Arginine as a protein stabilizer and destabilizer in liquid formulations.
Even though arginine monohydrochloride (ArgHCl) is a useful additive for protein stabilization, its mechanism is not yet fully elucidated. Moreover, there is a concern that ArgHCl may be a protein denaturant since it decreases transition melting temperature (Tm) of certain proteins. It contains a guanidinium group, a critical structure for denaturating activity of guanidine hydrochloride (GndHCl). Effects of ArgHCl, GndHCl, and creatinine on a model protein, etanercept, were examined by biophysical analyses including DLS, DSC, FT-IR, microviscometer, and SEC. Accelerated storage stability of the protein was examined in the absence and presence of H2O2at different incubation temperatures with pH monitoring. ArgHCl reduced protein aggregation and retained monomer, but increased fragmentation at high temperature. Tm1 and Tm2 of the protein increased with ArgHCl, but slight decrease (>1°C) in Tm3 was observed. GndHCl and creatinine decreased all three Tms. In the presence of heat and H2O2, the effect of ArgHCl was significantly decreased compared to GndHCl and creatinine. In addition, it accelerated the loss of monomer and increased fragmentation with decreasing pH. ArgHCl differed from GndHCl in the mode of physical interaction with the protein, due to its unique balance of three steric functional groups (guanidinium, carboxylic acid, and carbon aliphatic straight chain). In contrast, ArgHCl acted as a protein denaturant at high temperature since NOxgenerated from the amine group at the 3-carbon aliphatic straight chain and it is supported by GndHCl which did not change the pH nor accelerated the monomer loss after oxidation by H2O2at high temperature.
College of Pharmacy, Dongguk University-Seoul, Gyeonggi 410-820, Republic of Korea.
Nam Ah
NA
Kim
College of Pharmacy, Dongguk University-Seoul, Gyeonggi 410-820, Republic of Korea.
Sharvron
S
Hada
College of Pharmacy, Dongguk University-Seoul, Gyeonggi 410-820, Republic of Korea.
Ritu
R
Thapa
College of Pharmacy, Dongguk University-Seoul, Gyeonggi 410-820, Republic of Korea. Electronic address: shjeong@dongguk.edu.
Seong Hoon
SH
Jeong
D001120:Arginine / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical; D003404:Creatinine / Q000737:chemistry*; D004355:Drug Stability; D004356:Drug Storage; D000068800:Etanercept / Q000008:administration & dosage / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry*; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation / Q000187:drug effects; D044366:Transition Temperature
D001120:Arginine; D003404:Creatinine; D006861:Hydrogen Peroxide; D019791:Guanidine; D000068800:Etanercept
Arginine oxidation; DSC; Etanercept; NO(x); Protein aggregation; Protein denaturant