Mullick A, Flickinger M C
Biological Process Technology Institute, University of Minnesota, St. Paul, Minnesota, USA.
Biotechnol Bioeng. 1999 Nov 5;65(3):282-90. doi: 10.1002/(sici)1097-0290(19991105)65:3<282::aid-bit5>3.0.co;2-g.
The adsorption of proteins from high cell density yeast suspensions on mixed-mode fluoride-modified zirconia (FmZr) particles (38 to 75 microm, surface area of 29 m(2)/g and density of 2.8 g/cm(3)) was investigated using human serum albumin (HSA) added to Saccharomyces cerevesiae as the model expression host. Because of the high density of the porous zirconia particles, HSA (4 mg/mL) can be adsorbed from a 100 g dry cell weight (DCW)/L yeast suspension in a threefold-expanded bed of FmZr. The expanded bed adsorption of any protein from a suspension containing >50 g DCW/L cells has not been previously reported. The FmZr bed expansion characteristics were well represented by the Richardson-Zaki correlation with a particle terminal velocity of 3.1 mm/s and a bed expansion index of 5.4. Expanded bed hydrodynamics were investigated as a function of bed expansion using residence time distribution studies with sodium nitrite as the tracer. The adsorption of HSA on FmZr exhibited features of multicomponent adsorption due to the presence of dimers. The protein binding capacity at 5% breakthrough decreased from 22 mg HSA/mL settled bed void volume for 20 g DCW/L yeast to 15 mg HSA/mL settled bed void volume for 40 g DCW/L yeast and remained unchanged for the higher yeast concentrations (60 to 100 g DCW/L). However, the batch (or equilibrium) binding capacity decreased monotonically as a function of yeast concentration (20 to 100 g DCW/L) and the binding capacity at 100 g DCW/L yeast was fivefold lower compared with that at 20 g DCW/L yeast. The lower batch binding capacity at high cell concentrations resulted from the adsorption of cells at the surface of the particles restricting access of HSA to the intraparticle surface area. Batch (or equilibrium) and column HSA adsorption results indicated that the adsorption of HSA on FmZr occurred at a time scale that may be much faster than that of yeast cells. The zirconia particles were cleaned of adsorbed HSA and yeast with a total of 1500 to 2000 column volumes (over many cycles) of 0. 25 M NaOH, without any significant effect on the chromatographic performance.
以添加了人血清白蛋白（HSA）的酿酒酵母作为模型表达宿主，研究了高细胞密度酵母悬浮液中的蛋白质在混合模式氟化物修饰氧化锆（FmZr）颗粒（粒径38至75微米，表面积29平方米/克，密度2.8克/立方厘米）上的吸附情况。由于多孔氧化锆颗粒密度高，在FmZr的三倍膨胀床中，HSA（4毫克/毫升）可从100克干细胞重量（DCW）/升的酵母悬浮液中被吸附。此前尚未有从细胞密度大于50克DCW/升的悬浮液中对任何蛋白质进行膨胀床吸附的报道。FmZr床层膨胀特性可用Richardson-Zaki关联式很好地描述，颗粒终端速度为3.1毫米/秒，床层膨胀指数为5.4。以亚硝酸钠为示踪剂，通过停留时间分布研究，考察了膨胀床流体力学随床层膨胀的变化情况。由于二聚体的存在，HSA在FmZr上的吸附表现出多组分吸附的特征。5%穿透时的蛋白质结合容量从20克DCW/升酵母时的22毫克HSA/毫升沉降床空隙体积降至40克DCW/升酵母时的15毫克HSA/毫升沉降床空隙体积，而对于更高的酵母浓度（60至100克DCW/升）则保持不变。然而，分批（或平衡）结合容量随酵母浓度（20至100克DCW/升）单调下降，100克DCW/升酵母时的结合容量比20克DCW/升酵母时低五倍。高细胞浓度下较低的分批结合容量是由于颗粒表面细胞的吸附限制了HSA进入颗粒内表面积。分批（或平衡）和柱上HSA吸附结果表明，HSA在FmZr上的吸附时间尺度可能比酵母细胞快得多。用总共1500至2000柱体积（经过多个循环）的0.25 M NaOH清洗氧化锆颗粒上吸附的HSA和酵母，对色谱性能没有任何显著影响。
