Controlling the elution order of insulin and its analogs in sub‐/supercritical fluid chromatography using methanesulfonic acid and 18‐crown‐6 as mobile phase additives

Abstract

The purity analysis of therapeutic peptides can often be challenging, demanding the application of more than a single analytical technique. Supercritical fluid chromatography nowadays is a promising alternative to reversed‐phase liquid chromatography, providing orthogonal and complementary information. This study investigated its applicability for the separation of human insulin, its analogs and degradation products. A previously published method development protocol for peptides up to 2000 Da was successfully applied to the higher molecular weight insulins (6 kDa). A single gradient method was optimized for all insulins using a Torus DEA column (100 × 3.0 mm, 1.7 μm), carbon dioxide and a modifier consisting of methanol/acetonitrile/water/methanesulfonic acid (65:35:2:0.1, v/v/v/v). Consecutively, the crown ether 18‐crown‐6, which is well known to complex charged lysine sidechains and other amino functionalities, was added to the modifier to evaluate its impact on selectivity. A decreased retention and a shift in the elution order for the insulins were observed. An inverse effect on retention was found when combined with a neutral stationary phase chemistry (Viridis BEH).

Retention modeling of therapeutic peptides in sub‐/supercritical fluid chromatography

“…Thus, the terC/tG/T model is considered a suitable alternative model for SFC. A ternary modifier composition (carbon dioxide, MeOH, and ACN) improved the peptide fingerprint resolution in our previous works [17, 19]. In addition, the terC was found to alter the elution order of the analytes when combined with an aromatic SP (2‐PIC) and was now supplemented by a non‐aromatic SP (BEH).…”

“…In recent years, sub‐/supercritical fluid chromatography (SFC) extended its potential to the separation of complex biomolecules, such as peptides [17–19], providing orthogonal selectivity to RP‐LC [20–24]. Thus, the application of modeling software packages in SFC is desirable.…”

“…No data is available on the high‐modifier SFC mode used for peptide and biopharmaceutical separations. Previously, the differing composition of MeOH and acetonitrile (ACN) used as the modifier turned out to be an efficient tool to improve peak separation and to control the peak order of peptides when combined with varying SP chemistries providing π‐π or hydrogen bonding interactions [17, 19].…”

Advances in Therapeutic Peptides Separation and Purification

“…However, accurate predictions were successfully validated through verification experiments [73]. The authors subsequently applied their developed model and method development approach using design of experiments (DoE) principles to human serum albumin (HSA) and six analogues with molecular weights up to 6 kDa [74]. Their results from SFC provided sufficient resolution, suggesting it could serve as a potential alternative or complementary method for determining impurities in this mass range [74].…”