Effect of 'pH' on the rate of asparagine deamidation in polymeric formulations: 'pH'-rate profile.
The rate of Asn deamidation of a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala) was measured as a function of effective pH ('pH') in glassy and rubbery polymeric solids containing poly(vinyl pyrrolidone) (PVP) and in solution controls at 70 degrees C. The reaction exhibited pseudo-first-order kinetics in all samples over a wide 'pH' range (0.5 < 'pH' < 12); the formation of similar products suggests that the reaction mechanism is unaffected by matrix type. Rates of deamidation were comparable for the polymeric and solution samples in the acidic range ('pH' < 4). Solution-state rates were faster than those in polymeric solids at neutral 'pH' (6 < 'pH' < 8), increasing to a > 10,000-fold difference in the basic range ('pH' > 8). Specific base catalysis was observed in solution and in the polymeric solids under neutral conditions (6 < 'pH' < 8). In solution, the reaction exhibited general base catalysis for 'pH' > 8, whereas the reaction was 'pH'-independent in the polymeric solids in this range. The 'pH'-rate profile and supporting buffer catalysis data are consistent with a change in the rate-determining step in the basic range from 'pH'-dependent attack of the deprotonated backbone amide nitrogen on the Asn side chain in solution to 'pH'-independent ammonia expulsion in the polymeric solids. The results suggest that polymer matrix incorporation not only affects the magnitude of the deamidation rate constant but also the 'pH' dependency of the reaction and the rate-determining step in the basic 'pH' range.
Song
Y
Y
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, USA.
Schowen
R L
RL
Borchardt
R T
RT
Topp
E M
EM
D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D002021:Buffers; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011108:Polymers / Q000737:chemistry*
D000577:Amides; D002021:Buffers; D011108:Polymers; D001216:Asparagine