Comparative study of trastuzumab modification analysis using mono/multi-epitope affinity technology with LC-QTOF-MS

Abstract

Dynamic tracking analysis of monoclonal antibodies (mAbs) biotransformation in vivo is crucial, as certain modifications could inactivate the protein and reduce drug efficacy. However, a particular challenge (i.e. immune recognition deficiencies) in biotransformation studies may arise when modifications occur at the paratope recognized by the antigen. To address this limitation, a multi-epitope affinity technology utilizing the metal organic framework (MOF)@Au@peptide@aptamer composite material was proposed and developed by simultaneously immobilizing complementarity determining region (CDR) mimotope peptide (HH24) and non-CDR mimotope aptamer (CH1S-6T) onto the surface of MOF@Au nanocomposite. Comparative studies demonstrated that MOF@Au@peptide@aptamer exhibited significantly enhanced enrichment capabilities for trastuzumab variants in comparison to mono-epitope affinity technology. Moreover, the higher deamidation ratio for LC-Asn-30 and isomerization ratio for HC-Asn-55 can only be monitored by the novel bioanalytical platform based on MOF@Au@peptide@aptamer and liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS). Therefore, multi-epitope affinity technology could effectively overcome the biases of traditional affinity materials for key sites modification analysis of mAb. Particularly, the novel bioanalytical platform can be successfully used for the tracking analysis of trastuzumab modifications in different biological fluids. Compared to the spiked phosphate buffer (PB) model, faster modification trends were monitored in the spiked serum and patients' sera due to the catalytic effect of plasma proteins and relevant proteases. Differences in peptide modification levels of trastuzumab in patients' sera were also monitored. In summary, the novel bioanalytical platform based on the multi-epitope affinity technology holds great potentials for in vivo biotransformation analysis of mAb, contributing to improved understanding and paving the way for future research and clinical applications.

Keywords: Biotransformation analysis; Breast cancer; LC-QTOF-MS; Monoclonal antibody; Multi-epitope affinity technology.

Graphical abstract

Fig. 1

Peptide mapping analysis of trastuzumab incubated in phosphate buffer for 21 days. (A) Total ion chromatogram for the mass spectrometry (MS) and tandem mass spectrometry (MS/MS). (B) Extracted ion chromatogram (XIC) of ASQDVNTAVAWYQQKPGK+3 and the corresponding light chain (LC) N30 deamidation peptide in complementarity-determining region (CDR) of the variable region. (C) XIC of IYPTNGYTR+2 and the corresponding heavy chain (HC) N55 isomerization and deamidation peptides in CDR of the variable region. (D) XIC of WGGDGFYAMDYWGQGTLVTVSSASTK+3 and the corresponding HC D102 isomerization peptide in CDR of the variable region. (E) XIC of EVQLVESGGGLVQPGGSLR+3 and the corresponding HC E1 cyclization peptide in non-CDR of the variable region. (F) XIC of NTAYLQMNSLR+2 and the corresponding HC N84 isomerization and deamidation peptides in non-CDR of the variable region.

Fig. 2

Tandem mass spectrometry (MS/MS) spectra of the analyzed peptides. (A, B) MS/MS spectra of IYPTNGYTR+2 and the corresponding heavy chain (HC) N55 deamidation (Dea) peptide. (C, D) MS/MS spectra of NTAYLQMNSLR+2 and the corresponding HC M83 oxidation (Oxi) peptide. (E, F) MS/MS spectra of EVQLVESGGGLVQPGGSLR+3 and the corresponding HC E1 cyclization (Cyc) peptide.

Fig. 3

Schematic illustration of comparative study of multi-epitope versus mono-epitope affinity technologies.

Fig. 4

Comparative study of multi-epitope affinity technology versus mono-epitope affinity technologies. (A) The key modification sites of trastuzumab selected for analysis. (B, C) Comparisons of recovery (B) and sequence coverage (C) of trastuzumab captured by metal organic framework (MOF)@Au@peptide, MOF@Au@aptamer and MOF@Au@peptide@aptamer. (D–I) Comparative results of light chain (LC) N30 deamidation (Dea) (D–F) and heavy chain (HC) N55 isomerization (Iso) (G–I) ratios of trastuzumab incubated for 7, 14 and 21 days after pretreatment with MOF@Au@peptide, MOF@Au@aptamer, MOF@Au@peptide@aptamer, with none as the control.

Fig. 5

Variants analyses of the incubated trastuzumab from the intact protein level. (A) Capillary zone electrophoresis analysis and (B) circular dichroism spectra of trastuzumab incubated in phosphate buffer for 0, 4, 7, 14 and 21 days.

Fig. 6

Specificity of the multi-epitope affinity technology. (A) Workflow of the analysis in complex biological fluids. (B, C) Enrichment of trastuzumab in blank (B) and spiked sera (C) using metal organic framework (MOF)@Au@peptide@aptamer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis (reducing condition) with different lanes as follows: LF: loading fraction; FT: flow through; W: washing fractions; E: elution fractions; M: protein marker; S: standard trastuzumab.

Fig. 7

Evaluation of the bioanalytical platform based on the multi-epitope affinity technology. Total ion chromatograms for the mass spectrometry (MS) and tandem mass spectrometry (MS/MS) of (A) blank and (B) spiked sera. Extracted ion chromatograms for the complementarity-determining region (CDR) peptides of (C) blank and (D) spiked sera. Extracted ion chromatograms for the non-CDR peptides of (E) blank and (F) spiked sera. All the blank and spiked sera were pretreated with metal organic framework (MOF)@Au@peptide@aptamer.

Fig. 8

Application of the bioanalytical platform based on the multi-epitope affinity technology. (A, B) Biotransformation analysis for complementarity-determining region (CDR) (A) and non-CDR (B) amino acid sites of trastuzumab incubated in serum over 21 days. (C) Comparative results for the modification trends of trastuzumab incubated in phosphate buffer (PB) and serum over 21 days. (D) Biotransformation analysis of trastuzumab in breast cancer patients' sera. LC: light chain; HC: heavy chain; Dea: deamidation; Iso: isomerization; Oxi: oxidation; Cyc: cyclization.