基于Au纳米粒子新型SPR技术的抗体药物C225的定量分析
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摘要
表面等离子共振(SPR)技术是上世纪90年代发展起来的一种用于生物分子间相互作用分析的新技术。近些年来在生物医学研究领域得到越来越广泛的应用。利用SPR技术,生析物间相互作用分析不需要对样品进行标记和前处理。此外,其自动化操作和实时性检测特点也优于酶联免疫检测法(ELISA)和其他生物检测法。但是,将SPR技术应用于定量分析时灵敏度较低是其主要局限。其主要原因是固定于SPR传感芯片表层的葡聚糖层在生物分子发生相互作用过程中能够阻碍芯片表面质量传输,并诱导产生空间位阻效应,这些因素将导致基于SPR技术的定量分析方法较在溶液中直接进行的经典分析方法具有较低的灵敏度。因此,提高SPR技术在定量分析中的灵敏度是亟待解决的关键问题之一。
此次研究,SPR信号通过载有CM5芯片的BIAcore 3000蛋白测定仪进行采集。本实验室曾成功利用BIAcore 3000系统对猕猴血清中西妥昔单抗(C225)进行定量分析,此方法简单快速并已成功应用于猕猴体内C225临床前药代动力学研究。但是,该方法最低定量下限仅为0.05μg/mL,在进行C225低浓度检测时灵敏度较低,因此,本实验目的是将胶体金纳米粒子(Au NPs)引入SPR系统以提高SPR响应信号,建立猕猴血清中C225定量分析新方法,并将此方法应用于C225在猕猴体内临床前的药代动力学研究。
首先,利用柠檬酸还原HAuCl4自行制备胶体金纳米颗粒,并将其与羊抗人IgG进行偶联。C225是人/鼠嵌合型IgG1单克隆抗体,对表皮生长因子受体(EGFR)胞外配体结构域有高度的特异性和亲和力。利用氨基偶联试剂盒,将EGFR偶联于CM5芯片葡聚糖基质层表面。用HBS-EP缓冲液新鲜配制10μg/mL C225储备液,并用20%猕猴血清(HBS-EP缓冲液稀释)进行系列稀释,产生如下系列标准曲线浓度:0.0125,0.025,0.05,0.1,0.4,0.8,1.6和3.2μg/mL。C225质控样品浓度为0.025,0.4和1.6μg/mL。首先,注射20μL C225血清样品于芯片表面,过程中C225分子会与EGFR分子进行结合。其次,一定体积缓冲液流过芯片表面清洗掉游离C225后,注射60μL羊抗人IgG-Au NPs偶联溶液(HBS-EP缓冲液5倍稀释)于芯片表面,过程中抗体与捕获EGFR的C225分子进行结合。游离的羊抗人IgG-Au NPs偶联分子在HBS-EP缓冲液流动作用下清洗掉。最后,注射10μL glycine-HCL(pH 2.0)于CM5芯片表面进行芯片再生以进行下一循环检测。实验中对每一浓度标准曲线样品进行重复检测分析,结果取平均值。此方法标准曲线符合四参数Logistic拟合模型。将固定EGFR的Fc1通道设为检测通道,未固定EGFR的Fc2通道设为参比通道。通过羊抗人IgG-Au NPs偶联物捕获C225分子,Au NPs成功结合于芯片表面并明显提高BIAcore仪器的响应信号。这个检测过程某种程度上类似于双抗体夹心免疫反应。检测过程中,样品流过固定EGFR的芯片表面时,将检测通道的信号减去参比通道信号以补偿折射率,非特异性吸附,及微小温度变化的影响。此外,还将该定量分析方法按照中国FDA生物分析方法准则进行特异性、准确度、精密度、线性范围、最低定量下限以及稳定性等指标的方法学考察。
经过方法学的建立和确证,对此基于BIAcore技术的新方法同样进行了猕猴体内C225药代动力学研究。三组猕猴分别单次静脉滴注7.5 mg.kg-1、24 mg.kg-1、75 mg.kg-1三个剂量C225各60分钟,比较不同剂量组间药代动力学参数差异。实验结果显示按照此方法制备的10nm胶体金纳米微粒形状圆润,大小均一,100个Au NPs平均直径为11.2±2.4 nm。紫外可见光谱结果显示Au NPs和羊抗人IgG-Au NPs偶联物的最大吸收峰分别为518.5 nm和527.0 nm。此红移现象表明有蛋白层形成于Au NPs表面,并且胶体金偶联蛋白后紫外可见光吸收性质发生了改变。形成的偶联物用于下一步实验。
