Alsenaidy Mohammad A, Okbazghi Solomon Z, Kim Jae Hyun, Joshi Sangeeta B, Middaugh C Russell, Tolbert Thomas J, Volkin David B
Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas 66047, USA.
J Pharm Sci. 2014 Jun;103(6):1613-1627. doi: 10.1002/jps.23975. Epub 2014 Apr 16.
The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH 2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0-6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies.
测定了由毕赤酵母产生的具有不同糖基化位点占据情况（二糖基化、单糖基化和非糖基化）的各种IgG1-Fc蛋白的结构完整性和构象稳定性。此外，还研究了三种不同形式的非糖基化Fc分子（由于Fc糖型的点突变和酶切作用，CH2结构域中第297位氨基酸残基不同）的物理稳定性概况。通过使用多种技术结合数据可视化工具（三个指标的经验相图和雷达图）的高通量生物物理分析，研究了这些IgG1-Fc糖蛋白的物理稳定性随pH值和温度的变化情况。在4.0-6.0的pH范围内，IgG1-Fc的二糖基化和单糖基化形式分别表现出最高和最低的物理稳定性水平，非糖基化形式的稳定性则根据溶液pH值处于中间水平。在无糖基化的Fc蛋白中，第297位引入天冬氨酸（D）残基（QQ型与DN型与DD型）会导致结构完整性和物理稳定性根据溶液pH值发生更细微的变化。在分析可比性研究的背景下，讨论了评估各种IgG1-Fc糖蛋白之间构象稳定性概况差异的实用性。
