Qualitative analysis of antibody–drug conjugates (ADCs): an experimental comparison of analytical techniques of cysteine-linked ADCs

Abstract

Antibody-drug conjugates (ADCs) are an emerging type of biotherapeutics that utilize multiple tissuespecific antibodies combined with a range of linker designs to enable the transportation and selective release of cytotoxic drugs in close proximity to tumours. Consisting of antibodies conjugated to small drug molecules through a variety of linkers, ADCs are chemically complex analytes. Here we present a unique experimental comparison of four techniques for ADC analysis: hydrophobic interaction chromatography (HIC-UV/Vis), reversed phase liquid chromatography mass spectrometry (RPLC-MS), using either a QToF or an Orbitrap analyser, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Four different ADCs consisting of Trastuzumab, monomethyl auristatin E (MMAE) and a peptidic linker moiety differing in their respective stoichiometric ratios in regard to drugto-antibody ratio (DAR) were used for the comparison. We found that the determined DAR from all techniques was comparable, while the accuracy of the molecular weights for the conjugated light and heavy chain differed more extensively. This indicates that the choice of a mass analyser is more crucial for determining the accurate weights of the light and heavy chains than to evaluate the DAR of a given batch.However, ambiguous DAR assignment in HIC-UV/Vis or bias for either the light or heavy chain fragments in the mass spectrometry-based techniques can influence the obtained average DAR value and the use of complementary techniques is advisable. Out of the four techniques evaluated, HIC-UV/Vis and MALDI required less time to obtain an average DAR value and would therefore be good for initial screenings in the early stages of the discovery phase of new ADCs. † Electronic supplementary information (ESI) available: S-1. LC-chromatograms for HIC-UV/Vis and both RPLC-MS with consecutive blank runs depicting carry over effects, further information on MALDI samples and further comparison of mass accuracy of the different techniques (PDF). See

Application of triple quadrupole mass spectrometry for the characterization of antibody–drug conjugates

“…As has been reported previously [ 27 ], the average DAR value for a given ADC production batch may vary up to ± 0.3 DAR units between analytical techniques and a variability of 0.01–0.4 DAR units between different injections of the same ADC for a specific technique. For the triple quadrupole mass analyzer, DAR varied between injections up to 0.5 DAR units for one batch of ADCs, but was below 0.2 DAR units for all other batches, and on average within 0.32 DAR units from HIC-UV/Vis-derived values and was therefore in good agreement with previous findings.…”

A Purification Strategy Utilizing Hydrophobic Interaction Chromatography to Obtain Homogeneous Species from a Site-Specific Antibody Drug Conjugate Produced by AJICAP™ First Generation

“…RP-HPLC analysis was also carried out for DAR determination ( Supplementary Materials Figures S9-S12), however slightly lower DAR results were obtained. It has been reported in a previous paper that the DAR value from RP-HPLC may be slightly lower than that measured from HIC analysis [10,20,23]. RP-HPLC analysis requires relatively high temperature and denaturing pretreatment with a combination of urea and DTT.…”

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

“…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.…”