• •
马烨玮1(), 孙艳娜1, 高栋2, 王海彬2, 姚善泾1, 林东强1()
收稿日期:
2024-05-30
修回日期:
2024-07-15
出版日期:
2024-07-16
通讯作者:
林东强
作者简介:
马烨玮(1999—),女,硕士研究生,mayewei2022@163.com
基金资助:
Yewei MA1(), Yanna SUN1, Dong GAO2, Haibin WANG2, Shanjing YAO1, Dongqiang LIN1()
Received:
2024-05-30
Revised:
2024-07-15
Online:
2024-07-16
Contact:
Dongqiang LIN
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。
中图分类号:
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, DOI: 10.11949/0438-1157.20240583.
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, DOI: 10.11949/0438-1157.20240583.
步骤 | 溶液 | 保留时间(min) | 体积(CV) | 时间(min) |
---|---|---|---|---|
冲洗 | 0.025 M Tris + 0.025 M NaCl 缓冲液(pH 7.70) | 6 | 4 | 24 |
淋洗 | 0.5 M 磷酸盐缓冲液(pH 6.00) | 6 | 5 | 30 |
平衡 | 0.025 M Tris + 0.025 M NaCl 缓冲液(pH 7.70) | 6 | 3 | 18 |
洗脱 | 0.15 M 醋酸缓冲液(pH 2.80) | 6 | 4 | 24 |
再生 | 0.1 M NaOH 溶液 | 6 | 5 | 30 |
再平衡 | 0.025 M Tris + 0.025 M NaCl 缓冲液(pH 7.70) | 6 | 3 | 18 |
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 M Tris + 0.025 M NaCl 缓冲液(pH 7.70) | 6 | 4 | 24 |
淋洗 | 0.5 M 磷酸盐缓冲液(pH 6.00) | 6 | 5 | 30 |
平衡 | 0.025 M Tris + 0.025 M NaCl 缓冲液(pH 7.70) | 6 | 3 | 18 |
洗脱 | 0.15 M 醋酸缓冲液(pH 2.80) | 6 | 4 | 24 |
再生 | 0.1 M NaOH 溶液 | 6 | 5 | 30 |
再平衡 | 0.025 M Tris + 0.025 M NaCl 缓冲液(pH 7.70) | 6 | 3 | 18 |
图2 模型计算与实验穿透曲线比较(a)不同上样浓度;(b)不同保留时间
Fig.2 Model calculated and experimental breakthrough curves at varying load concentrations (a) and varying residence times (b)
模型参数 | 参数值 |
---|---|
ε | 0.38 |
εp | 0.52 |
qmax (g/L) | 130 |
kd (g/L) | 0.08 |
Dax (10-7 m2/s) | 30 |
kf (10-6 m2/s) | 20 |
Ds (10-14 m2/s) | 0.5 |
Dp (10-12 m2/s) | 4.9 |
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 | 参数值 |
---|---|
ε | 0.38 |
εp | 0.52 |
qmax (g/L) | 130 |
kd (g/L) | 0.08 |
Dax (10-7 m2/s) | 30 |
kf (10-6 m2/s) | 20 |
Ds (10-14 m2/s) | 0.5 |
Dp (10-12 m2/s) | 4.9 |
图3 CaptureSMB连续捕获过程性能比较注: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(a) c0=0.5 g/L; (b) c0=6 g/L; (c) c0=15 g/L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
图4 3C-PCC连续捕获过程性能比较注: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(a) c0=0.5 g/L; (b) c0=3 g/L; (c) c0=10 g/L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
图5 4C-PCC连续捕获过程性能比较注: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(a) c0=1 g/L; (b) c0=5 g/L; (c) c0=10 g/L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
图6 不同操作模式下CU与Pmax*随上样浓度的变化情况(a) CU; (b) Pmax*
Fig.6 Capacity utilization and optimal productivities of batch, CaptureSMB, 3C-PCC and 4C-PCC at varying c0
批次 | 连续 | |
---|---|---|
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-1·h-1) | 11.17 | 14.21 |
介质利用率/ % | 59.9 | 89.9 |
缓冲液消耗/ (L·g-1) | 0.55 | 0.35 |
表3 批次与连续的分离效果比较
Table 3 Comparison of batch and continuous separation performance
批次 | 连续 | |
---|---|---|
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-1·h-1) | 11.17 | 14.21 |
介质利用率/ % | 59.9 | 89.9 |
缓冲液消耗/ (L·g-1) | 0.55 | 0.35 |
1 | David L, Schwan P, Lobedann M, et al. Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies[J]. Biotechnology and Bioengineering, 2020, 117(4): 1024-1036. |
2 | Mahal H, Branton H, Farid S S. End-to-end continuous bioprocessing: Impact on facility design, cost of goods, and cost of development for monoclonal antibodies[J]. Biotechnology and Bioengineering, 2021, 118(9): 3468-3485. |
