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Review
. Aug/Sep 2019;11(6):1101-1112.
doi: 10.1080/19420862.2019.1618675. Epub 2019 Jun 4.

Using Bispecific Antibodies in Forced Degradation Studies to Analyze the Structure-Function Relationships of Symmetrically and Asymmetrically Modified Antibodies

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Free PMC article
Review

Using Bispecific Antibodies in Forced Degradation Studies to Analyze the Structure-Function Relationships of Symmetrically and Asymmetrically Modified Antibodies

Adam R Evans et al. MAbs. .
Free PMC article

Abstract

Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.

Keywords: Fc; Monoclonal antibody; asymmetric antibodies; duobody®; mass spectrometry; methionine oxidation; protein A separation; single chain modification; structure function characterization.

Figures

Figure 1.
Figure 1.
The process for creating asymmetrically oxidized bispecific molecules (BsAb) is presented. (a) Peracetic acid treatment of mAb1 (blue) or mAb2 (peach) resulted in methionine oxidation (illustrated as red lines) throughout the molecule. (b) Combinations of non-oxidized (A) and/or oxidized (B) mAb1 and mAb2 were pooled prior to Fab arm exchange to generate control BsAb1 (AA), asymmetrically oxidized BsAb2 (AB) and BsAb3 (BA), and symmetrically oxidized BsAbs (BB). All BsAbs were purified to the final product prior to analytical characterization.
Figure 2.
Figure 2.
HIC chromatograms of BsAb products. Chromatograms for post-FAE reaction intermediates and final-purified products are shown in the top and bottom panels, respectively.
Figure 3.
Figure 3.
Subunit MS analysis of mAb and BsAb. (a) The deconvoluted spectra for control and oxidized mAb1 and mAb2 are shown in the left and right panels, respectively. (b) The deconvoluted spectra for BsAb1, BsAb2, BsAb3, and BsAb4 are shown in order from top to bottom. mAb1, mAb2, and methionine oxidation are shown with blue fill, peach fill, and red lines, respectively.
Figure 4.
Figure 4.
Protein A affinity chromatography chromatograms for BsAb1, BsAb2, BsAb3, and BsAb4 intact IgG. Protein absorbance was monitored at 280 nm.
Figure 5.
Figure 5.
The process for creating bispecific antibodies with asymmetric deamidation at Asn330 is presented. (a) mAb1 (blue) and mAb2 (peach) were stressed at 40°C at pH 5.5 to induce selective deamidation at the Asn330 residue in the Fc Region (brown with red lines). Combinations of native or stressed mAb1 and mAb2 were pooled prior to Fab-arm exchange to generate control BsAb5, asymmetrically deamidated BsAb6 and BsAb7, and symmetrically deamidated BsAb8. (b) The bar plot illustrates the levels of FcγRIIIa binding of BsAb6, BsAb7, and BsAb8 relative to the control BsAb5 sample. The HC Asn330 deamidation values for the mAb1 and mAb2 arms of each BsAb are shown above the corresponding bar.

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