Liu Dingjiang, Ren Da, Huang Holly, Dankberg Jane, Rosenfeld Robert, Cocco Melanie J, Li Luke, Brems David N, Remmele Richard L
Department of Pharmaceutics, Amgen, Inc., Thousand Oaks, California 91320, USA.
Biochemistry. 2008 May 6;47(18):5088-100. doi: 10.1021/bi702238b. Epub 2008 Apr 12.
The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fc's structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fc's physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.
Fc区域有两个高度保守的甲硫氨酸残基，即Met 33（C(H)3结构域）和Met 209（C(H)3结构域），它们对Fc的结构和生物学功能很重要。为了了解甲硫氨酸氧化对在大肠杆菌中表达的人IgG1 Fc的结构和稳定性的影响，我们使用生物物理方法（差示扫描量热法、圆二色性和核磁共振）和生物分析方法（尺寸排阻色谱和反相高效液相色谱-质谱）对完全氧化的Fc进行了表征。甲硫氨酸氧化导致通过圆二色性测量可检测到的二级和三级结构改变。核磁共振数据进一步支持了这一点。异核单量子相干谱的变化表明C(H)2和C(H)3结构域的结构都受到甲硫氨酸氧化的影响。甲硫氨酸氧化后人IgG1 Fc的C(H)2结构域的解链温度（Tm）显著降低，而C(H)3结构域的解链温度仅受到轻微影响。C(H)2结构域Tm的变化取决于Met 33和Met 209的氧化程度。这通过对两个位点特异性甲硫氨酸突变体的甲硫氨酸氧化样品的差示扫描量热分析得到了证实。当在45℃孵育时，氧化的Fc表现出增加的聚集速率。此外，氧化的Fc在位于C(H)2结构域的Asn 67和Asn 96位点处显示出增加的脱酰胺（在pH 7.4时）速率，而其他残基的脱酰胺速率不受影响。甲硫氨酸氧化导致Fc的结构和稳定性发生变化，这些变化主要局限于C(H)2结构域。这些变化会影响Fc的物理和共价稳定性以及潜在的生物学功能；因此，在蛋白质治疗药物的生产和储存过程中监测和控制甲硫氨酸氧化至关重要。
