Comparison of Analytical Methods for Antibody–Drug Conjugates Produced by Chemical Site-Specific Conjugation: First-Generation AJICAP

Abstract

Antibody–drug conjugates (ADCs) have become a major class of oncology biopharmaceuticals. Traditional ADCs have a stochastic distribution of cytotoxic drugs attached at several different sites on the antibody. The heterogeneous nature of stochastic ADCs results in a complex compositional analysis. To improve on traditional ADC technology, we have developed a chemical conjugation platform termed “AJICAP” for the site-specific modification of native antibodies using a class of IgG Fc affinity reagents. Here we report further investigation focusing on several analyses of a first-generation AJICAP-ADC (Angew. Chem., Int. Ed. 2019, 58, 5592–5597). For drug–antibody ratio (DAR) determination, we examined and compared six different analytical methods. To the best of our knowledge, this is the first report of a comparison of analytical techniques to measure the DAR for ADCs produced by a site-specific technology such as AJICAP. Furthermore, a rapid analytical process for confirmation of the site selectivity of AJICAP conjugation was established by SEC–Q-TOF-MS. The analytical strategy reported here can be applied to the DAR determination of site-specific ADCs.

Insight into Temperature Dependency and Design of Experiments towards Process Development for Cysteine‐Based Antibody‐Drug Conjugates

“…Free thiols generated in the resulting reduced antibody is an essential factor for DAR of the resulting ADCs [17] . This theory was also confirmed in previous analytical investigations where the free thiols per antibody post-reduction were comparable and predictive of the DAR values measured from the final ADCs [18,19] . Since the TCEP reduction step is critical for the final DAR value in this type of ADC conjugation, our DOE focused on the reduction reaction step by evaluating three different antibodies (Table 2) [20] at different temperatures and times (Scheme 1, Figure 1).…”

Analytical Comparison of Antibody-drug Conjugates Based on Good Manufacturing Practice Strategies

“…31 Purified DAR ADC analysis provides clear DAR results with no significant differences using six analytical methods, as shown in our previous report. 8 To establish an ideal analytical approach for characterizing ADCs, many tasks and challenges remain.…”

“…DAR from RP-HPLC was slightly lower than HIC results in all investigations, which was also observed in our previous studies for site-specific ADCs. 8 In many cases, HIC does not provide sufficient peak separation including positional isomers because this native analysis requires highly concentrated salt in the mobile phases which affect peak separation. 22 The introduction of organic solvents like isopropyl alcohol (IPA) sometimes improve peak shape, and in our previous study mobile phases that included IPA provided better resolution than organic solvent-free running conditions, 8 however, that may not be sufficient in this present study.…”

“…Previously, we reported analytical comparison for site-specific ADCs using six analytical techniques to establish the appropriate analytical method for each phase in the project cycle. 8 The results indicated that a site-specific ADC is preferred model to demonstrate new analytical techniques due to its simple composition. However, comparative studies regarding multiple types of analysis for DAR determination using a single ADC batch are extremely limited in a peer-reviewed setting.…”

“…However, comparative studies regarding multiple types of analysis for DAR determination using a single ADC batch are extremely limited in a peer-reviewed setting. [8][9][10][11][12] Moreover, no examination to compare HPLC results among different ADCs prepared by various TCEP equivalents was reported to best of our knowledge. TCEP titration is the most crucial investigation to determine the appropriate equivalents to obtain cysteine-based ADCs with target DAR.…”