Wang Wei, Kelner Drew N
Analytics & Formulation, Process Sciences, Biotechnology, Bayer Corp., 800 Dwight Way, Berkeley, California 94701, USA.
Pharm Res. 2003 Apr;20(4):693-700. doi: 10.1023/a:1023271405005.
This study was designed to investigate the stability of recombinant FVIII (rFVIII) in solution at different pHs and to probe the cause(s) of rFVIII inactivation under accelerated storage conditions.
Aqueous stability samples of full-length rFVIII at different pHs were incubated at 40 degrees C for several days and analyzed by the one-stage clotting assay. SEC-HPLC, SDS-PAGE, and UV spectrophotometry.
Incubation of liquid rFVIII at 40 degrees C inactivated the protein rapidly and linearly with time on a semi-log scale at all pHs, suggesting a first order or pseudo first order process. A U-shaped relationship was found between the rate constant for loss of rFVIII activity and the solution pH. The minimal rate of inactivation was found between pH 6.6 and 7.0 with a half-life of approximately 4 days. The SEC-HPLC results indicated pH-dependent aggregation of rFVIII during incubation. It was found that the disappearance of monomeric rFVIII by SEC-HPLC correlated with the loss of rFVIII activity (r2 = 0.97). Both the SDS-PAGE and UV results confirmed the aggregation pathway of rFVIII. In addition, the SDS-PAGE results suggest involvement of three aggregation mechanisms--disulfide-bond formation/exchange, non-reducible crosslinking, and physical interactions.
The full-length rFVIII is unstable in solution at 40 degrees C and loses activity rapidly through a first order or pseudo first order aggregation process, which consists of both physical and chemical pathways. SEC-HPLC may be used in monitoring rFVIII stability studies in lieu of the clotting assay under the incubation conditions used in this study.
本研究旨在调查重组凝血因子VIII（rFVIII）在不同pH值溶液中的稳定性，并探究加速储存条件下rFVIII失活的原因。
将不同pH值的全长rFVIII的水性稳定性样品在40℃孵育数天，并通过一步凝血试验、尺寸排阻色谱-高效液相色谱（SEC-HPLC）、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和紫外分光光度法进行分析。
在所有pH值下，将液体rFVIII在40℃孵育会使蛋白质迅速失活，且在半对数尺度上随时间呈线性变化，表明这是一个一级或准一级过程。发现rFVIII活性丧失的速率常数与溶液pH值之间呈U形关系。在pH 6.6至7.0之间发现失活速率最小，半衰期约为4天。SEC-HPLC结果表明孵育期间rFVIII存在pH依赖性聚集。发现通过SEC-HPLC检测到的单体rFVIII的消失与rFVIII活性的丧失相关（r2 = 0.97）。SDS-PAGE和紫外结果均证实了rFVIII的聚集途径。此外，SDS-PAGE结果表明涉及三种聚集机制——二硫键形成/交换、不可还原交联和物理相互作用。
全长rFVIII在40℃的溶液中不稳定，通过一级或准一级聚集过程迅速丧失活性，该过程包括物理和化学途径。在本研究使用的孵育条件下，SEC-HPLC可用于监测rFVIII稳定性研究，以替代凝血试验。
