Size exclusion chromatography as a characterization tool for quality control of marine biopharmaceutical keyhole...

Keyhole limpet hemocyanin is a large metalloprotein present in hemolymph of the mollusk Megathura crenulata. This protein is highly immunogenic, making it very attractive to the biopharmaceutical industry, where it is used as a carrier protein for haptens, an adjuvant, or as a stand-alone biologic for the treatment of various cancer diseases. The structural complexity of such immunogenic protein makes its characterization and quality control very challenging. Here, we developed, optimized and validated a size exclusion chromatography approach for quality control of keyhole limpet hemocyanin products. The separation procedure was performed on an Acquity UPLC BEH450 SEC column with pore size of 450 Å in a mobile phase composed of 25 mmol L^{- 1} phosphate buffer (pH 6.8) with 250 mmol L^{- 1} NaCl. These conditions led to appropriate elution of the analysed protein. The developed method showed very promising values of validation parameters, such as coefficient of de termination (R ² = 0.9997), sensitivity (limit of detection 10.21 μg mL-1, limit of quantitation 31.25 μg mL^{- 1}), precision (repeatability 0.3-2.5 %, intermediate precision 1.1-4.2 %), and accuracy (100.4-102.8 %). The developed method was used to determine the content of keyhole limpet hemocyanin in three commercially available samples obtained from various suppliers. The determined content ranged from 20.08 to 22.91 mg per vial.