Development of a routine analysis method for liposome encapsulated recombinant interleukin-2

Area of Science:

• Analytical Chemistry
• Biochemistry
• Pharmaceutical Sciences

Background:

• Accurate quantification of therapeutic proteins like recombinant interleukin-2 (rIL-2) in complex delivery systems such as liposomes is crucial for drug development.
• Existing analytical methods may face challenges with phospholipid interference common in liposomal formulations.

Purpose of the Study:

• To develop and validate a robust isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method for routine analysis of rIL-2 in liposome samples.
• To integrate a lipid-extraction procedure to mitigate interference from phospholipids without compromising rIL-2 integrity.

Main Methods:

• Development of an isocratic RP-HPLC method using a C4 column at 30°C with a mobile phase of 52.5% acetonitrile in water, containing 100 mM NaClO4 and 10 mM HClO4.
• Incorporation of a lipid-extraction step prior to chromatographic analysis to remove interfering phospholipids.
• Validation of the method for linearity, precision, reproducibility, and robustness, including assessment of recovery and capacity factor (k').

Main Results:

• The developed method demonstrated linearity over a concentration range of 1-100 µg/ml for rIL-2.
• The method showed good precision with relative standard deviations (RSD) within approximately 5%, except for a higher intra-day RSD (17.8%) at 10 µg/ml.
• High recovery of rIL-2 (94±9%) was achieved throughout the liposome preparation and analysis process, with a capacity factor (k') of 5.8.

Conclusions:

• The developed and validated isocratic RP-HPLC method, coupled with lipid extraction, is suitable for the routine quantitation of rIL-2 in liposomal formulations.
• The method effectively addresses phospholipid interference, ensuring reliable and accurate analysis for quality control and development of rIL-2-based liposomal products.