Misaki Ryo, Fukura Natsuko, Kajiura Hiroyuki, Yasugi Mayo, Kubota-Koketsu Ritsuko, Sasaki Tadahiro, Momota Masatoshi, Ono Ken-Ichiro, Ohashi Takao, Ikuta Kazuyoshi, Fujiyama Kazuhito
International Center for Biotechnology, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871, Japan; The Japan Science and Technology Agency/Japan International Cooperation Agency, Science and Technology Research Partnership for Sustainable Development, Tokyo, Japan.
International Center for Biotechnology, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871, Japan.
Biologicals. 2016 Sep;44(5):394-402. doi: 10.1016/j.biologicals.2016.05.006. Epub 2016 Jul 25.
In previous studies, hybridomas producing human immunoglobulin G, the antibodies 5E4 and 5A7 against influenza A and B virus were established using a novel human lymphocyte fusion partner, SPYMEG. In the present study, we succeeded in achieving the recombinant production and secretion of 5E4 and 5A7 in Chinese hamster ovary cells. Our N-glycan analysis by intact-mass detection and liquid chromatography mass spectrometry showed that recombinant 5E4 and 5A7 have one N-glycan and the typical mammalian-type N-glycan structures similar to those in hybridomas. However, the glycan distribution was slightly different among these antibodies. The amount of high-mannose-type structures was under 10% of the total N-glycans of recombinant 5E4 and 5A7, compared to 20% of the 5E4 and 5A7 produced in hybridomas. The amount of galactosylated N-glycans was increased in recombinants. Approximately 80% of the N-glycans of all antibodies was fucosylated, and no sialylated N-glycan was found. Recombinant 5E4 and 5A7 neutralized pandemic influenza A virus specifically, and influenza B virus broadly, quite similar to the 5E4 and 5A7 produced in hybridomas, respectively. Here we demonstrated that recombinants of antibodies identified from hybridomas fused with SPYMEG have normal N-glycans and that their neutralizing activities bear comparison with those of the original antibodies.
在先前的研究中，利用新型人类淋巴细胞融合伙伴SPYMEG建立了产生抗甲型和乙型流感病毒的人免疫球蛋白G抗体5E4和5A7的杂交瘤。在本研究中，我们成功地在中国仓鼠卵巢细胞中实现了5E4和5A7的重组生产和分泌。我们通过完整质量检测和液相色谱质谱联用进行的N-聚糖分析表明，重组5E4和5A7具有一个N-聚糖以及与杂交瘤中相似的典型哺乳动物型N-聚糖结构。然而，这些抗体之间的聚糖分布略有不同。与杂交瘤产生的5E4和5A7中20%的高甘露糖型结构相比，重组5E4和5A7的高甘露糖型结构在总N-聚糖中的比例低于10%。重组体中半乳糖基化N-聚糖的数量增加。所有抗体中约80%的N-聚糖被岩藻糖基化，未发现唾液酸化的N-聚糖。重组5E4和5A7分别特异性中和甲型大流行性流感病毒，广泛中和乙型流感病毒，这与杂交瘤产生的5E4和5A7非常相似。在此我们证明，由与SPYMEG融合的杂交瘤鉴定出的抗体重组体具有正常的N-聚糖，并且它们的中和活性与原始抗体相当。
