Figure 6. To determine if multiple freeze/thaw cycles would affect the amount of PCSK9 measured in the assay, samples with drug:ligand at varying molar ratios were subjected to four freeze/thaw cycles. As shown in Figure 6 there was no significant change in the recovery of PCSK9 across the four freeze/thaw cycles for all samples tested. Three independent aliquots were analyzed for each sample and at each test point the CV s were all ≤ 5.3% demonstrating robustness of the data sets. Error bars within the graph indicate STD of the n = 3 independent measurements at each test point. The %bias from the mean ng/mL PCSK9 for each test sample was within ± 8.5% indicating there was no significant increase in free ligand. The CV for the calculated means across the test series were ≤ 5.8%.