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-MSE 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.

Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry

“…Specifically, the removal of the carbohydrate by the heavy chain N297G mutation eliminated an extra deglycosylation step before LC-MS analyses. Additionally, the deletion of the heavy chain C-terminal lysine residue (ΔK447) circumvented the heterogeneity that may result from proteolytic removal of this lysine 23 . As a result, simpler IgG mass spectra could be obtained immediately following protein A chromatography.…”

“…Previously, quadrupole time-of-flight (Q-TOF) LC-MS analyses have been used successfully to measure the relative amounts of different IgG species 23,24 . For example, Woods et al.…”

“…For example, Woods et al. 23 coupled a C4 reverse phase LC system with an electrospray ionization (ESI) Q-TOF mass spectrometer to quantify homodimers and associated half-antibody impurities in BsIgG samples. The limit of quantification of antibody impurities was estimated as 2% based upon spiking of standards into purified heterodimer.…”

Improving biophysical properties of a bispecific antibody scaffold to aid developability

“…To further characterize the two chains and establish the limit of detection of the homodimeric and monomeric species, we independently expressed and characterized (by LC-MS) each of the heavy chains separately with the light chain. 50 In these independent expressions of both chain A and chain B, the product formed is greater than 70% monomers. This ability of the individual heavy chains to preferentially exist as stable monomers over homodimers is a critical design attribute and has important implication for the ability to select and develop a stable cell line producing the heterodimeric protein with minimal amount of contaminants.…”

“…An alternative purity assessment method using the Acquity UPLCXevo G2 QToF MS system was also developed. 50 Glycosylation of the heterodimeric antibodies was evaluated by trypsin digest followed by nano LC-MS/MS at the glycopeptide level. The LC-MS spectra of variants ZW2 and ZW3 are shown in the Figure S6.…”