Development Of Colloidal Au-enhanced SPR Technology For Quantitation Of Antibody Drug Of C225

Surface plasmon resonance (SPR) was introduced in the early 1990s as the underlying technology for biomolecular interaction analysis. SPR technology has been used recently for analysis and characterization in the biomedical sciences. With SPR biosensor technology, the reactants are measured without the need to label and pre-treat. In addition, automated operation and real-time measurement characteristics are improved over enzyme-linked immunosorbent assay (ELISA) or other bioassays. Nevertheless, SPR biosensors also have disadvantages, and low sensitivity is the primary drawback. Because on the surface of SPR sensor chip, a carboxymethylated dextran layer was used as the interface, which allowed for protein immobilization onto the gold surface of the SPR sensor. This 100-nm thick interface is responsible for mass-transfer resistance and inducing steric hindrance during the biomolecular interactions that occur at the sensor surface. These interactions may result in the low sensitivity of the SPR technology compared to the classical quantification method in solution. Therefore, enhancing the sensitivity of the quantification with the SPR technology is a crucial task to resolve.In our work, SPR signals were measured using a BIAcore 3000 instrument with CM5 sensor chips. We previously developed a method for measuring the concentration of cetuximab (C225) in monkey serum using the BIAcore system. This method was simple and fast and was successfully applied to a preclinical pharmacokinetic study of C225 in rhesus monkeys. However, the lower limit of quantification (LLOQ) of this method was 0.05μg/mL and the sensitivity for detecting C225 at low concentrations was low. Therefore, this work aims to explore a new method where colloidal gold nanoparticles (Au NPs) are used to enhance SPR signals during C225 concentration detection in monkey serum, and applied to a preclinical pharmacokinetic study of C225 in rhesus monkeys.The Au NPs were self-assembly synthesized by reducing HAuCl4 using sodium citrate as the reducing agent and conjugated with goat anti-human IgG. As a chimeric, murine-human derivative-IgG1 monoclonal antibody, C225 can bind the extracellular domain of epidermal growth factor receptor (EGFR) with high affinity. Using an amine coupling kit, the EGFR was covalently coupled to the carboxymethylated dextran matrix by primary amine groups onto the CM5 sensor chip. A 10μg/mL C225 stock solution in HBS-EP buffer was freshly prepared and was serially diluted with 20% rhesus monkey serum (diluted with HBS-EP buffer) to generate concentrations of 0.0125, 0.025, 0.05, 0.1, 0.4, 0.8, 1.6, and 3.2μg/mL. Quality controls (QC) were prepared at 0.025, 0.4 and 1.6μg/mL for C225. First, 20μL of the C225 serum sample was injected onto the sensor to enable the C225 molecules to combine with the EGFR. Second, after the sensor was washed with the appropriate buffer, 60μL of goat anti-human IgG-Au NPs solvent (diluted five-fold with HBS-EP buffer) was injected onto the sensor chip surface to enable the antibodies to bind to the captured C225 analyte. The unbound goat anti-human IgG-Au NPs were washed off by HBS-EP buffer. Third, regeneration of the CM5 sensor surface was performed by injecting 10μL of glycine-HCL (pH 2.0) for repeated measurements. The calibration sample analyses at each concentration were performed in duplicate.The calibration curve was fitted to the parameter Logistic model. Fc1, which was immobilized with the EGFR, was set as the test channel, and Fc2 (without the EGFR) served as the reference channel. By using goat anti-human IgG-conjugated Au NPs to capture the C225 molecules, the Au particles combined with the sensor chip surface and enhanced the BIAcore responses significantly. This detect course is similar with sandwich immunoassay at some level . After the sample flowed over the EGFR-immobilized sensor surface, the signal from the reference channel was subtracted from that of the test channel to compensate for changes in the bulk refractive index, non-specific binding, and minor temperature fluctuations. In addition, the specificity, accuracy, precision, calibration range, LLOQ, and stability of this assay were validated according to FDA guidelines.After development and validation, this new BIAcore -based method has also been used to characterize the serum pharmacokinetic behavior of C225 in monkey serum. Groups of rhesus monkeys received 7.5, 24, 75 mg/kg for 60 minites in single intravenous infusion test. Comparing the pharmacokinetic parameters of the different groups.Results show that 10nm Au NPs yielded by this method were relatively spherical and homogeneous. The mean diameter of the 100 Au NPs was 11.2±2.4 nm. The UV-vis spectra shows the maximum absorption peak of the Au NPs and goat anti-human IgG-Au NPs complex were 518.5 nm and 527.0 nm, respectively. This red shift phenomenon indicated that a protein layer formed on the surface of the Au NPs, and the UV-vis absorption properties of the colloidal Au was modified after the conjugation. The formed