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A Novel Hybridization LC-MS/MS Methodology for Quantification of siRNA in Plasma, CSF and Tissue Samples
用于血浆、脑脊液和组织样本中 siRNA 定量的新型杂交 LC-MS/MS 方法

by 1,*,
by 1,*
2 and
1,* 2
2
1
Drug Metabolism and Pharmacokinetics, Biogen, 225 Binney St., Cambridge, MA 02142, USA
美国马萨诸塞州剑桥市宾尼街 225 号生物基因公司药物代谢与药代动力学部 02142
2
Altasciences, 575 Armand-Frappier Blvd., Laval, QC H7V 4B3, Canada
*
Author to whom correspondence should be addressed.
通讯作者
Molecules 2023, 28(4), 1618; https://doi.org/10.3390/molecules28041618
Molecules2023,28(4), 1618;https://doi.org/10.3390/molecules28041618
Submission received: 18 January 2023 / Revised: 1 February 2023 / Accepted: 7 February 2023 / Published: 8 February 2023
收到投稿:2023 年 1 月 18 日/修订:2023 年 2 月 1 日/接受: 2023 年 2 月7/出版 :2023 年 2 月8 2023 年 2 月 7 日/出版:2023 年 2 月 8 日
(This article belongs to the Special Issue Bioorganic Chemistry: Current and Future Perspectives)
(本文属于《生物有机化学》特刊:当前与未来展望)

Abstract 摘要

Therapeutic oligonucleotides, such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA), are a new class of therapeutics rapidly growing in drug discovery and development. A sensitive and reliable method to quantify oligonucleotides in biological samples is critical to study their pharmacokinetic and pharmacodynamic properties. Hybridization LC-MS/MS was recently established as a highly sensitive and specific methodology for the quantification of single-stranded oligonucleotides, e.g., ASOs, in various biological matrices. However, there is no report of this methodology for the bioanalysis of double-stranded oligonucleotides (e.g., siRNA). In this work, we investigated hybridization LC-MS/MS methodology for the quantification of double-stranded oligonucleotides in biological samples using an siRNA compound, siRNA-01, as the test compound. The commonly used DNA capture probe and a new peptide nucleic acid (PNA) probe were compared for the hybridization extraction of siRNA-01 under different conditions. The PNA probe achieved better extraction recovery than the DNA probe, especially for high concentration samples, which may be due to its stronger hybridization affinity. The optimized hybridization method using the PNA probe was successfully qualified for the quantitation of siRNA-01 in monkey plasma, cerebrospinal fluid (CSF), and tissue homogenates over the range of 2.00–1000 ng/mL. This work is the first report of the hybridization LC-MS/MS methodology for the quantification of double-stranded oligonucleotides. The developed methodology will be applied to pharmacokinetic and toxicokinetic studies of siRNA-01. This novel methodology can also be used for the quantitative bioanalysis of other double-stranded oligonucleotides.
治疗性寡核苷酸,如反义寡核苷酸 (ASO) 和小干扰 RNA (siRNA),是一类在药物发现和开发中迅速发展起来的新型疗法。要研究寡核苷酸的药代动力学和药效学特性,灵敏可靠的生物样本寡核苷酸定量方法至关重要。杂交 LC-MS/MS 是一种高灵敏度和特异性的方法,可用于定量分析各种生物基质中的单链寡核苷酸(如 ASO)。然而,目前还没有这种方法用于双链寡核苷酸(如 siRNA)生物分析的报道。在这项工作中,我们以 siRNA 复合物 siRNA-01 为测试化合物,研究了用于定量生物样品中双链寡核苷酸的杂交 LC-MS/MS 方法。在不同条件下杂交提取 siRNA-01 时,比较了常用的 DNA 捕获探针和新型多肽核酸(PNA)探针。与DNA探针相比,PNA探针的提取回收率更高,尤其是在高浓度样品中,这可能是因为PNA探针具有更强的杂交亲和力。优化后的 PNA 探针杂交方法成功地定量检测了猴子血浆、脑脊液(CSF)和组织匀浆中 2.00-1000 ng/mL 范围内的 siRNA-01。本研究首次报道了用于定量双链寡核苷酸的杂交 LC-MS/MS 方法。所开发的方法将应用于 siRNA-01 的药代动力学和毒代动力学研究。这种新方法也可用于其他双链寡核苷酸的定量生物分析。

