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A stability-indicating reversed-phase high-performance liquid chromatographic method was developed and validated as per the International Conference on Harmonization (ICH) guidelines to evaluate the reproducibility of batches of synthetic peptides included in a stability program, in particular cholecystokinin (CCK-4) peptide. Both isothermal and nonisothermal approaches were used to determine stability under experimental conditions and the resulting degradation products were identified by liquid chromatography-mass spectrometry (LC-MS). The principal degradation product was the cyclic dimer, although another two products derived from it were also detected, due to the loss of one or two Phe-NH(2) residues. The dimerization follows first-order kinetics, whereas the hydrolytic cleavage implies both consecutive and in-parallel processes. The linear Arrhenius plot indicates that the degradation mechanism and kinetics do not change with temperature or the batch, but the degradation rate does depend on the batch, for example, the shelf-life at 25 degrees C was 2.54 days for batch 3, which is 13-times lower than batch 2. This variability is caused by a change in the synthesis process introduced by the manufacturer. The combination of these two elements: the analytical and stability-evaluating methods provide enough data to establish a stability-indicating profile, as required by the guideline ICH-Q6B for biotechnological/biological products.

The molecular weight of biopolymers such as peptides or proteins is vital information for understanding their physical/chemical properties. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and size-exclusion chromotography–multi-angle laser light scattering (SEC–MALLS) techniques each with its advantages and limitations were used for molecular weight determination of the SPf66 peptide. The precision of each method was studied using a two-factor fully-nested design with all the analyses performed by the same operator on a single instrument. The overall precision corresponded to the time-different intermediate precision (i.e. time and repeatability conditions). In the case of SEC–MALLS, all potential uncertainty components were carefully evaluated in an Ishikawa diagram, then included and mathematically combined with the uncertainty arising from the accuracy assessment to provide the overall uncertainty. In this case, the refractive-index increment with solute concentration value (dn/dc) provided the most significant contribution to the combined uncertainty. A method for its quantitation is proposed. The accuracy of method B (SEC–MALLS) against reference method A (MALDI-TOF-MS) was assessed using the interval testing hypothesis to limit the risk of unacceptable bias. The results indicate that the bias of B is higher than the limit established at 5%, and is therefore not traceable to A under the studied conditions.