Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies
Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies
复制标题
DOI：
10.1002/prot.21243
复制
发表时间：
2007-03-01
影响因子：
2.9
通讯作者：
Philo, John S.
中科院分区：
文献类型：
--
作者：
Ejima, Daisuke;Tsumoto, Kouhei;Philo, John S.
抗体暴露于低pH对于纯化和病毒清除通常是不可避免的。用圆二色谱（CD）、差示扫描量热法（DSC）和沉降速率法研究了两种抗不同抗原的人源化单克隆抗体（hIgG 4-A和hIgG 4-B）和一种鼠源化单克隆抗体（mIgG 1）在0.1M柠檬酸盐溶液中的构象和稳定性。近-和远-UV CD光谱表明，这些抗体暴露于pH 2.7-3.9仅诱导有限的构象变化，虽然在较低的pH值的变化更大。然而，酸构象是远离未折叠或所谓的熔融球结构。在pH 2.7和3.5、4 ℃下孵育hIgG 4-A 24 h后，近紫外CD光谱几乎没有变化，表明酸性构象稳定。沉降速度显示，hIgG 4-A在pH 2.7和3.5以及pH 6.0下主要是单体。在这些低pH值下孵育后，沉降曲线未发生时间依赖性变化，与CD观察到的构象稳定性一致。在pH 2.7或3.5的单体的沉降系数再次表明，在这些pH值没有发生总的构象变化。抗体的DSC分析显示在pH 2.7-3.9以及在pH 6.0下的热解折叠，但在较低pH下具有降低的解链温度。这些结果与抗体经历有限的构象变化的观点一致，并且在4 ℃下在低pH下孵育不会导致时间依赖性构象变化。从pH 3.5至6.0滴定hIgG 4-A导致天然单体蛋白的回收，其CD和DSC曲线类似于原始样品的那些。然而，从pH 2.7滴定导致单体抗体的回收率较低，表明在该pH下观察到的较大构象变化不能通过简单的pH滴定完全逆转为天然结构。
Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.