title: Effect of pH and excipients on structure, dynamics, and long-term stability of a model IgG1 monoclonal antibody upon freeze-drying.
abstract: OBJECTIVE
  To investigate the mechanism of IgG1 mAb stabilization after freeze-drying and the interdependence of protein structural preservation in the solid state, glassy state dynamics and long-term storage stability under different formulation conditions.
  
  
  METHODS
  IgG1 mAb was formulated with mannitol at pH 3.0, 5.0, and 7.0 in the presence and absence of sucrose and stability was monitored over 1 year at different temperatures. Physical and covalent degradation of lyophilized formulation was monitored using SEC, CEX, and light obscuration technique. Secondary and tertiary structure of the protein in the solid state was characterized using FTIR and fluorescence spectroscopy respectively. Raman spectroscopy was also used to monitor changes in secondary and tertiary structure, while SS-NMR (1)H relaxation was used to monitor glassy state dynamics.
  
  
  RESULTS
  IgG1 mAb underwent significant secondary structural perturbations at pH 3.0 and conditions without sucrose, while pH 5.0 condition with sucrose showed the least structural change over time. The structural changes correlated with long-term stability with respect to protein aggregate formation and SbVP counts. SS-NMR data showed reduced relaxation time at conditions that were more stable.
  
  
  CONCLUSIONS
  Native state protein structural preservation and optimal solid-state dynamics correlate with improved long-term stability of the mAb in the different lyophilized formulations.
authors.affiliation: Amgen Inc., Process and Product Development, Thousand Oaks, California, 91320, USA.
authors.forename: Jihea
authors.initials: J
authors.lastname: Park
authors.forename: Karthik
authors.initials: K
authors.lastname: Nagapudi
authors.forename: Camille
authors.initials: C
authors.lastname: Vergara
authors.forename: Ranjini
authors.initials: R
authors.lastname: Ramachander
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Laurence
authors.forename: Sampathkumar
authors.initials: S
authors.lastname: Krishnan
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004356:Drug Storage; D005079:Excipients / Q000737:chemistry*; D005612:Freeze Drying; D007074:Immunoglobulin G / Q000737:chemistry*; D008353:Mannitol / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013395:Sucrose / Q000737:chemistry; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D007074:Immunoglobulin G; D008353:Mannitol; D013395:Sucrose