Antibody-drug conjugates (ADCs) are on the brink of widespread use for the targeted treatment of cancer. ADCs manage the toxicity of drugs with unacceptable narrow therapeutic windows by guiding highly toxic compounds to the target cells, thereby sparing healthy cells. Drug-to-antibody ratio (DAR) is an important quality attribute of ADC drugs, which represents the average number of antibody-conjugated small molecule cytotoxic drugs. Lower DAR will reduce the potency of ADC, but higher DAR will affect the ADC analysis pharmacokinetics and toxicity. In addition, the conjugation method is an important factor affecting DAR. Only by selecting a suitable conjugation technology can the ADC toxin be conjugated to the antibody uniformly and stably.
Drug distribution can be differentiated because conjugation reaction at different amino acid residues of mAbs leads to different types of ADCs, such as lysine conjugation, cysteine conjugation, or engineered conjugation. Different drug distribution may contribute to very different pharmacokinetics and toxicological properties, directly affecting the efficacy and safety of ADCs. Therefore, drug distribution must be characterized to ensure the efficiency and safety of ADCs. The DAR calculation is closely relevant to the drug load distribution. Normally, analysis of the average DAR as well as its drug load distribution should consider several factors, including conjugation strategies, linkers, small-molecule payloads and platform applications.
UV/Vis spectroscopy is a simple and convenient method to determine protein concentrations as well as the average number of drugs that are conjugated to the antibody in an ADC. This method requires that the UV/VIS spectra of the drug and antibody have different Amax values. The average DAR can be determined by using the measured ADC absorbance and the extinction coefficient of mAb at Amax and the extinction coefficient of drug at Amax. Currently, this technology has been widely used in the detection of various cytotoxic ADC drugs, such as maytansinoid DM1, methotrexate, CC-1065 analogues, adriamycin, doxorubicin, calicheamicin analogues and vc-MMAE.
Fig. 1. Analysis method for DAR determination by UV/Vis (Chem. Pharm. Bull. 2021, 69(10): 970-983).
HIC is an analytical technique for separating molecules based on differences in their hydrophobic properties. Currently, HIC is considered as the standard technique for studying drug distribution, naked antibody content, and average DAR of cysteine-conjugated ADC products. Since the disulfide bond of lysine-conjugated ADCs is not opened by drug loading, lysine-conjugated ADCs elutes with HIC, and a major peak will appear in the chromatogram, which cannot characterize DAR and drug distribution. However, HIC can elute intact cysteine-conjugated ADCs, DAR can be directly characterized by peak area.
Fig. 2. HIC profile of an ADC sample (Anal. Chem. 2013, 85: 1699-1704).
RPLC is mostly frequently used to estimate the average DAR. The main difference between HIC and RPLC is that proteins are denatured under RPLC conditions. Therefore, cysteine-conjugated ADCs cannot be eluted intact owing to the disruption of interchain disulfide bonds between the L and H chains of the mAb. RPLC is mostly used to separate the DAR species of reduced ADC related to H and L, but can also be applied on the native ADC to separate L, H, HH, HL and HHL fragments. In RPLC, interaction with the stationary phase is mediatedpredominantly through hydrophobic interactions between the nonpolar amino acid residues of proteins and the immobilizedn-alkyl ligands. Solutes are eluted in their order of increasingmolecular hydrophobicity.
Fig. 3. RP-HPLC analysis of cysteine-based ADCs (Chem. Pharm. Bull. 2021, 69(10): 970-983).
LC-MS has been widely used for ADC analysis. Preprocessing is required for ADC analysis using LC-MS, and several recent studies have focused on optimizing preprocessing in LC-MS. For example, deglycosylation by peptide-N-glycosidase F (PNGaseF) is generally used to eliminate the complexity of peak appearance. Using the reducing agent DL-dithiothreitol (DTT), heavy and light chains of the ADC were obtained, and alkylation is generally used after reduction to prevent the re-formation of disulfide-linked structures. Considering the method and integrity of proteins, protein characterization by LC-MS is further divided into four types, as shown in Fig. 4. Among these methods, middle-up and middle-down are more suitable for ADC analysis.
Fig. 4. Four types of ADC characterized by LC-MS (J. Pharm. Biomed. Anal. 2020, 179: 112991).
SDS is mainly used to separate ADCs with intact or H and L chain forms. Unlike size exclusion chromatography, SDS can also be used to separate sFc and Fd fragments. When CE-SDS is applied for lysine-conjugated ADCs, it separates loaded H or L chains because of the disulfide bridge interaction. However, the main peak is the intact antibody represented by H2L2, accounting for about 96%. In contrast, when cysteine-conjugated ADCs are analyzed, intact H2L2 comprises only 8.1%. Therefore, CE-SDS cannot be used to characterize the DAR of hinge cysteine-linked ADCs. In general, CE-SDS can be used to determine lot consistency of ADC conjugates.
Fig. 5. Characterization of ADCs by capillary electrophoresis (Humana, New York, NY, 2020: 251-262).
Data obtained from our DAR and drug distribution analysis services can be used to examine possible hazards and advantages of medication candidates, improve drug formulations, and choose the best dosing schedules. This data is essential for the development and optimization of novel medications as well as for ensuring the efficacy and safety of currently available medications.