1. Introduction 1.导言

Therapeutic oligonucleotides are short synthetic single- or double-stranded nucleic acid strands usually in length of 15–30 base pairs. Oligonucleotides have been rapidly growing as a new class of therapeutics and are gaining increasing attention in drug discovery and development across various disease areas [1]. Antisense oligonucleotides (ASO) and small interfering RNA (siRNA) are two major types of therapeutic oligonucleotides. An ASO is a single-stranded oligonucleotide that can specifically bind to a complementary messenger RNA (mRNA) via Watson–Crick base pairings. ASO can modulate gene expression through various mechanisms, such as RNase H mediated cleavage of mRNA and steric blocking for splicing modulation. siRNA is a short double-stranded RNA that can specifically hybridize to its target RNA. siRNA usually induces the mRNA degradation via the RNA-induced silencing complex (RISC). Many disease targets lack a specific binding pocket, which is required for developing traditional small molecule drugs or protein therapeutics, and thus are considered ‘undruggable’. Oligonucleotides do not depend on a binding pocket to modulate the target, and therefore, oligonucleotide therapeutics can be developed to treat the diseases that otherwise could not be addressed [2]. There have been 13 oligonucleotide drugs approved by the US Food and Drug Administration (FDA) and/or the European Medicines Agency (EMA) by 2021 [1]. Nine out of the thirteen approved oligonucleotide drugs are ASOs and the other four are siRNA. There are also approximately 200 oligonucleotide drug candidates under clinical development, and more than 500 under preclinical development [2].
治疗性寡核苷酸是短的合成单链或双链核酸链,长度通常为 15-30 个碱基对。寡核苷酸作为一类新的治疗药物发展迅速,在各种疾病领域的药物发现和开发中日益受到关注[1]。反义寡核苷酸(ASO)和小干扰 RNA(siRNA)是两种主要的治疗性寡核苷酸。反义寡核苷酸是一种单链寡核苷酸,可通过沃森-克里克碱基配对与互补的信使核糖核酸(mRNA)特异性结合。siRNA 是一种短的双链 RNA,能特异性地与目标 RNA 杂交。siRNA 通常通过 RNA 诱导的沉默复合体(RISC)诱导 mRNA 降解。许多疾病靶点缺乏开发传统小分子药物或蛋白质疗法所需的特异性结合口袋,因此被认为是 "不可药用 "的。寡核苷酸不依赖于结合袋来调节靶点,因此可以开发寡核苷酸疗法来治疗其他疗法无法治疗的疾病[2]。到 2021 年,美国食品药品管理局(FDA)和/或欧洲药品管理局(EMA)已经批准了 13 种寡核苷酸药物[1]。获批的 13 种寡核苷酸药物中有 9 种是 ASO,另外 4 种是 siRNA。此外,还有约 200 种候选寡核苷酸药物正在进行临床开发,500 多种候选寡核苷酸药物正在进行临床前开发[2]。
Quantitative bioanalysis of oligonucleotides plays a critical role in evaluating and understanding the pharmacokinetic (PK), toxicokinetic (TK), pharmacological, and toxicological properties of the oligonucleotide drug candidates, and the success of their drug research and development [3]. The rapid growth of oligonucleotides in drug discovery and development has resulted in significantly increased demands for quantitative bioanalysis of oligonucleotides. A bioanalytical methodology that can achieve accurate, sensitive, and reliable quantification of oligonucleotides in various biological matrices (plasma, cerebrospinal fluid, tissues, etc.) is much desired. There are three major types of approaches for oligonucleotide bioanalysis: hybridization-based immunoassays, liquid chromatography (LC) based methods, and quantitative polymerase chain reaction (qPCR) methods [4,5,6]. Conventionally, hybridization immunoassay, e.g., hybridization enzyme linked immunosorbent assay (HELISA), has been the most used methodology for the quantitative bioanalysis of oligonucleotides [4,5]. Hybridization immunoassays have the advantages of high sensitivity (single digit ng/mL or even pg/mL sensitivity), good assay performance, and minimal sample preparation. However, one major limitation of hybridization immunoassay is its lack of specificity: it is often difficult to differentiate the full-length oligonucleotide analyte from its truncated metabolites (for instance, N-1, N-2 metabolites) [4,7]. In addition, hybridization assays have relatively narrow dynamic ranges and may be affected by the presence of anti-drug antibodies in the samples [5]. qPCR is the most sensitive method for oligonucleotide bioanalysis: it can often achieve sensitivity at pg/mL level. qPCR also has the advantage of wide dynamic range. However, like hybridization immunoassays, it often lacks the specificity to mitigate the cross-reactivity with truncated metabolites. [8,9] In addition, the qPCR method requires significant method development to improve the accuracy, precision and robustness of the assay, and its application in regulated bioanalysis is limited. In recent years, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been gaining increasing attention and interests for the quantification of oligonucleotides due to its unique advantages of good specificity, wide dynamic range, and ability to simultaneously measure multiple analytes (e.g., oligonucleotide drug and its metabolites) [10,11,12,13]. However, the significantly lower sensitivity of LC-MS/MS assay compared to hybridization immunoassay limited its wider applications. To overcome the sensitivity limitation of LC-MS/MS assay, recently, a novel hybridization LC-MS/MS methodology was developed for the quantification of oligonucleotides in biological samples [14,15,16,17,18,19]. This methodology utilized a capture probe (a DNA strand complementary to the target oligonucleotide) to specifically hybridize to the oligonucleotide to achieve highly efficient sample purification. The hybridization sample preparation generated much cleaner sample extracts with improved extraction recovery compared to conventional liquid-liquid extraction (LLE) or solid phase extraction (SPE) methods. As a result, hybridization LC-MS/MS method can achieve greatly improved sensitivity that is comparable to immunoassay (below 1 ng/mL), while maintaining its advantage of high specificity. Although the truncated metabolites may still be extracted during the hybridization sample preparation process, the LC-MS/MS analysis can differentiate them from the full-length oligonucleotide analyte based on their different retention time or monitored ions. In addition, it has been demonstrated that microflow LC can further increase the sensitivity for LC-MS/MS bioanalysis of ASO [20]. The cleanness of the sample plays an important role in improving sensitivity by microflow LC: the cleaner the sample extract, the more the sensitivity improvement. Hybridization, with its highly specific and efficient sample cleanup and good recovery, is the preferred sample extraction method to be used with microflow LC to achieve the maximal sensitivity improvement.
寡核苷酸的定量生物分析在评价和了解寡核苷酸候选药物的药代动力学(PK)、毒代动力学(TK)、药理学和毒理学特性及其药物研发成功与否方面发挥着至关重要的作用[3]。寡核苷酸在药物发现和开发中的快速增长导致对寡核苷酸定量生物分析的需求大幅增加。人们迫切希望有一种生物分析方法能对各种生物基质(血浆、脑脊液、组织等)中的寡核苷酸进行准确、灵敏、可靠的定量分析。寡核苷酸生物分析主要有三种方法:基于杂交的免疫分析法、基于液相色谱法(LC)的方法和基于聚合酶链反应(qPCR)的定量方法[4,5,6]。传统上,杂交免疫测定(如杂交酶联免疫吸附测定法(HELISA))是寡核苷酸定量生物分析中最常用的方法[4,5]。杂交免疫测定具有灵敏度高(灵敏度为个位数纳克/毫升甚至皮克/毫升)、测定性能好、样品制备量少等优点。然而,杂交免疫测定的一个主要局限性是缺乏特异性:通常难以区分全长寡核苷酸分析物与其截短代谢物(如 N-1、N-2 代谢物)[4,7]。 此外,杂交测定的动态范围相对较窄,可能会受到样品中存在的抗药性抗体的影响[5]。qPCR 是寡核苷酸生物分析中最灵敏的方法:其灵敏度通常可达到 pg/mL 级。然而,与杂交免疫测定一样,它往往缺乏特异性,无法减轻与截短代谢物的交叉反应。[8,9]此外,qPCR 方法需要大量的方法开发来提高检测的准确度、精密度和稳健性,因此在规范生物分析中的应用有限。近年来,液相色谱-串联质谱法(LC-MS/MS)因其特异性好、动态范围宽、可同时测定多种分析物(如寡核苷酸药物及其代谢物)等独特优势,在寡核苷酸的定量分析中越来越受到关注和重视[10,11,12,13]。然而,与杂交免疫测定法相比,LC-MS/MS 法的灵敏度明显较低,这限制了它的广泛应用。为了克服 LC-MS/MS 检测灵敏度的限制,最近开发了一种新型杂交 LC-MS/MS 方法,用于定量检测生物样品中的寡核苷酸[14,15,16,17,18,19]。 该方法利用捕获探针(与目标寡核苷酸互补的 DNA 链)与寡核苷酸特异性杂交,实现了高效的样品纯化。与传统的液液萃取(LLE)或固相萃取(SPE)方法相比,杂交样品制备产生的样品提取物更纯净,萃取回收率更高。因此,杂交 LC-MS/MS 方法的灵敏度大大提高,可与免疫测定法相媲美(低于 1 ng/mL),同时保持了其高特异性的优势。虽然在杂交样品制备过程中仍可能提取到截短的代谢物,但 LC-MS/MS 分析可根据其不同的保留时间或监测离子将其与全长寡核苷酸分析物区分开来。此外,微流液相色谱法还能进一步提高 ASO 的 LC-MS/MS 生物分析灵敏度[20]。样品的洁净度对微流液相色谱灵敏度的提高起着重要作用:样品提取物越洁净,灵敏度的提高就越大。杂交法具有特异性强、样品净化效率高、回收率高的特点,是微流液相色谱法提高灵敏度的首选样品提取方法。
To date, all reported hybridization LC-MS/MS methods are for the quantification of single-stranded oligonucleotides, such as small oligonucleotide [14], ASO [15,17,18,21], and microRNA [16]. No application of this methodology has been reported for the bioanalysis of double-stranded oligonucleotides (e.g., siRNA). The main challenge of applying hybridization LC-MS/MS to double-stranded oligonucleotides is the presence of a competing strand to the capture probe in the samples during the hybridization process, which could severely impact the extraction recovery. For example, if a DNA strand complementary to the antisense strand of the targe siRNA is used as the capture probe, the sense strand of the siRNA will compete with the capture probe during hybridization since they have the same sequence.
迄今为止,所有报道的杂交 LC-MS/MS 方法都是用于定量分析单链寡核苷酸,如小寡核苷酸[14]、ASO[15,17,18,21]和 microRNA[16]。目前还没有将这种方法应用于双链寡核苷酸(如 siRNA)生物分析的报道。将杂交 LC-MS/MS 应用于双链寡核苷酸的主要挑战是在杂交过程中样品中存在与捕获探针竞争的链,这会严重影响提取回收率。例如,如果使用与 targe siRNA 的反义链互补的 DNA 链作为捕获探针,那么 siRNA 的有义链就会在杂交过程中与捕获探针竞争,因为它们具有相同的序列。
In this paper, we describe the investigation of the hybridization LC-MS/MS methodology for the quantification of double-stranded oligonucleotides in biological samples. An siRNA compound (siRNA-01) was used as the model compound. We evaluated the use of the commonly utilized DNA capture probe for the hybridization extraction. We also investigated the use of peptide nucleic acid (PNA, a synthetic oligomer that mimics DNA) as the capture probe and compared it with the DNA probe for the extraction of siRNA. For PNA, the deoxyribose phosphate backbone is substituted with a pseudo-peptide polymer consisting of N-(2-amino-ethyl) glycyl units [22]. Since PNA does not contain charged phosphate groups, and thus lacks electrostatic repulsion, the hybridization affinity between PNA and its complementary DNA strand is higher than between DNA/DNA or DNA/RNA strands [22]. The higher affinity enables the PNA capture probe to achieve higher extraction recovery of siRNA or other double-stranded oligonucleotides. Another potential advantage of a PNA probe is that the melting temperature (Tm, the temperature at which half of the oligonucleotides in solution are in the double-stranded state and half single-stranded) of a PNA/DNA or PNA/RNA duplex generally is higher than that of the corresponding DNA/DNA or DNA/RNA duplex (roughly an increase of about 1 °C per base pair). Thus, the PNA probe can anneal with the siRNA antisense strand (or sense strand) at a higher temperature to form the PNA-RNA duplex, while the competing sense strand (or antisense strand) of the siRNA remains single-stranded in the solution at the higher temperature resulting in less competition to the hybridization process. This may also help to significantly increase the hybridization extraction recovery of the PNA probe. Because of these advantages, we included the PNA probe in addition to the DNA probe in the hybridization sample preparation evaluation. We investigated and optimized the capture probe and hybridization conditions. A sensitive and reliable hybridization LC-MS/MS method was successfully developed and qualified for the quantification of siRNA-01 in various biological matrices, including plasma, cerebrospinal fluid (CSF), and tissues. To our knowledge, this is the first-time a hybridization LC-MS/MS methodology was successfully applied to quantitative bioanalysis of double-stranded oligonucleotides. The developed hybridization methodology can also be applied to other siRNAs or double-stranded oligonucleotides.
本文介绍了用于生物样品中双链寡核苷酸定量的杂交 LC-MS/MS 方法的研究。一种 siRNA 复合物(siRNA-01)被用作模型化合物。我们评估了杂交提取中常用 DNA 捕获探针的使用情况。我们还研究了使用肽核酸(PNA,一种模拟 DNA 的合成寡聚体)作为捕获探针,并将其与 DNA 探针进行比较,以提取 siRNA。对于 PNA 来说,脱氧核糖磷酸骨架被一种由 N-(2-氨基乙基)甘氨酰单位组成的假肽聚合物所取代[22]。由于 PNA 不含带电的磷酸基团,因此缺乏静电排斥力,PNA 与其互补 DNA 链之间的杂交亲和力高于 DNA/DNA 或 DNA/RNA 链之间的亲和力[22]。较高的亲和力可使 PNA 捕获探针获得更高的 siRNA 或其他双链寡核苷酸提取回收率。PNA 探针的另一个潜在优势是,PNA/DNA 或 PNA/RNA 双链的熔化温度(Tm,即溶液中一半寡核苷酸处于双链状态,一半处于单链状态的温度)通常高于相应的 DNA/DNA 或 DNA/RNA 双链的熔化温度(每对碱基大约增加 1 ℃)。因此,PNA 探针可在较高温度下与 siRNA 反义链(或有义链)退火,形成 PNA-RNA 双链,而 siRNA 的竞争有义链(或反义链)在较高温度的溶液中仍为单链,从而减少了杂交过程中的竞争。 这也有助于大大提高 PNA 探针的杂交提取回收率。由于这些优点,我们在杂交样品制备评估中除了 DNA 探针外,还加入了 PNA 探针。我们研究并优化了捕获探针和杂交条件。我们成功地建立了一种灵敏可靠的杂交 LC-MS/MS 方法,用于定量检测各种生物基质(包括血浆、脑脊液(CSF)和组织)中的 siRNA-01。据我们所知,这是首次将杂交 LC-MS/MS 方法成功应用于双链寡核苷酸的生物定量分析。所开发的杂交方法也可应用于其他 siRNA 或双链寡核苷酸。

2. Results and Discussion
2.结果与讨论

2.1. Selection of the Surrogate Analyte for Quantification of siRNA
2.1.选择用于 siRNA 定量的替代分析物

It is challenging to directly analyze the intact siRNA duplex molecule as the analyte for LC-MS/MS assay because: (1) the sample preparation process will break the Watson–Crick base pairings between the siRNA strands; (2) the large size of siRNA (doubled compared to ASO) and the highly hydrophilic and negatively charged properties of siRNA make it difficult to achieve a good chromatographic behavior; and (3) the large size and highly charged properties also will result in poor sensitivity during MS/MS detection. For siRNA or other double-stranded oligonucleotides, either its antisense strand or sense strand can be analyzed as the surrogate analyte representing the intact duplex molecule [23]. The antisense strand of the siRNA is the pharmacologically active strand of the siRNA drug. Therefore, we chose to monitor AS1, the antisense strand of the siRNA-01, as the surrogate analyte for the quantification of siRNA-01. LC-MS/MS conditions were optimized using AS1 as the standard compound. The calibration curve and QC samples were prepared using siRNA-01, the intact siRNA duplex.
直接分析完整的 siRNA 双链分子作为 LC-MS/MS 检测的分析物具有挑战性,因为:(1)样品制备过程会破坏 siRNA 双链之间的 Watson-Crick 碱基配对;(2)siRNA 的大尺寸(比 ASO 大一倍)和高亲水性及带负电荷的特性使其难以获得良好的色谱性能;(3)大尺寸和高电荷特性也会导致 MS/MS 检测的灵敏度较低。对于 siRNA 或其他双链寡核苷酸,可将其反义链或有义链作为代表完整双链分子的替代分析物进行分析[23]。siRNA 的反义链是 siRNA 药物的药理活性链。因此,我们选择监测 siRNA-01 的反义链 AS1 作为 siRNA-01 定量的替代分析物。以AS1为标准化合物对LC-MS/MS条件进行了优化。使用完整的 siRNA 双链 siRNA-01 制备校准曲线和质控样品。

