title: A critical evaluation of Tm(FTIR) measurements of high-concentration IgG1 antibody formulations as a formulation development tool.
abstract: OBJECTIVE
  Fourier-transform infrared (FTIR) spectroscopy was applied for the determination of protein melting temperature (Tm(FTIR)) and to assess the stability predictability of a 100-mg/mL liquid IgG1 antibody formulation.
  
  
  METHODS
  Tm(FTIR) values of various formulations (different pH, buffers, excipients) were compared to the results of a stability study under accelerated conditions (40 degrees C/75% relative humidity), using size-exclusion high-performance liquid chromatography (SE-HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the detection of soluble aggregates and covalent modifications.
  
  
  RESULTS
  The highest Tm(FTIR) was achieved at pH 5.5, and, similarly, SE-HPLC and SDS-PAGE results suggested a pH optimum between 5.5 and 6.0. Transition temperatures were comparable for all tested buffers. However, the decrease in the monomer fraction upon thermal storage was the lowest for citrate buffers. Whereas sugars and polyols resulted in an increase in Tm(FTIR) and enhanced monomer fraction after storage, amino acids showed a destabilization according to SE-HPLC analysis, albeit no change or even an increase in the melting temperature was observed.
  
  
  CONCLUSIONS
  All examples gave evidence that Tm(FTIR) values did not necessarily correspond to the storage stability at 40 degrees C analyzed by means of SE-HPLC and SDS-PAGE. Tm values, e.g., determined by FTIR, should only be employed as supportive information to the results from both real-time and accelerated stability studies.
authors.affiliation: Merck KGaA, Global Pharmaceutical Development, Darmstadt, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Matheus
authors.forename: Hanns-Christian
authors.initials: HC
authors.lastname: Mahler
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Friess
mesh_terms: D002021:Buffers; D002626:Chemistry, Pharmaceutical; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D015203:Reproducibility of Results; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013395:Sucrose / Q000737:chemistry; D013678:Technology, Pharmaceutical / Q000379:methods; D044366:Transition Temperature; D014199:Trehalose / Q000737:chemistry
chemical_list: D002021:Buffers; D005079:Excipients; D007074:Immunoglobulin G; D013395:Sucrose; D014199:Trehalose