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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2021, Vol. 41 Issue (6): 38-44    DOI: 10.13523/j.cb.2102024
    
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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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     
Received: 22 February 2021      Published: 06 July 2021
ZTFLH:  Q819  
Corresponding Authors: Ling ZHANG     E-mail: linglong121@126.com
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Ling ZHANG
Xiao-dan CAO
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Wen-lei LI
Cite this article:

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.

URL:

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

Fig.1 CadenceTM BioSBM PD system(a)and example of a circular chronogram(b)
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
Table 1 EXP1 chromatography process
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
Table 2 EXP2 chromatography process
Fig.2 Breakthrough curve of the resin
Fig.3 Required Contact Time in a BioSMB Process
Fig. 4 Elution profiles in the BioSMB system (a) EXP1 Cycle 1-3 (b) EXP2 Cycle 1-3 (c) Cycle 4-6 (d) Cycle 7-10 (e) Cycle11-13 The graph shows every 3 cycles of each figure
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
Table 3 Analysis results of batch chromatography and continuous chromatography compare
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×
Table 4 Scale-up evaluation calculation table
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