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. Nov-Dec 2018;10(8):1226-1235.
doi: 10.1080/19420862.2018.1511198. Epub 2018 Sep 20.

A Systematic Approach for Analysis and Characterization of Mispairing in Bispecific Antibodies With Asymmetric Architecture

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Free PMC article

A Systematic Approach for Analysis and Characterization of Mispairing in Bispecific Antibodies With Asymmetric Architecture

Chunlei Wang et al. MAbs. .
Free PMC article

Abstract

Immunoglobulin G-like bispecific antibodies with asymmetric architecture are among the most widely used bispecific antibody formats for diagnostic and therapeutic applications. The primary technical challenge for this format is how to achieve correctly paired assembly of four unique polypeptide chains. Advances in protein engineering and process development are being used to overcome these challenges and are driving a corresponding demand for sensitive analytical tools to monitor and control mispaired species. Here, we report a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)-based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)-based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

Keywords: Bispecific antibody; DuetMab; hydrophobic interaction chromatography; mispairing; two-dimensional liquid chromatography.

Figures

Scheme I.
Scheme I.
Correctly paired bispecific antibody and potential mispaired species.
Figure 1.
Figure 1.
LC-MS analysis of Bis-A samples obtained from purification processes as shown in Scheme II. (A) Protein A pool; (B) LFS flow-through/protein A; (C) LFS elute; (D) kappa flow-through; (E) kappa elute; (F) polishing step washing; (G) polishing step elute. Detailed mass data for each sample are provided in Supplemental Table S1.
Scheme II.
Scheme II.
Downstream process used to purify bispecific antibodies.
Figure 2.
Figure 2.
LC-MS subunit analysis. (A) Subunits generated from correctly paired DuetMab; (B) subunits generated from LC swap mispair species; (C) LC-MS analysis results.
Figure 3.
Figure 3.
HIC method development for Bis-B. (A) Salt screening; (B) pH screening; (C) salt concentration screening. Seven peaks were separated in the final method, which are labeled in panel C. The HIC profile for final purified materials is also shown in panel C.
Figure 4.
Figure 4.
Intact mass measurements for Bis-B from the 2D LC-MS experiment for (A) peak 1, (B) peak 2, (C) peak 4, (D) peak 5, (E) peak 6, and (F) peak 7 from the HIC method (Figure 3C, final method).
Figure 5.
Figure 5.
Subunit analysis data for Bis-B from the 2D LC-MS experiment for (A) peak 1, (B) peak 2, (C) peak 4, (D) peak 5, (E) peak 6, and (F) peak 7 from the HIC method (Figure 3C, final method). Detailed mass data for each peak are provided in Supplemental Table S2.

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This study was supported by MedImmune, the global biologics R&D arm of AstraZeneca. All authors are employees of MedImmune and have stock interests and/or options in AstraZeneca.

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