添加SNS能显著提高CHT填料使用寿命的研究

SNS: An Effective Strategy for Extending Ceramic Hydroxyapatite Column Life

陶瓷羟基磷灰石广泛用于各种生物分子的分离和纯化,但在层析应用上,纯化洗脱过程中填料表面氢原子的释放容易导致其性能和寿命降低。研究了一种可以延长CHT填料使用寿命的方法,通过在洗脱步骤前加入一种称为表面中和体系(SNS)的溶液,结合释放的氢原子,使得整个体系处于一个接近中性pH的状态。先筛选出有效调整pH的SNS条件,通过钙离子流失的监测证实该条件的有效性。再通过在WLB304单克隆抗体纯化工艺中加入SNS之后,对羟基磷灰石填料载量、pH和压力的变化、杂质的去除能力进行考察。结果表明,加入SNS可以有效减少钙离子流失,同时并不会影响CHT层析工艺的效果。

Ceramic Hydroxyapatite（ CHT） has been widely used in the manufacturing of a wide variety of monoclonal antibodies（ m Abs）,vaccines,and other protein therapeutics. It is an effective tool for the removal of residual host cell proteins, nucleic acids, viruses, endotoxins, and product-related contaminants such as aggregates and degradation fragments. However,damage of the CHT media and increased column back pressure are often observed at production scale-up. A transient p H excursion during elution with increased salt concentration leads to Ca^（2 ＋）depletion from the CHT column. In the present study,the effect of surface neutralization system（ SNS） on the life cycles of CHT media is demonstrated. Following post-loading wash,a buffer with slightly elevated p H is applied onto the column so as to prevent significant p H drop during target protein elution. The level of Ca（2 ＋）depletion in response to p H and salt concentration of the SNS buffer have been investigated,and the optimal buffer composition for maintaining the p H of column effluent near neutral been identified. This newly developed protocol has been validated for the polish purification of m Ab WLB304. The dynamic binding capacity of WLB304,impurity clearance efficiency,leached Ca（2 ＋）level,p H excursion and column back pressure during purification process were closely monitored. Data indicate that the implementation of SNS can significantly reduce Ca（2 ＋）depletion and effectively extend the life time of a CHT column,without compromising its chromatography performance.