Anti-Idiotype DNA Aptamer Affinity Purification–High-Temperature Reversed-Phase Liquid Chromatography: A Simple, Accurate, and Selective Bioanalysis of Bevacizumab

Abstract

This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purification–high-temperature reversed-phased liquid chromatography (HT-RPLC) with fluorescence detection. Bevacizumab in plasma samples was purified using magnetic beads immobilized with an anti-idiotype DNA aptamer for bevacizumab. The purified bevacizumab was separated with HT-RPLC and detected with its native fluorescence. Using aptamer affinity beads, bevacizumab was selectively purified and detected as a single peak in the chromatogram. HT-RPLC achieved good separation for bevacizumab with a sharp peak within 10 min. The calibration curves of the two monoclonal antibodies ranged from 1 to 50 μg/mL and showed good correlation coefficients (r2 > 0.999). The limit of detection (LOD) and lower limit of quantification (LLOQ) values for bevacizumab were 0.15 and 0.51 μg/mL, respectively. The proposed method was successfully applied to the bioanalysis of the plasma samples obtained from the patients with lung cancer and may be extended to plan optimal therapeutic programs and for the evaluation of biological equivalencies in the development of biosimilars.

Aptamers against Immunoglobulins: Design, Selection and Bioanalytical Applications

“…The same aptamer was used in affinity purification-high temperature reversed phase liquid chromatography (HP-RTLC) with fluorescence detection for bevacizumab determination from plasma samples from patients with lung cancer. A sharp, single peak was detected within 10 min, when bevacizumab was pre-extracted with anti-idiotype DNA aptamer-modified magnetic beads [80]. However, the limit of detection was 0.15 µg/mL (1 nM), which is ca.…”

Sensitive Method for LC Analysis of Therapeutic Monoclonal Antibodies Using a Centrifugal Filtration Device with Adsorption Suppression Treatment

“…However, the column was too short to separate therapeutic mAbs or human endogenous IgG. High-temperature reversed-phase LC [10,11,15,16], which we have already reported, can be applied here as it allows simultaneous analysis of multiple antibody drugs and bioanalysis. Table 1 shows the peak heights and concentration factors before and after centrifugal concentration of the trastuzumab solutions.…”

“…We have recently reported a bioanalysis method for therapeutic mAbs that combined immunoaffinity purification or aptamer affinity purification with LC-fluorescence detection [10,11]. In addition, we reported a high-sensitivity LC analysis method in which bevacizumab was concentrated using a centrifugal filtration device, and direct LC analysis was performed [12].…”

High-Temperature Reversed-Phase LC Separation of Heavy and Light Chain Fragments of Therapeutic Monoclonal Antibodies and Antibody-Drug Conjugate Produced by Chemical Reduction

“…Most of the experimental parameters for HT-RPLC were the same as in previous studies [5][6][7]. We used the Prominence UFLC liquid chromatography system (Shimadzu), which consisted of a CBM-20A system controller, a SIL-20AC autosampler, two LC-20AD pumps, a DGU-20A online degasser, a CTO-20AC column oven, an SPD-M20A PDA detector, and an RF10A XL fluorescence spectrometer equipped with a 12 µL flow cell.…”

“…We have also reported bioanalytical methods for the direct analysis of therapeutic mAbs in human blood samples. The method was a combination of high-temperature reversed-phase LC (HT-RPLC) [5] and immunoaffinity [6] or DNA aptamer affinity [7,8] purification.…”