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Utility of High Resolution NMR Methods to Probe the Impact of Chemical Modifications on Higher Order Structure of Monoclonal Antibodies in Relation to Antigen Binding.
Majumder, Subhabrata; Saati, Andrew; Philip, Shibu; Liu, Lucy L; Stephens, Elaine; Rouse, Jason C; Alphonse Ignatius, Arun.
Afiliação
  • Majumder S; Pharmaceutical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, 63017, USA.
  • Saati A; Analytical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, 01810, USA.
  • Philip S; Analytical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, 01810, USA.
  • Liu LL; Analytical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, 01810, USA.
  • Stephens E; Analytical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, 01810, USA.
  • Rouse JC; Analytical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, 01810, USA.
  • Alphonse Ignatius A; Pharmaceutical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, 63017, USA. arun.alphonseignatius@pfizer.com.
Pharm Res ; 36(9): 130, 2019 Jul 01.
Article em En | MEDLINE | ID: mdl-31264003
ABSTRACT

PURPOSE:

An understanding of higher order structure (HOS) of monoclonal antibodies (mAbs) could be critical to predicting its function. Amongst the various factors that can potentially affect HOS of mAbs, chemical modifications that are routinely encountered during production and long-term storage are of significant interest.

METHODS:

To this end, two Pfizer mAbs were subjected to forced deamidation stress for a period of eight weeks. Samples were aliquoted at various time points and high resolution accurate mass liquid chromatography-mass spectrometry (LC-MS/MS) was performed using low-artifact trypsin digestion (LATD) peptide mapping to identify and quantify chemical modifications. 2D backbone amide and sidechain methyl NMR spectra were acquired to gauge the effect of HOS changes upon chemical modification. Differential scanning calorimetry was also performed to assess the effect of thermal stability of mAbs upon modification. Finally, functional studies via target-binding based ELISA were performed to connect HOS changes to any loss of potency.

RESULTS:

The extent of deamidation in the mAb domains were quantified by LC-MS/MS. The HOS changes as obtained from 2D NMR were mostly localized around the affected sites leaving the overall structure relatively unchanged. The antigen-antibody binding of the mAbs, in spite of deamidation in the Fab region, remains unchanged.

CONCLUSION:

This case study provides an integrated approach of relating chemical modifications in mAb domains with possible changes in HOS. This can be potentially used to assess a possible loss of potency within the structure-function paradigm of proteins in an orthogonal manner.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Anticorpos Monoclonais / Complexo Antígeno-Anticorpo Tipo de estudo: Prognostic_studies Idioma: En Revista: Pharm Res Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Anticorpos Monoclonais / Complexo Antígeno-Anticorpo Tipo de estudo: Prognostic_studies Idioma: En Revista: Pharm Res Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Estados Unidos