DAR is defined as the number of drug molecules per mAb. DAR plays a definitive role in developing ADCs, as it determines the dose needed to produce the desired effect in patients. There is a limited number of drug molecules that can be efficiently delivered to the target site and drug loading significantly contributes to the pharmacokinetic profile of ADC. Hamblett and co-workers showed that the effect of drug distribution on the different properties like therapeutic window, pharmacokinetic properties, and maximum tolerated dose of cAC10-MMAE conjugates. Decreasing the DAR resulted in a superior therapeutic window of cAC10-MMAE conjugates, proving that drug loading as a decisive parameter for designing ADCs. Although cAC10-MMAE conjugates with DAR ~2–4 were less active in in vitro studies, but their results in in vivo studies were found to be equivalently potent (DAR~4) and better tolerated than the conjugate with higher DAR ~8. Similar observations were found with regards to pharmacokinetic properties [
70]. If fewer drug molecules are conjugated per mAb, the ADC system will not be effective clinically. On the other hand, conjugating too many drug molecules per mAb will make the ADC unstable, toxic and may lead to aggregation and immunogenic reactions [
79,
80]. Hydrophobic MMAE conjugates using interchain cysteines with higher DAR are found to be physically unstable [
79]. Normally ADCs contain different species with differing DAR values and every species has its own distinct pharmacokinetics. ADCs with heavily loaded drugs are more rapidly cleared from the system. In general, an average DAR of 3–4 is used to achieve optimum effect in ADCs, depending upon potency of the payload [
70,
81]. However, a recently developed poly-1-hydroxymethylethylene hydroxymethylformal (PHF) polymer-based ADC with a higher DAR of ~20 challenged this conventional concept. With vinca alkaloid as the payload and trastuzumab as the targeting mAb, the newly developed platform not only showed promising activity in xenograft tumor models, but also demonstrated good pharmacokinetic properties [
82]. Conjugations through side-chain lysine residues are highly heterogeneous leading to inconsistent DAR values and different conjugation sites in the antibody. In case of Kadcyla
® where the drug DM1 was conjugated with the trastuzumab through the side chain lysine residues, an average DAR was found to be ~3.5 [
83]. Side chain cysteine conjugation employs a controlled reduction of four intrachain disulfide bonds that allows conjugation of 0–8 drug molecules per antibody [
84]. Common analytical methods for determining DAR are UV-Vis spectroscopy, hydrophobic interaction chromatography (HIC), LC-ESI-MS and rpHPLC. UV visible spectroscopy exploits the dissimilarities in maximum wave length absorbance of payload and mAb for determining respective concentrations [
85]. UV-Vis spectroscopic method is widely employed to characterize huN901-DM1, 791T/36-methotrexate and cAC10-MMAE conjugates [
70,
86,
87]. HIC uses a column consisting of a hydrophobic stationary phase and a mobile phase with gradient salt concentration to separate ADC species based on hydrophobic interactions. Mostly the ADC payloads are hydrophobic in nature, and hydrophobic conjugated species are retained in the column, whereas unconjugated species elute first in neutral pH and non-denaturizing conditions [
88]. This method is more compatible with ADCs with cysteine conjugation sites on mAb, while LC-ESI-MS method was developed for characterizing lysine-conjugated ADCs [
89,
90]. LC-MS is advantageous over HIC or UV-Vis spectroscopic characterization as it not only gives information on DAR or drug distribution but also gives crucial structural insights of ADCs at the molecular level [
91]. Wagner-Rousset and co-workers designed a simple and fast method of DAR determination based on antibody-fluorophore conjugates (AFCs) with the same linker and conjugation chemistry as ADCs. Instead of toxic payloads, a non-toxic dansyl sulfonamide ethyl amine payload was used. AFCs were subjected to digestion by
Streptococcus pyogenes (IdeS) accompanied by DTT reduction, which generated seven easily ionizable fragments (Fd0, Fd1, Fd2, Fd3. L0, L1, Fc/2) of ~25 kDa. These resultant fragments were analyzed by LC-ESI-TOF-MS method. This method is advantageous over single step reduction as it not only gives routine information like DAR and drug distribution but also provides crucial structural details like
N-glycosylation profiling, C-terminal lysine truncation, pyroglutamylation, oxidation and degradation products [
92].