Česká společnost pro hmotnostní spektrometrii

(CEEPC/IPM/CMSC 2022 - ThS-03)
RPLC-UV and HILIC-UV characterisation of on-site produced Ramucirumab-DTPA immunoconjugate

Denis K Naplekov ^1 *, Pavel Bárta ², František Trejtnar ³, Hana Sklenářová ¹, Juraj Lenčo ¹

1. Dept. of Analytical Chemistry, Faculty of Pharmacy, Charles University, Hradec Kralove
2. Dept. of Biophysics and Physical Chemistry, Faculty of Pharmacy, Charles University, Hradec Kralove
3. Dept. of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Hradec Kralove

Abstrakt

Conjugates of antibodies with chelating linkers, such as diethylenetriamine pentaacetate (DTPA), represent a specific class of biopharmaceuticals often produced on-site by research groups for scientific purposes. The conjugation reaction is usually carried out within a long period of time, leading to the stochastic linkage of the chelating agent and highly heterogeneous products [1]. In turn, these conjugates represent a real challenge for characterization by liquid chromatography because the separation of heterogeneous products is imperative for monitoring the linker attachment sites and their quantification. Reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) have demonstrated the potency to suffice a need of this kind at intact, subunit, and peptide levels [2]. In turn, RPLC and HILIC analyses are still complicated for free and chiefly on-site conjugated mAbs. Yet, such separation methods must be fast and simple but reliable. We found optimal conditions for analyses of ramucirumab and its DTPA-conjugated variant at intact, reduced, subunit, and peptide levels. The conjugation efficiency has been confirmed by the differences between the resulting peak of ramucirumab-DTPA against the peak of free ramucirumab using both RPLC and HILIC. However, none of those modes could baseline separate the mixture of conjugated ramucirumab molecules. Our pilot data suggest that conjugation reaction can be monitored online in RPLC-UV mode to select the optimal reaction time, which could limit the attachment of the chelating linker to desired amino acid residues only.

* Korespondující autor: naplekod@faf.cuni.cz

Literatura

1. Joubert N. et al.: J. Pharmaceuticals 13(9), 245 (2020).
2. Zhu X. et al.: J. Pharm. Anal. 10(3), 209-220 (2020).

Poděkování:

The study was supported by the STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000465) cofounded by ERDF. and by specific research project No. SVV 260548.