Multiplex Bioanalytical Methods for Comprehensive Characterization and Quantification of the Unique Complementarity-Determining-Region Deamidation of MEDI7247, an Anti-ASCT2 Pyrrolobenzodiazepine Antibody-Drug Conjugate

Abstract

Deamidation, a common post-translational modification, may impact multiple physiochemical properties of a therapeutic protein. MEDI7247, a pyrrolobenzodiazepine (PBD) antibody-drug conjugate (ADC), contains a unique deamidation site, N102, located within the complementarity-determining region (CDR), impacting the affinity of MEDI7247 to its target. Therefore, it was necessary to monitor MEDI7247 deamidation status in vivo. Due to the low dose, a sensitive absolute quantification method using immunocapture coupled with liquid chromatography-tandem mass spectrometry (LBA-LC-MS/MS) was developed and qualified. We characterized the isomerization via Electron-Activated Dissociation (EAD), revealing that deamidation resulted in iso-aspartic acid. The absolute quantification of deamidation requires careful assay optimization in order not to perturb the balance of the deamidated and nondeamidated forms. Moreover, the selection of capture reagents essential for the correct quantitative assessment of deamidation was evaluated. The final assay was qualified with 50 ng/mL LLOQ for ADC for total and nondeamidated antibody quantification, with qualitative monitoring of the deamidated antibody. The impact of deamidation on the pharmacokinetic characteristics of MEDI7247 from clinical trial NCT03106428 was analyzed, revealing a gradual reduction in the nondeamidated form of MEDI7247 in vivo. Careful quantitative biotransformation analyses of complex biotherapeutic conjugates help us understand changes in product PTMs after administration, thus providing a more complete view of in vivo pharmacology.

Keywords: antibody–drug conjugate (ADC); biotransformation; deamidation; hybrid liquid chromatography–mass spectrometry (LC-MS) assay; post-translational modification (PTM).

Figure 1

(A) MEDI7247 structure, with the N102, conjugation site and N320 enlarged; (B) ion exchange chromatography for the antibody (INT-001) and MEDI7247 of a representative lot, with the main peak and the two pre-peaks as labeled.

Figure 2

Method development flowchart for the monitoring of MEDI7247 in vivo deamidation assay.

Figure 3

EAD MS² spectra of deamidated peptide of MEDI7247 trypsin digestion sample. (A) EAD MS² mass spectrum of 200–3000 m/z. Zoomed-in spectrum of 1800–3000 m/z is shown as an insert. (B) EAD MS² spectrum of c₄+57 signature ion of deamidation peptide. Sequence and cleavages are noted on the insets.

Figure 4

Evaluation of the capture reagents. (A) anti-PBD antibody shows significant difference in capture capability for the pre-peaks, compared with the main peak; (B) anti-ID m1H10.9 and (C) anti-ID r5A11.3 were not affected by the deamidation of the reference material.

Figure 5

(A) The low deamidation % of N320 indicates that little deamidation was introduced during sample preparation, while the deamidation % of the N102 in the main peak, pre-peak 1 and pre-peak 2 increased. (B) The miscleavage % of the N320 peptide is below 1%, while that of the D320 peptide is around 25%; (C) the miscleavage% of the N102 peptide is undetectable, while that of the D102 peptide results in ~80% for the miscleavage peptide.

Figure 6

Representative chromatograms of the tryptic digestion peptides for (A) N320 and (B) N102. The top panel is nondeamidated peptides. The lower panel is the deamidated version of the corresponding top panel peptides.The red arrows point to the specific peak in the chromatograms corresponding with the label.

Figure 7

(A) Average plasma concentration–time plot of MEDI7247 ADC (red), nondeamidated Ab (blue) and total antibody (black) for eleven patients from one of the DLBCL cohorts; (B) the AUC_0-last (h × g/mL) for total antibody, nondeamidated antibody and ADC; (C) the percentage of nondeamidated Ab in total Ab decreasing with time.