The selection of highly specific and selective aptamers using modified SELEX and their use in process analytical techniques for Lucentis bioproduction

Abstract

Aptamers for Lucentis were selected using 10 rounds of a modified and highly stringent SELEX process. Affinity column chromatography was used for the binding, partitioning, and elution steps, and the regeneration of ssDNA was performed via asymmetric PCR in the SELEX process. The interaction of aptamers with Lucentis was studied by means of the HADDOCK web server docking program. In addition, the secondary structures of aptamers were interrogated using the mfold web server to check common regions responsible for better affinity towards Lucentis. The two best aptamers for Lucentis (aptamers 1 and 25) were found to have dissociation constant (K _d) values between 23 and 35 nM by means of thermofluorimetric and non-faradaic impedance spectroscopy (NFIS) analysis. The low dissociation constants in the nanomolar range showed the high specificities of the aptamers for Lucentis. Selectivity tests were also performed using both aptamers with different proteins in which negligible responses were obtained from interfering proteins with respect to Lucentis. Although neither of the two aptamers showed prominent responses to the interfering proteins, slightly better selectivity was shown by aptamer 1. The same aptamers were tested for their application in the detection of Lucentis in spiked and real media broth samples. For this detection test, interdigitated (IDT) gold electrodes on a glass substrate were fabricated using standard photolithography and thermal deposition techniques. NFIS measurements were used for the label-free detection of Lucentis in samples. The linear ranges of detection for aptamers 1 and 25 were found to be 22-100 nM and 40-100 nM, respectively. The LODs for aptamers 1 and 25 were calculated to be 22 nM and 40 nM, respectively, which were significantly better than the values from a HPLC-based detection method (about 240 nM). The real sample analysis results were cross-checked via a standard HPLC method, and better correlation was found between the HPLC and aptamer 1 results than the aptamer 25 results; hence, aptamer 1 can be further analyzed and tested for use in affinity column chromatography and detection-kit/chip-based PAT for Lucentis bioproduction.

Fig. 1. A schematic illustration of the steps involved in the SELEX process.

Fig. 2. The gel electrophoresis of asymmetric PCR products at different primer ratios (left to right): lane 1: 50 bp ladder; lane 2: pure ssDNA library; lanes 3–4: PCR products of ssDNA library; lanes 5–8: asymmetric PCR products at primer ratios of 10 : 1, 20 : 1, 100 : 1, and 500 : 1, respectively, on 4% agarose gel.

Fig. 3. The ssDNA yield eluted in each SELEX cycle for Lucentis.

Fig. 4. Chart showing the difference between the melting peak height of the aptamer () and the aptamer–Lucentis complex (), obtained using thermofluorimetric analysis.

Fig. 5. Nyquist plots toward 13 nM (), 33 nM (), 66 nM (), 333 nM (), 666 nM (), and 3500 nM () Lucentis for (a) aptamer 1 and (b) aptamer 25. (c) Calibration curves between the capacitive component and concentration of Lucentis for the calculation of dissociation constants for aptamers 1 () and 25 () using NFIS.

Fig. 6. The secondary structures of (a) aptamer 1 and (b) aptamer 25 obtained using the mfold web server.

Fig. 7. The impedimetric responses of aptamers 1 () and 25 () following incubation with different proteins, where Luc stands for Lucentis.

Fig. 8. Nyquist plots from a gold electrode (), an aptamer-immobilized gold electrode/blank () dipped in phosphate buffer at pH 5.7, and aptamer-immobilized electrodes dipped in 1 nM (), 10 nM (), 50 nM (), 100 nM (), and 500 nM () Lucentis for (a) aptamer 1 and (b) aptamer 25. (c) Calibration curves between the capacitive component and concentration of Lucentis for aptamers 1 and 25.

Fig. 9. Real sample analysis of Lucentis using aptamer 1 (), aptamer 25 (), and HPLC ().

Fig. 10. The immobilization of aptamers on gold sensing electrodes and the impedance spectroscopy detection setup. (a) Bare gold electrode on glass substrate. (b) Formation of thiol layer on gold electrode after aminoethanethiol treatment. (c) Aptamer immobilization on gold electrode using NHS & EDC. (d) Impedance spectroscopy detection setup.