2.2. Optimization of LC-MS/MS Conditions
2.2.优化 LC-MS/MS 条件

A Q1 full scan was conducted to obtain the MS scan ion spectra of AS1. The ions at m/z 629.8 (−11 charged) and 692.8 (−10 charged) were the two most abundant ions and were selected as the precursor ions. For oligonucleotides with phosphorothioate backbones, m/z 95 (phosphorothioate ion) is a commonly used product ion [17]. This ion also offered the best sensitivity (signal-to-noise ratio) for the analysis of AS1. After further comparison of m/z 629.8 and 692.8 as the precursor ions, MRM transition of m/z 629.8 → 95 was selected for the detection of AS1 for its better sensitivity and selectivity.
对 AS1 进行了 Q1 全扫描,以获得 MS 扫描离子谱。m/z629.8(带 -11 电荷)和 692.8(带 -10 电荷)的离子是含量最高的两个离子,被选为前体离子。对于以硫代磷酸酯为骨架的寡核苷酸,m/z95(硫代磷酸酯离子)是常用的产物离子[17]。该离子也为分析 AS1 提供了最佳灵敏度(信噪比)。在对m/z629.8 和 692.8 作为前体离子进行进一步比较后,由于m/z629.8 → 95 的灵敏度和选择性更好,因此选择其作为检测 AS1 的 MRM 过渡。
Ion-pair chromatography with a combination of alkylamine and fluoroalcohol is commonly used for the LC separation of oligonucleotides to improve the chromatographic retention and peak shape, as well as to enhance the MS response [24]. We adopted the same strategy and used LC conditions modified from previously published methods [17,21]. A combination of 10 mM DMCHA and 25 mM HFMIP (final concentration during LC separation) was used to achieve the best chromatographic separation and MS response.
烷基胺和氟乙醇的离子对色谱法常用于寡核苷酸的液相色谱分离,以改善色谱保留和峰形,并提高质谱响应[24]。我们采用了相同的策略,并根据之前发表的方法[17,21]对 LC 条件进行了修改。10 mM DMCHA 和 25 mM HFMIP(LC 分离过程中的最终浓度)的组合可实现最佳的色谱分离和 MS 响应。

2.3. Optimization of Hybridization Extraction Conditions
2.3.优化杂交提取条件

2.3.1. Capture Probes 2.3.1.捕获探针

Single-stranded DNA and PNA with complementary sequences to the antisense strand of siRNA-01 were evaluated as the capture probes. To maximize the binding affinity and therefore achieving maximized extraction recovery, 100% complementary sequences were used for the DNA capture probe. Similarly, for the PNA capture probe, we used a 19-mer PNA with complementary sequence to AS1 to maximize the binding affinity. The PNA probe has two less bases than AS1: one removed each from the 3′ and 5′ end. This is to reduce the purine content and therefore improve the solubility of the probe. In addition, two lysines were added to the 3′-end of the probe to further improve its solubility. In previous work, 3′- and 5′-biotin labeled capture probes showed no difference in the performance of extraction recovery and matrix effect [17]. Therefore, in this work, we did not compare 3′- and 5′-biotin labeling and directly used 5′-biotin labeling for both the DNA and PNA capture probes.
单链 DNA 和与 siRNA-01 的反义链具有互补序列的 PNA 被用作捕获探针。为了最大限度地提高结合亲和力,从而最大限度地提高提取回收率,DNA捕获探针使用了100%的互补序列。同样,对于 PNA 捕获探针,我们使用了与 AS1 有互补序列的 19-merPNA,以最大限度地提高结合亲和力。PNA 探针比 AS1 少两个碱基:3′端和 5′端各去掉一个碱基。这是为了减少嘌呤含量,从而提高探针的溶解度。此外,还在探针的 3′ 端添加了两个赖氨酸,以进一步提高其溶解度。在之前的研究中,3′-生物素和 5′-生物素标记的捕获探针在提取回收率和基质效应方面的表现没有差异[17]。因此,在这项工作中,我们没有比较 3′-生物素和 5′-生物素标记,而是直接使用 5′-生物素标记 DNA 和 PNA 捕获探针。

2.3.2. Temperature and Salt Concentration on the Extraction Recovery of Antisense Strand and siRNA Duplex
2.3.2.温度和盐浓度对反义链和 siRNA 双链提取回收率的影响

We first evaluated the effect of temperature and salt concentration of the hybridization buffer on the extraction recovery of AS1. Hybridization temperature at room temperature (22 °C), 30, 40, 50 and 60 °C, and salt (NaCl) concentration at 10, 50, 500 and 1000 mM were compared for both the DNA and the PNA capture probe for the extraction of ULOQ samples. As shown in Figure 1, high recovery (approximately 80% or higher) of AS1 was achieved for both probes. No significant difference in recovery was observed under the different hybridization temperature and salt concentrations tested. The recovery using the DNA probe was slightly higher than using the PNA probe which may be due to experimental variation; the difference was not considered significant. The result is as expected and is consistent with previous recovery results of ASOs [17,21]: hybridization extraction of AS1, a single-stranded oligonucleotide, should be similar to the extraction of ASOs.
我们首先评估了杂交缓冲液的温度和盐浓度对 AS1 提取回收率的影响。我们比较了 DNA 和 PNA 捕获探针在室温(22 °C)、30、40、50 和 60 °C 下的杂交温度以及 10、50、500 和 1000 mM 下的盐浓度(NaCl)对 ULOQ 样品提取的影响。如图 1 所示,两种探针都实现了 AS1 的高回收率(约 80% 或更高)。在测试的不同杂交温度和盐浓度下,没有观察到明显的回收率差异。使用 DNA 探针的回收率略高于使用 PNA 探针的回收率,这可能是由于实验差异造成的;差异并不显著。这一结果符合预期,也与之前 ASO 的回收结果一致[17,21]:AS1 是一种单链寡核苷酸,其杂交提取应该与 ASO 的提取相似。
Figure 1. Effect of temperature and salt concentration on the recovery of AS1 using DNA probe and PNA probe.
图 1.温度和盐浓度对使用 DNA 探针和 PNA 探针回收 AS1 的影响。
Similar evaluation of temperature and salt concentration was also conducted for the extraction of siRNA-01 using ULOQ samples. When the capture temperature was in the range of 22 °C to 40 °C, the recoveries stayed very low at <5% for both the DNA and the PNA probes (Figure 2). The recoveries started to increase to 10–20% when the temperature was increased to 50 °C, and significantly increased to approximately 50–60% when the temperature increased to 60 °C (NaCl concentration 10 or 50 mM). At lower temperatures, siRNA-01 is predominantly present in duplex form which could not be hybridized by the capture probes. At higher temperatures, the siRNA duplex started to break, and the resulting single-stranded antisense strand annealed with the capture probes and was extracted from the samples. The higher the temperature, the more antisense strand in the single-stranded state, the better the recovery. This requirement of higher temperature for good recovery is significantly different from the extraction of single-stranded oligonucleotides (e.g., ASOs), for which room temperature usually is enough to achieve good recoveries [17,21]. The concentration of NaCl in the hybridization buffer also affected the recovery. The recovery of siRNA-01 decreased with increased salt concentration: the recovery was reduced to about half when the concentration of NaCl was increased from 10 mM to 1 M. Best recoveries were achieved at 60 °C and 10 mM NaCl for both probes. The recovery using the PNA probe was slightly (approximately 10%) higher than using the DNA probe. The temperature and salt concentration were further fine-tuned with additional temperatures at 55 and 65 °C and salt concentration at 0 and 1 mM tested. As shown in Figure 3, the condition of 65 °C and 10 mM NaCl achieved best recovery for both probes, with the recovery using PNA probe around 80% and DNA probe around 55%. Higher temperatures (75, 85, and 95 °C) were also tested. The recovery of AS1 and siRNA-01 started to decrease when the temperature was increased to 75 °C, and decreased to almost no recovery when increased to 95 °C for both probes. Therefore, the temperature of 65 °C and salt concentration of 10 mM NaCl was selected as the optimized hybridization condition for both probes for later experiments.
在使用 ULOQ 样品提取 siRNA-01 时,也对温度和盐浓度进行了类似的评估。当捕获温度在 22 °C 至 40 °C 之间时,DNA 和 PNA 探针的回收率都很低,仅为 %3-C5%(图 2)。当温度升高到 50 ℃ 时,回收率开始增加到 10-20%,当温度升高到 60 ℃ 时(NaCl 浓度为 10 或 50 mM),回收率大幅增加到约 50-60%。在较低温度下,siRNA-01 主要以双链形式存在,无法与捕获探针杂交。在较高温度下,siRNA 双链开始断裂,产生的单链反义链与捕获探针退火,并从样品中提取出来。温度越高,单链状态的反义链越多,回收效果越好。这种对较高温度的要求与单链寡核苷酸(如 ASO)的提取要求明显不同,后者通常在室温下就能达到很好的回收率[17,21]。杂交缓冲液中 NaCl 的浓度也会影响回收率。siRNA-01 的回收率随着盐浓度的增加而降低:当 NaCl 浓度从 10 mM 增加到 1 M 时,回收率降低到一半左右。使用 PNA 探针的回收率略高于 DNA 探针(约 10%)。对温度和盐浓度进行了进一步的微调,测试温度分别为 55 和 65 °C,盐浓度分别为 0 和 1 mM。 如图 3 所示,在 65 °C 和 10 mM NaCl 的条件下,两种探针的回收率均达到最佳,PNA 探针的回收率约为 80%,DNA 探针的回收率约为 55%。还测试了更高的温度(75、85 和 95 ℃)。当温度升高到 75 ℃ 时,AS1 和 siRNA-01 的回收率开始下降,当温度升高到 95 ℃ 时,两种探针的回收率几乎为零。因此,温度为 65 ℃、盐浓度为 10 mM NaCl 被选为两种探针的优化杂交条件,用于后面的实验。
Figure 2. Effect of temperature and salt concentration on the recovery of siRNA-01 using DNA probe and PNA probe.
图 2.温度和盐浓度对使用 DNA 探针和 PNA 探针回收 siRNA-01 的影响。
Figure 3. Optimization of temperature and salt concentration for the recovery of siRNA-01 using DNA probe and PNA probe.
图 3.使用 DNA 探针和 PNA 探针回收 siRNA-01 时温度和盐浓度的优化。
The higher recovery using the PNA probe than using the DNA probe is as expected, since the PNA probe has stronger binding affinity and higher melting temperature than the DNA probe. One major concern for using DNA probe is the competition between the siRNA sense strand in the samples and the DNA probe during the hybridization process, which could severely impact the extraction recovery. It was surprising that the DNA probe also achieved reasonable recovery (around 50%) of the siRNA duplex at higher temperature (65 °C). There are two main possible reasons for this. First, the amount of the DNA capture probe is approximately 20-fold excess compared to the ULOQ of the siRNA, and even more excess for lower concentration samples. The much excessive amount of capture probe ensured that the antisense strand in the samples mainly hybridized to the capture probe, and only a small percentage may hybridize to the competing sense strand. Second, the capture probe is immobilized on the surface of the streptavidin beads. This solid support (beads-based) approach may offer better hybridization efficiency for the capture probe with the antisense strand than the competing sense strand in the solution.
由于 PNA 探针比 DNA 探针具有更强的结合亲和力和更高的熔化温度,因此使用 PNA 探针比使用 DNA 探针的回收率更高。使用 DNA 探针的一个主要顾虑是杂交过程中样品中 siRNA 有义链与 DNA 探针之间的竞争,这会严重影响提取回收率。令人惊讶的是,在较高温度(65 °C)下,DNA 探针也能实现 siRNA 双链的合理回收(约 50%)。这可能有两个主要原因。首先,DNA 捕获探针的量比 siRNA 的 ULOQ 多出约 20 倍,在低浓度样品中甚至更多。过量的捕获探针确保了样品中的反义链主要与捕获探针杂交,只有一小部分可能与竞争的有义链杂交。其次,捕获探针固定在链霉亲和素珠子表面。与溶液中的竞争有义链相比,这种固态支持(基于珠子)的方法可以提供更好的捕获探针与反义链的杂交效率。

