隨著生物科技產業的發展，追求高經濟效益的趨勢也逐年成長，廣泛應用在傳統產業的連續式生產與操作也開始被套用在生技製造上。相較於傳統的批次式生產，連續式操作提供穩定的生產與更高的產能。以生技產業下游純化工程來說，連續式的純化流程能夠縮小既有的設備體積，並同時生產多樣化高經濟價值的生技製劑，除了能降低工廠的生產成本，還能應用於個人化治療平台的開發。在本項研究中，我們以單元操作的觀念為基礎建構連續式蛋白質純化平台，透過微流體裝置的技術設計多向閥系統來控制流體流向，藉此提升連續式的操作程序。
我們透過繪圖軟體設計裝置原型，並以先進微加工技術結合雷射切割與逐層建構技術製作出多重材料結合之微流體裝置。此純化平台主要包含：多層流向控制閥，多功能儲存槽，純化層析管柱。流體控制系統是由微型蠕動幫浦與閥控制裝置所構成。各層析管柱在平台中能同時進行不同的純化步驟，純化後的產物則依序蒐集自各管柱，透過微流閥裝置切換各管柱之純化步驟可以簡化操作流程並達到連續化的生產。各項純化裝置與元件可手動組裝成可攜帶式大小的平台。
首先，我們透過有色墨水來模擬純化步驟與微流閥的操作效果，證明系統的可行性。而後再將多層析管柱平台的連續式純化結果和單一管柱純化結果相比較並用凝膠電泳來分析蛋白質的純度。為了達到蛋白質純化的即時監測，我們首次使用AvaSpec連續式探測光譜儀做初步的測試，針對波長595奈米與280奈米進行訊號的量測與蛋白的定量。未來會評估蛋白質純化後的回收率並加以改良現有裝置並結合自動化系統。我們期許此純化平台能結合更多下游工程技術以利產線多樣化。

The development of a continuous process for protein purification is growing rapidly due to the current trend of cost-effective manufacturing in biological industries. The continuous purification process has a more stable operation and higher volumetric productivity compared to the conventional batch process. Additionally, the integration of continuous purification process can reduce the equipment size and produce more diverse and high values of proteins, eventually leading to the personalized therapy application. To achieve this goal, the implementation of a novel unit-operation concept is a key principle to develop an innovative purification process. In this study, we demonstrated a microfluidic multi-positions valves system to improve continuous operation. The highly integrated microfluidic-based platform was fabricated using a rapid prototyping technique based on the combination of laser subtractive manufacturing and lamination additive manufacturing. The switching valves and microfluidic channels were designed using Solid Edge 2D software and patterned using laser cutter on acrylic, polycarbonate and polyethylene terephthalate sheets. Each layer was aligned by aligned gigs and followed by assembling using biocompatible tapes to complete the purification system. The system mainly included four parts: (1) a multi-flow control device with fluid networks; (2) a multi-functional buffer reservoir; (3) customized peristaltic pumps; (4) four chromatography columns. The purification columns can be operated simultaneously after the integration of microfluidic valve controlling platform. Each column went through four steps: loading, washing, elution, and regeneration alternatively at the same time. Every unit was connected by pharmaceutical silicon tubes and was driven by computer-controlled pumps to simplify operation procedures. The multi-position valves provided multi-functional control for uninterrupted purification. Moreover, every single component can be connected and combined into a shoe-box sized platform allowing the system to be portable. The four-column purification results were comparable to the single-column operation process. For real-time protein measurement, AvaSpec absorbance system was connected to the output of chromatography column. Preliminary results showed the potential of this absorbance applied to this research. The purification system can be fully automatic in the future by integrating automatic controllers and intelligent analysis. This multi-position valves platform may become a powerful tool to control other downstream purification processes (e.g. ion exchange, ultrafiltration, dialysis filtration) potentially suitable for diverse product pipelines.

1. Chaudhary, R.S., A. Pazhayattil, and J. Spes, Continuous Manufacturing: A Generic Industry Perspective. 2017.
2. Warikoo, V., et al., Integrated continuous production of recombinant therapeutic proteins. Biotechnol Bioeng, 2012. 109(12): p. 3018-29.
3. Godawat, R., et al., End-to-end integrated fully continuous production of recombinant monoclonal antibodies. J Biotechnol, 2015. 213: p. 13-9.
4. Estes, K.A. and E.J.B.I. Longer, Update on Continuous Bioprocessing: From the Industry's Perception to Reality. 2017. 30(6): p. 10-12.
5. Cramer, S.M. and M.A. Holstein, Downstream bioprocessing: recent advances and future promise. Current Opinion in Chemical Engineering, 2011. 1(1): p. 27-37.
6. Gerontas, S., et al., Integration of scale-down experimentation and general rate modelling to predict manufacturing scale chromatographic separations. J Chromatogr A, 2010. 1217(44): p. 6917-26.
7. Mahajan, E., A. George, and B. Wolk, Improving affinity chromatography resin efficiency using semi-continuous chromatography. J Chromatogr A, 2012. 1227: p. 154-62.
8. Godawat, R., et al., Periodic counter-current chromatography—Design and operational considerations for integrated and continuous purification of proteins. Vol. 7. 2012.
9. Zhang, C., et al., Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography. Protein Expr Purif, 2016. 128: p. 29-35.
10. Perez-Pinera, P., et al., Synthetic biology and microbioreactor platforms for programmable production of biologics at the point-of-care. Nat Commun, 2016. 7: p. 12211.
11. Kitson, P.J., et al., Digitization of multistep organic synthesis in reactionware for on-demand pharmaceuticals. 2018. 359(6373): p. 314-319.
12. MICROFLUIDICS: A GENERAL OVERVIEW OF MICROFLUIDICS. Available from: https://www.elveflow.com/microfluidic-tutorials/microfluidic-reviews-and-tutorials/microfluidics/.
13. Niimi, M., et al., Virus purification and enrichment by hydroxyapatite chromatography on a chip. Sensors and Actuators B: Chemical, 2014. 201: p. 185-190.
14. Whitesides, G.M. and A.D.J.P.T. Stroock, Flexible methods for microfluidics. 2001. 54(6): p. 42-48.
15. Klank, H., J.P. Kutter, and O. Geschke, CO(2)-laser micromachining and back-end processing for rapid production of PMMA-based microfluidic systems. Lab Chip, 2002. 2(4): p. 242-6.
16. Li, H., et al., Fabrication of polystyrene microfluidic devices using a pulsed CO 2 laser system. 2012. 18(3): p. 373-379.
17. Bonner, P., Protein purification. 2007: Taylor & Francis.
18. Bornhorst, J.A. and J.J. Falke, [16] Purification of Proteins Using Polyhistidine Affinity Tags. Methods in enzymology, 2000. 326: p. 245-254.
19. NTA vs. IDA: a tale of two ligands. 2013; Available from: https://cube-biotech.com/background-tips-and-tricks/nta-vs-ida.
20. Protino® technology and products. 2018; Available from: http://www.mn-net.com/Support/Technologies/Protinotechnologyandproducts/tabid/10755/language/en-US/Default.aspx.
21. Harrison, R.G., et al., Bioseparations science and engineering. 2015: Oxford University Press, USA.
22. Pace, C.N., et al., How to measure and predict the molar absorption coefficient of a protein. 1995. 4(11): p. 2411-2423.
23. Spectrophotometry.
: GE Healthcare Life Science.
24. Enlightening Spectroscopy. Available from: https://www.avantes.com/.