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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (6): 38-44    DOI: 10.13523/j.cb.2102024
技术与方法     
连续流层析技术在亲和层析中的应用及生产放大评估
张玲(),曹小丹,杨海旭,李文蕾
天士力生物医药股份有限公司 天津 300410
The Application of Continuous Purification in Affinity Chromatography and Evaluation of Production Scale-up
ZHANG Ling(),CAO Xiao-dan,YANG Hai-xu,LI Wen-lei
Tasly Biophamaceuticals Co., Ltd., Tianjin 300410, China
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摘要：

生物制药行业迅速发展,尤其是上游表达量的增加和规模的扩大,促使上游培养采用连续灌流方式,同时也推动了下游纯化生产工艺相应的采取连续纯化策略。以灌流培养的Fc融合蛋白为例,采用 BioSMB PD设备,对比了下游工艺亲和层析捕获步骤中单柱批次纯化和连续流层析纯化的样品纯度和收率,并在此基础上进行小试工艺放大和生产实际用量成本计算评估。连续流层析实现了上游灌流培养与下游亲和层析连续化的可行性,工艺稳定,回收率与批次纯化接近,但相比批次纯化,生产效率明显提高,填料载量提高,同时填料使用效率提高,生产成本显著降低。
关键词： 亲和层析;  连续流层析;  批次纯化    
Abstract:

The rapid development of the biopharmaceutical industry, especially the increase in upstream expression and the expansion of scale, had promoted the perfusion culture for upstream. This also promoted the continuous purification strategy for the downstream purification process. In order to compare the purity and yield between batch purification and continuous purification, taking the perfusion of Fc fusion protein as an example, the experiment was carried out with BioSMB PD equipment, and on this basis, process scale-up and production cost evaluation were carried out. The continuous purification realized the continuity of perfusion culture and affinity chromatography. Compared with batch purification, the continuous purification process has many advantages. It can observably reduce the amount of the chromatography resin, improve the efficiency of packing, and reduce buffer used. The production efficiency is significantly improved. The production costs are significantly reduced.
Key words: Affinity chromatograph    Continuous purification    Batch purification
收稿日期: 2021-02-22 出版日期: 2021-07-06
ZTFLH:  Q819  
通讯作者: 张玲     E-mail: linglong121@126.com
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引用本文:

张玲,曹小丹,杨海旭,李文蕾. 连续流层析技术在亲和层析中的应用及生产放大评估[J]. 中国生物工程杂志, 2021, 41(6): 38-44.

ZHANG Ling,CAO Xiao-dan,YANG Hai-xu,LI Wen-lei. The Application of Continuous Purification in Affinity Chromatography and Evaluation of Production Scale-up. China Biotechnology, 2021, 41(6): 38-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2102024    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I6/38

图1  连续层析工艺开发系统(a)和环形系统运行(b)示意图
Step Buffer Volume Flow rate
/(mL/min)
loopback — 48.10 CV 3.4
Load Cell harvest 40.1 CV 2.8
Wash 1 20 mmol/L PB,150 mmol/L NaCl, pH 7.4 8.00 CV 2.5
Wash 2 20 mmol/L citric acid buffer, pH 5.5 5.00 CV 2.3
Elution 20 mmol/L citric acid buffer, pH 3.0 6.00 CV 2.5
CIP 0.1 mol/L NaOH 3.00 CV 1.0
Equilibration 20 mmol/L PB,150 mmol/L NaCl, pH 7.4 5.00 CV 2.5
表1  EXP1层析工艺流程
Step Buffer Volume Flow rate
/(mL/min)
loopback — 58.00 CV 7.5
Load Cell harvest 50.0 CV 6.5
Wash 1 20 mmol/L PB,150 mmol/L NaCl, pH 7.4 8.00 CV 3.6
Wash 2 20 mmol/L citric acid buffer, pH 5.5 5.00 CV 5.6
Elution 20 mmol/L citric acid buffer, pH 3.0 6.00 CV 6.6
CIP 0.1 mol/L NaOH 3.00 CV 2.8
Equilibration 20 mmol/L PB,150 mmol/L NaCl, pH 7.4 5.00 CV 5.5
表2  EXP2层析工艺流程
图2  穿透曲线图
图3  CadenceTM BioSMB 响应曲线
图4  洗脱层析图谱
Process contrast Batch EXP1
Cycle 1-3 		EXP2
Cycle 1-3 		EXP2
Cycle 4-6 		EXP2
Cycle 7-10 		EXP2
Cycle 11-13
Feed concentration/(mg/mL) 1.2 1.25 1.03 1.08 1.08 1.01
Load volume/mL 105 1 850 5 620 5 660 6 443 5 623
Column volume/mL 5 5 12 12 12 12
DBC/(mg/mL) 25 51 54 57 48 52
RT/min 3.7 3.7 3.7 3.7 3.7 3.7
Cycle(include washdown) 1 4 4 4 5 4
Number of columns 1 3 3 3 3 3
Yield/% 96.5 87.0 94.8 85.9 92.0 102.5
Purity/% 99.6 98.1-98.9 98.0-99.6 97.2-99.0 96.8-98.5 97.0-98.3
Aggregates/% 0.4 1.9 -1.1 0.4-2.0 1.0-2.8 2.5-3.2 2.7-3.0
Production efficiency/[g/(L·h)] 6.5 12.5 11 10.5 9.5 11.6
表3  批次层析与连续流层析结果分析
Process contrast Batch Continuous
Process of scale/L 400 400
Feed concentration/(g/L) 1.5 1.5
Process time/h 3.8×2 12
Number of columns 1柱×2批次 3柱×3批
Retention time/min 3.7 3.7
Dynamic binding capacity/(g/L) 25 51
Time of each cycle/h 3.8 4
Column diameter/cm 40 14
Column height/cm 9.5 8.5
Column volume/L 12 1.3
Total resin volume/L 12 4
Resin cost saving(one time)/% — -66.70
Resin cost saving(total)/% — -50
Buffer saving/% — -50
Production efficiency/[g/(L·h)] 6.5 12.5
Production efficiency multiple — 1.92×
表4  放大评估计算表
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