2.3.3. DNA Probe Concentration and Incubation Time on the Recovery
2.3.3.DNA 探针浓度和孵育时间对回收率的影响

The linearity of the method was tested with a calibration curve of siRNA-01 in monkey plasma (2.00–1000 ng/mL) extracted using DNA probes at 10, 37.5, and 75 pmoles/sample. Slight lack of linearity was observed at the high end of the curve indicating that the recoveries of high concentration standard samples were lower compared to that of the low concentration standards. To improve the recovery of DNA probe for high concentration samples, we tested the recoveries of LLOQ and ULOQ samples using higher probe concentrations (150 and 300 pmoles/sample vs. 75 pmoles/sample), as well as longer incubation time (180 min vs. 90 min). As shown in Figure 4, the recovery of ULOQ samples were approximately 30% lower than the recovery of LLOQ samples using probe concentration of 75 pmoles/sample and 90 min incubation time. Increasing the probe concentration to 150 and 300 pmoles/sample did not improve the recovery of ULOQ samples. On the contrary, there was a trend of decreased recovery with the increase of probe concentration. When the incubation time was increased from 90 min to 180 min, there was no improvement in recoveries either under all the tested conditions. This may because the probe concentration of 75 pmoles/sample is already approximately 20-fold in excess to the ULOQ sample and therefore high enough for maximizing the hybridization recovery. Further increase of the probe concentration may cause increased loss of the analyte due to non-specific binding to the probes/beads.
用 10、37.5 和 75 pmoles/样品的 DNA 探针提取猴子血浆(2.00-1000 ng/mL)中的 siRNA-01,用校准曲线测试了该方法的线性度。在曲线的高端观察到轻微的线性度不足,表明高浓度标准样品的回收率低于低浓度标准样品的回收率。为了提高高浓度样品 DNA 探针的回收率,我们测试了 LLOQ 和 ULOQ 样品的回收率,使用了更高的探针浓度(150 和 300 pmoles/样品对 75 pmoles/样品)以及更长的孵育时间(180 分钟对 90 分钟)。如图 4 所示,在探针浓度为 75 pmoles/样品、孵育时间为 90 分钟的情况下,ULOQ 样品的回收率比 LLOQ 样品的回收率低约 30%。将探针浓度提高到 150 和 300 pmoles/样品并没有提高 ULOQ 样品的回收率。相反,随着探针浓度的增加,回收率呈下降趋势。当培养时间从 90 分钟增加到 180 分钟时,在所有测试条件下回收率都没有提高。这可能是因为探针浓度为 75 毫摩尔/样品时,已经超出超低定量样品约 20 倍,因此足够高,可以最大限度地提高杂交回收率。进一步提高探针浓度可能会由于探针/探针珠的非特异性结合而增加分析物的损失。
Figure 4. Effect of DNA probe concentration and incubation time on the recovery of siRNA-01.
图 4.DNA 探针浓度和孵育时间对 siRNA-01 回收率的影响。

2.3.4. PNA Probe vs. DNA Probe
2.3.4.PNA 探针与 DNA 探针

PNA probe alone (75 pmoles/sample), DNA probe alone (75 pmoles/sample), or mixtures of various concentrations of DNA (37.5 or 75 pmoles/sample) and PNA probes (37.5 or 75 pmoles/sample) were compared for the recovery of LLOQ and ULOQ samples. As shown in Figure 5, similar recovery between ULOQ and LLOQ samples were achieved using PNA alone or mixtures of PNA and DNA probes. Lower recovery of ULOQ samples was observed when using DNA probe alone, which was consistent with previous results. Best performance (>85% recovery for both LLOQ and ULOQ) was achieved using 75 pmoles/sample of PNA probe. The use of a mixture of 37.5 or 75 pmoles/sample of PNA probe with 37.5 pmoles/sample of DNA probe also achieved satisfactory recovery (>80%). These results indicated that the PNA probe was more effective than the DNA probe in extracting high concentration samples, which is consistent with our hypothesis that the stronger hybridization affinity and higher melting temperature of the PNA probe may help to improve the recovery.
比较了单独的 PNA 探针(75 pmoles/样品)、单独的 DNA 探针(75 pmoles/样品)或不同浓度的 DNA(37.5 或 75 pmoles/样品)和 PNA 探针(37.5 或 75 pmoles/样品)的混合物对 LLOQ 和 ULOQ 样品的回收率。如图 5 所示,单独使用 PNA 或 PNA 与 DNA 探针的混合物可获得相似的 ULOQ 和 LLOQ 样品回收率。单独使用 DNA 探针时,ULOQ 样品的回收率较低,这与之前的结果一致。使用 75 pmoles/样品的 PNA 探针可获得最佳性能(LLOQ 和 ULOQ 的回收率均大于 85%)。使用 37.5 或 75 pmoles/pample的 PNA 探针与 37.5 pmoles/pample的DNA探针的混合物也达到了令人满意的回收率(>80%)。这些结果表明,在提取高浓度样品时,PNA 探针比 DNA 探针更有效,这与我们的假设一致,即 PNA 探针更强的杂交亲和力和更高的熔化温度可能有助于提高回收率。
Figure 5. Comparison of different combination of DNA and PNA probes for recovery of siRNA-01.
图 5.不同 DNA 和 PNA 探针组合回收 siRNA-01 的比较。
To confirm the performance of the PNA probe, a calibration curve (2.00–1000 ng/mL) and QCs at 2.00, 6.00, 500, and 750 ng/mL in monkey plasma were extracted using 75 pmoles/sample PNA probe or 37.5 pmoles/sample each of PNA and DNA probes. The assay showed good linearity with similar accuracy and precision under both extraction conditions (Table 1). The use of 75 pmoles/sample of PNA probe was selected as the final method for its simplicity.
为了确认 PNA 探针的性能,使用 75 pmoles/样品的 PNA 探针或 37.5 pmoles/样品的 PNA 和 DNA 探针提取猴子血浆中 2.00、6.00、500 和 750 ng/mL 的校准曲线(2.00-1000 ng/mL)和 QC。在两种提取条件下,检测结果均显示出良好的线性关系,准确度和精密度相似(表 1)。最终选择使用 75 pmoles/样本的 PNA 探针进行检测,因为这种方法简便易行。
Table 1. Comparison of different capture probes for the analysis of siRNA-01 QCs in monkey plasma.
表 1.用于分析猴子血浆中 siRNA-01 QCs 的不同捕获探针的比较。

2.4. Equivalence of the Assay for the Analysis of siRNA-01 Duplex and Single-Stranded AS1
2.4.分析 siRNA-01 双链和单链 AS1 的等效性

In in vivo samples, the siRNA may exist in both duplex and single-stranded form. It is critical that the developed method can accurately quantify both forms in the samples. Theoretically, during sample preparation, the duplex form of the siRNA in the samples will be converted to the single-stranded form after incubation at high temperature which will then be hybridized by the capture probe and then further processed for LC-MS/MS analysis. As a result, the hybridization LC-MS/MS method should be able to accurately quantify both the duplex and single-stranded form equivalently. To investigate if this holds true, we prepared low and high QCs of siRNA-01, AS1, and 1:1 mixture of siRNA-01 and AS1 in monkey plasma (concentrations of AS1 were normalized to equal molar of the siRNA-01 concentrations). A siRNA-01 calibration curve (2.00–1000 ng/mL of siRNA-01 in plasma) was used to analyze all the prepared QC samples. As shown in Table 2, the accuracy and %CV of siRNA-01, AS1 and siRNA-01/AS1 mixture QCs at low and high concentrations were all within ±15%, demonstrating that the developed hybridization LC-MS/MS method can accurately quantify both the duplex and single-stranded form of siRNA across the assay range.
在体内样本中,siRNA 可能以双链和单链两种形式存在。所开发的方法必须能准确定量样品中这两种形式的 siRNA。从理论上讲,在样品制备过程中,样品中双链形式的 siRNA 经过高温孵育后会转化为单链形式,然后与捕获探针杂交,再进一步进行 LC-MS/MS 分析。因此,杂交 LC-MS/MS 方法应能准确量化双链和单链形式。为了研究这一点是否成立,我们制备了猴子血浆中 siRNA-01、AS1 以及 siRNA-01 和 AS1 1:1 混合物(AS1 浓度归一化为等摩尔 siRNA-01 浓度)的低和高 QC。siRNA-01 校准曲线(血浆中 siRNA-01 浓度为 2.00-1000 ng/mL)用于分析所有制备的质控样品。如表 2 所示,低浓度和高浓度 siRNA-01、AS1 和 siRNA-01/AS1 混合物质控物的准确度和 %CV 均在±15%以内,表明所开发的杂交 LC-MS/MS 方法可以在检测范围内准确定量 siRNA 的双链和单链形式。
Table 2. Equivalence of the hybridization LC-MS/MS assay for the quantification of siRNA-01 and AS1.
表 2.杂交 LC-MS/MS 法对 siRNA-01 和 AS1 定量的等效性。

2.5. Qualification and Performance of the Hybridization LC-MS/MS Method
2.5.杂交 LC-MS/MS 方法的鉴定和性能

To support a toxicity study of siRNA-01 in monkeys, the optimized method was qualified for the quantification of siRNA-01 in monkey plasma, CSF, and tissue homogenates prepared from various types of tissues. Since similar method performances were achieved for monkey plasma, CSF, and tissue homogenate samples, the results of the monkey plasma method were described and discussed below as representative.
为了支持猴子 siRNA-01 的毒性研究,对优化后的方法进行了鉴定,以定量检测猴子血浆、脑脊液和各种组织匀浆中的 siRNA-01。由于猴子血浆、脑脊液和组织匀浆样品的方法性能相似,因此猴子血浆的方法结果作为代表在下文中进行描述和讨论。

2.5.1. Accuracy, Precision, and Curve Linearity
2.5.1.准确度、精确度和曲线线性度

Accuracy and precision of the optimized method for siRNA-01 in monkey plasma were assessed for QCs at four concentration levels in two runs. Table 3 summarizes the accuracy and precision data of siRNA-01 QCs in monkey plasma. Based on the three levels of analytical QCs (low, mid, and high), the within-run precision was within 9.3% CV, and the accuracy was 92.1–99.3% of the nominal concentration for siRNA-01, all well within the 15% acceptance criteria. A quadratic 1/x2 weighted regression model (y = ax2 + bx + c) provided the best statistical fit for siRNA-01 over the range of 2.00 to 1000 ng/mL with coefficient of determination (R2) ≥ 0.9932 in both runs. All the calibration standards in the runs passed the acceptance criteria of within ± 15% of nominal concentrations (within ± 20% nominal at the LLOQ level). These results demonstrated the good accuracy and precision of the method for the analysis of siRNA-01 in monkey plasma.
对猴子血浆中四种浓度水平的质控品进行了两次运行,评估了 siRNA-01 优化方法的准确度和精密度。表 3总结了猴子血浆中 siRNA-01 质控品的准确度和精密度数据。根据分析质控的三个水平(低、中、高),运行内精密度在 9.3% CV 范围内,准确度为 siRNA-01 标称浓度的 92.1-99.3%,均在 15%的接受标准范围内。二次1/x2加权回归模型(y =ax2+ bx + c)为 siRNA-01 在 2.00 至 1000 ng/mL 范围内提供了最佳统计拟合,两次运行的决定系数 (R2) 均≥ 0.9932。运行中的所有校准标准品均通过了标称浓度±15%以内(LLOQ水平标称浓度±20%以内)的验收标准。这些结果表明该方法分析猴子血浆中的 siRNA-01 具有良好的准确度和精密度。
Table 3. Accuracy and precision of siRNA-01 QCs in monkey plasma.
表 3.猴血浆中 siRNA-01 质控品的准确度和精密度。

