specific surface area (BET) and the particle size, as measured by laser light scattering,


RESULTS
Protein particles with an average diameter of 300 nm and 100% enzyme activity upon reconstitution (for LDH) were formed by TFF. Droplets of protein solutions, 3.6 mm in diameter, spread upon impact with 223 and 133 K metal surfaces to form cylindrical disks with thicknesses of 200-300 microm. Calculated cooling rates of the disks of 10(2) K/s were confirmed experimentally with infrared measurements.


CONCLUSIONS
The cooling rates of 10(2) K/s, intermediate to those in lyophilization (1 K/min) and spray freeze-drying (SFD) (10(6) K/s), were sufficiently fast to produce sub-micron protein particles with surface areas of 31-73 m2/g, an order of magnitude higher than in lyophilization. In addition, the low surface area/volume ratio (32-45 cm(-1)) of the gas-liquid interface led to minimal protein adsorption and denaturation relative to SFD.
Engstrom
Joshua D
JD
Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA.
Lai
Edwina S
ES
Ludher
Baltej S
BS
Chen
Bo
B
Milner
Thomas E
TE
Williams
Robert O
RO
Kitto
G Barrie
GB
Johnston
Keith P
KP
D005615:Freezing; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D010316:Particle Size; D013678:Technology, Pharmaceutical
D007770:L-Lactate Dehydrogenase