抗体药物LC-MS法生物分析进展

在抗体药物的生物分析中,液相色谱-质谱联用(LC-MS)技术已经成为传统配体结合分析(LBA)的一种重要的替代或补充方法。其方法开发中替代肽的选择、样品制备、灵敏度和定量准确性是值得关注的问题。本文对抗体药物LC-MS法生物分析的进展进行综述,介绍LC-MS法分析抗体药物的原理,说明LC-MS法定量抗体过程中面临的挑战与当前可用于应对这些挑战的新策略,包括:替代肽的选择、样品纯化与富集、提高酶解效率、肽段富集与应用低速LC等方面。本文最后展示了LC-MS技术在抗体药物生物分析中的应用实例,并对抗体药物LC-MS法的发展提出了展望。     

靶向c-Met抗体偶联药物(RC108)在食蟹猴中PK生物分析方法学研究

抗体偶联药物(antibody-drug conjugate,ADC)有着低毒、高效的药物特点,在癌症治疗中发挥着重要作用。但是因其结构的复杂性,带来药代动力学(pharmacokinetic,PK)生物分析的困难。本研究建立一种应用配基结合分析法(ligand-binding assay,LBA)和液相色谱与串联质谱联用法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)检测食蟹猴血浆中ADC (RC108)的分析方法,用于分析定量食蟹猴血浆中的总抗体、结合抗体和游离药物。基于LBA法,在96孔板预包被rabbit anti-RC108 Fab和mouse anti-MMAE m Ab分别作为总抗体和结合抗体的试剂,加入待测样品,随后依次加入检测试剂goat anti-human Ig G (H+L)-HRP和显色液四甲基联苯胺(tetramethylbenzidine,TMB),H₂SO₄终止反应后,酶标仪450 nm/630 nm波长处读取数据;LC-MS/MS分析方法定量...     

液质联用技术在肿瘤诊断与监测中的应用进展

液相色谱串联质谱法(LC-MS/MS)是小分子代谢物的重要分析工具，随着其广泛应用，发现了大量新型可靠的潜在生物标志物用以辅助肿瘤的早期诊断，为预测肿瘤的侵袭和转移、提高肿瘤的诊断率提供了强有力的技术支持。此外，基于LC-MS/MS的治疗药物监测(TDM)为肿瘤的预防、干预和治疗提供了重要依据，在个性化药物治疗中发挥着关键作用。该文就LC-MS/MS在肿瘤标志物的筛选、检测和TDM中的进展进行综述，以期为临床筛选可靠的肿瘤标志物和个性化药物治疗提供参考。     

蛋白质组学与卵巢癌研究

随着后基因组时代的到来，蛋白质组学已成为一个发展迅速、日益重要的研究领域，高分辨率的双向聚丙烯酰胺凝胶电泳（two-dimensional polyacrylamide gel electrophoreasis．2-D PAGE）与质谱分析技术（mass spectrometry，MS）和生物信息学的结合使蛋白质组分析及其在临床疾病中的应用成为可能。恶性肿瘤是一种多基因参与的复杂疾病。由于蛋白质组学研究是直接定位于蛋白质水平，因此，通过对肿瘤蛋白质的整体、动态、定量的研究，     

串联质谱在生物样品手性药物立体选择性定量测定中的应用

液相色谱串联质谱(LC-MS/MS)的选择性和灵敏度较高,广泛应用于直接拆分或定量测定生物样品中的手性药物.超临界流体色谱(SFC)因其在质谱兼容和分析速度方面优于液相色谱,使得SFC-MS/MS成为一种新的手性药物立体选择性定量测定技术.本文综述了LC-MS/MS法和SFC-MS/MS法在生物样品手性药物立体选择性定量测定中的应用.     

LC-MS/MS技术及其在天然产物分析中的应用

目的:研究LC-MS/MS技术及其在天然产物分析中的应用;方法:通过分析Lc-MS接口的基本原理详细研究LC-MS/MS技术,并分别介绍其在天然产物分析中微量天然物质分析、天然产物异构体的分离和鉴定、天然产物筛选和中药指纹图谱四个方面的应用.结果和结论:LC-MS/MS在技术及应用方面取得了很大进展,相信在天然产物分析中必将发挥越来越重要的作用.     

