Fig 2. Mean serum concentrations of WT anti-PCSK9 antibody (F0016A) and the Fc variants (F0016B, C, D) versus time after a single intravenous dose of 5 mg/kg to cynomolgus monkeys.

First, 96-well plates were coated with mouse anti-His tag antibody at 1:300 dilution for 18–24 h at 2–8°C. Recombinant human PCSK9 with a His₆ tag was added at 3μg/mL and incubated for 45 ± 5 min at 37°C. Serially diluted standards (0.625–60 ng/mL) as well as diluted cynomolgus monkey serum samples (minimum 1:40 dilution, day 1 ~ day 31) were added and incubated for 60 ± 5 min at 37°C. Bound drug was detected by incubation with HRP-conjugated sheep anti-human IgG Ab for 2 h ± 5 min at 37°C. PCSK9 mAb concentrations were calculated using a four or five parameter logistic fit regression model by SoftMax Pro 6.2.1. The PK assay was validated, the limit of detection (LOD) was 0.625 ng/ml, the lower limit of quantitation (LLOQ) was 1.25ng/ml, and the upper limit of quantitation (ULOQ) was 40 ng/ml. Points, mean of two animals. Points, mean of two animals.