Table 1.
SEC-HPLC system conditions.
Table 2.
Summary of FFF-MALS system conditions.
Table 3.
SEC-HPLC test results of AMP-glucagon and ELI-glucagon.
Fig 1.
A representative SEC-HPLC chromatogram for expired AMP-glucagon: A dimer peak was observed at approximately 5.8 minutes.
Fig 2.
A representative SEC-HPLC chromatogram for recently released AMP- glucagon: No dimer peaks were detected.
Fig 3.
A representative SEC-HPLC chromatogram for expired ELI-glucagon: A dimer peak was observed at approximately 5.8 minutes.
Fig 4.
A representative SEC-HPLC chromatogram for non-expired ELI-glucagon: A dimer peak was observed at approximately 5.8 minutes.
Table 4.
Equivalence evaluation of aggregation of AMP-glucagon vs. ELI-glucagon By SEC-HPLC.
Fig 5.
Typical chromatograms of LS2 signal for glucagon samples: No peak differences were observed between new (a, b) and old (c, d) lots in the two glucagon products.
Fig 6.
A Representative FFF chromatogram for AMP-glucagon: The molecular weight of glucagon was approximately 3,504 Da.
Fig 7.
A representative FFF chromatogram for ELI-glucagon. The molecular weight of glucagon was approximately 3,720 Da.
Table 5.
Glucagon samples’ weighted-average molar masses by FFF/UV-MALS.
Table 6.
BSA standard sample MW summary.
Fig 8.
Detection limit of glucagon samples: A reconstituted glucagon sample was diluted to 0.1% and 0.5% of its original concentration.
For the 0.1%, the UV, LS2, and RID, no glucagon signal was observed at 7 minutes. For the 0.5%, a very low signal was found at 7 minutes, indicating that the FFF-MALS test is unable to detect glucagon at contents lower than 0.5%.
Fig 9.
Detection limit of BSA (positive control): The 2 mg/mL BSA sample was diluted to 0.4 and 0.2 mg/mL as positive control samples which is equivalent to 10% and 5% of glucagon sample loaded respectively.
The BSA peak should appear at 13–16 minutes based on the UV signals.