液-质联用法测定大鼠血浆中索他洛尔的浓度及其药动学研究

目的：建立大鼠血浆中索他洛尔的LC-MS/MS定量分析方法,考察口服给药后,索他洛尔在大鼠体内的药动学特征。方法：大鼠口服给药,分别在给药前和给药后不同的时间点从眼底静脉丛取血。采用LC-MS/MS分析方法,以阿替洛尔为内标测定大鼠血浆中索他洛尔的含量,并运用DAS1.0软件计算待测药物各药动学参数。结果：索他洛尔在5~5 000 ng·mL^-1范围内线性关系良好;日内日间精密度（RSD）范围为2.99%~5.75%,准确度（RE）为98.56~105.71%;提取回收率为92.50%~97.16%,基质效应为96.59%~102.10%。结论：所建立的LC-MS/MS分析方法准确、灵敏,可用于大鼠血浆中的索他洛尔的含量测定和药动学研究。     

液相色谱-串联质谱与化学衍生化及在线氢／氘交换结合药物代谢物的特性策略

介绍了应用高效液相色谱．串联质谱（LC-MS／MS）与化学衍生化、在线氢／氘（H／D）交换技术联合研究粗提样品中药物代谢物结构的策略。讨论了子离子扫描、恒定中性丢失扫描、母离子扫描、多级的MS^n和精确质量测量的质谱扫描技术。尤其重要的是，只需微量的粗提样品就足以满足LC-MS／MS的分析要求，且可以免去从复杂生物样品中纯化低含量代谢产物的困难，从而减少工作强度。     

生物样品分析中采用单孔法进行配体结合分析的进展和技术要求

复孔分析是生物样品分析中配体结合分析(LBA)的标准操作方式，其目的是为了降低分析的变异性。随着分析仪器技术的进步和试剂质量的提升，特别是自动化应用，LBA的精密度得到显著提升，生物分析行业提出LBA是否也可以像LC-MS一样采用单孔分析。2019年3月发布的ICH M10(征求意见稿)已接受单孔分析的方式，目前已有多个行业组织或研究机构开展了单孔分析与复孔分析的对比研究，结果表明采用单孔分析并不显著影响毒代动力学(TK)和/或药代动力学(PK)关键参数、抗药抗体(ADA)的临界值以及灵敏度。本文总结现有文献的研究进展，提出单孔分析应用的技术要求，以期为单孔分析应用于TK/PK、以及ADA研究提供参考和借鉴，进一步促进药品注册技术标准与国际接轨。     

高速逆流色谱技术及其在药物分离分析中的应用

简单介绍了高速逆流色谱（HSCCC）技术及其在药物分离分析中的应用。通过参阅国内外的文献报道，综述了HSCCC的仪器类型、特点、溶剂系统、技术发展及近年来在分离分析天然产物、蛋白质、抗生素、手性化合物等领域的研究和应用进展。结果表明，HSCCC适用于药物有效成分的定性分析与分离，与其他色谱技术联用有望用于中药定量分析。HSCCC技术在药物研发中有广阔应用前景。     

Optimized Approaches for Quantification of Drug Transporters in Tissues and Cells by MRM Proteomics

Drug transporter expression in tissues (in vivo) usually differs from that in cell lines used to measure transporter activity (in vitro). Therefore, quantification of transporter expression in tissues and cell lines is important to develop scaling factor for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated drug disposition. Since traditional immunoquantification methods are semiquantitative, targeted proteomics is now emerging as a superior method to quantify proteins, including membrane transporters. This superiority is derived from the selectivity, precision, accuracy, and speed of analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Moreover, LC-MS/MS proteomics has broader applicability because it does not require selective antibodies for individual proteins. There are a number of recent research and review papers that discuss the use of LC-MS/MS for transporter quantification. Here, we have compiled from the literature various elements of MRM proteomics to provide a comprehensive systematic strategy to quantify drug transporters. This review emphasizes practical aspects and challenges in surrogate peptide selection, peptide qualification, peptide synthesis and characterization, membrane protein isolation, protein digestion, sample preparation, LC-MS/MS parameter optimization, method validation, and sample analysis. In particular, bioinformatic tools used in method development and sample analysis are discussed in detail. Various pre-analytical and analytical sources of variability that should be considered during transporter quantification are highlighted. All these steps are illustrated using P-glycoprotein (P-gp) as a case example. Greater use of quantitative transporter proteomics will lead to a better understanding of the role of drug transporters in drug disposition.     

Analysis of polar metabolites by hydrophilic interaction chromatography--MS/MS

Increasing emphasis has been placed on quantitative characterization of drug metabolites during drug discovery and development. Due to the more polar nature of drug metabolites, quantitative analysis using traditional reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS) can be quite challenging. As an alternative chromatographic mode, hydrophilic interaction chromatography (HILIC) offers unique advantages for analysis of polar metabolites, providing better retention/separation, higher sensitivity, higher efficiency and potential for ultra-fast analysis to improve throughput. In this article, selected case studies from the authors' own laboratory, and examples from current literature, will be discussed to demonstrate some practical considerations for method development of HILIC-MS/MS assays. The effectiveness of using HILIC-MS/MS for mitigating analytical challenges associated with quantitation of polar metabolites, including phase I and II metabolites of drugs, as well as endogenous metabolites, will be exhibited.     