3 | Rathore A S, Thakur G, Kateja N. Continuous integrated manufacturing for biopharmaceuticals: A new paradigm or an empty promise?[J]. Biotechnology and Bioengineering, 2023, 120(2): 333-351. |
4 | 鲁伟, 应国清, 杨晓明, 等. 单克隆抗体工业生产中蛋白A亲和层析步骤的成本分析[J]. 高校化学工程学报, 2023, 37(2): 276-284. |
Lu W, Ying G Q, Yang X M, et al. Cost analysis of protein A affinity chromatography in industrial production of monoclonal antibody[J]. Journal of Chemical Engineering of Chinese Universities, 2023, 37(2): 276-284. | |
5 | FDA. Quality considerations for continuous manufacturing[EB/OL]. [2024-05-30]. . |
6 | ICH. Continuous manufacturing of drug substances and drug products Q13[EB/OL]. [2024-05-30]. . |
7 | 史策, 虞骥, 高栋, 等. 单抗制备的过程模拟和经济性分析[J]. 化工学报, 2018, 69(7): 3198-3207. |
Shi C, Yu J, Gao D, et al. Process simulation and economic evaluation of monoclonal antibody production[J]. CIESC Journal, 2018, 69(7): 3198-3207. | |
8 | Bielser J M, Wolf M, Souquet J, et al. Perfusion mammalian cell culture for recombinant protein manufacturing – A critical review[J]. Biotechnology Advances, 2018, 36(4): 1328-1340. |
9 | Matanguihan C, Wu P. Upstream continuous processing: recent advances in production of biopharmaceuticals and challenges in manufacturing[J]. Current Opinion in Biotechnology, 2022, 78: 102828. |
10 | 高宗晔, 史策, 姚善泾, 等. 双柱连续流层析亲和分离抗体的过程设计与应用[J]. 高校化学工程学报, 2019, 33(1): 117-127. |
Gao Z Y, Shi C, Yao S J, et al. Process design and application of twin-column continuous chromatography for antibody affinity separation[J]. Journal of Chemical Engineering of Chinese Universities, 2019, 33(1): 117-127. | |
11 | Nicoud R M. The amazing ability of continuous chromatography to adapt to a moving environment[J]. Industrial & Engineering Chemistry Research, 2014, 53(10): 3755-3765. |
12 | 荆淑莹, 史策, 姚善泾, 等. 连续流层析及用于抗体分离的新进展[J]. 高校化学工程学报, 2021, 35(1):1-12. |
Jing S Y, Shi C, Yao S J, et al. Progress on continuous chromatography and its application in antibody separation[J]. Journal of Chemical Engineering of Chinese Universities, 2021, 35(1):1-12. | |
13 | ChromaCon. Contichrom CUBE – versatile benchtop process development tool[EB/OL]. [2024-05-30]. . |
14 | Cytica. ÄKTA pcc and BioProcess pcc continuous chromatography systems[EB/OL]. [2024-05-30]. . |
15 | Sartorius. Continuous Chromatography – BIOSMB PD and BIOSMBProcess[EB/OL]. [2024-05-30]. . |
16 | Novasep. BioSC continuous chromatography for biologics[EB/OL]. [2024-05-30]. . |
17 | Semba. Octave 10 Chromatography System[EB/OL]. [2024-05-30]. . |
18 | Mahajan E, George A, Wolk B. Improving affinity chromatography resin efficiency using semi-continuous chromatography[J]. Journal of Chromatography A, 2012, 1227: 154-162. |
19 | Angarita M, Müller-Späth T, Baur D, et al. Twin-column CaptureSMB: A novel cyclic process for protein A affinity chromatography[J]. Journal of Chromatography A, 2015, 1389: 85-95. |
20 | Gao Z Y, Zhang Q L, Shi C, et al. Antibody capture with twin-column continuous chromatography: Effects of residence time, protein concentration and resin[J]. Separation and Purification Technology, 2020, 253: 117554. |
21 | Lin D Q, Zhang Q L, Yao S J. Model-assisted approaches for continuous chromatography: Current situation and challenges[J]. Journal of chromatography A, 2021, 1637: 461855. |
22 | Shi C, Chen X J, Jiao B, et al. Model-assisted process design for better evaluation and scaling up of continuous downstream bioprocessing[J]. Journal of Chromatography A, 2022, 1683: 463532. |
