Purpose: Superoxide dismutases 3 (SOD3) is a major extracellular antioxidant enzyme that catalyze the conversion of superoxide anion radicals into oxygen and hydrogen peroxide. The main role of SOD3 is to protect organs and tissues from oxidative stress, and, thus, plays a role in maintaining homeostasis and normal organ function. A many of studies have reported on the connection between SOD3 activity and expression levels and pathological conditions. Considering this pathological relevance, along with its function, stability, and mobility throughout the plasma, SOD3 could be a promising biopharmaceutical candidate for the treatment of various diseases. To support the early development of SOD3 as a biopharmaceutical, a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry method was developed and validated for the determination of recombinant human SOD3 (rhSOD3) levels in the plasma of ICR mice after the bolus intravenous administration.
Methods: Because rhSOD3 contains an N′-terminal poly histidine tag, the mouse plasma containing rhSOD3 and internal standard was firstly purified with a nickel (Ni)-based purification method. And then, purified rhSOD3 was suffered trypsin digestion to produce signature peptide for quantification. After purification with Ni-NTA magnetic beads and digestion with trypsin, SOD3 signature peptides and internal standard signature peptide (ISP) were separated via high performance liquid chromatography using a Zorbax C18 column (2.1 × 50 mm, 3.5 μm) and a mobile phase consisting of 10 mM ammonium formate, 0.1% formic acid, and acetonitrile. The analyte and ISP were detected via a tandem mass spectrometer in electrospray ionization and multiple reaction monitoring modes to select both the signature peptide for SOD3 at m/z 669 to 969 and the ISP at m/z 655 to 941 in the positive ion mode. Established analytical method was validated under the guidance of FDA and EMA validation guidance. Investigational validation parameters contained selectivity, precision, accuracy, matrix effect, purification recovery, process efficiency, and stability.
Results: The calibration curves were linear (r > 0.99) between 5 and 1,000 μg/mL with a lower limit of quantification of 5 μg/mL. The relative standard deviation ranged from 3.08 to 8.83% while the relative error ranged from -0.13 to -9.56%. Matrix effect, purification recovery, and process efficiency were met the acceptable criteria of FDA and EMA guidance. SOD3 was stable in mouse plasma at all experimental conditions, and stability.
Conclusion: A reliable bioanalytical method for the determination rhSOD3 in mouse plasma samples was developed and validated employing his-tag purification, tryptic digestion, and LC-MS/MS. This method was successfully applied to a pharmacokinetic study in mice for the first time and convenient owing to its efficient sample preparation.
Jong-Woo Jeong
– Research assistant, ChungNam National University, Yuseong-guJong-Woo Jeong
– Research assistant, ChungNam National University, Yuseong-guTae-Sung Koo
– ChungNam National UniversityTae-Sung Koo
– ChungNam National UniversityJong-Woo Jeong
– Research assistant, ChungNam National University, Yuseong-gu236 Views