模型辅助的单抗连续捕获工艺分析和过程优化

doi:10.11949/0438-1157.20240583

摘要/Abstract

摘要：

连续流层析具有提高过程产率和介质利用率、降低生产成本等显著优势，在抗体药物生产中具有良好的应用前景。但是，连续流层析模式多样，影响因素众多，传统的基于实验的过程开发方法存在困难。将模型辅助方法引入到连续流层析亲和捕获过程，建立了模型辅助过程优化方法，系统比较了两柱、三柱和四柱连续捕获模式，确定了最佳模式和操作条件，并经实验验证。结果表明：模型预测与实验结果基本一致；与批次层析相比，四柱连续捕获的过程产率提高了27.2%，介质利用率提高了50.1%，且产品质量稳定。由此说明，模型辅助方法有助于确定最佳连续捕获模式和操作条件，促进过程优化，加速抗体药物连续生产过程的工业实现。

关键词: 连续流层析, 抗体捕获, 模型辅助方法, 蛋白A亲和层析, 过程开发

Abstract:

Continuous chromatography is a promising technology for antibody production, which has significant advantages of improving process productivity, saving operation cost and enhancing product quality. However, continuous chromatography modes are diverse and there are many influencing factors, making traditional experiment-based process development methods difficult. In this study, model-assisted process development and optimization method was proposed and applied to continuous capture of monoclonal antibody. Different continuous capture modes (twin columns, three columns and four columns) were compared systematically, and the best mode and operating conditions were determined and further verified by experiments. It was found that the model prediction was consistent with the experimental results. Compared with batch chromatography, process productivity of continuous capture was increased by 27.2%, and the resin capacity utilization was increased by 50.1%. Moreover, there were no significant changes in product quality. The results demonstrated that model-assisted process development is useful to determine the optimal operating mode and conditions for continuous capture, promote process optimization, and accelerate the industrial applications of continuous process.

Key words: continuous chromatography, antibody capture, model-assisted, protein A affinity chromatography, process development

中图分类号:

TQ 028.8

马烨玮, 孙艳娜, 高栋, 王海彬, 姚善泾, 林东强. 模型辅助的单抗连续捕获工艺分析和过程优化[J]. 化工学报, 2024, 75(11): 4274-4285.

Yewei MA, Yanna SUN, Dong GAO, Haibin WANG, Shanjing YAO, Dongqiang LIN. Model-assisted process evaluation and optimization of continuous chromatography for antibody capture[J]. CIESC Journal, 2024, 75(11): 4274-4285.

图/表 11

图1 4C-PCC连续捕获流程

Fig.1 Operation mode of 4C-PCC continuous capture process

表1 洗脱和再生过程的工艺参数

Table 1 Operation parameters of recovery and regeneration process units

步骤	溶液	保留时间/min	体积/CV	时间/min
冲洗	0.025 mol/L Tris + 0.025 mol/L NaCl缓冲液（pH 7.7）	6	4	24
淋洗	0.5 mol/L磷酸盐缓冲液（pH 6.0）	6	5	30
平衡	0.025 mol/L Tris + 0.025 mol/L NaCl缓冲液（pH 7.7）	6	3	18
洗脱	0.15 mol/L醋酸缓冲液（pH 2.8）	6	4	24
再生	0.1 mol/L NaOH溶液	6	5	30
再平衡	0.025 mol/L Tris + 0.025 mol/L NaCl缓冲液（pH 7.7）	6	3	18

图2 模型计算与实验穿透曲线比较

Fig.2 Model calculated and experimental breakthrough curves

表2 蛋白A亲和层析模型参数汇总

Table 2 Model parameters for protein A affinity chromatography

模型参数	数值
ε	0.38
ε_p	0.52
Q_max/(g/L)	130
K_d/(g/L)	0.08
D_ax/(10^-7 m²/s)	30
k_f/(10^-6 m/s)	20
D_s/(10^-14 m²/s)	0.5
D_p/(10^-12 m²/s)	4.9

图3 CaptureSMB连续捕获过程性能比较［彩色等高线图为过程产率变化，黑色实线为介质利用率变化，红色虚线为上样保留时间（对应流速）上限，蓝色星点为最优操作点］

Fig.3 Process performance of CaptureSMB continuous capture（Color contour maps show the changes of productivity; Black-line contour maps show the changes of capacity utilization; Red dash lines represent the upper flow rate of the resin; Star points are the optimal operation points）

图4 3C-PCC连续捕获过程性能比较（彩色等高线图为过程产率变化，黑色实线为介质利用率变化，红色虚线阶段Ⅰ-1和阶段Ⅰ-2分界线，蓝色虚线为阶段Ⅱ和阶段Ⅲ分界线，蓝色星点为最优操作点）

Fig.4 Process performance of 3C-PCC continuous capture（Color contour maps show the changes of productivity; Black-line contour maps show the changes of capacity utilization; Red dash line represents the boundary of phase Ⅰ‍-1 and phase Ⅰ‍-2; Blue dash line represents the boundary of phase Ⅱ and phase Ⅲ; Star points are the optimal operation points）

图5 4C-PCC连续捕获过程性能比较（彩色等高线图为过程产率变化，黑色实线为介质利用率变化，红色虚线阶段Ⅰ-1和阶段Ⅰ-2分界线，蓝色虚线为阶段Ⅱ和阶段Ⅲ分界线，蓝色星点为最优操作点）

Fig.5 Process performance of 4C-PCC continuous capture（Color contour maps show the changes of productivity; Black-line contour maps show the changes of capacity utilization; Red dash line represents the boundary of phase Ⅰ-1 and phase Ⅰ-2; Blue dash line represents the boundary of phase Ⅱ and phase Ⅲ; Star points are the optimal operation points）

图6 不同操作模式下CU与Pmax*随上样浓度的变化情况

Fig.6 Capacity utilization and optimal productivities of batch, CaptureSMB, 3C-PCC and 4C-PCC at varying c0

图7 4C-PCC连续捕获的洗脱过程UV变化

Fig.7 UV curves of elution procedure of 4C-PCC continuous capture process

表3 批次与连续的分离效果比较

Table 3 Comparison of batch and continuous separation performance

项目	批次层析	连续捕获
纯度/%	97.3	99.3
收率/%	92.2	91.1
聚集体/%	2.2	0.7
HCP LRV	2.33	2.57
hcDNA LRV	4.04	2.36
过程产率/(g/(L·h))	11.17	14.21
介质利用率/%	59.9	89.9
缓冲液消耗/(L/g)	0.55	0.35

图8 模型辅助优化路线

Fig.8 Model-assisted optimization roadmap

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