LC-MS characterization and purity assessment of a prototype bispecific antibody

Abstract

Bispecific IgG asymmetric (heterodimeric) antibodies offer enhanced therapeutic efficacy, but present unique challenges for drug development. These challenges are related to the proper assembly of heavy and light chains. Impurities such as symmetric (homodimeric) antibodies can arise with improper assembly. A new method to assess heterodimer purity of such bispecific antibody products is needed because traditional separation-based purity assays are unable to separate or quantify homodimer impurities. This paper presents a liquid chromatography-mass spectrometry (LC-MS)-based method for evaluating heterodimeric purity of a prototype asymmetric antibody containing two different heavy chains and two identical light chains. The heterodimer and independently expressed homodimeric standards were characterized by two complementary LC-MS techniques: Intact protein mass measurement of deglycosylated antibody and peptide map analyses. Intact protein mass analysis was used to check molecular integrity and composition. LC-MS(E) peptide mapping of Lys-C digests was used to verify protein sequences and characterize post-translational modifications, including C-terminal truncation species. Guided by the characterization results, a heterodimer purity assay was demonstrated by intact protein mass analysis of pure deglycosylated heterodimer spiked with each deglycosylated homodimeric standard. The assay was capable of detecting low levels (2%) of spiked homodimers in conjunction with co-eluting half antibodies and multiple mass species present in the homodimer standards and providing relative purity differences between samples. Detection of minor homodimer and half-antibody C-terminal truncation species at levels as low as 0.6% demonstrates the sensitivity of the method. This method is suitable for purity assessment of heterodimer samples during process and purification development of bispecific antibodies, e.g., clone selection.

Keywords: LC-MS; bispecific antibody; deglycosylation; heterodimeric antibody; impurity assessment; intact protein mass; peptide map; purity assay.

Figure 1. UV₂₈₀ chromatograms for Hetero-AB, Homo-A, and Homo-B.

Figure 2.(A) Raw and (B) MaxEnt1 deconvoluted mass spectra of Hetero-AB.

Figure 3. Mass spectra for Homo-A: (A) Raw mass spectrum; (B) MaxEnt1 deconvoluted spectrum for the intact antibody charge envelope; (C) MaxEnt1 deconvoluted spectrum for the half-antibody charge envelope.

Figure 4. Mass spectra for Homo-B: (A) Raw mass spectrum; (B) MaxEnt1 deconvoluted spectrum for the intact antibody charge envelope; (C) MaxEnt1 deconvoluted spectrum for the half-antibody charge envelope.

Figure 5. LC-MS^E results identifying heavy chain A- and B-specific sequences for tryptic peptide T36: (A) Total ion chromatogram (TIC) with zoomed mass spectra shown in insets; (B) MS^E fragment ions confirming the sequence difference.

Figure 6. Raw mass spectra for Hetero-AB containing low levels of spiked homodimer standards, zoomed to show two charge states. (A) Hetero-AB containing 30% Homo-B standard. (B) Hetero-AB containing 9% Homo-A standard.

Figure 7. Deconvoluted mass spectra (intact antibody charge envelope) and homodimer intensity response curves for Hetero-AB mixed with varying amounts of Homo-A or Homo-B standards. (A) Overlaid spectra for Hetero-AB containing Homo-A, normalized to the AB-2Lys peak. (B) Overlaid spectra for Hetero-AB containing Homo-B, normalized to the AB-2Lys peak. (C) Plot of AA-2Lys intensity (relative to the sum of AA-2Lys and AB-2Lys intensities) vs. Homo-A relative concentration. (D) Plot of BB-2Lys intensity (relative to the sum of BB-2Lys and AB-2Lys intensities) vs. Homo-B relative concentration.

Figure 8. Deconvoluted mass spectra from the half-antibody charge envelope and half-antibody intensity response curves for Hetero-AB mixed with varying amounts of Homo-B. (A) Overlaid half-antibody spectra for Hetero-AB containing Homo-B. (B) Plot of B-Lys intensity relative to the sum of B-Lys and AB-2Lys vs. Homo-B relative concentration.