Regulated LC-MS/MS bioanalysis technology for therapeutic antibodies and Fc-fusion proteins using structure-indicated approach

In recent studies, the development of bioanalysis technologies using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has attracted attention. Our developed nano-surface and molecular-orientation limited (nSMOL) proteolysis enables Fab-specific proteolysis and is optimal for LC-MS/MS analysis of antibody drugs and Fc-fusion proteins in biological samples. In this nSMOL method, antibodies and Fc-fusion proteins are held in pores of the particle and the subsequent proteolysis is carried out with protease-immobilized nanoparticles. The Fab of antibodies or fused region of Fc-fusion protein can be held to orient toward the reaction solution. The access of the immobilized protease is limited to a part in the structure of protein substrate on the particle surface. Thus, nSMOL proteolysis reacts selectively at the Fab complementarity-determining region of antibodies or N-terminal specific domain of Fc-fusion proteins and can be applied to both types of drugs. We have already evaluated drug concentrations in biological samples pretreated with nSMOL proteolysis using LC-MS/MS for more than twenty drugs, of which ten drugs have been fully validated and published. In this review, we discuss the development and application of LC-MS/MS bioanalysis, which enables the bioanalysis of therapeutic antibodies and Fc-fusion proteins by focusing on a structure-based approach.     

'Large Meets Small': connecting the bioanalytical community around peptide and protein bioanalysis with LC-MS(/MS)

This conference report provides an overview of the discussions at the 2nd European Bioanalysis Forum (EBF) Focus Meeting 'Large Meets Small' held on 20 and 21 June 2011 in Brussels. The meeting discussed scientific progress in the bioanalysis of peptides and proteins with MS-based techniques. Bioanalytical experts in ligand-binding assays (LBAs) and MS from industry and academia presented at the meeting or joined the discussion. The conference hosted sessions on technology developments, validation requirements, cutting edge (bio)analytical approaches for both proteins and peptides and discussions on the analytical challenge presented by the metabolism of peptides or proteins. The engagement of the scientists as well as the bioanalytical challenges identified were real: by shifting peptide or protein analysis from the LBAs laboratory into the LC-MS laboratory, the bioanalytical scientist is moving into partially uncharted territory. The conference delegates strongly shared the feeling that success in overcoming the challenges of peptide and protein bioanalysis will require further integration of the expertise of LBAs and LC-MS/MS experts.     

Column-switching procedures for the fast analysis of drugs in biologic samples

Liquid chromatography coupled with mass spectrometry in single and dual mode (LC-MS and LC-MS/MS) is the method of choice for the quantification of drugs and their metabolites in biologic fluids. Following the new challenges encountered in the process of drug development, liquid chromatography-mass spectrometry has been found to achieve high-throughput analysis. With this impressive tool, the sample preparation step before analysis is simplified, and the analytic process speeded up. Several generic approaches have recently been developed for the sample extraction coupled on line with a LC-MS system. In this paper, different extraction supports allowing the direct injection of biologic fluids were investigated, namely, restricted-access media, large-size particle, and monolithic phases. In the column-switching configuration, these supports, coupled with microbore analytic columns, were found suitable for the fast analysis (total analysis time of less than 10 minutes) of different drugs and their metabolites in biologic matrices at the nanogram per milliliter level.     

Characterization of protein therapeutics by mass spectrometry: recent developments and future directions

Mass spectrometry (MS) has become a powerful technology in the discovery and development of protein therapeutics in the biopharmaceutical industry. This review article describes recent developments and future trends in the characterization of protein therapeutics using MS. We discuss top-down MS for the characterization of protein modifications, hydrogen/deuterium exchange MS and ion mobility MS methods for higher order protein structure studies. Quantitative analysis of protein therapeutics (in vivo) by MS as an orthogonal approach to immunoassay for pharmacokinetics studies will also be illustrated.     

Recent development in software and automation tools for high-throughput discovery bioanalysis

Bioanalysis with LC-MS/MS has been established as the method of choice for quantitative determination of drug candidates in biological matrices in drug discovery and development. The LC-MS/MS bioanalytical support for drug discovery, especially for early discovery, often requires high-throughput (HT) analysis of large numbers of samples (hundreds to thousands per day) generated from many structurally diverse compounds (tens to hundreds per day) with a very quick turnaround time, in order to provide important activity and liability data to move discovery projects forward. Another important consideration for discovery bioanalysis is its fit-for-purpose quality requirement depending on the particular experiments being conducted at this stage, and it is usually not as stringent as those required in bioanalysis supporting drug development. These aforementioned attributes of HT discovery bioanalysis made it an ideal candidate for using software and automation tools to eliminate manual steps, remove bottlenecks, improve efficiency and reduce turnaround time while maintaining adequate quality. In this article we will review various recent developments that facilitate automation of individual bioanalytical procedures, such as sample preparation, MS/MS method development, sample analysis and data review, as well as fully integrated software tools that manage the entire bioanalytical workflow in HT discovery bioanalysis. In addition, software tools supporting the emerging high-resolution accurate MS bioanalytical approach are also discussed.     

