Bioanalytical Challenge Kumar, King, Clark & Gorovits
tional to DAR [1]. To better understand the change in DAR species over time, LC–MS measurement of aver- age DAR is also often measured and the DAR distri- bution data are combined with the total antibody and conjugated antibody data to select optimal candidates. During the
late drug development stage, when
the IND enabling studies are conducted for the lead ADC candidate, validated DAR-insensitive LBAs may be needed for collection of PK data so that all DAR species are measured equally to aid in defining and understanding potential toxicity and TK of the lead candidate. This is because of a need to meet regulated validation expectations and because of our limited cur- rent understanding on which DAR species can provide best correlation for exposure–response relationship for safety of ADCs. The recommendations for assay development and
validation for total antibody and conjugated antibody analytes of ADCs has been discussed elsewhere [8]. The heterogeneous mixture of ADCs containing various DAR species and diverse conjugation sites may have different binding affinities for the critical reagents used in the total antibody and conjugated antibody assays. Thus depending on the reagents used, the low avid- ity and the steric hindrance observed during critical reagent binding may lead to inaccurate estimation of low and high DAR species, respectively. For DAR- insensitive assays, it is recommended that the assay sensitivity to DAR values be evaluated early on dur- ing assay development by comparing the recovery of enriched or individually purified DAR species against the reference standard containing average DAR to ensure that the chosen assay format and critical reagents recover all DAR species equally [8,9]. The enriched DAR species represent the hetero-
geneous mixture of DAR species that has relatively higher abundance of a certain DAR species in the mix- ture. They are used when the DAR heterogeneity in ADC is too complex to isolate individual DAR spe- cies, and are prepared by either crude fractionation of the ADC reference standard or employing conjugation procedures designed to produce DARs either higher or lower than the reference standard DAR [9,15]. The availability of enriched or purified DAR species may depend on the chemistry of conjugation. For instance, isolation and purification of heterogeneous ADC mix- ture to individual DAR species may be challenging for conventional lysine-based conjugation chemistry that yields DAR values of 0–8 and generates greater than 1 million different ADC species due to conjugation sites located at approximately 20 different lysine residues on both the heavy and light chain [30] compared with novel site-specific conjugation chemistries that yield site-specific ADCs with DAR values of two or four [31].
1610 Bioanalysis (2015) 7(13) An overview of bioanalytical considerations and
challenges associated with LBA-based assessment of total antibody and conjugated antibody analytes are discussed below.
Total antibody assay Total antibody assay as the name implies measures total antibody (DAR greater than or equal to 0, sum of con- jugated and unconjugated forms) analyte of ADC. It monitors antibody component of ADC irrespective of whether cytotoxic small molecule drug is conjugated to the antibody or not. The total antibody concentration is commonly used to assess the antibody-dependent PK characteristics (half-life, clearance) and the overall in vivo stability of the ADC. The conjugation of small molecule drug to the antibody moiety may negatively influence its PK characteristics such as shorter half-life and faster clearance in vivo compared with the parent antibody. For this reason, during early drug discovery stage, the total antibody assay is the primary choice of analyte for comparing ADC candidates with various linker–cytotoxic small molecule drug combinations. In addition, during early discovery stage, in the absence of a conjugated antibody LBA or antibody conjugated small molecule drug LC–MS assay, the data from total antibody assay in combination with DAR measure- ments could be used for assessing conjugated small molecule drug concentration. The critical reagents typically employed in the total
antibody assay bind to the antibody component of ADC regardless of its DAR value (Figure 1). Though these reagents do not directly bind to the small mol- ecule drug, due to steric hindrance the small molecule drug can indirectly influence binding of these reagents to the antibody component of ADC. This interference might be more prominent for higher DAR species. As a result, the assay may not accurately estimate all expected DAR species in systemic circulation, thereby, affecting the observed overall PK characteristics of the ADC. It can be particularly challenging to address this potential issue in early discovery when multiple ADCs with different conjugations sites are analyzed from a single study with the same total antibody assay. Industry recommends determining if the total anti-
body assay is DAR-insensitive by evaluating recovery of the samples prepared with the Ab as well as the samples prepared with the enriched or individually purified DAR species against the ADC reference standard with aver- age reported DAR [8]. If a total antibody assay is DAR- insensitive, both the Ab and the enriched or purified DAR species should yield percentage recovery within the acceptable range of the assay (e.g., ±20% of nominal). Both specific reagents such as targeted tumor anti- gens or anti-Id or anti-CDR monoclonal antibodies,
future science group
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