23 | Narayanan H, Luna M, Sokolov M, et al. Hybrid models based on machine learning and an increasing degree of process knowledge: Application to capture chromatographic step[J]. Industrial & Engineering Chemistry Research, 2021, 60(29): 10466-10478. |
24 | Chopda V, Gyorgypal A, Yang O, et al. Recent advances in integrated process analytical techniques, modeling, and control strategies to enable continuous biomanufacturing of monoclonal antibodies[J]. Journal of Chemical Technology & Biotechnology, 2022, 97(9): 2317-2335. |
25 | Guo J, Jin M, Kanani D. Optimization of single-column batch and multicolumn continuous protein A chromatography and performance comparison based on mechanistic model[J]. Biotechnology Journal, 2020, 15(10): e2000192. |
26 | Shi C, Zhang Q L, Jiao B, et al. Process development and optimization of continuous capture with three-column periodic counter-current chromatography[J]. Biotechnology and Bioengineering, 2021, 118(9): 3313-3322. |
27 | Shi C, Gao Z Y, Zhang Q L, et al. Model-based process development of continuous chromatography for antibody capture: A case study with twin-column system[J]. Journal of Chromatography A, 2020, 1619: 460936. |
28 | Sun Y N, Shi C, Zhang Q L, et al. Model-based process development and evaluation of twin-column continuous capture processes with Protein A affinity resin[J]. Journal of Chromatography A, 2020, 1625: 461300. |
29 | Sun Y N, Shi C, Zhang Q L, et al. Comparison of Protein A affinity resins for twin-column continuous capture processes: Process performance and resin characteristics[J]. Journal of Chromatography A, 2021, 1654: 462454. |
30 | Sun Y N, Shi C, Zhong X Z, et al. Model-based evaluation and model-free strategy for process development of three-column periodic counter-current chromatography[J]. Journal of Chromatography A, 2022, 1677: 463311. |
31 | 史策. 模型辅助的连续流层析过程开发和抗体分离应用研究[D]. 杭州: 浙江大学, 2021. |
Shi C. Model-assisted process development of continuous chromatography and its applications for antibody separation[D]. Hangzhou: Zhejiang University, 2021. |
[1] | 刘克润, 于源. 涡流空气分级流场中团聚体破碎规律研究[J]. 化工学报, 2024, (): 1-11. |
[2] | 苏彬, 董浩伟, 罗振敏, 邓军, 王涛, 程方明. 气粉两相体系爆炸动力学特性及机理研究进展[J]. 化工学报, 2024, 75(6): 2109-2122. |
[3] | 代艳辉, 熊启钊, 房强, 杨东晓, 王毅, 陈杨, 李晋平, 李立博. 原位蒸汽辅助法用于一步制备多级孔Cu-BTC[J]. 化工学报, 2024, (): 1-9. |
[4] | 顾天宇, 陈献富, 王思琪, 徐鹏, 邱鸣慧, 范益群. 膜技术在杜仲有效成分分离纯化中的应用研究进展[J]. 化工学报, 2024, (): 1-15. |
[5] | 唐宇昊, 张迎迎, 赵智伟, 鲁梦悦, 张飞飞, 王小青, 杨江峰. 弱极性超微孔Sc/In-CPM-66A用于CH4/N2吸附分离性能的研究[J]. 化工学报, 2024, (): 1-9. |
[6] | 赵光耀, 杨明磊, 钱锋. 基于降方差采样策略的随机重构法[J]. 化工学报, 2024, 75(5): 1939-1950. |
[7] | 张文焱, 刘浩, 宋伟龙, 赵频, 王新华. 不同粒径UiO-66混掺改性TFN-FO膜的构建及性能评价[J]. 化工学报, 2024, 75(5): 1920-1928. |
[8] | 刘帆, 张芫通, 陶成, 胡成玉, 杨小平, 魏进家. 歧管式射流微通道液冷散热性能[J]. 化工学报, 2024, 75(5): 1777-1786. |
[9] | 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. |
[10] | 张梦婷, 王书林, 桑熙, 元兴昊, 徐刚. 人工Cu-TM1459金属酶催化不对称迈克尔加成反应[J]. 化工学报, 2024, (): 1-12. |
[11] | 刘亚超, 谭晓杰, 李旭东, 王瑞, 王慧, 韩璇, 赵青山. DES合成高活性CoCO3纳米片及析氧反应性能研究[J]. 化工学报, 2024, (): 1-10. |
[12] | 周文轩, 刘珍, 张福建, 张忠强. 高通量-高截留率时间维度膜法水处理机理研究[J]. 化工学报, 2024, (): 1-12. |
[13] | 曹佳蕾, 孙立岩, 曾德望, 尹凡, 高子翔, 肖睿. 双流化床化学链制氢反应器的数值模拟[J]. 化工学报, 2024, (): 1-10. |
[14] | 张颂红, 赵欣怡, 楼小玲, 沈绍传, 贠军贤. 阳离子交换纳晶胶分离乳过氧化物酶的研究[J]. 化工学报, 2024, (): 1-9. |
[15] | 童永祺, 程杰, 林海, 陈曦, 赵海波. 10MWth化学链燃烧反应装置的CPFD模拟[J]. 化工学报, 2024, (): 1-10. |
阅读次数 | ||||||
全文 |
|
|||||
摘要 |
|
|||||