离子交换层析分离单抗电荷异质体的模型辅助过程优化

doi:10.11949/0438-1157.20231246

摘要/Abstract

摘要：

针对单抗电荷异质体分离，采用离子交换层析机理模型，预测洗脱分离行为，辅助工艺条件优化。设计了校准实验，拟合得到模型参数，模型计算与实验吻合良好，具有良好的预测能力。利用模型分析比较了不同洗脱方式，得到最优的两步阶跃洗脱方案，具有较高的收率，但发现该分离过程对盐浓度极为敏感。进一步针对第一步洗脱盐浓度进行过程稳健性约束的过程优化，发现盐浓度为108.5 mmol/L时过程稳健性增强。经实验验证，两步阶跃洗脱收率最高可达到85.3%，稳健约束优化后第一步等度洗脱盐浓度操作区间增大为98.9~117.5 mmol/L。结果表明，模型辅助的工艺优化可以进行复杂条件分析，促进难分离体系的分离过程优化，并能够针对过程稳健性给出合理解决方案。

关键词: 单克隆抗体, 电荷异质体, 分离, 离子交换层析, 层析模型, 过程优化

Abstract:

For the separation of monoclonal antibody charge heterogeneous species, an ion-exchange chromatography mechanism model is used to predict the elution and separation behavior and assist in the optimization of process conditions. The calibration experiments were designed to estimate the model parameters and the model simulation matched well with the experiments, demonstrating the model has good predictive ability. The model was used to compare and evaluate the influences of different elution modes and conditions. The optimal two-step elution was obtained to achieve high yield. However, it was found that the separation efficiency was highly sensitive to the salt concentration of first-step elution solution. Therefore, the process robustness was considered further to optimize the salt concentration, and it was found the salt concentration of 108.5 mmol/L could greatly enhance the process robustness. The results of validation experiments showed that the highest yield of two-step elution process reached 85.3%, and the operating range of the salt concentration of first-step elution was widened to 98.9—117.5 mmol/L. The results demonstrated that model-assisted process optimization could facilitate the complex condition analysis, promote the optimization and provide reasonable solutions for process robustness.

Key words: monoclonal antibody, charge variant, separation, ion-exchange chromatography, chromatography model, process optimization

中图分类号:

TQ 028.8

许茹枫, 陈煜成, 高丹, 焦静雨, 高栋, 王海彬, 姚善泾, 林东强. 离子交换层析分离单抗电荷异质体的模型辅助过程优化[J]. 化工学报, 2024, 75(5): 1903-1911.

Rufeng XU, Yucheng CHEN, Dan GAO, Jingyu JIAO, Dong GAO, Haibin WANG, Shanjing YAO, Dongqiang LIN. Model-assisted process optimization of ion-exchange chromatography for monoclonal antibody charge variant separation[J]. CIESC Journal, 2024, 75(5): 1903-1911.

图/表 13

表1 层析系统和层析柱参数

Table 1 Chromatographic system and column parameters

参数类别	参数名称	参数符号	数值	单位
层析柱参数	柱径	d_col	10	mm
	床高	L	196	mm
	总空隙率	ε_t	0.91	—
	离子交换容量	Λ	0.58	mol/L
操作条件参数	单抗上样浓度	c_inj	3.16	mg/ml
	线性流速	u	0.467	mm/s
	表观轴向分散系数	D_app	0.0989	mm²/s

图1 实验洗脱曲线和模型计算结果比较

Fig.1 Comparison of experimental elution curve and model calculation

表2 SMA模型参数

Table 2 SMA model parameters

参数名称	参数符号	A1	A2	M
特征电荷数	υ	11.58	11.75	11.89
平衡常数	k_eq	4.23×10^-7	6.20×10^-7	7.34×10^-7
空间因子	σ	10.47	10.97	11.70
动力学常数	k_kin	0.35×10^-8	0.20×10^-8	0.10×10^-8

表3 不同洗脱方式的优化结果

Table 3 Process optimization of different elution methods

洗脱方式		最大收率/%（纯度为89%）
单步洗脱	等度洗脱	59.2
单步洗脱	梯度洗脱	61.9
两步洗脱	两步阶跃	65.5
	等度-梯度	64.2
	梯度-等度	65.6
	两步梯度	64.1
三步洗脱	三步阶跃	65.5
	等度-梯度-等度	65.1
	梯度-等度-梯度	65.0

图2 优化后不同洗脱方式的洗脱曲线（阴影部分为收集区间）

Fig.2 Elution curves of different optimized elution modes (the shaded parts are the collection ranges)

图3 不同盐浓度的第一步阶跃洗脱曲线

Fig.3 First-step elution curves of different salt concentrations

图4 第一步洗脱盐浓度对两步阶跃洗脱的纯度和收率的影响

Fig.4 Effects of first-step elution salt concentration on purity and yieldof two-step stepwise elution

图5 收集区间恒定的稳健优化两步阶跃洗脱曲线

Fig.5 Elution curve of optimized two-step stepwise elution under the robust constraints and fixed collection range

图6 第一步洗脱盐浓度对稳健优化及收集区间恒定的两步阶跃洗脱纯度和收率的影响

Fig.6 Effects of first-step elution salt concentration on purity and yield of optimized two-step stepwise elution under the robust constraints and fixed collection range

图7 收集区间变化的稳健优化两步阶跃洗脱曲线

Fig.7 Elution curve of optimized two-step stepwise elution under the robust constraints and changed collection range

图8 第一步洗脱盐浓度对稳健优化及收集区间变化的两步阶跃洗脱收集起点和收率的影响

Fig.8 Effects of first-step elution salt concentration on purity and yield of optimized two-step stepwise elution under the robust constraints and changed collection range

表4 层析分离验证实验与模型预测结果比较

Table 4 Comparison of chromatographic separation verification experiments and model predicted results

序号	洗脱总长度/CV	收集起点/CV	第一步等度洗脱		纯度			收率
序号	洗脱总长度/CV	收集起点/CV	洗脱长度/CV	盐浓度/(mmol/L)	预测值/%	实验值/%	偏差/%	预测值/%	实验值/%	偏差/%
Ⅰ	15.0	13.80	14.42	103.4	89.0	92.2	3.47	65.5	64.2	2.02
Ⅱ	15.0	11.00	14.38	108.5	89.0	91.1	2.31	63.4	59.8	6.02
Ⅲ	15.0	16.06	14.38	98.9	89.0	91.7	2.94	43.8	43.5	0.69
Ⅳ	15.0	4.40	14.38	117.5	89.0	89.0	0	60.0	57.5	4.35

图9 层析分离验证实验与模型预测结果比较

Fig.9 Comparison of chromatographic separation verification experiments and model predicted results

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