通过将BIAcore仪器响应信号(RU)对分析物浓度对数进行作图并建立九个时间点标准曲线,利用四参数Logistic模型拟合曲线,结果显示C225标准曲线线性范围为0.0125–3.2μg/mL。方法日内精密度在4.33%–13.11%之间,日内准确度在-3.72%–4.84%之间。方法日间精密度在2.16%–11.77%之间,准确度在-0.15%–5.79%之间。特异性实验结果表明基质中加入人血清和SD大鼠血清对该检测方法无明显影响。交叉实验结果显示C225猕猴血清样品中加入嵌合重组抗CD20多克隆抗体,人γ-球蛋白和嵌合重组her2抗体对分析物的测定无明显影响。为评价芯片基线稳定性,于CM5芯片表面进行100次再生循环,结果表明基线响应值没有明显的改变(变化值小于7.8%)。这些结果表明,利用SPR技术联合Au NPs定量C225的新方法所有方法学确证结果均在规定的检测范围以内。本次实验,利用BIAcore仪器结合信号放大方法进行猕猴血清内C225定量分析,最低定量下限为0.0125μg/mL。较以前未引入Au NPs,直接利用BIAcore系统进行猕猴血清内C225定量分析(定量下限为0.05μg/mL),灵敏度提高了4倍。此次研究过程中,同时开发了针对猕猴血清内C225进行浓度检测的ELISA方法,并对该方法进行了方法学确证。ELISA方法浓度检测范围为0.0125–0.4μg/mL。但是,基于BIAcore仪器进行定量分析的新方法较此ELISA方法具有更宽的浓度检测范围(新方法检测范围为0.0125–3.2μg/mL)。此外,文章对引入Au NPs后SPR系统的信号提高机制也进行了描述。Au NPs结合于BIAcore感应芯片,通过Au NPs本身的表面等离子波与BIAcore芯片本身金膜表面等离子波间的电子耦合作用,芯片表面折射率的变化得以放大。因此,BIAcore仪器响应信号得以提高,检测方法的灵敏度和最低定量下限同时有所提高。检测过程中,CM5芯片表面折射率的变化引起相应RU值的变化,Au NPs表面蛋白包被量与BIAcore系统共振角的变化成线性相关。因此,共振角的变化与待分析物的浓度直接相关。经过以上因素的综合作用,BIAcore系统的灵敏度和特异性得以改善,猕猴血清内C225定量分析方法的最低定量下限也进一步降低。
三组猕猴分别单次静脉滴注7.5,24和75 mg·kg-1三个剂量组国产C225后,均在拔针时间点血清药物浓度达到峰值,并且各时间点的血清药物浓度随着滴注剂量的增高而增高。所有相同时间点在低-中、中-高和高-低剂量组之间具有统计学差异。末端相消除半衰期随着剂量的增加而显著延长。低剂量组血药浓度在药物注射后312小时后降到峰浓度值得1/20,中剂量组和高剂量组分别在注射216和456小时后血药浓度降至峰浓度的1/20。对各组间药代动力学参数进行比较,平均驻留时间随着剂量的增加而明显延长,低、中、高剂量组MRT分别为105.11±4.78h;118.89±1.19h和152.84±6.83h。低剂量组AUC(0-t)为16390.54±1201.80μg·h·mL-1,中剂量组AUC(0-t)为51453.00±1199.80μg·h·mL-1,高剂量组AUC(0-t)为263091.21±30698.14μg·h·mL-1。低、中、高三剂量组清除率分别为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。全身清除率CL随剂量增加而降低,呈典型的非线性动力学特征,同时也说明国产C225在高剂量给药后呈饱和消除特征。结果证明:在7.5-75mg·kg-1剂量范围内,C225在猕猴体内呈非线性药代动力学特征。
总之,本研究描述了一种简单的药物定量分析方法,包括Au NPs制备及蛋白偶联物的合成,以及引入Au NPs利用BIAcore进行C225定量分析新模式。此方法制备的Au纳米颗粒形状相对圆润,大小较均一。定量分析新方法具有良好的精密度、准确度和足够低的最低定量下限(0.0125μg/mL)。此方法的最低定量下限较之前方法有很大程度的降低。另外,利用此SPR技术进行C225定量分析,较ELISA方法具有更宽的浓度检测范围。该方法可靠,可应用于猕猴血清内C225定量分析。未来该新技术应发展成为一种通用的药代动力学研究方法。
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.
