Allen Martin J, Guo Amy, Martinez Theresa, Han Mei, Flynn Gregory C, Wypych Jette, Liu Yaoqing Diana, Shen Wenyan D, Dillon Thomas M, Vezina Christopher, Balland Alain
Process and Product Development, Amgen, Inc., Seattle, Washington 98119, USA.
Biochemistry. 2009 May 5;48(17):3755-66. doi: 10.1021/bi8022174.
Human IgG2 exists as a mixture of disulfide-linked structural isoforms that can show different activities. To probe the contribution of specific cysteine residues to the formation of structural isoforms, we characterized a series of Cys-->Ser mutant IgG2 recombinant monoclonal antibodies, focused on the first C(H)1 cysteine and the first two hinge cysteines. These residues participate in the formation of structural isoforms that have been noted by nonreduced capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, and cation exchange chromatography. We show that single Cys-->Ser mutants can greatly reduce heterogeneous disulfide bonding in human IgG2 and maintain in vitro activity. The data demonstrate the feasibility of applying site-directed mutagenesis to reduce disulfide bond heterogeneity in human IgG2 while preserving the activity of this therapeutically important class of human antibodies.
人IgG2以二硫键连接的结构异构体混合物形式存在，这些异构体可表现出不同的活性。为了探究特定半胱氨酸残基对结构异构体形成的贡献，我们对一系列Cys→Ser突变型IgG2重组单克隆抗体进行了表征，重点关注第一个C(H)1半胱氨酸和前两个铰链区半胱氨酸。这些残基参与了通过非还原毛细管十二烷基硫酸钠聚丙烯酰胺凝胶电泳、反相高效液相色谱和阳离子交换色谱所观察到的结构异构体的形成。我们表明，单个Cys→Ser突变体可极大地减少人IgG2中异质二硫键的形成，并保持体外活性。数据证明了应用定点诱变减少人IgG2中二硫键异质性同时保留这类具有治疗重要性的人抗体活性的可行性。