conjugates were used in the next work.A nine-point calibration curve was constructed by plotting the signal of the BIAcore instrument (RU) to the log of the analyte concentration. The curve was then fitted to a four-parameter Logistic model. The results indicated that the calibration curves for C225 were linear from 0.0125 to 3.2μg/mL. The intra-day assay precision ranged from 4.33% to 13.11%, and the intra-day assay accuracy ranged from -3.72% to 4.84%. The inter-day precision ranged from 2.16% to 11.77%, and the accuracy ranged from -0.15% to 5.79%. The specificity results show that human serum and Sprague-Dawley rat serum exhibited no interference with the proposed assay method. The cross test indicated that the addition of the chimeric recombinant anti-CD20 monoclonal antibody, humanγ-globulin and chimeric recombinant her2 antibody in the sample did not affect analyte detection. To evaluate the baseline stability of the sensor chip, 100 regeneration cycles were performed on the surface of CM5 chip, and test showed that there was no significant changes in baseline (less than 7.8%). These results indicate that all of the validation data were within the required limits. In this test, C225 in the monkey serum was detected at a concentration as low as 0.0125μg/mL by the BIAcore immunoassay combined with signal amplification. This concentration indicates that a four-fold magnitude enhancement was achieved compared to the BIAcore system without Au NPs, which has been used in previous reports (at a concentration of 0.05μg/mL). In our study, an ELISA method was also developed and validated for quantifying C225 in monkey serum. The ELISA assay can detect C225 from 0.0125-0.4μg/mL. However, the BIAcore -based method described here measured a much broader range of C225 concentrations (0.0125-3.2μg/mL). In addition, the mechanism of this enhancement was also described. By combining Au NPs with the BIAcore sensor chip, changes in the refractive index were amplified by the electronic coupling interaction between the localized surface plasmon of the Au particles and the surface plasmon wave associated with the BIAcore gold film. Thus, the BIAcore signal enhancement was achieved, and the sensitivity and LLOQ of the assay were improved. The changes in the relative RU value were improved by altering the amplification of the refractive index on the CM5 sensor chip surface. The amount of proteins coated onto the Au NP surface showed a linear correlation with the resonance angle changes in the BIAcore system. Therefore, changes in the resonance angle are directly associated with analyte concentration. Through these factors, the sensitivity and specificity of the BIAcore system were improved, and the C225 LLOQ in monkey serum was lowered further.After single intravenous infusion to rhesus monkeys, the drug concentration in serum reached peak at the needle-withdrawing time for three different dosage, and the concentration was parallel to the dosage of injected drug. All same time points has statistical variances among the low-medium, medium-high, high-low dosage groups. The terminal phase half lives significantly extended with the increase of dosages. The drug concentration of low dosage group decreased to 1/20 of peak concentration at 312 hours after drug injection and the middle dosage group concentration decreased to 1/20 of peak concentration at 216 hours after drug injection. While the high dosage group needed 456 hours to reach to 1/20 of peak concentration. Comparing the parameters of the different groups, the average residence time significantly extended with the increase of dosage, the MRT was 105.11±4.78h, 118.89±1.19h and 152.84±6.83h for the three different dosage (7.5, 24, 75 mg/kg) separately. The AUC(0-t) value of low dosage group was 16390.54±1201.80μg·h·mL-1, The AUC(0-t) value of middle dosage group was 51453.00±1199.80μg·h·mL-1, while the hige dosage group AUC(0-t) value was 263091.21±30698.14μg·h·mL-1. The dose of CL were 0.459±0.03 mL·h-1·kg-1, 0.466±0.01mL·h-1·kg-1, 0.29±0.03mL·h-1·kg-1 separately. The clearance decreased with the increasing dose. This shows a typical non-linear kinetic characters, and indicates that a saturated clearance reached at high doses. All results indicate that between the dose range(7.5-75 mg/kg), the C225 presented non-linear kinetic character in rhesus monkey.In conclusion, this paper describes a straightforward quantification method. Involving yield of Au NPs and synthesis of gold conjugates as well as a new amplification model that further enhances the sensitivity of the BIAcore-based quantification of C225. The Au NPs yielded by this method are relatively spherical and homogeneous. The proposed method exhibits good precision, accuracy, and sufficiently low LLOQ (0.0125μg/mL). The LLOQ obtained in the current work is much lower than in previous reports. Moreover, this SPR strategy can be used to determine a much broader range of C225 concentrations than the ELISA assay. The method was reliable and can quantify C225 concentrations in monkey serum. This new method may develop into a common method for pharmacokinetic studies in the future.