此次研究,SPR信号通过载有CM5芯片的BIAcore 3000蛋白测定仪进行采集。本实验室曾成功利用BIAcore 3000系统对猕猴血清中西妥昔单抗(C225)进行定量分析,此方法简单快速并已成功应用于猕猴体内C225临床前药代动力学研究。但是,该方法最低定量下限仅为0.05μg/mL,在进行C225低浓度检测时灵敏度较低,因此,本实验目的是将胶体金纳米粒子(Au NPs)引入SPR系统以提高SPR响应信号,建立猕猴血清中C225定量分析新方法,并将此方法应用于C225在猕猴体内临床前的药代动力学研究。
首先,利用柠檬酸还原HAuCl4自行制备胶体金纳米颗粒,并将其与羊抗人IgG进行偶联。C225是人/鼠嵌合型IgG1单克隆抗体,对表皮生长因子受体(EGFR)胞外配体结构域有高度的特异性和亲和力。利用氨基偶联试剂盒,将EGFR偶联于CM5芯片葡聚糖基质层表面。用HBS-EP缓冲液新鲜配制10μg/mL C225储备液,并用20%猕猴血清(HBS-EP缓冲液稀释)进行系列稀释,产生如下系列标准曲线浓度:0.0125,0.025,0.05,0.1,0.4,0.8,1.6和3.2μg/mL。C225质控样品浓度为0.025,0.4和1.6μg/mL。首先,注射20μL C225血清样品于芯片表面,过程中C225分子会与EGFR分子进行结合。其次,一定体积缓冲液流过芯片表面清洗掉游离C225后,注射60μL羊抗人IgG-Au NPs偶联溶液(HBS-EP缓冲液5倍稀释)于芯片表面,过程中抗体与捕获EGFR的C225分子进行结合。游离的羊抗人IgG-Au NPs偶联分子在HBS-EP缓冲液流动作用下清洗掉。最后,注射10μL glycine-HCL(pH 2.0)于CM5芯片表面进行芯片再生以进行下一循环检测。实验中对每一浓度标准曲线样品进行重复检测分析,结果取平均值。此方法标准曲线符合四参数Logistic拟合模型。将固定EGFR的Fc1通道设为检测通道,未固定EGFR的Fc2通道设为参比通道。通过羊抗人IgG-Au NPs偶联物捕获C225分子,Au NPs成功结合于芯片表面并明显提高BIAcore仪器的响应信号。这个检测过程某种程度上类似于双抗体夹心免疫反应。检测过程中,样品流过固定EGFR的芯片表面时,将检测通道的信号减去参比通道信号以补偿折射率,非特异性吸附,及微小温度变化的影响。此外,还将该定量分析方法按照中国FDA生物分析方法准则进行特异性、准确度、精密度、线性范围、最低定量下限以及稳定性等指标的方法学考察。
经过方法学的建立和确证,对此基于BIAcore技术的新方法同样进行了猕猴体内C225药代动力学研究。三组猕猴分别单次静脉滴注7.5 mg.kg-1、24 mg.kg-1、75 mg.kg-1三个剂量C225各60分钟,比较不同剂量组间药代动力学参数差异。实验结果显示按照此方法制备的10nm胶体金纳米微粒形状圆润,大小均一,100个Au NPs平均直径为11.2±2.4 nm。紫外可见光谱结果显示Au NPs和羊抗人IgG-Au NPs偶联物的最大吸收峰分别为518.5 nm和527.0 nm。此红移现象表明有蛋白层形成于Au NPs表面,并且胶体金偶联蛋白后紫外可见光吸收性质发生了改变。形成的偶联物用于下一步实验。
通过将BIAcore仪器响应信号(RU)对分析物浓度对数进行作图并建立九个时间点标准曲线,利用四参数Logistic模型拟合曲线,结果显示C225标准曲线线性范围为0.0125–3.2μg/mL。方法日内精密度在4.33%–13.11%之间,日内准确度在-3.72%–4.84%之间。方法日间精密度在2.16%–11.77%之间,准确度在-0.15%–5.79%之间。特异性实验结果表明基质中加入人血清和SD大鼠血清对该检测方法无明显影响。交叉实验结果显示C225猕猴血清样品中加入嵌合重组抗CD20多克隆抗体,人γ-球蛋白和嵌合重组her2抗体对分析物的测定无明显影响。