Engstrom Joshua D, Lai Edwina S, Ludher Baltej S, Chen Bo, Milner Thomas E, Williams Robert O, Kitto G Barrie, Johnston Keith P
Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA.
Pharm Res. 2008 Jun;25(6):1334-46. doi: 10.1007/s11095-008-9540-4.
Highly stable, submicron lactate dehydrogenase (LDH) and lysozyme particles may be produced by thin film freezing (TFF) of aqueous solutions followed by lyophilization.
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,
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.
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.
通过水溶液的薄膜冷冻（TFF）然后冻干，可以制备出高度稳定的亚微米级乳酸脱氢酶（LDH）和溶菌酶颗粒。
通过测量丙酮酸转化为乳酸反应中NADH吸光度随时间的下降来测定LDH活性。对于溶菌酶，通过扫描电子显微镜（SEM）确定颗粒形态，并与通过激光散射测量的比表面积（BET）和粒径进行比较。
通过TFF形成了平均直径为300 nm且复溶后具有100%酶活性的蛋白质颗粒（对于LDH）。直径为3.6 mm的蛋白质溶液液滴在与223 K和133 K金属表面碰撞时铺展，形成厚度为200 - 300微米的圆柱形盘。通过红外测量实验证实了计算得出的盘的冷却速率为10² K/s。
10² K/s的冷却速率介于冻干（1 K/min）和喷雾冷冻干燥（SFD）（10⁶ K/s）之间，足够快以产生比表面积为31 - 73 m²/g的亚微米级蛋白质颗粒，比冻干高一个数量级。此外，气液界面的低表面积/体积比（32 - 45 cm⁻¹）导致相对于SFD而言蛋白质吸附和变性最小。
