Formulation development and primary degradation pathways for recombinant human nerve growth factor.

Abstract

The chemical and physical stabilities of recombinant human nerve growth factor (NGF) in aqueous solution were investigated between 5 and 37 degrees C and at pH 4.2-5.8. NGF chemical stability decreased with a decrease in pH due to Asp60-Pro61 cleavage, with the stability being greater in acetate buffer than in succinate buffer at each pH investigated. Aggregation was a significant degradation pathway at 37 degrees C, with the aggregation rate being greatest in succinate buffer at pH 5.8. Quantitation of NGF degradation by cation-exchange chromatography was complicated by the rearrangement of the NGF monomer variants into various mixed dimers over time. Treatment with dilute acid brought the dimer distribution rapidly to equilibrium, allowing NGF degradation to be accurately quantitated. An acetate-buffered formulation at pH 5.5 was investigated in more detail. To assist in degradation product identification, NGF degradation was accelerated with base, hydrogen peroxide, and temperature. These degradation products were shown to coelute on RP-HPLC with the variants found when the protein was stored at -70, 5, and 25 degrees C. By electrospray mass spectrometry, peptide maps, and LC/MS, these degradation products were shown to be monooxidized (Met37) and dioxidized (Met37 and Met92) NGF, with Met37 being more labile, deamidated NGF (Asn45), and NGF with Asp93 isomerized to beta-Asp93. NGF can be stored in pH 5.5 acetate buffer at 5 degrees C for 1.5 years with less than 10% conversion to these degradation products, with Asp93 isomerization being the primary degradation pathway.