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Table 8 Ion-exchange chromatography (IEX) in proinsulin (PI) and insulin purification

From: Downstream processing of recombinant human insulin and its analogues production from E. coli inclusion bodies

Protein AEX/CEX Media Comments References
Folded preproinsulin AEX DEAE-Sepharose fast flow/ Source 30 Q Flow-through mode at pH 8.3, 6.1 mS/cm, in which the preproinsulin was not bound to the gel but washed through the column with the permeate
The higher molecular weight impurities were adsorbed
Thurow et al. (2010)
Folded preproinsulin CEX Source 30 S Elution with linearly increasing NaCl gradient Thurow et al. (2010)
Refolded PI AEX Q-Sepharose Fast Flow Equilibration with 20 mM glycine–NaOH, pH 10.0
NaCl elution in 20 mM glycine–NaOH, pH 10.0
Chen et al. (2016)
Sulfonated PI AEX DEAP-Spheronit NaCl elution in 7.5 M urea
Yield: 95%
Purity: 70–95% (order of chromatography affects purity)
Tikhonov et al. (2001)
Sulfonated PI AEX Mono-Q HR Binding and elution buffers contain 7 M urea
NaCl elution
Cowley and Mackin (1997)
Sulfonated PI AEX Q-Sepharose Fast Flow NaCl elution in 8 M urea Castellanos-Serra et al. (1996)
Sulfonated PI CEX S-Sepharose Eluted at a rate of 3 mL/min using a linear gradient of 0.5 M NaCl in 7 M urea/20 mM formic acid buffer (pH 4.0) for 50 min Redwan et al. (2007)
Sulfonated PI CEX SP Sepharose Fast Flow NaCl elution
Yield: 90%
Petrides et al. (1995)
Insulin AEX DEAE Purification after citraconylation and trypsin digestion step Mikiewicz et al. (2017)
Insulin AEX DEAE-Sepharose Purification after citraconylation and trypsin digestion step
Elution with Tris pH 8.6 and 30% isopropanol (conductivity 6 mS)
Zieliński et al. (2019)
Insulin AEX Q Purification after citraconylation and trypsin digestion step Mikiewicz et al. (2017)
Insulin AEX Q-Sepharose Elution with Tris pH 8.6 and 30% isopropanol (conductivity 3 mS) Zieliński et al. (2019)
Insulin AEX Source 30Q The load is in 30% ethanol, pH 7.5, < 3 mS/cm and contains Zn-ions in an amount of 2 zinc atoms per six insulin molecules
Elution with ammonium acetate, ethanol, triethanolamine at pH 6.4, 6.8 and 7.2
At pH 6.4, the peak is fronting (flat front and steep tail), and a baseline separation is seen between the impurity and the product peak
Yield: > 90%
Mollerup and Frederiksen (2016)
Insulin CEX BioSepra CM Loading diluent and elution solution contain hexylene glycol
2 washes with NaCl
Isocratic elution with NaCl
Yield: 60–85%; Purity: > 90%
Coleman et al. (2019)
Insulin CEX Capto SP ImpRes Subsequent purification after enzymatic conversion
Binding buffer: Na acetate buffer pH 4, 10% ethanol
Elution with 47.5% ethanol and 128 mM NaCl
Yield: 102%; Purity: 63–94%
GE application note 29–0018-56 AB
Insulin CEX SP Sepharose Fast Flow Capture of glargine insulin
NaCl elution
Hwang et al. (2016)
Insulin NA NA NaCl elution
Yield: 95%
Petrides et al. (1995)
  1. NA information not available