An Imaged Capillary Isoelectric Focusing Separation of the Linear and Cyclic Variants of a Mimotope of the Cancer‐Related CD20 Antigen–Validation and Statistical Evaluation

Imaged capillary isoelectric focusing was successfully applied for separating an in-house synthesized closely related peptide pair, that is, a linear 12-mer (Rp5-L) and its cyclic 15-mer variant (Rp5-C). Rp5-L represents a mimotope, that is, an epitope mimicking peptide, of the CD20 antigen, which is over-expressed in B-cell-related tumors. Peptide identity-including the successful disulfide bond formation in Rp5-C-was confirmed with matrix-assisted laser desorption ionization-time of flight mass spectrometry. The purity of synthesized products was determined by a reversed-phase high-performance liquid chromatographic method with ultraviolet detection. The apparent isoelectric point (pI) of cyclic Rp5-C and Rp5-L was 5.99 and 6.47, respectively. An appropriate combination of carrier ampholytes allowed for their baseline separation with an analysis time of <20 min. Method validation was done for the synthesized peptides and three flanking pI markers covering, for example, repeatability and intermediate precision. Calibrations on different days resulted in identical slopes for Rp5-L and Rp5-C, respectively, as statistically confirmed by Welch's t-test and pooled t-test over 8 days. The calibration data of mimotopes and pI markers were evaluated for outliers, normality, homoscedasticity, and autocorrelation with complementary statistical procedures, which identified an otherwise unnoticed outlier for a pI marker. The linearity of calibration for Rp5-L, Rp5-C, and the pI markers was tested with Mandel's fitting test and lack-of-fit test. For Rp5-L and Rp5-C, the calculated limits of detection and limits of quantification were ≤0.31 and ≤0.96 µmol/L, respectively.