Advances in LC-MS bioanalysis for drug research and development 液 相 质 谱 生 物 样 品 分 析 技 术 在 新 药 研 发 中 的 新 进 展

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1 Advances in LC-MS bioanalysis for drug research and development 液 相 质 谱 生 物 样 品 分 析 技 术 在 新 药 研 发 中 的 新 进 展 张 杰 DMPK, Novartis Institutes for BioMedical Research ( 诺 华 ), New Jersey 2014 南 京 国 际 药 代 会 议, June 27-28, 2014

2 Focused areas in LC-MS bioanalysis in recent years 生 物 样 品 定 量 分 析 技 术 热 点 领 域 Global harmonization of bioanalytical practice and new BMV guidance from FDA and EMA 方 法 验 证 全 球 协 调 Quantitation of Antibody Drug Conjugate (ADC), Bispecific Antibody (BsMAb), MAb (intact and large fragment characterization, PEGylated), peptides, sirna, etc. 抗 体 药 物 复 合 物, 多 肽 及 蛋 白 药 物, 短 干 扰 核 糖 核 酸 等 类 的 定 量 分 析 Dried Blood Spot (DBS)/microsampling 干 血 斑 点 / 微 量 样 品 Unstable compound bioanalysis, whole blood stability, lipemic plasma matrix effects 不 稳 定 的 化 合 物 分 析, 全 血 稳 定 性, 脂 血 血 浆 基 质 效 应 Electronic Laboratory Notebooks (ELN) 电 子 实 验 室 笔 记 本 Incurred sample reanalysis/stability (ISR/ISS) 实 际 样 品 再 分 析 / 稳 定 性 Biomarker quantification; Fit-for-Purpose validation 生 物 标 志 物 定 量 Free & total measurement 非 蛋 白 键 合 及 总 和 的 药 物 定 量 High resolution MS in regulated bioanalysis 高 分 辨 质 谱 生 物 定 量 分 析 2 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

3 Major meetings and publications in bioanalysis 生 物 样 品 分 析 领 域 的 主 要 会 议 和 出 版 物 Conferences/Meetings/Workshops 3 American Society for Mass Spectrometry (ASMS) Conference on Mass Spectrometry and Allied Topics Workshop on Recent Issues in Bioanalysis (WRIB) European Bioanalysis Forum (EBF) Open Symposium Land O Lakes Bioanalytical Conference Applied Pharmaceutical Analysis (APA) American Association of Pharmaceutical Scientists (AAPS) Annual Meeting and Exposition Journals Bioanalysis Analytical Chemistry Journal of Chromatography A/B Biomedical Chromatography Rapid Communications in Mass Spectrometry Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

4 Handbook of LC-MS Bioanalysis 最 近 二 十 年 来 最 全 面 完 整 的 生 物 样 品 分 析 参 考 书 Edited by Wenkui Li, Jie Zhang & Francis Tse (Novartis US) (Wiley in August of 2013) Key highlights >70 contributors/51 chapters/700 pages Bioanalysis overview Current regulations 4 Technical details including instrumentation, method development, validation, stability assessment, DBS, etc. Representative experimental protocols: pro-drugs acylglucurorides N-oxides, reactive compounds photosensitive and autooxidative compounds inter/intra-convertible compounds Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only proteins and antibodies nucleosides and nucleotides oligonucleotides chiral compounds endogenous compounds steroids, etc.

5 Selected topics to be discussed in this presentation Dried Blood Spot (DBS) sampling and bioanalysis 干 血 斑 (DBS) 的 取 样 和 生 物 分 析 Working with unstable compounds in LC-MS/MS bioanalysis 不 稳 定 化 合 物 生 物 样 品 分 析 sirna LC-MS assay Development 短 干 扰 核 糖 核 酸 生 物 样 品 分 析 方 法 Electronic Laboratory Notebooks (ELN) 电 子 实 验 室 笔 记 本 5 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