2.5.2. Specificity and Sensitivity
2.5.2.特异性和灵敏度

Assay specificity was evaluated using three different lots of monkey plasma. No significant interfering peaks were observed at the retention time of either the analyte or the IS for all the different lots of blank monkey plasma samples, indicating good specificity of the assay. Representative MRM chromatograms of siRNA-01 in a blank monkey plasma, a LLOQ, and a ULOQ samples are presented in Figure 6. The signal-to-noise ratio of the LLOQ sample was much greater than 5. The intra-run accuracy and precision all met the 20% acceptance criteria (Table 3), which demonstrated the establishment of LLOQ at 2.00 ng/mL in monkey plasma. The same LLOQ of 2.00 ng/mL was also established for siRNA-01 in treated monkey CSF and tissue homogenates.
使用三种不同批次的猴血浆对测定的特异性进行了评估。在所有不同批次的空白猴血浆样品中,分析物或 IS 的保留时间处均未观察到明显的干扰峰,表明该检测方法具有良好的特异性。图 6 是 siRNA-01 在空白猴血浆、LLOQ 和 ULOQ 样品中的 MRM 色谱图。LLOQ 样品的信噪比远远大于 5。运行中的准确度和精密度均符合 20% 的验收标准(表 3),这表明猴血浆中 2.00 ng/mL 的 LLOQ 已经确定。在经处理的猴子脑脊液和组织匀浆中,siRNA-01的最低检出限同样为2.00 ng/mL。
Figure 6. Representative MRM chromatograms of siRNA-01 (left) and the internal standard ASO-002 (right) in a blank monkey plasma, a monkey plasma spiked with the analyte at the LLOQ concentration (2.00 ng/mL), and ULOQ (1000 ng/mL) in monkey plasma.
图 6.siRNA-01(左)和内标 ASO-002(右)在空白猴血浆、添加了 LLOQ 浓度(2.00 纳克/毫升)分析物的猴血浆和 ULOQ(1000 纳克/毫升)猴血浆中的 MRM 色谱图。

2.5.3. Extraction Recovery and Matrix Effect
2.5.3.提取回收率和基质效应

The extraction recovery of siRNA-01 was determined at 6.00 ng/mL (low QC) and 750 ng/mL (high QC) by comparing the analyte to IS response ratios in monkey plasma samples, which were spiked with the analyte before the extraction, with those spiked after the extraction. The recovery of siRNA-01 at low and high QC was 93.5%, and 86.3%, respectively, indicating good extraction recovery of the analyte from plasma across the assay range.
通过比较猴子血浆样品中分析物与 IS 的反应比,确定 siRNA-01 在 6.00 ng/mL(低 QC)和 750 ng/mL(高 QC)时的提取回收率。在低和高QC条件下,siRNA-01的回收率分别为93.5%和86.3%,表明在整个检测范围内血浆中分析物的提取回收率良好。
The matrix effect, which is the ion suppression or enhancement of the analyte by coeluting matrix components, is expressed as matrix factor (MF). The MF was determined by calculating the ratio of the analyte response in plasma extract spiked post-extraction to the response of analyte spiked in elution solution. The MF of the IS was calculated similarly. The IS-normalized MF was determined by dividing the analyte MF by the IS MF. Three different lots of monkey plasma were evaluated in triplicate for the MF at 6.00 and 750 ng/mL. As shown in Table 4, the MF of the analyte at 6.00 ng/mL was 0.92–1.02, the MF of the IS was 0.96–1.03, and the IS-normalized MF was 0.92–1.03. The %CV for the IS-normalized MF of three lots of plasma was 4.1%. At 750 ng/mL, the MF of the analyte, the IS, and the IS-normalized MF were 0.95–1.08, 0.92–1.02, and 0.98–1.08, respectively, and the %CV for the IS-normalized MF was 3.9%. These results demonstrated that there was minimal matrix effect on the analysis of the analyte indicating a highly selective extraction of the analyte by this method resulting in very clean sample extract.
基质效应是指共聚基质成分对分析物离子的抑制或增强作用,用基质因子(MF)表示。基质因子是通过计算萃取后血浆提取物中分析物的响应与洗脱液中分析物的响应之比来确定的。IS 的 MF 计算方法类似。IS 归一化 MF 是用分析物 MF 除以 IS MF 得出的。在 6.00 毫微克/毫升和 750 毫微克/毫升时,对三个不同批次的猴血浆进行了一式三份的 MF 评估。如表 4 所示,分析物在 6.00 纳克/毫升时的 MF 为 0.92-1.02,IS 的 MF 为 0.96-1.03,IS 归一化 MF 为 0.92-1.03。三批血浆的 IS 归一化 MF 的 %CV 为 4.1%。在 750 纳克/毫升时,分析物、IS 和 IS 归一化 MF 分别为 0.95-1.08、0.92-1.02 和 0.98-1.08,IS 归一化 MF 的 %CV 为 3.9%。这些结果表明,基质对分析物的影响极小,表明该方法对分析物的萃取具有高度选择性,样品萃取物非常干净。
Table 4. Matrix effect of the analysis of siRNA-01 in monkey plasma.
表 4.分析猴子血浆中 siRNA-01 的矩阵效应。

2.5.4. Stability 2.5.4.稳定性

The bench-top (room temperature), frozen storage (−80 °C), and freeze–thaw stability of siRNA-01 in monkey plasma were evaluated. siRNA-01 was stable in monkey plasma for at least 8 h at room temperature, 7 days at −80 ºC, and after 3 freeze–thaw cycles.
对 siRNA-01 在猴子血浆中的台式(室温)、冷冻储存(-80 °C)和冻融稳定性进行了评估。siRNA-01 在猴子血浆中的稳定性分别为室温下至少 8 小时、-80 °C下 7 天和 3 次冻融循环。

2.5.5. Metabolite Interference
2.5.5.代谢物干扰

siRNA duplex compounds usually are metabolized by endonucleases and exonucleases ubiquitously presented in vivo and form truncated metabolites of its antisense or sense strand [23]. To ensure there is no interference from the potential metabolites (e.g., n-1 truncated metabolite) to the quantification of siRNA-01, the putative 3′ n-1 metabolite was synthesized and assessed in a metabolite interference test. As the expected metabolite level was <20% of the intact antisense strand and the antisense strand would be the most abundant species present in the samples, the 3′ n-1 metabolite was spiked into siRNA-01 low QC (6.00 ng/mL) and high QC (750 ng/mL) samples at 20% of the corresponding siRNA-01 concentrations. The spiked QCs were analyzed in quadruplicate, and the average %Bias was at −5.9% and −1.6%, and the %CV at 6.0% and 3.1% for low and high QC, respectively, which indicated the absence of metabolite interference. We did not test n-2, n-3, or other shorter truncated metabolites in this work. These metabolites usually will not cause interference to the analysis of the intact antisense strand [21], since their much smaller size would make them easily differentiated from the analyte by LC-MS/MS.
siRNA 双链化合物通常会被体内普遍存在的内切酶和外切酶代谢,形成其反义或有义链的截短代谢物[23]。为确保潜在代谢物(如 n-1 截短代谢物)不干扰 siRNA-01 的定量,合成了推测的 3′ n-1 代谢物,并在代谢物干扰测试中进行了评估。由于预期的代谢物水平为<20 data-dl-uid="1">21],因为它们的体积更小,很容易通过 LC-MS/MS 与分析物区分开来。

2.5.6. Dilution Integrity and Nonspecific Binding Test
2.5.6.稀释完整性和非特异性结合试验

Dilution integrity of the assay was evaluated by analyzing dilution QC samples (2000 ng/mL in monkey plasma, n = 4) extracted with 10-fold dilution. The accuracy was 107.1% of the nominal concentration, and the precision (%CV) was 5.8%, all within 15% acceptance criteria, demonstrating satisfactory dilution integrity.
通过分析经 10 倍稀释提取的稀释质控样本(猴子血浆中浓度为 2000 ng/mL,n= 4),评估了该检测方法的稀释完整性。准确度为标称浓度的 107.1%,精密度(%CV)为 5.8%,均在 15%的接受标准范围内,表明稀释完整性令人满意。
Nonspecific binding loss of siRNA-01 in monkey plasma was tested by analyzing the low QC samples (6.00 ng/mL, n = 4) after 5 × transfers in polypropylene tubes. There was no decrease (%difference 2.9%) in siRNA-01 concentration (analyte/IS response ratio) for the QCs after 5 × transfers compared to the QCs without any transfer, indicating no loss of siRNA-01 due to nonspecific binding.
猴子血浆中 siRNA-01 的非特异性结合损失是通过分析在聚丙烯试管中转移 5 次后的低质量控制样品(6.00 ng/mL,n= 4)来检测的。与未转移的质控品相比,转移 5 次后的质控品 siRNA-01 浓度(分析物/IS 响应比)没有下降(差异率为 2.9%),表明 siRNA-01 没有因非特异性结合而损失。

2.6. CSF Assay 2.6.CSF 检测

Non-specific binding (NSB) loss of analyte is a well-known challenge for the analysis of oligonucleotides, especially for a protein deficient matrix such as CSF. To prevent the potential NSB loss during sample preparation and analysis, we used a previously developed anti-adsorption treatment solution [21], which is a combination of nonionic surfactant and protein (2% Tween 80 and 100 mg/mL BSA in TBS), to treat the CSF samples 1:1 (v/v). Due to the limited availability of monkey CSF, we used artificial CSF (aCSF) as a surrogate matrix for the CSF assay. The aCSF was treated the same way using the anti-adsorption treatment solution to prevent the NSB and minimize the matrix difference between aCSF and real CSF samples. Nonspecific binding loss of siRNA-01 in treated monkey CSF and aCSF was tested by analyzing the low QC samples after 5 × transfers in polypropylene tubes. No nonspecific binding loss of siRNA-01 was observed after 5 × transfers (%difference −1.9% and 0.7% in treated monkey CSF and treated aCSF, respectively).
分析物的非特异性结合(NSB)损失是寡核苷酸分析面临的一个众所周知的挑战,尤其是对于 CSF 这种缺乏蛋白质的基质。为了防止样品制备和分析过程中可能出现的非特异性结合损失,我们使用了之前开发的抗吸附处理液[21],即非离子表面活性剂和蛋白质(2% Tween 80 和 100 mg/mL BSA 在 TBS 中的组合),以 1:1(v/v) 的比例处理 CSF 样品。由于猴子 CSF 的供应有限,我们使用人工 CSF(aCSF)作为 CSF 检测的替代基质。为了防止非特异性结合损失(NSB)并尽量减少人工 CSF 和真实 CSF 样品之间的基质差异,我们用抗吸附处理液对人工 CSF 进行了同样的处理。通过分析在聚丙烯试管中转移 5 次的低质量控制样品,检测了经处理的猴 CSF 和 aCSF 中 siRNA-01 的非特异性结合损失。经过 5 次转移后,未观察到 siRNA-01 的非特异性结合损失(在经处理的猴 CSF 和经处理的 aCSF 中的差异率分别为-1.9%和 0.7%)。

