Sepax HIC-Phenyl Columns for Low DAR ADC Process Analytics

Sepax summary from the paper:

Process Analytical Technology for Real-Time Monitoring of Pharmaceutical Bioconjugation Reactions

https://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.oprd.4c00399&ref=pdf

Published as part of Organic Process Research & Development special issue “Analytical Control Strategies for

Process Chemists”.

Nicole M. Ralbovsky,* Gunjan Dixit,δ Justin P. Lomont,δ Jay Desai,δ Cristina Butu, Anumita Saha-Shah, Emily Costello, Janelle Lukens, Michael Mazur, Patrick M. McHugh, Rodell C. Barrientos, Andrew Semple, Gregory J. Hughes, Rebecca Chmielowski, Sheng-Ching Wang, Bhumit A. Patel, and Joseph P. Smith

PAT (process analytical technology) is a valuable method for monitoring the manufacturing of a drug ensuring a safe and reliable product. Such characterization is invaluable to assessing the impact of each process step before moving to the next. It can also be used to trace any minor deviations that can occur in the final product or drug substance. The researchers of the paper accessed two different types of conjugates, one of which uses Sepax HIC columns. Proteomix HIC phenyl is probably the most hydrophobic HIC resin on the market. This hydrophobicity is due to the ligand and the polystyrenic backbone. The unique aspect of this HIC column enables detection of low DAR conjugates.

The small molecule conjugation was performed in DMSO with a reduced mAb paired with a maleimide linker with small, medium, and high payloads (PD). The free cysteine from the reduced mAb interacts with the maleimido group forming a thioester bond known as the thiol-maleimide reaction. The amount of drug linked maleimide to mAb dictates the DAR ratio.

Method:

HPLC Column: Proteomix HIC Phenyl-NP5 (p/n: 433NP5?4610

Mobile phase A: 3 M ammonium acetate, 50 mM potassium phosphate, 5% acetonitrile at pH7.0. Mobile Phase B: 5% acetonitrile Column Temperature : 30C Injection Volume: 5 μL Sample Amount: 50 μg Detector: UV/VIS 280nm Flow Rate: 0.25 mL min/ml Gradient: 20% B - 0.0?2.0 min; 95% B from 32.0?37.0 min Re-equilibration back to 20% B from 37.1 to 45.0 min. System: Waters Patrol UPLC system (Waters, Milford, MA)

Small Molecule- Protein in-line DAR:

The chromatogram below shows at-line analytical results of the three species. The latest eluting peak is the mAb that has the highest DAR due to its hydrophobicity.

The yellow star is DAR0, the cyan star represents DAR1, and the purple star corresponds to DAR2. The average DAR is calculated by the percentage of each peak area. This method has been proven to monitor the at-line ADC conjugation for the 3 DAR species. The assay can measure the payload equivalence ~ real time to achieve the desired target DAR. The reaction could monitor full reaction completion if DAR 2 was the end goal. The method can also monitor DAR distribution over time further aiding in efficacy and reliability.