Two-Dimensional Isoelectric Focusing OFFGEL, Micro-Fluidic Lab-on-Chip Electrophoresis and FTIR for Assessment of Long-Term Stability of rhG-CSF Formulation

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been increasingly recognized from among one of the most abundant families of biosimilars. Upon long-term storage, the rhG-CSF is subject to subtle chemical modifications that rapidly occur and, in particular, produce deaminated variants with divergent charge. Indeed, changes in charge from glutamine deamination may alter the way rhG-SCF will refold and the structure of resulting molecule. To assess this charge heterogeneity, 2-D gel electrophoresis has limited application. Recent micro-fluidic based technical advances offer a great alternative method to better control liquid volumes on a minute scale. Here, we used IEF OFFGEL-lab-on-chip electrophoresis for 2-D separation of the rhG-CSF peptides according to their isoelectric point (pI) and molecular weight (kDa). We used an rhG-CSF commercial therapeutic formulation, kept refrigerated 24 months after expiry. The samples were analyzed for particulate matter and charge variants. Subsequently, the secondary structure was assessed by FTIR spectroscopy and residual biological activity was recorded. Interestingly, we showed an additional band in the acidic gel area above and below the most intense protein band (fractions 10, 11, and 12 at 22.84s). This observation reveals the presence of the rhG-CSF variant charges without any additional high molecular weight impurity or biological activity decrease. We conclude that after two years of storage, the rhG-CSF solution maintained its native secondary structure with little β-sheet deviation, as reflected in the 1622 cm-1 and 1695 cm-1. These data demonstrated that a combined strategy is a more suitable and accurate analytical assessment of the rhG-CSF and recombinant protein-based biosimilars.