2.7. Tissue Assay 2.7.组织化验

To support the toxicity study, tissue assays need to be developed and qualified for the quantification of siRNA-01 in eight different types of tissues, including brain, kidney, liver, heart, lung, spleen, colon, and spinal cord. For traditional LBA or LC-MS/MS approaches, due to the significantly different matrix effect between different tissues, multiple methods may need to be developed for individual tissues and samples may be analyzed separately. Hybridization LC-MS/MS, with its highly selective and efficient sample extraction, usually generates clean extract with minimal matrix effect. Thus, a surrogate matrix approach can be used for the bioanalysis of different types of tissues [21]. Here, we used a combined tissue homogenate from different tissues (equal parts brain, kidney, liver, heart, lung, spleen, and colon tissue homogenates) as the surrogate matrix to prepare the calibration curve. The method was qualified with QCs prepared in individual type of tissues. Initially, monkey tissues were homogenized in a ratio of 1:12.5 (w/v) using the homogenization buffer (20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% NP-40, pH 8.0). However, variabilities were still observed between different types of tissue, indicating the presence of matrix effect. After further diluting the tissues during the homogenization step using a 1:37.5 ratio, good accuracy and precision were achieved for siRNA-01 in all different types of tissue homogenates over the range of 2.00–1000 ng/mL. Matrix effect (matrix factor) was assessed using low (6.00 ng/mL) and high (750 ng/mL) QC samples prepared in individual tissue homogenate (three difference lots, each lot in triplicates). The mean IS normalized MF was 0.97 (%CV 3.9%) and 1.04 (%CV 5.4%) for low and high QC, respectively. Figure 7 showed the accuracy and precision results of individual tissue QCs at low and high concentrations. The mean accuracy (%Bias) was within ±13.0% and %CV (n = 3) was within 11.4% for all eight different types of tissue homogenate QCs at both low and high concentrations. The results demonstrated that the tissue assay had minimal matrix effect and excellent accuracy and precision. This surrogate matrix method was successfully qualified for the analysis of eight different types of tissues which greatly saved the resources and improved the efficiency.
为支持毒性研究,需要开发组织检测方法,并对八种不同类型组织(包括脑、肾、肝、心、肺、脾、结肠和脊髓)中的 siRNA-01 进行定量。对于传统的 LBA 或 LC-MS/MS 方法,由于不同组织间的基质效应存在显著差异,可能需要针对单个组织开发多种方法,并对样品进行单独分析。杂交 LC-MS/MS 具有高度的选择性和高效的样品提取能力,通常能产生基质效应最小的清洁提取物。因此,代基质方法可用于不同类型组织的生物分析[21]。在此,我们采用不同组织(等份的脑、肾、肝、心、肺、脾和结肠组织匀浆)的组合组织匀浆作为替代基质,制备校准曲线。用不同组织的质控品对该方法进行了鉴定。首先,使用匀浆缓冲液(20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% NP-40, pH 8.0)以 1:12.5 的比例(w/v)匀浆猴组织。然而,不同类型的组织之间仍存在差异,这表明存在基质效应。在匀浆步骤中以 1:37.5 的比例进一步稀释组织后,所有不同类型组织匀浆中 siRNA-01 的准确度和精密度在 2.00-1000 ng/mL 的范围内都达到了很好的水平。基质效应(基质因子)是用低(6.00 ng/mL)和高(750 ng/mL)质控样品制备的单个组织匀浆(三个不同批次,每个批次三重)进行评估的。平均 IS 归一化 MF 为 0.97(%CV 3.9%)和 1.04(%CV 5.0%)。4%)。图 7显示了低浓度和高浓度下单个组织质控物的准确度和精密度结果。所有八种不同类型的组织匀浆质控品在低浓度和高浓度下的平均准确度(%Bias)均在±13.0%以内,%CV(n= 3)在11.4%以内。结果表明,组织检测法的基质效应极小,准确度和精密度极高。这种替代基质方法成功地用于八种不同类型组织的分析,大大节省了资源,提高了效率。
Figure 7. Accuracy and precision of siRNA-01 in monkey liver, kidney, heart, brain, spinal cord, lung, spleen, and colon homogenates (calibration curve prepared in a combined tissue homogenate).
图 7.siRNA-01 在猴子肝脏、肾脏、心脏、大脑、脊髓、肺脏、脾脏和结肠匀浆中的准确度和精确度(在合并组织匀浆中制备的校准曲线)。

3. Materials and Methods 3.材料与方法

3.1. Chemicals, Reagents, Materials, and Instrumentation
3.1.化学品、试剂、材料和仪器

The analyte siRNA-01, the antisense strand of siRNA-01 (AS1, a 21-mer oligonucleotide), the analogue internal standard (IS) ASO-002, and 3′ n-1 truncated metabolite of AS1 were proprietary compounds obtained from Biogen (Cambridge, MA, USA) and its collaborator (see Table 5 for the basic compound information). The biotinylated DNA capture probe (5′-BiotinTEG-DNA, full sequence reverse-complementary to AS1) was synthesized by Integrated DNA Technologies (Coralville, IA, USA). The biotinylated peptide nucleic acid (PNA) capture probe (5′-Biotin-OO-PNA-KK, 19-mer reverse-complementary to AS1) was synthesized by PNA Bio (Newbury Park, CA, USA).
分析物 siRNA-01、siRNA-01 的反义链(AS1,21-mer 寡核苷酸)、类似物内标(IS)ASO-002 和 AS1 的 3′n-1 截短代谢物均为从 Biogen 公司(美国马萨诸塞州剑桥市)及其合作者处获得的专利化合物(化合物基本信息见表 5)。生物素化 DNA 捕获探针(5′-BiotinTEG-DNA,与 AS1 全序列反向互补)由 Integrated DNA Technologies 公司(Coralville, IA, USA)合成。生物素化的肽核酸(PNA)捕获探针(5′-生物素-OO-PNA-KK,与 AS1 反向互补的 19 个单体)由 PNA Bio 公司(Newbury Park, CA, USA)合成。
Table 5. siRNA-01 antisense strand, AS1, and its internal standard, ASO-002.
表 5siRNA-01 反义链 AS1 及其内标 ASO-002。
Acetonitrile (ACN), methanol (MeOH), N,N-dimethylcyclohexylamine (DMCHA), 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFMIP), ethylenediaminetetraacetic acid (EDTA), 10 N sodium hydroxide (NaOH), tris(Hydroxymethyl)aminomethane (Tris), Tween 20, sodium chloride (NaCl), and DL-dithiothreitol (DTT) were obtained from MilliporeSigma (Burlington, MA, USA). Clarity OTX Lysis-Loading Buffer v 2.0 was purchased from Phenomenex (Torrance, CA, USA). Proteinase K, Dynabeads MyOne Streptavidin C1, and Blocker BSA in TBS (10×) Concentrate (100 mg/mL) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). NONIDET P40 (NP-40) was purchased from Accurate Chemical & Scientific (Carle Place, NY, USA). LoBind Eppendorf 1 mL round bottom 96-well plates were obtained from Eppendorf (Enfield, CT, USA). Control rat plasma (K2EDTA), monkey plasma (K2EDTA), monkey CSF, and monkey tissues (liver, brain, kidney, heart, spleen, colon, lung, and spinal cord) were purchased from BioIVT (Westbury, NY, USA). Artificial CSF (aCSF) was purchased from Tocris Bioscience (Toronto, ON, Canada).
乙腈 (ACN)、甲醇 (MeOH)、N,N-二甲基环己胺 (DMCHA)、1,1,1,3,3,3-六氟-2-甲基-2-丙醇 (HFMIP)、乙二胺四乙酸 (EDTA)、10 N 氢氧化钠(NaOH)、三(羟甲基)氨基甲烷(Tris)、吐温 20、氯化钠(NaCl)和 DL-二硫苏糖醇(DTT)取自 MilliporeSigma(美国马萨诸塞州伯灵顿)。Clarity OTX Lysis-Loading Buffer v 2.0 购自 Phenomenex(美国加利福尼亚州托兰斯)。蛋白酶 K、Dynabeads MyOne Streptavidin C1 和 Blocker BSA in TBS (10×) Concentrate (100 mg/mL)购自 Thermo Fisher Scientific (Waltham, MA, USA)。NONIDET P40 (NP-40) 购自 Accurate Chemical & Scientific (Carle Place, NY, USA)。LoBind Eppendorf 1 mL 圆底 96 孔板购自 Eppendorf 公司(Enfield, CT, USA)。对照组大鼠血浆(K2EDTA)、猴血浆(K2EDTA)、猴 CSF 和猴组织(肝、脑、肾、心、脾、结肠、肺和脊髓)购自 BioIVT(Westbury,NY,USA)。人工 CSF(aCSF)购自 Tocris Bioscience 公司(加拿大安大略省多伦多市)。
A Shimadzu Nexera X2 UHPLC system (Shimadzu, Columbia, MD, USA) equipped with three LC-30AD pumps was used for the LC separation. A Sciex TripleQuad 6500+ mass spectrometer (Sciex, Framingham, MA, USA) with Analyst software v 1.6.3 was used for the mass spectrometric detection. A KingFisher Flex Purification System (ThermoFisher Scientific, Waltham, MA, USA) was used for the hybridization sample preparation.
采用岛津 Nexera X2 超高效液相色谱系统(Shimadzu, Columbia, MD, USA)进行液相分离,该系统配备了三个 LC-30AD 泵。质谱检测使用了 Sciex TripleQuad 6500+ 质谱仪(Sciex,Framingham,MA,USA)和 Analyst 软件 v 1.6.3。杂交样品制备使用了 KingFisher Flex 纯化系统(ThermoFisher Scientific, Waltham, MA, USA)。

