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Assessment of Physical Stability of an Antibody Drug Conjugate by Higher Order Structure Analysis: Impact of Thiol- Maleimide Chemistry

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ABSTRACT

Purpose

To provide a systematic biophysical approach towards a better understanding of impact of conjugation chemistry on higher order structure and physical stability of an antibody drug conjugate (ADC).

Methods

ADC was prepared using thiol-maleimide chemistry. Physical stabilities of ADC and its parent IgG1 mAb were compared using calorimetric, spectroscopic and molecular modeling techniques.

Results

ADC and mAb respond differently to thermal stress. Both the melting temperatures and heat capacities are substantially lower for the ADC. Spectroscopic experiments show that ADC and mAb have similar secondary and tertiary structures, but these are more easily destabilized by thermal stress on the ADC indicating reduced conformational stability. Molecular modeling calculations suggest a substantial decrease in the conformational energy of the mAb upon conjugation. The local surface around the conjugation sites also becomes more hydrophobic in the ADC, explaining the lower colloidal stability and greater tendency of the ADC to aggregate.

Conclusions

Computational and biophysical analyses of an ADC and its parent mAb have provided insights into impact of conjugation on physical stability and pinpointed reasons behind lower structural stability and increased aggregation propensity of the ADC. This knowledge can be used to design appropriate formulations to stabilize the ADC.

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Abbreviations

ADC:

Antibody drug conjugate

ANS:

8-Anilino-1-naphthalenesulfonic acid

CD:

Circular dichroism

DAR:

Drug: antibody ratio

DCVJ:

4-(dicyanovinyl)Julolidine

DMSO:

Dimethyl sulfoxide

DSC:

Differential scanning calorimetry

DSF:

Differential scanning fluorimetry

Fab:

Fragment antigen binding

Fc:

Fragment crystallizable

HC:

Heavy chain

LC:

Light chain

LP:

Linker plus payload

mAb:

Monoclonal antibody

OD:

Optical density

PDB:

Protein Data Bank

RMSD:

Root mean square deviation

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Authors and Affiliations

  1. Biotherapeutics Pharmaceutical Sciences, Pharmaceutical R&D, Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO, 63017, USA

    Jianxin Guo, Sandeep Kumar & Satish K. Singh

  2. Biotherapeutics Pharmaceutical Sciences, Bioprocess R&D, Pfizer Inc., 401 N Middletown Rd, Pearl River, NY, 10965, USA

    Amarnauth Prashad

  3. Biotherapeutics Pharmaceutical Sciences, Analytical R&D, Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO, 63017, USA

    Jason Starkey

Authors
  1. Jianxin Guo
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  2. Sandeep Kumar
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  3. Amarnauth Prashad
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  5. Satish K. Singh
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Correspondence to Satish K. Singh.

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Guo, J., Kumar, S., Prashad, A. et al. Assessment of Physical Stability of an Antibody Drug Conjugate by Higher Order Structure Analysis: Impact of Thiol- Maleimide Chemistry. Pharm Res 31, 1710–1723 (2014). https://doi.org/10.1007/s11095-013-1274-2

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  • DOI: https://doi.org/10.1007/s11095-013-1274-2

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