Simultaneous assessment of Asp isomerization and Asn deamidation in recombinant antibodies by LC-MS following incubation at elevated temperatures.
Simultaneous assessment of Asp isomerization and Asn deamidation in recombinant antibodies by LC-MS following incubation at elevated temperatures.
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DOI：
10.1371/journal.pone.0030295
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Bulau P
中科院分区：
文献类型：
--
作者：
Diepold K;Bomans K;Wiedmann M;Zimmermann B;Petzold A;Schlothauer T;Mueller R;Moritz B;Stracke JO;Mølhøj M;Reusch D;Bulau P
通过天冬酰胺脱酰胺和天冬氨酸异构化降解蛋白质是重组抗体的几种化学降解途径之一。在这项研究中，我们已经确定了两个溶剂可及的降解位点（轻链天冬氨酸-56和重链天冬氨酸-99/101）的重组IgG 1抗体的互补决定区在高温条件下易受异构化。对于这两个热点，发现异构化程度显著高于保守CH 3区域中天冬酰胺-（387，392，393）的脱酰胺化程度，在先前的研究中，已将其鉴定为溶剂可及且对化学降解敏感。为了缩短潜在天冬酰胺脱酰胺和天冬氨酸异构化位点的同时鉴别和功能评价时间，开发了采用加速温度条件和蛋白水解肽图谱结合定量UPLC-MS的试验系统。该方法占用制剂缓冲液系统组氨酸/HCl（20 mM; pH 6.0）进行变性/还原/消化，并消除了烷基化步骤。天冬酰胺脱酰胺和天冬氨酸异构化的实现程度足以通过结合研究确定功能性结果。总之，本文提出的方法通过监测位于重组抗体互补决定区的敏感标记肽，极大地促进了对抗体天冬酰胺脱酰胺和天冬氨酸异构化的发酵、纯化、配制和储存条件的评价。
The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.
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影响因子：
2.9
作者：
Cacia, J;Keck, R;Frenz, J
通讯作者：
Frenz, J
影响因子：
8
作者：
POTTER, SM;HENZEL, WJ;ASWAD, DW
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ASWAD, DW
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作者：
Cournoyer, JJ;Pittman, JL;O'Connor, PB
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O'Connor, PB
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作者：
Schmid, DG;von der Mülbe, F;Jung, G
通讯作者：
Jung, G
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7.4
作者：
Xiao, Gang;Bondarenko, Pavel V.;Chelius, Dirk
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