Abstract
Purpose
To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO).
Methods
Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins.
Results
The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified.
Conclusion
The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.
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Abbreviations
- i.d.:
-
Inner diameter
- ICP-MS:
-
Inductively coupled plasma-mass spectrometry
- mAb:
-
Monoclonal antibody
- MCO:
-
Metal catalyzed oxidation
- MFI:
-
Micro-Flow Imaging
- o.d.:
-
Outer diameter
- PBMC:
-
Peripheral blood mononuclear cells
- PDB:
-
Protein Data Bank
- ppb:
-
Parts per billion
- rCE-SDS:
-
Reduced capillary electrophoresis-sodium dodecyl sulfate
- SAS:
-
Solvent accessible surface
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ACKNOWLEDGMENTS AND DISCLOSURES
The authors wish to thank Diana Woehle for the preparation of mAb2, and Daniel Weinbuch for the preparation of MCO treated rituximab.
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Supplemental Figure 1
Comparison of particle number and size distribution of micron particles generated by MCO via different detection techniques. mAb1, mAb2, and rituximab untreated and MCO samples were examined by A) HIAC/light obscuration and B) MFI to determine the number and size range of particles present. For each sample, the differential particle counts per ml for each size range is shown. (JPG 132 kb)
Supplemental Figure 2
Comparison of the biophysical and chemical properties of the mAb2 antibody treated by three different MCO methods. MCO methods 1, 2 and 3 are described in detail in the materials and methods section. A) mAb2 untreated and MCO samples were examined by HIAC to determine the number and size range of particles present. For each sample, the differential particle counts per ml for each size range is shown. B) Particle images were captured on a MFI system. Representative images of the largest particles detected are shown. The size threshold (equivalent circular diameter) indicates the lower size limit of the particles that were used for comparison. C) The level of elemental copper (ppb) in each sample was determined by ICP-MS. The ratio shown represents the calculated ratio of the molecules of antibody (mAb) to molecules of copper. D) The percent oxidation of each amino acid of mAb2 was determined by peptide map. (JPG 192 kb)
Supplemental Figure 3
HCD MS/MS spectrum of the His 272 oxidized peptide H20 (A), His272 oxidized peptide H21 (B) and His 314 oxidized peptide H24 (C). (JPG 186 kb)
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Narhi, L.O., Luo, Q., Wypych, J. et al. Chemical and Biophysical Characteristics of Monoclonal Antibody Solutions Containing Aggregates Formed during Metal Catalyzed Oxidation. Pharm Res 34, 2817–2828 (2017). https://doi.org/10.1007/s11095-017-2262-8
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DOI: https://doi.org/10.1007/s11095-017-2262-8