Insulin preparations are classified into six categories by type of action, including rapid-acting type, short-acting type, and long-acting type, and inlusilin aspart is a part of rapid-acting type. According to the United States Pharmacopeia and the National Formulary (USP40-NF35*), the European Pharmacopoeia (EP 9.0*), and the Japanese Pharmacopoeia (Supplement I, JP XVII*), analysis of high molecular weight proteins for "Insulin Aspart" should be carried out with a column filled with hydrophilic silica gel (USP-NF L20). The PROTEIN KW-802.5, a column filled with L20 packing material, confirmed the requirements were met.

System Suitability RequirementsUSP-NF (”LIMIT OF HIGH MOLECULAR WEIGHT PROTEINS" for GENERAL CHAPTERS "<121.1> PHYSICOCHEMICAL ANALYTICAL PROCEDURES FOR INSULINS"）
Retention times (Resolution solution for USP-NF)
  Polymeric insulin aspart complexes: 13 - 17 min
  Insulin aspart dimer: about 17.5 min
  Insulin aspart monomer: 18 - 22 min
  Salts: eluted after the insulin monomer
Peak-to-valley ratio** (Resolution solution for USP): ≥ 2.0

EP (Impurities with molecular masses greater than that of insulin aspart)
Retention times (Resolution solution for EP)
  Insulin aspart polymers: 13 - 17 min
  Insulin aspart dimer: about 17.5 min
  Insulin aspart monomer: about 20 min
  Salts: about 22 min
Peak-to-valley ratio** (Resolution solution for EP): ≥ 2.0

JP ((2) High-molecular proteins）
Peak area percentage of insulin aspart dimer peak obtained the two-fold Solution for system suitability test for JP: 80 - 120% of peak area percentage of insulin aspart dimer peak obtained Solution for system suitability test for JP
Retention times (Solution for system suitability test for JP)
  Insulin aspart polymers: 13 - 17 min
  Insulin aspart dimer: about 17.5 min
  Insulin aspart monomer: 18 - 20 min
Peak-to-valley ratio** (System suitability solution for JP): ≥ 2.0
Relative standard deviation (RSD) of peak area of insulin aspart monomer (Solution for system suitability test for JP): ≤ 2.0％ (repeated 6 times)

*The version at the time of the application acquisition.
**The ratio of the height of the dimer peak to the height of the valley between the dimer peak and the monomer peak.

Sample: 100 µL
(Solution for system suitability test) 4 mg/mL of Insulin aspart containing about 0.4 mg of high molecular  weight proteins in 0.01 M HCl aq. (prepared according to JP method)

1. 1.Insulin aspart dimer
2. 2.Insulin aspart monomer