The five selected anti-IDs were labeled with biotin and Sulfo-tag and used as both capture and detection reagent in the next screening step (Figure ). These labeled reagents were used to determine the best pairing for a PK assay resulting in 25 different possible pairing combinations. Additionally, a generic mouse anti-human IgG4 was used as a detection reaction in combination with the 5 different capture reagents for a total of 30 pairing combinations. A standardized plate map was again utilized to enable use of automated liquid handlers and the automated scoring tool (Figure ). The anti-ID screening for PK assay development has historically been done on a 'fit-for-purpose approach' wherein the anti-IDs are typically screened in a pre-defined assay format at a single concentration . Once the reagent pair is identified by the pre-defined assay format, other assay formats are tested to further optimize the performance of the assay. However, a limitation exists, in that the best reagent pair selected by the predefined assay format is not necessarily the best pair of reagents for other assay conditions. Given the interdependence of reagents combination, concentration and assay formats, a more comprehensive approach was developed with 30 anti-ID pairings that were tested with two different concentrations (0.25 µg/mL and 1.0 µg/mL) in six different PK assay formats (Figure ). These pairings of anti-IDs were screened across >2 orders of magnitude of the mAb +/-soluble antigen. A second scoring system was developed to determine both the best pairing and optimal PK assay format. The parameters measured and scored include: 1) background signal, 2) signal to noise, 3) sensitivity LLT (lower therapeutic concentration counts), 4) sensitivity ULT (upper therapeutic concentration counts), and 5) soluble antigen in matrix (Figure ). Parameters 1 to 4 were assessed in the presence and absence of soluble antigen. The cumulative scores for both concentrations of anti-IDs across all PK assay formats tested are displayed in Figure . The generic-tag detection reagent was the best detection reagent regardless of the anti-ID capture or concentration as the top five scores all had the generic-tag for detection (Figure ; Supplementary . Further, the anti-ID #6 had the greatest cumulative score across all PK assay formats tested with anti-ID 17 selected as the back-up with the second top score. The pairing of anti-ID #6-Biotin/Generic-Tag had the maximal score, 20, in both concentrations tested, while the pairing of anti-ID #17-Biotin/Generic-Tag had the maximal score at 0.25 µg/mL and a score of 18 at 1.0 µg/mL due to soluble antigen interference at 10x drug (Supplementary Table ). These anti-IDs were both selected to generate large-scale recombinant proteins for future use in the development and validation of the clinical PK assay. Assay format 4 had the greatest cumulative score for all anti-IDs across both concentrations tested (Figure ; Supplementary Table , 5) and was selected as the optimal PK assay format.