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.

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.

Emergency department presentation of heart transplant recipients with acute heart failure.

With an increasing number of heart transplants being performed around the world and the improvement in survival rates, more transplant recipients may present to the emergency department with comorbidities unique to the transplanted heart and related immunosuppression, including heart failure. This article is aimed at enabling the emergency department physician identify and better manage this unique group of patients for whom time is life.