Chung Cheng-Yu, Wang Qiong, Yang Shuang, Ponce Sean A, Kirsch Brian J, Zhang Hui, Betenbaugh Michael J
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland.
Department of Pathology, Johns Hopkins University, Baltimore, Maryland.
Biotechnol Bioeng. 2017 Dec;114(12):2848-2856. doi: 10.1002/bit.26375. Epub 2017 Sep 19.
One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications.
单克隆抗体的关键质量属性之一是聚糖模式和分布。两个末端半乳糖残基通常仅占抗体总聚糖的一小部分。然而，具有特定糖基化特性（包括增强的半乳糖基化）的抗体已被证明在这些重要的生物医学模式中表现出改变的特性。在本研究中，将中国仓鼠卵巢（CHO）细胞中的两种α-2,3唾液酸转移酶（ST3GAL4和ST3GAL6）的破坏与Fc区域的蛋白质工程相结合，以产生含有80%双半乳糖基化和岩藻糖基化（G2F）糖型的IgG。在具有岩藻糖基转移酶（FUT8）加ST3GAL4和ST3GAL6三基因敲除的CHO细胞中表达相同的单氨基酸突变体（F241A）IgG，将半乳糖基化糖谱降低至65%的双半乳糖基化G2聚糖。然而，具有四个氨基酸取代的IgG的过表达使G2糖型组成恢复到约80%。在CHO细胞中结合基因组和蛋白质工程将提供一个新的抗体生产平台，使生物技术人员能够为特定的生物医学和生物技术应用生成糖型标准。
