Editors’ Series: Streamlined Characterization of Antibody–Drug Conjugates by Two-Dimensional (2D) and Four Dimensional (4D) LC/MS

Register free: http://www.chromatographyonline.com/lcgc_w/two_dimensional

Event Overview:
This presentation describes the characterization of an antibody-drug conjugate (ADC) using size-exclusion chromatography (SEC) or hydrophobic interaction chromatography (HIC) using a multidimensional LC–MS system. In order to understand the effect of the different drug-to-antibody ratio (DAR) species on aggregate formation, the size variants of a cysteine-linked ADC were characterized at both the intact and reduced antibody levels. For this purpose, the ADC product and stressed samples were analyzed. The dimeric ADC species were baseline resolved from the main peak (Rs = 2.7) by SEC under non-denaturing conditions using a buffered mobile phase containing 5% 2-propanol. A 2D-LC (SEC-HIC) method was then developed to compare the average DAR values of the main peak species versus the aggregates at the antibody intact level. A 4D-LC–MS method (SEC-reduction-digestion-RPLC) was also developed to determine levels of potential critical quality attributes (pCQAs) including aggregation, average DAR at the antibody reduced level, oxidation, and deamidation, in a 2 h run. An average DAR value of 3.5–3.6 was found for the main peak using both 2D-LC and 4D-LC methods, and these values were consistent with DAR determined by the in-house reference HIC method. The multidimensional LC approaches also showed an increase in the content of high-DAR species in the SEC fractions containing the aggregates. The on-line and automated 2D- and 4D-LC–MS fraction collection and subsequent analysis was performed within a day in comparison to multiple days required with the traditional offline approaches.

Key Learning Objectives:

• The latest methods for characterization of antibody-drug conjugates (ADCs)

• How 2D and 4D LC–MS can be used for streamlined and more effective ADC characterization

• A new 2D LC method to compare the average DAR values of the main peak species to the aggregates

• How a 4D LC method for ADC characterization can be used to determine levels of potential critical quality attributes (pCQAs) including aggregation, average DAR, oxidation, and deamidation, in a two-hour run

Speakers: Cinzia Stella, PhD, Senior Scientist and Team Leader, Genentech, South San Francisco, California

Alexandre Goyon, PhD, Associate Scientist, Genentech Research and Early Development

Time and Date: Tuesday, Dec. 12, 2019 at 10am EST | 9am CST | 3pm GMT | 4pm CET

On demand available after final airing until Dec. 12, 2020

Sponsor: Tosoh Bioscience

Register free: http://www.chromatographyonline.com/lcgc_w/two_dimensional