Application of CYP102A1M11H as a tool for the generation of protein adducts of reactive drug metabolites

Covalent binding of reactive metabolites (RMs) to proteins is considered to be one of the important mechanisms by which drugs can cause tissue damage. To facilitate the study of drug-protein adducts, we developed a potentially generic method for producing high levels of covalently modified proteins. A highly active drug metabolizing P450 BM3 mutant (CYP102A1M11H) is used for drug bioactivation. Because of its His-tag, CYP102A1M11H is easily removed by nickel affinity chromatography, facilitating subsequent characterization of the modified target protein. The applicability of our procedure is demonstrated by the trapping of RMs of acetaminophen (APAP), clozapine (CLOZ), and troglitazone (TGZ) with human glutathione-S-transferase P1-1 (hGST P1-1) as the model target protein. Tryptic digests of hGST P1-1 were subjected to analysis by LC-MS/MS and modified peptides identified by the comparative analysis of tryptic peptides of adducted and nonadducted hGST P1-1. Characteristic MS/MS ions of drug-modified peptides were identified by first searching for expected adduct-masses. Unanticipated drug-peptide adducts were subsequently identified in an unbiased manner by screening for diagnostic MS/MS ions of modified peptides. Reactive intermediates of APAP and CLOZ adducted to cysteine-47 and mass shifts corresponded to the alkylation of N-acetyl-p-benzoquinone imine (NAPQI) and the CLOZ nitrenium ion, respectively. Adduction of TGZ appeared more complex, yielding three different types of adducts to cysteine-47, two adducts to cysteine-14, and a single adduct to cysteine-101. Together, these findings show that P450 BM3 mutants with high capacity to activate drugs into relevant RMs can be employed to produce protein adducts to study the nucleophilic selectivity of highly reactive electrophiles.     

Getting to the core of protein pharmaceuticals – Comprehensive structure analysis by mass spectrometry

Protein pharmaceuticals are the fastest growing class of novel therapeutic agents, and have been a major research and development focus in the (bio)pharmaceutical industry. Due to their large size and structural diversity, biopharmaceuticals represent a formidable challenge regarding analysis and characterization compared to traditional small molecule drugs. Any changes to the primary, secondary, tertiary or quaternary structure of a protein can potentially impact its function, efficacy and safety. The analysis and characterization of (structural) protein heterogeneity is therefore of utmost importance. Mass spectrometry has evolved as a powerful tool for the characterization of both primary and higher order structures of protein pharmaceuticals. Furthermore, the chemical and physical stability of protein drugs, as well as their pharmacokinetics are nowadays routinely determined by mass spectrometry.Here we review current techniques in primary, secondary and tertiary structure analysis of proteins by mass spectrometry. An overview of established top-down and bottom-up protein analyses will be given, and in particular the use of advanced technologies such as hydrogen/deuterium exchange mass spectrometry (HDX-MS) for higher-order structure analysis will be discussed. Modification and degradation pathways of protein drugs and their detection by mass spectrometry will be described, as well as the growing use of mass spectrometry to assist protein design and biopharmaceutical development.     

Miniaturized formats for efficient mass spectrometry-based proteomics and therapeutic development

Off-line miniaturized "nano-spray" formats for electrospray ionization mass spectrometry (ESI-MS) enable the routine identification of femtomole quantities of protein or peptide. Even greater strides have been achieved using on-line miniaturized ESI-MS methods, such as nanobore LC-MS and CE-MS. On-line methods enable greater sensitivity (sub-attomole limit of detection), dynamic range, and throughput. In either off- or on-line methods for protein analysis, samples are typically isolated and digested enzymatically, with MS analysis of the peptide fragments, yielding 5-50% sequence coverage, in a "bottom-up" approach. Obtaining biologically relevant (structure/function) information (such as the localization of regions of error or post-transnational modifications) often demands 100% sequence coverage and this may be obtained by analyzing intact proteins by MS with a "top-down" methodology. Proteome wide success with top-down methods will require the development of novel miniaturized approaches for sample preparation along with new tools for bioinformatics. As these miniaturized formats continue to power proteomics applications, they will undoubtedly pollinate "cross-over" applications in LC-MS ranging from drug discovery to development. An example of metabolite identification using an order of magnitude less sample than usually required, with a concurrent order of magnitude increase in signal, illustrates the potential of miniaturized formats in lead characterization activities.