目的:单克隆抗体中的化学修饰可以改变疏水性,电荷异质性以及构象,最终会影响它们的物理稳定性。在这项研究中,研究了在下游纯化过程中使用的两种不同的缓冲条件下各个电荷变体对物理稳定性和聚集倾向的影响。
方法:使用半制备型阳离子交换色谱法分离电荷变体,并在具有pH6.0和3.8的两种缓冲液中进行缓冲液交换。随后使用尺寸排阻色谱和动态光散射分析每个变体的尺寸异质性。构象稳定性,胶体稳定性,和加速稳定条件下的聚集行为。
结果:当在没有延长储存的情况下分析时,每个电荷变体中的大小变体在两种pH条件下相似。然而,构象稳定性在pH3.8低于pH6.0。所有电荷变体在pH6.0显示相似的表观解链温度。相比之下,在pH3.8时,变体A3,A5,B2,B3和B4显示较低的Tm,表明构象稳定性降低。Further,A2、A3和A5在pH3.8时表现出降低的胶体稳定性。总的来说,酸性变体比碱性变体更容易聚集。
结论:当今典型的工业实践是检查过程中的中间体稳定性,酸性物种和碱性物种分别作为一个类别。我们建议,由于不同的酸性或碱性物种具有不同的稳定性,因此可能需要在物种水平上进行稳定性评估,并且这些知识可用于下游过程的巧妙设计以实现稳定的产品。
OBJECTIVE: Chemical modifications in monoclonal antibodies can change hydrophobicity, charge heterogeneity as well as conformation, which eventually can impact their physical stability. In this study, the effect of the individual charge variants on physical stability and aggregation propensity in two different buffer conditions used during downstream purification was investigated.
METHODS: The charge variants were separated using semi-preparative cation exchange chromatography and buffer exchanged in the two buffers with pH 6.0 and 3.8. Subsequently each variant was analysed for size heterogeneity using size exclusion chromatography and dynamic light scattering, conformational stability, colloidal stability, and aggregation behaviour under accelerated stability conditions.
RESULTS: Size variants in each charge variant were similar in both pH conditions when analyzed without extended storage. However, conformational stability was lower at pH 3.8 than pH 6.0. All charge variants showed similar apparent melting temperature at pH 6.0. In contrast, at pH 3.8 variants A3, A5, B2, B3 and B4 display lower Tm, suggesting reduced conformational stability. Further, A2, A3 and A5 exhibit reduced colloidal stability at pH 3.8. In general, acidic variants are more prone to aggregation than basic variants.
CONCLUSIONS: Typical industry practice today is to examine in-process intermediate stability with acidic species and basic species taken as a single category each. We suggest that perhaps stability evaluation needs to be performed at specie level as different acidic or basic species have different stability and this knowledge can be used for clever designing of the downstream process to achieve a stable product.