Table 3.

Analytical characterization of ADC CQAs

Quality Attributes	Analytical Methods
	Cysteine Conjugates	Lysine Conjugates	Site-Specific Conjugates
DAR, DLD, and unconjugated species (DAR-0)	HIC Native IM-MS, native SEC-MS, or native sheathless CE-MS SEC-HIC two-dimensional HPLC (2D-LC) HIC-RPLC-MS or HIC-SEC-MS (2D-LC-MS) HIC-SEC-IM-MS (2D-LC-IM-MS)	UV/Vis spectroscopy (only for average DAR) CIEF	HIC RPLC RPLC-MS SEC-MS on deglycosylated ADC
Conjugation sites	RPLC-MS/MS or sheathless CE-MS/MS	RPLC-MS/MS (combining tryptic peptide mapping with Asp-N and/or Glu-C peptide mapping)	RPLC-MS/MS
Posttranslational modifications (PTMs)	HILIC-MS or sheathless CE-MS/MS on Fc-fragments HIC-RPLC-MS on reduced ADC	mCE-MS CEX-RPLC-MS	RPLC-MS/MS
Free drug species	RPLC on ADC-free sample SEC, SEC-RPLC, or solid phase extraction LC (SPE) coupled with RPLC-MS (SPE-RPLC-MS) analysis of untreated ADC sample	RPLC on ADC-free sample SEC or SEC-RPLC analysis of untreated ADC sample	RPLC on ADC-free sample
Size variants	SEC SEC-HIC or SEC-RPLC	SEC SEC-PRLC CE-SDS	SEC Analytical ultra-centrifugation (AUC) CE-SDS
Charge variants	CIEF	CIEF	IEX

The listed techniques have been used to characterize the CQAs of cysteine, lysine, and site-specific conjugates.

Antibody–drug conjugate (ADC); Analytical ultracentrifugation (AUC); Capillary isoelectric focusing (CIEF); Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS); Cation exchange chromatography (CEX); Hydrophobic interaction chromatography (HIC); Hydrophilic interaction liquid chromatography (HILIC); Ion mobility mass spectrometry (IM-MS); Microfluidic capillary electrophoresis (mCE); Mass spectrometry (MS); Reverse phase liquid chromatography (RPLC); Size exclusion chromatography (SEC); Solid phase extraction (SPE); Ultraviolet-visible spectroscopy (UV/Vis)