Cm. Hekman et al., Isolation and identification of cyclic imide and deamidation products in heat stressed pramlintide injection drug product, J PHARM B, 20(5), 1999, pp. 763-772
This report summarizes the identification of six cyclic imide [Asu] and two
deamidation products from a sample of pramlintide final drug product that
had been stressed at 40 degrees C for 45 days. The pramlintide degradation
products were isolated by cation exchange high-performance liquid chromatog
raphy (HPLC) followed by reversed-phase HPLC. The isolated components were
characterized by mass spectrometry (MS), tandem MS (MS/MS) and when necessa
ry, by enzymatic (thermolysin) digestion followed by liquid chromatography/
mass spectrometry (LC/MS) and sequence analysis. The isolated products were
identified as [Asu(14)]-pramlintide, [Asu(21)]-pramlintide, [Asu(22)]-pram
lintide, [Asu(35)]-pramlintide, [1-21]-succinimide-pramlintide, and [1-22]-
succinimide-pramlintide. Also identified were [Asp(35)]-pramlintide, the de
amidation product of pramlintide at Asn(35), and [Tyr(37)-OH]-pramlintide,
the deamidation product of the pramlintide amidated C-terminal Tyr. Togethe
r these data support those presented earlier (C. Hekman et al., Isolation a
nd identification of peptide degradation products of heat stressed pramlint
ide injection drug product. Pharm Res 1998;15:650-9) indicating that the pr
imary mechanism of degradation for pramlintide in this pH 4.0 formulation i
s deamidation, with six of the eight possible deamidation sites observed to
undergo deamidation. Gln-10 and Asn-31 are the only two residues subject t
o deamidation for which none is observed. The data indicate that the cyclic
imide products account for approximate to 20% of the total thermal degrada
tion while the deamidation products account for approximate to 64%. The rem
aining degradation is due to peptide backbone hydrolysis. (C) 1999 Elsevier
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