Table 9.
Reversed-phase chromatography (RP) in proinsulin (PI) and insulin purification
| 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) |
NA information not available, ACN acetonitrile, TFA trifluoroacetic acid