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Table 9 Reversed-phase chromatography (RP) in proinsulin (PI) and insulin purification

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

Protein C4/C8/C18 Media Comments References
Renatured PI C4 Vydac C4 Equilibration: 4% ACN, 0.1% TFA
Elution with a linear gradient of increasing ACN (0.88%/min)
Purity: 90%
Cowley and Mackin (1997)
Renatured PI C4 Vydac C4 Buffer A = 0.1% TFA in water, Buffer B = 0.1% TFA in 20% water/80% acetonitrile
Linear gradient increasing to 60% B over 40 min
Mackin (1999)
Renatured PI Polystyrene-divinylbenzene matrix SOURCE 15RPC Buffer A = 0.1% TFA in water, B = 0.1% TFA in 80% acetonitrile/20% water
Gradient elution of 30–50% B over 50 min
60% recovery
Recovered 95–98% pure DKP-hPI
Mackin and Choquette (2003)
Insulin C8 C8 prep HT Purification of glargine insulin
Elution with a linear gradient of 15% to 36% ACN (0.88%/min)
Final product purity: 98.11% (1.96% desamido insulin)
Hwang et al. (2016)
Insulin C8 Kromasil C8 Dissolving buffer contains acetone or ACN
Elution with n-propanol in buffered solvent comprising zwitterions, e.g., glycine or betaine
Product is virtually free from proteases and insulin acetylated at position A9
Dickhardt and Unger (1997)
Insulin C8 Kromasil C8 Elution with a gradient of 0 ± 22% buffer B (50% isopropanol in water, 1.5 mS ammonium sulfate, pH 3.0) Mikiewicz et al. (2017)
Insulin C8 Kromasil C8 Equilibration: 30% ACN, 0.25% pentafluoropropionic acid (PFPA)
Elution with a gradient of 30% to 50% ACN, 0.25% PFPA
Yield: 54%
Nilsson et al. (1996)
Insulin C8 Kromasil C8 Elution with a linear gradient of isopropanol Watson et al. (2018)
Insulin C8 Zorbax Process grade C8 Load are partially purified human insulin zinc crystals
Elution in linear gradient of 0.25 M acetic acid (eluent A) to 60% ACN (eluent B)
Yield: 82%; Purity: 98.5%
Ideal pH is in the region 3.0 to 4.0, which is below the isoelectric point of 5.4
Acidic mobile phase provided resolution of insulin from structurally similar insulin-like components while promoting insulin solubility
Kroeff et al. (1989)
Insulin C18 ACE 5 C18-300 Equilibration: 0.2 M sodium sulfate pH 2.3 and ACN in ratio of 4.5:1
Elution with 0.2 M sodium sulfate pH 2.3 and ACN in ratio of 1:1
Zieliński et al. (2019)
Insulin C4/C8/C18 C4/C8/C18 Isocratic or a shallow gradient elution with ACN in the presence of 200 mM sodium sulfate and 0.16% phosphate Zimmerman and Stokell (2010)
Insulin C8/C18 Kromasil C8; Lichrospher Select B, C8; Zorbax Pro10, C8; Nucleosil C18; Nucleosil C18-P Elution with n-propanol/ethanol gradient in buffered solvent, in the presence of zwitterions, e.g., glycine, glutamic acid or glycine betaine
The solvent mixture is within about one pH unit above or below the isoelectronic point of the insulin or insulin derivative to be purified
Dickhardt et al. (1993)
Insulin NA Kromasil Mobile phase: ACN/ 0.2 M ammonium acetate buffer, pH 4
Gradient: 0 min: 22%, 60 min: 32% ACN
Co-elution of human insulin with impurity
Kromasil (2021)
Insulin NA NA Elution with 25% ACN, 1.5% acetic acid, 73.5% water
Yield: 95%
Petrides et al. (1995)
  1. NA information not available, ACN acetonitrile, TFA trifluoroacetic acid