3.2. Preparation of Calibration Standard and Quality Control Samples
3.2.制备校准标准和质量控制样品

The intact siRNA-01 (duplex of the full-length oligonucleotides) was used for the preparation of calibration standard (STD) and quality control (QC) samples. The siRNA-01 stock solution (2958 µM in PBS) was diluted in ASO diluent (25 mM HFMIP, 10 mM DMCHA, 100 μM EDTA, and 0.05% rat plasma K2EDTA in water:ACN 90:10% v/v) to prepare the intermediate solution (1000 μg/mL). The intermediate solution was appropriately diluted in ASO diluent to prepare ten STD spiking solutions (200–100,000 ng/mL) and four QC spiking solutions (200, 600, 50,000, and 75,000 ng/mL). The ASO-002 IS stock solution was prepared at 500 μg/mL in water, which was further diluted in ASO diluent to obtain 20.0 ng/mL IS working solution (ISWS).
完整的 siRNA-01(全长寡核苷酸的双链)用于制备校准标准(STD)和质量控制(QC)样品。用 ASO 稀释剂(25 mM HFMIP、10 mM DMCHA、100 μM EDTA 和 0.05% 大鼠血浆 K2EDTA,水:乙腈:90:10%v/v)稀释 siRNA-01 原液(2958 µM,PBS),制备中间溶液(1000 μg/mL)。用 ASO 稀释剂适当稀释中间溶液,制备 10 种 STD 加标溶液(200-100,000 ng/mL)和 4 种 QC 加标溶液(200、600、50,000 和 75,000 ng/mL)。ASO-002 IS 原液在水中的浓度为 500 μg/mL,用 ASO 稀释剂进一步稀释,得到 20.0 ng/mL IS 工作溶液(ISWS)。
To prepare STD samples in monkey plasma, the STD spiking solutions were diluted 100-fold with blank monkey plasma to prepare STDs from 2.00 ng/mL (lower limit of quantitation, LLOQ) to 1000 ng/mL (upper limit of quantification, ULOQ). Similarly, QC samples, including LLOQ QC at 2.00 ng/mL, low QC at 6.00 ng/mL, mid QC at 500 ng/mL, and high QC at 750 ng/mL, were prepared in monkey plasma. A dilution QC at 2000 ng/mL was prepared by appropriate dilution of the intermediate solution.
为了制备猴血浆中的 STD 样品,STD 加标溶液用空白猴血浆稀释 100 倍,以制备从 2.00 纳克/毫升(定量下限,LLOQ)到 1000 纳克/毫升(定量上限,ULOQ)的 STD。同样,还在猴子血浆中制备了质控样本,包括 2.00 纳克/毫升的低定量质控样本、6.00 纳克/毫升的低质控样本、500 纳克/毫升的中质控样本和 750 纳克/毫升的高质控样本。通过适当稀释中间溶液,制备出 2000 纳克/毫升的稀释质控品。
Monkey tissue homogenate was prepared by homogenizing the individual tissues (brain, kidney, liver, heart, lung, spleen, colon, and spinal cord) in homogenization buffer (20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% NP-40, pH 8.0) in a ratio of 1:37.5 (w/v). A combined tissue homogenate was prepared by mixing brain, kidney, liver, heart, lung, spleen, and colon tissue homogenates at 1:1:1:1:1:1:1 ratio. The STD/QC samples in monkey CSF, artificial CSF (aCSF), and combined or individual tissue homogenate were prepared similarly to the preparation of plasma STD/QC samples using the corresponding matrices. For the preparation of monkey CSF and aCSF samples, after the spiking of the analyte, the samples were treated with equal volume (1:1, v/v) of the anti-adsorptive agent (2% Tween 80 in 100 mg/mL BSA in TBS).
在匀浆缓冲液(20 mM Tris、20 mM EDTA、100 mM NaCl、0.5% NP-40,pH 8.0)中以 1:37.5(w/v)的比例将各组织(脑、肾、肝、心、肺、脾、结肠和脊髓)匀浆,制备猴组织匀浆。将脑、肾、肝、心、肺、脾和结肠组织匀浆按 1:1:1:1:1:1:1 的比例混合,制备综合组织匀浆。猴脑脊液(CSF)、人工脑脊液(aCSF)、组合或单个组织匀浆中的 STD/QC 样品的制备方法与血浆 STD/QC 样品的制备方法类似,均使用相应的基质。在制备猴 CSF 和 aCSF 样品时,在添加分析物后,用等体积(1:1,v/v)的抗吸附剂(2%吐温 80 与 100 mg/mL BSA 在 TBS 中的混合液)处理样品。

3.3. Sample Preparation 3.3.样品制备

Streptavidin magnetic beads preparation: Dynabeads MyOne Streptavidin C1 magnetic beads (15 µL per sample) were washed 3 times with the washing buffer (5 mM Tris, 1 M NaCl, 0.5 mM EDTA and 0.05% Tween 20 in water). The beads were resuspended to the same buffer and incubated with capture probe (approximately 0.5 nmol probe per 100 µL of beads) at room temperature for 1 h. After incubation, the coated beads were washed 3 times and resuspended to the original volume of washing buffer.
链霉亲和素磁珠制备:用洗涤缓冲液(5 mM Tris、1 M NaCl、0.5 mM EDTA 和 0.05% Tween 20 水溶液)洗涤 Dynabeads MyOne Streptavidin C1 磁珠(每个样品 15 µL)3 次。将珠子重悬到相同的缓冲液中,并与捕获探针(每 100 µL 珠子约含 0.5 nmol 探针)在室温下孵育 1 小时。
Hybridization sample extraction: add samples (50 µL of plasma or tissue homogenate or 100 µL of treated CSF or aCSF), 100 µL of Lysis Buffer (Clarity OTX Lysis-Loading Buffer), 180 µL of Digestion Buffer (100 mM Tris, 250 mM NaCl, 10 mM EDTA and 10 mM DTT in water, prepared fresh daily), and 20 µL of Proteinase K (20 mg/mL) into wells of a 96-well plate. Briefly centrifuge the plate and then incubate for 120 min at 65 °C using a plate shaker at a speed of 850 rpm. Then, add 400 µL of Capture Buffer (10 mM Tris, 10 mM NaCl, 1 mM EDTA and 0.05% Tween 20 in water) and 15 µL of prepared streptavidin beads to the samples. Incubate the sample plate for 90 min at 65 °C. Then, on a KingFisher Flex Purification System, wash the samples twice at room temperature using 500 µL of 10 mM Tris, 10 mM NaCl, 1 mM EDTA and 0.05% Tween 20 in water. Then, add 125 µL of ISWS to the plate and incubate at 95 °C for 20 min for the elution. Transfer the sample eluent to a LoBind Eppendorf plate, centrifuge at 4612× g for 5 min, and then submit for LC-MS/MS analysis.
杂交样本提取:在 96 孔板的孔中加入样本(50 µL 血浆或组织匀浆或 100 µL 经处理的 CSF 或 aCSF)、100 µL 裂解缓冲液(Clarity OTX Lysis-Loading Buffer)、180 µL 消化缓冲液(100 mM Tris、250 mM NaCl、10 mM EDTA 和 10 mM DTT 水溶液,每日新鲜配制)和 20 µL 蛋白酶 K(20 mg/mL)。将平板简单离心,然后在 65 °C 下用平板振荡器以 850 rpm 的速度孵育 120 分钟。然后,向样品中加入 400 µL 捕获缓冲液(10 mM Tris、10 mM NaCl、1 mM EDTA 和 0.05% Tween 20 水溶液)和 15 µL 配制好的链霉亲和素珠子。将样品板在 65 °C 下孵育 90 分钟。然后,在 KingFisher Flex 纯化系统上,用 500 µL 10 mM Tris、10 mM NaCl、1 mM EDTA 和 0.05% Tween 20 的水在室温下清洗样品两次。然后,在平板中加入 125 µL ISWS,在 95 °C 下孵育 20 分钟进行洗脱。将样品洗脱液转移到 LoBind Eppendorf 平板上,4612×g离心 5 分钟,然后进行 LC-MS/MS 分析。

3.4. UHPLC-MS/MS Conditions
3.4.超高效液相色谱-质谱/质谱条件

Chromatographic separation was achieved using an Acquity UPLC Oligonucleotide BEH C18 column (2.1 × 50 mm, 1.7 µm particle size; Waters, Milford, MA, USA) coupled with an Acquity BEH C18 guard column (VanGuard 2.1 × 5 mm, 1.7 μm particle size; Waters, Milford, MA, USA). The column temperature was set at 70 °C. The mobile phases consisted of water (mobile phase A), ACN/water 90/10 (mobile phase B) and 125 mM HFMIP, 50 mM DMCHA in ACN/water 50/50 (mobile phase C). The gradient and flow-rate conditions are presented in Table 6. The total run time was 7 min. The injection volume was 10 µL.
色谱分离采用 Acquity UPLC Oligonucleotide BEH C18 色谱柱(2.1 × 50 毫米,1.7 微米粒径;Waters,Milford,MA,USA)和 Acquity BEH C18 保护柱(VanGuard 2.1 × 5 毫米,1.7 微米粒径;Waters,Milford,MA,USA)。色谱柱温度设定为 70 °C。流动相包括水(流动相 A)、ACN/水 90/10(流动相 B)和 125 mM HFMIP、50 mM DMCHA(ACN/水 50/50)(流动相 C)。梯度和流速条件见表 6。总运行时间为 7 分钟。进样量为 10 µL。
Table 6. LC gradient and flow rate conditions.
表 6.LC 梯度和流速条件
The mass spectrometric detection was conducted in electrospray negative ionization mode using the following parameters: ion spray voltage −4500 V; temperature 550 °C; curtain gas 30 units; ion source gas 1, 80 units; ion source gas 2, 60 units; dwell time 250 msec for AS1 and 50 msec for the IS; declustering potential −75 V for the analyte and −65 V for the IS; collision energy −140 eV for AS and −150 eV for the IS, respectively. The monitored multiple reaction monitoring (MRM) transitions were m/z 629.8 → 95 for AS1 and m/z 879.5 → 95 for ASO-002, the IS.
质谱检测采用电喷雾负离子模式,参数如下:离子喷雾电压-4500 V;温度 550 ℃;帘气 30 单位;离子源气体 1,80 单位;离子源气体 2,60 单位;AS1 的停留时间为 250 毫秒,IS 的停留时间为 50 毫秒;分析物的去簇电位为-75 V,IS 的去簇电位为-65 V;碰撞能量分别为 AS-140 eV 和 IS-150 eV。监测到的多反应监测(MRM)跃迁为:AS1 为m/z629.8 → 95,IS ASO-002 为m/z879.5 → 95。

