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Antibody structural integrity of site-specific antibody-drug conjugates investigated by hydrogen/deuterium exchange mass spectrometry
- Source :
- Analytical chemistry. 87(11)
- Publication Year :
- 2015
-
Abstract
- We present the results of a hydrogen/deuterium exchange mass spectrometric (HDX-MS) investigation of an antibody-drug conjugate (ADC) comprised of drug-linkers conjugated to cysteine residues that have been engineered into heavy chain (HC) fragment crystallizable (Fc) domain at position 239. A side-by-side comparison of the HC Ser239 wild type (wt) monoclonal antibody (mAb) and the engineered Cys239 mAb indicates that site directed mutagenesis of Ser239 to cysteine has no impact on the HDX kinetics of the mAb. According to the crystal structure of a homologous immunoglobulin G1 (IgG1) antibody (PDB: 1HZH ), the backbone amide of Ser239 is hydrogen-bonded to Val264 backbone amide in the wt-mAb studied here. Replacing Ser239 with a Cys residue does not alter the exchange kinetics of the backbone amide of Val264 suggesting that either Ser or Cys at position 239 has similar amide-hydrogen bonding with Val264. However, a small segment in CH2 domain of the ADC ((264)VDVS) was found to have a slightly increased HDX rate compared to the wt- and C239-mAb constructs. The slightly increased HDX rate of the segment (264)VDVS in ADCs indicates that the further modification of Cys239 with drug-linkers only attenuates the local backbone amide hydrogen-bonding network between Cys239 and Val264. All other regions which are proximal to the site of drug conjugation are unaffected. The results demonstrate that the site-specific drug conjugation at the engineered Cys residue at the position 239 of HC does not impact the structural integrity of antibodies. The results also highlight the utility of applying HDX-MS to ADCs to gain a molecular level insight into the impact of site-specific conjugation technologies on the higher-order structure (HOS) of mAbs. The methodology can be applied generally to site-specific ADC modalities to understand the individual contributions of site-mutagenesis and drug-linker conjugation on the HOS of therapeutic candidate ADCs.
- Subjects :
- Binding Sites
Immunoconjugates
Chemistry
Stereochemistry
Protein Data Bank (RCSB PDB)
Deuterium Exchange Measurement
Crystallography, X-Ray
Antibodies
Mass Spectrometry
Analytical Chemistry
Residue (chemistry)
chemistry.chemical_compound
Biochemistry
Pharmaceutical Preparations
Amide
Hydrogen–deuterium exchange
Binding site
Site-directed mutagenesis
Cysteine
Conjugate
Subjects
Details
- ISSN :
- 15206882
- Volume :
- 87
- Issue :
- 11
- Database :
- OpenAIRE
- Journal :
- Analytical chemistry
- Accession number :
- edsair.doi.dedup.....081ffb56ee784005e4f7b9f89d56440d