Characterization of humanized antibodies secreted by Aspergillus niger
Characterization of humanized antibodies secreted by Aspergillus niger
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DOI：
10.1128/aem.70.5.2567-2576.2004
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发表时间：
2004-05-01
影响因子：
4.4
通讯作者：
Wang, HM
中科院分区：
文献类型：
--
作者：
Ward, M;Lin, C;Wang, HM
通过尼日尔曲霉产生两种不同的人源化免疫球蛋白G1（κ）抗体和Fab'片段。抗体被分泌到培养上清液中。轻链和重链最初都是作为与天然葡糖淀粉酶的融合蛋白合成的。抗体组装后，A.尼日尔KexB蛋白酶允许释放游离抗体。通过疏水电荷诱导层析的纯化证明在去除葡糖淀粉酶保持附着的任何抗体方面是有效的。在重链Fc区的N297处观察到糖基化，但该位点在约50%的重链上未被占据。聚糖为高甘露糖型，存在一些半乳糖，大小范围为Hex（6）GlcNAc（2）至Hex（15）GlcNAc（2）。还产生了抗体的无糖基突变体形式。在亲和力、亲合力、药代动力学或抗体依赖性细胞毒性功能试验中，未观察到曲霉菌产生的糖基化抗体与哺乳动物细胞培养物产生的糖基化抗体之间存在显著差异。
Two different humanized immunoglobulin G1(kappa) antibodies and an Fab' fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex(6)GlcNAc(2) to Hex(15)GlcNAc(2). An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.