Separation and Purification Process of Recombinant Human Interferon α-2b by Reversed Phase Chromatography

doi:10.13523/j.cb.2301034

China Biotechnology

2023, Vol. 43

Issue (9): 9-18 DOI: 10.13523/j.cb.2301034

Separation and Purification Process of Recombinant Human Interferon α-2b by Reversed Phase Chromatography

WANG Wen-bo¹,LI Xiu-nan²,ZHAO Da-wei²,ZHANG Song-ping²,ZHANG Wan-zhong^1,^**(

),CHEN Chao^2,^**(

)

1 College of Pharmaceutical and Biological Engineering, Shenyang University of Chemical Technology, Shenyang 110142, China
2 State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

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Abstract

The 2020 edition of the Chinese Pharmacopoeia proposed new quality control standards for recombinant human interferon α-2b, increased the analysis and detection of related proteins and clarified the upper limit of related proteins. However, the related proteins are only slightly different from recombinant human interferon α-2b in structure, and it is difficult to remove them by conventional separation methods. A reversed phase chromatographic purification process of recombinant human interferon α-2b was established in this paper to separate recombinant human interferon α-2b from related proteins. First, the reversed-phase chromatographic packing and the concentration of acetonitrile in the eluent and the loading amount were screened and optimized. Next, the reversed-phase chromatographic separation and purification process of recombinant human interferon α-2b was determined, which include chromatographic packing: HPLCONE-10C18C3 (300 ?, 10 μm); elution conditions: 0.1% TFA-48% acetonitrile aqueous solution equivalent elution; loading amount: no more than 1.6 times of column volume; detection wavelength: 210 nm. Second, the scale up test was carried out. The results showed that the prepared recombinant human interferon α-2b stock solution, with high activity and purity greater than 99%, met the quality control standards of the 2020 edition of the Chinese Pharmacopoeia. The process has the advantages of good separation results, low cost, simple preparation technology and easy commercial production, which provides a valuable reference for the production enterprises of interferons and other biological macromolecular drugs.

Key words： Recombinant human interferon alpha-2b Purification Reverse phase chromatography Pilot-scale

Received: 19 January 2023 Published: 08 October 2023

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	Chao CHEN

Cite this article:

WANG Wen-bo, LI Xiu-nan, ZHAO Da-wei, ZHANG Song-ping, ZHANG Wan-zhong, CHEN Chao. Separation and Purification Process of Recombinant Human Interferon α-2b by Reversed Phase Chromatography. China Biotechnology, 2023, 43(9): 9-18.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2301034 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I9/9

Table 1 The packings used in reversed-phase chromatography

Table 2 Procedure for gradient elution in recombinant human interferon α-2b related protein

Table 3 Separation and purification of recombinant human interferon α-2b by different types of reversed phase chromatographic packings

Table 4 Screening of different acetonitrile concentration eluates for reverse phase chromatography of recombinant human interferon α-2b

Fig.1 Relationship between capacity factor and resolution (a)and relationship between resolution and peak width (b) of reversed-phase chromatography of recombinant human interferon α-2b eluted with different concentrations of acetonitrile

Fig.2 Reverse phase chromatography of recombinant human interferon α-2b eluting at different acetonitrile concentrations Acetonitrile concentration(a)40%,(b)44%,(c)48%,(d)52%,(e)56%,(f)60%.Ⅰ:Methionine oxidized recombinant human interferon α-2b;Ⅱ:Recombinant human interferon α-2b;Ⅲ:Intermediate

Table 5 Optimization of loading amount of recombinant human interferon α-2b

Table 6 Pilot-scale process validation results

Fig.3 Initial purification before reverse phase chromatography ( a ) and the recombinant human interferon α-2b stock solution after reverse phase chromatography ( b ) were analyzed by HPLC


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