为评价芯片基线稳定性,于CM5芯片表面进行100次再生循环,结果表明基线响应值没有明显的改变(变化值小于7.8%)。这些结果表明,利用SPR技术联合Au NPs定量C225的新方法所有方法学确证结果均在规定的检测范围以内。本次实验,利用BIAcore仪器结合信号放大方法进行猕猴血清内C225定量分析,最低定量下限为0.0125μg/mL。较以前未引入Au NPs,直接利用BIAcore系统进行猕猴血清内C225定量分析(定量下限为0.05μg/mL),灵敏度提高了4倍。此次研究过程中,同时开发了针对猕猴血清内C225进行浓度检测的ELISA方法,并对该方法进行了方法学确证。ELISA方法浓度检测范围为0.0125–0.4μg/mL。但是,基于BIAcore仪器进行定量分析的新方法较此ELISA方法具有更宽的浓度检测范围(新方法检测范围为0.0125–3.2μg/mL)。此外,文章对引入Au NPs后SPR系统的信号提高机制也进行了描述。Au NPs结合于BIAcore感应芯片,通过Au NPs本身的表面等离子波与BIAcore芯片本身金膜表面等离子波间的电子耦合作用,芯片表面折射率的变化得以放大。因此,BIAcore仪器响应信号得以提高,检测方法的灵敏度和最低定量下限同时有所提高。检测过程中,CM5芯片表面折射率的变化引起相应RU值的变化,Au NPs表面蛋白包被量与BIAcore系统共振角的变化成线性相关。因此,共振角的变化与待分析物的浓度直接相关。经过以上因素的综合作用,BIAcore系统的灵敏度和特异性得以改善,猕猴血清内C225定量分析方法的最低定量下限也进一步降低。
三组猕猴分别单次静脉滴注7.5,24和75 mg·kg-1三个剂量组国产C225后,均在拔针时间点血清药物浓度达到峰值,并且各时间点的血清药物浓度随着滴注剂量的增高而增高。所有相同时间点在低-中、中-高和高-低剂量组之间具有统计学差异。末端相消除半衰期随着剂量的增加而显著延长。低剂量组血药浓度在药物注射后312小时后降到峰浓度值得1/20,中剂量组和高剂量组分别在注射216和456小时后血药浓度降至峰浓度的1/20。对各组间药代动力学参数进行比较,平均驻留时间随着剂量的增加而明显延长,低、中、高剂量组MRT分别为105.11±4.78h;118.89±1.19h和152.84±6.83h。低剂量组AUC(0-t)为16390.54±1201.80μg·h·mL-1,中剂量组AUC(0-t)为51453.00±1199.80μg·h·mL-1,高剂量组AUC(0-t)为263091.21±30698.14μg·h·mL-1。低、中、高三剂量组清除率分别为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。全身清除率CL随剂量增加而降低,呈典型的非线性动力学特征,同时也说明国产C225在高剂量给药后呈饱和消除特征。结果证明:在7.5-75mg·kg-1剂量范围内,C225在猕猴体内呈非线性药代动力学特征。
总之,本研究描述了一种简单的药物定量分析方法,包括Au NPs制备及蛋白偶联物的合成,以及引入Au NPs利用BIAcore进行C225定量分析新模式。此方法制备的Au纳米颗粒形状相对圆润,大小较均一。定量分析新方法具有良好的精密度、准确度和足够低的最低定量下限(0.0125μg/mL)。此方法的最低定量下限较之前方法有很大程度的降低。另外,利用此SPR技术进行C225定量分析,较ELISA方法具有更宽的浓度检测范围。该方法可靠,可应用于猕猴血清内C225定量分析。未来该新技术应发展成为一种通用的药代动力学研究方法。
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.
引文
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