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

Abstract

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.