(M0930-01-05) Novel Light Scattering Solutions for Accelerating Development of Antibody-Drug Conjugates

Purpose: Antibody-drug conjugates (ADCs) combine the best of both worlds for effective biotherapeutics: the specificity and targeting ability of monoclonal antibodies (mAbs) and the cytotoxicity of a small molecule drug attached via a linker. Liquid chromatography (LC) and mass spectrometry (MS) provide excellent quantitation of critical quality attributes (CQAs) such as the drug-antibody ratio (DAR), however it is more suitable for later stages of characterization as it requires specific tuning to achieve sensitivity and detail. Additionally, MS can miss aggregation information as it not often under native conditions. For initial stages of CQA characterization, a platform method such as Size Exclusion Chromatography coupled with Multi Angle Light Scattering (SEC-MALS) is ideal for routine analysis and can provide valuable bridging data. Here we describe an easy-to-implement method to characterize ADC CQAs such as average DAR, molar mass, and aggregation using LC combined with MALS, UV absorption, and differential refractive index (RI) detection.

Methods: Commercial ADCs, Kadcyla^TM Drug Substance (DS) and Adcetris^TM DS were analyzed using a Waters ACQUITY^TM Premier system with a Photodiode array (PDA) optical detector, a microDAWN^TM (MALS) instrument, and microOptilab^TM (dRI) detector. The autosampler was kept at 6 °C and the columns were kept at 25 °C. UV data were collected at 280 and 252 nm for Kadcyla and 280 and 248 nm for Adcetris. SEC-MALS separation was performed using an ACQUITY Premier Protein SEC column, 1.7 µm, 250Å, 4.6 x 150 mm. The mobile phase was Dulbecco’s PBS (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 6.7). The flow rate was 0.35 mL/min. For each measurement, 1 µL of Kadcyla and 2µL of Adcetris were injected. Data was collected and analyzed using the ASTRA^TM software.

Results: SEC-MALS analysis of the two ADC drug substances (DS), Kadcyla and Adcetris, enabled simultaneous analysis of multiple quality attributes. The ASTRA software determines the concentration of the mAb and the drug independently based on their unique extinction coefficients and refractive index increment (dn/dc). The average DAR measured was 4.1 for Adcetris and 3.4 for Kadcyla which was close to their expected values of 4.2 and 3.5 respectively. SEC-MALS analysis enabled identification and quantification of aggregates and  fragments as well as their molar mass heterogeneity. We identified a dimer population in both ADCs with a weight fraction of 1.5 and 2.4 % (w/w) for Kadcyla and Adcetris respectively. The dimer population had a DAR of 5.7 in Adcetris and 3.5 in Kadcyla. We also identified a high molecular weight species present in Adcetris with a DAR of 7.2 and weight fraction of 9% (w/w). SEC-MALS allows use of the formulation buffer as the mobile phase which will provide a true native environment. We analyzed Kadcyla using DPBS (physiological conditions) and ammonium acetate ( a semi-native MS compatible mobile phase). We observed the same DAR for the monomer peak under both conditions. However, the dimer species, with the same DAR, was only observed in the DPBS condition. This data suggests that the dimer species may be missed under native SEC-MS. SEC-MALS is thus the optimal choice for providing bridging data.

Conclusion: SEC-MALS is an easy-to-implement platform method for characterization of ADCs in early development. This approach enables detailed analysis of key attributes such as the average DAR, aggregation, and molar mass. Data obtained via SEC-MALS provides valuable bridging data for orthogonal techniques such as SEC-MS.


Table 1: Measured attributes for Adcetris DS and Kadcyla DS


Figure. 1: SEC-MALS analysis of (left) Kadcyla drug substance (DS) and (right) Adcetris DS. UV chromatogram is shown with measured molar masses overlaid.


Figure 2: SEC-MALS analysis of Kadcyla using DPBS and ammonium acetate mobile phase. UV chromatogram is shown with measured molar masses overlaid.