Twice-daily dosing results in near-complete target coverage over the dosing interval. BTK target occupancy enzyme-linked immunosorbent assay was performed on PBMC lysates using a biotin-tagged covalent analog of acalabrutinib as a molecular probe. Drug trough was 12 or 24 hours after dosing for the twice-daily (BID) and once-daily (QD) regimens, respectively. Box and whiskers show the median using the Tukey method for whisker range. Statistical significance was determined using an unpaired, parametric, 2-tailed Student t test.