4. Conclusions and Future Perspectives
4.结论和未来展望

A hybridization LC-MS/MS method was successfully developed and qualified for the quantification of siRNA-01, an investigational siRNA drug candidate, in monkey plasma, CSF, and tissue homogenate over the range of 2.00–1000 ng/mL. The use of the PNA probe as the capture probe achieved satisfactory recovery (around 90%) for the hybridization extraction of siRNA-01. This is the first report of applying hybridization methodology for the quantitative bioanalysis of a double-stranded oligonucleotide. The developed methodology will be used to support pharmacokinetic, toxicokinetic, and biodistribution studies of siRNA-01 in monkeys. The methodology can also be applied to the bioanalysis of other siRNAs or double-stranded oligonucleotides.
成功开发并鉴定了一种杂交LC-MS/MS方法,用于定量检测猴子血浆、脑脊液和组织匀浆中候选siRNA药物siRNA-01的含量,范围为2.00-1000 ng/mL。使用 PNA 探针作为捕获探针,杂交提取 siRNA-01 的回收率令人满意(约 90%)。这是首次应用杂交方法对双链寡核苷酸进行定量生物分析的报告。所开发的方法将用于支持 siRNA-01 在猴子体内的药代动力学、毒代动力学和生物分布研究。该方法也可用于其他 siRNA 或双链寡核苷酸的生物分析。
With the rapid growth in oligonucleotide therapeutics, sensitive and reliable bioanalytical methods are in increasing demand to characterize the PK, TK, and biodistribution of oligonucleotide drug candidates and therefore support their research and development. Hybridization LC-MS/MS combines the advantages of hybridization immunoassay and LC-MS/MS, and has been proven to be able to provide accurate, sensitive and specific bioanalysis of single-stranded oligonucleotides. In this work, the application of this strategy to double-stranded oligonucleotides, such as siRNA, has also been successfully demonstrated. Thus, hybridization LC-MS/MS as a novel platform is able to be more widely applied to the bioanalysis of all types of oligonucleotides, including ASO, siRNA, or other oligonucleotides, in various biological matrices. In combination with other advanced technologies in chromatography and mass spectrometry, the sensitivity and selectivity/specificity of hybridization LC-MS/MS methods can be further improved (e.g., the use of microflow LC to increase the sensitivity and high-resolution mass spectrometry to improve specificity). In addition, since oligonucleotides are composed with the same nucleic acid bases or modifications, they have similar properties. The developed method and the learnings for one oligonucleotide can be easily transferred/applied to other oligonucleotides. This will significantly save the time and efforts and increase the efficiency of method development. This unique advantage, along with its good sensitivity and selectivity, makes hybridization LC-MS a superior platform for wider application in oligonucleotide bioanalysis, covering both single-stranded and double-stranded oligonucleotides.
随着寡核苷酸疗法的迅速发展,人们越来越需要灵敏可靠的生物分析方法来表征寡核苷酸候选药物的 PK、TK 和生物分布,从而为其研发提供支持。杂交 LC-MS/MS 结合了杂交免疫测定和 LC-MS/MS 的优点,已被证明能够对单链寡核苷酸进行准确、灵敏和特异的生物分析。在这项工作中,这一策略还成功地应用于 siRNA 等双链寡核苷酸。因此,杂交 LC-MS/MS 作为一种新型平台,能够更广泛地应用于各种生物基质中各类寡核苷酸(包括 ASO、siRNA 或其他寡核苷酸)的生物分析。结合色谱和质谱方面的其他先进技术,杂交 LC-MS/MS 方法的灵敏度和选择性/特异性可得到进一步提高(例如,使用微流 LC 提高灵敏度,使用高分辨率质谱提高特异性)。此外,由于寡核苷酸由相同的核酸碱基或修饰组成,因此具有相似的性质。针对一种寡核苷酸开发的方法和学到的知识可以很容易地转移/应用到其他寡核苷酸上。这将大大节省时间和精力,提高方法开发的效率。这一独特的优势,加上其良好的灵敏度和选择性,使杂交 LC-MS 成为一个卓越的平台,可广泛应用于寡核苷酸生物分析,包括单链和双链寡核苷酸。

Author Contributions 作者供稿

Conceptualization, L.Y.; methodology, L.Y. and K.M.; investigation, L.Y.; J.-F.D. and K.M.; writing—original draft preparation, L.Y.; review and editing, L.Y., J.-F.D. and K.M. All authors have read and agreed to the published version of the manuscript.
构思,L.Y.;方法,L.Y.和K.M.;调查,L.Y.;J.-F.D.和K.M.;写作-原稿准备,L.Y.;审阅和编辑,L.Y.、J.-F.D.和K.M.。所有作者均已阅读并同意手稿的出版版本。

Funding 资金筹措

This research received no external funding.
本研究未获得外部资助。

Institutional Review Board Statement
机构审查委员会声明

Not applicable. 不适用。

Informed Consent Statement
知情同意声明

Not applicable. 不适用。

Data Availability Statement
数据可用性声明

Not applicable. 不适用。

Acknowledgments 致谢

The authors thank Patrick Trapa for his helpful review of the manuscript.
作者感谢 Patrick Trapa 对稿件进行的有益审阅。

Conflicts of Interest 利益冲突

The authors declare no conflict of interest.
作者声明没有利益冲突。

Sample Availability 样品供应

Not available. 不详。

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Figure 1. Effect of temperature and salt concentration on the recovery of AS1 using DNA probe and PNA probe.
Molecules 28 01618 g001
Figure 2. Effect of temperature and salt concentration on the recovery of siRNA-01 using DNA probe and PNA probe.
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Figure 3. Optimization of temperature and salt concentration for the recovery of siRNA-01 using DNA probe and PNA probe.
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Figure 4. Effect of DNA probe concentration and incubation time on the recovery of siRNA-01.
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Figure 5. Comparison of different combination of DNA and PNA probes for recovery of siRNA-01.
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Figure 6. Representative MRM chromatograms of siRNA-01 (left) and the internal standard ASO-002 (right) in a blank monkey plasma, a monkey plasma spiked with the analyte at the LLOQ concentration (2.00 ng/mL), and ULOQ (1000 ng/mL) in monkey plasma.
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Figure 7. Accuracy and precision of siRNA-01 in monkey liver, kidney, heart, brain, spinal cord, lung, spleen, and colon homogenates (calibration curve prepared in a combined tissue homogenate).
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Table 1. Comparison of different capture probes for the analysis of siRNA-01 QCs in monkey plasma.
PNA Probe
75 pmols/Sample
LLOQ QC
2.00 ng/mL
Low QC
6.00 ng/mL
Mid QC
500 ng/mL
High QC
750 ng/mL
Measured Conc.2.14 5.78 502.18 758.45
2.03 6.32 417.41 726.41
1.79 6.21 446.34 726.46
1.95 6.00 472.78 788.82
Mean1.986.08459.68750.03
S.D.0.150.2436.2529.94
N4444
% CV7.54.07.94.0
% Nominal98.7101.391.9100.0
PNA and DNA Probes 37.5 pmols Each/SampleLLOQ QC
2.00 ng/mL
Low QC
6.00 ng/mL
Mid QC
500 ng/mL
High QC
750 ng/mL
Measured Conc.1.98 6.25 526.62751.12
2.09 6.07 457.77 745.37
2.24 6.04 533.94 752.48
2.01 6.42 456.61 742.90
Mean2.086.19493.73747.97
S.D.0.120.1842.314.57
N4444
% CV5.62.88.60.6
% Nominal103.9103.298.799.7
Table 2. Equivalence of the hybridization LC-MS/MS assay for the quantification of siRNA-01 and AS1.
siRNA-01
Low QC
6.00 ng/mL
AS1
Low QC
6.00 ng/mL
siRNA-01 + AS1
Low QC
3.00 + 3.00 ng/mL
siRNA-01
High QC
750 ng/mL
AS1
High QC
750 ng/mL
siRNA-01 + AS1
High QC
375 + 375 ng/mL
6.466.275.80696.41752.41733.57
6.555.995.99677.39813.93705.49
6.926.505.10782.65761.66680.36
5.625.535.41799.26759.01822.64
Mean6.396.075.58738.93771.75735.52
S.D.0.550.420.4060.9528.3962.02
n444444
% C.V.8.66.97.28.23.78.4
% Nominal106.5101.292.998.5102.998.1
Table 3. Accuracy and precision of siRNA-01 QCs in monkey plasma.
Run 1LLOQ QC
2.00 ng/mL
Low QC
6.00 ng/mL
Mid QC
500 ng/mL
High QC
750 ng/mL
Measured Conc.1.985.87527.11681.34
1.946.50441.75674.75
2.055.91464.04756.63
2.295.56469.18753.45
Mean2.065.96475.52716.54
S.D.0.160.4036.4044.55
n4444
% CV7.76.67.76.2
% Nominal103.199.395.195.5
Run 2LLOQ QC
2.00 ng/mL
Low QC
6.00 ng/mL
Mid QC
500 ng/mL
High QC
750 ng/mL
Measured Conc.1.785.83493.31746.88
1.825.06460.57754.49
1.725.12471.69721.32
1.756.09492.21702.19
Mean1.775.53479.45731.22
S.D.0.040.5116.0424.00
n4444
% CV2.49.33.33.3
% Nominal88.392.195.997.5
Table 4. Matrix effect of the analysis of siRNA-01 in monkey plasma.
Low QC (6.00 ng/mL)High QC (750 ng/mL)
Analyte
Matrix Factor
IS
Matrix Factor
IS-Normalized
Matrix Factor
Analyte
Matrix Factor
IS
Matrix Factor
IS-Normalized
Matrix Factor
Lot 10.920.970.951.001.010.99
0.951.020.930.980.981.00
1.011.011.000.940.950.99
Lot 20.921.000.921.041.021.02
0.971.000.970.950.921.03
0.990.961.031.081.001.08
Lot 31.021.011.010.990.981.01
0.981.030.950.981.000.98
0.941.010.931.040.961.08
Mean 0.97 1.02
S.D. 0.040 0.040
n 9 9
% C.V. 4.1 3.9
Table 5. siRNA-01 antisense strand, AS1, and its internal standard, ASO-002.
NameMW (kDa)Sequence LengthChemistry
AS16.921Mixed backbone with 2′-OMe and 2′-F modification
ASO-0027.920Uniform MOE with PS backbone
2′-OMe: 2′-O-methylation. MOE: 2′-O-(2-Methoxyethyl)-oligoribonucleotides. PS: phosphorothioate linkage.
Table 6. LC gradient and flow rate conditions.
Time (min)ModuleEventsParameters
0PumpsPump B Conc.0
0PumpsA/B Total Flow 0.400
0PumpsPump C Flow0.100
0.3PumpsPump B Conc.0
3.0PumpsPump B Conc.25
3.0PumpsA/B Total Flow 0.400
3.0PumpsPump C Flow0.100
3.1PumpsPump C Flow0
3.2PumpsPump B Conc.100
3.2PumpsA/B Total Flow 0.600
3.6PumpsPump B Conc.100
3.7PumpsPump B Conc.25
4.1PumpsPump B Conc.25
4.4PumpsPump B Conc.100
4.9PumpsA/B Total Flow 0.600
4.9PumpsPump B Conc.100
5.0PumpsPump C Flow0
5.1PumpsPump B Conc.0
5.1PumpsA/B Total Flow 0.400
5.1PumpsPump C Flow0.100
7.0ControllerStop
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Yuan, L.; Dupuis, J.-F.; Mekhssian, K. A Novel Hybridization LC-MS/MS Methodology for Quantification of siRNA in Plasma, CSF and Tissue Samples. Molecules 2023, 28, 1618. https://doi.org/10.3390/molecules28041618

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Yuan L, Dupuis J-F, Mekhssian K. A Novel Hybridization LC-MS/MS Methodology for Quantification of siRNA in Plasma, CSF and Tissue Samples. Molecules. 2023; 28(4):1618. https://doi.org/10.3390/molecules28041618

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Yuan, Long, Jean-François Dupuis, and Kevork Mekhssian. 2023. "A Novel Hybridization LC-MS/MS Methodology for Quantification of siRNA in Plasma, CSF and Tissue Samples" Molecules 28, no. 4: 1618. https://doi.org/10.3390/molecules28041618

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