6 What is DBS and why? Microsampling technique ( µl per spot) Capillary blood via a simple prick (heel, finger, toe or tail) or venous blood from human and animal Spotted onto a paper card for the analysis of the analyte(s) First used in newborn screening ( 新 生 儿 筛 查 ) in 1963 for inherited metabolic disorders Further applications in clinical testing (infectious disease etc.), human general health study (DNA, RDA, etc.), and TDM (therapeutic drug monitoring) Ethical ( 人 道 ) benefits - simplified sample collection and handling (less invasive and pain, sample taken from home, etc) and reduced sample volume (suitable for pediatrics and elderly patient and use less animals) Economical ( 成 本 ) benefits - less cost in sample storage and shipping 6 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

7 DBS handling procedures in Bioanalytical lab DBS 生 物 样 品 分 析 程 序 7 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

8 Challenges and considerations of DBS Hematocrit effects and spot homogeneity 红 细 胞 比 容 影 响 及 斑 点 均 匀 性 New tools to be developed for accurately spotting and easy to use Innovations in card technology are needed Accurate blood spotting and analyzing full spot Liquid microsampling (e.g. dried plasma spot) Plasma is more suitable matrix for PK than blood Micro-plasma sampling technique is under evaluation (e.g. ~75 µl blood for ~30 µl plasma on card) Limitation in assay sensitivity Limitation for unstable compounds 8 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

9 Common causes of analyte instability in LC-MS/MS Bioanalysis* 药 物 在 生 物 样 品 中 不 稳 定 的 常 见 原 因 Enzyme-activated degradation Hydrolysis of conjugated metabolites Decomposition of N-oxidation metabolites Chemical reactivity Photochemical degradation and autooxidation Inter-conversion during storage, processing, extraction and/or chromatography Fragmentation and/or transformation in MS Source * Wenkui Li, Jie Zhang and Francis L. S. Tse. Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices. Biomed. Chromatogr. 2011; 25: Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

10 Approaches to working with unstable analytes 解 决 药 物 在 生 物 样 品 中 不 稳 定 问 题 的 常 用 方 法 Sample collection, processing and storage. Selection of matrix and anticoagulant Temperature control Light protection Instability vs non-specific binding Instability vs temperature dependent re-equilibration of analyte between plasma and erythrocytes Sample preparation Chromatographic separation Suitable MS tuning Identification and confirmation of analyte instability using incurred samples in addition to spiked QC Samples 10 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

11 Approaches to working with unstable analytes 解 决 药 物 在 生 物 样 品 中 不 稳 定 问 题 的 特 殊 方 法 Addition of enzyme inhibitors ph control Addition of antioxidants Derivitization of unstable analyte(s) during sample collection Storage of biological sample in dried form on cellulose card/paper 11 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

12 sirna therapeutics and delivery techniques 短 干 扰 核 糖 核 酸 药 物 及 输 送 系 统 sirna 短 干 扰 RNA 或 沉 默 RNA 双 链 RNA 分 子 (sense and anti-sense),18-25 个 碱 基 对 干 扰 基 因 表 达 Cyclodextrin polymer nanoparticles 环 糊 精 聚 合 物 纳 米 粒 子 Lipid nanoparticles 脂 质 纳 米 粒 Conjugate delivery systems 共 轭 输 送 系 统 12 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only Zhou et al., Pharmaceuticals (Basel). 2013; 6(1): 85

13 Techniques for sirna bioanalysis 生 物 样 品 分 析 技 术 概 述 Hybridization-ELISA 杂 交 酶 联 免 疫 吸 附 测 定 based on the measurement of radioactivity of 3 H, 14 C, 35 S or 125 I- labeled OGNs Reverse transcription qpcr 反 转 录 定 量 PCR unmatched sensitivity and superior dynamic range (low pg/ml high ng/ml range) LC UV/fluorescence 液 相 色 谱 紫 外 / 荧 光 widely used for quality controls of synthesized OGNs and other DNA/RNA products for purity verification due to its great availability and ease of operation LC MS/MS 液 相 串 联 质 谱 based on not only the retention time, but also the molecular structure information 13 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

14 Typical LC MS assay of sirna sirna 分 析 技 术 要 点 Sample preparation LLE with phenol:chloroform:isoamyl alcohol 苯 酚 - 氯 仿 - 异 戊 醇 Mixed-mode SPE (weak-anion SPE and reversed phase, Phenomenex Clarity -OXT, Waters Oasis) LLE-SPE combination for better sample clean up HPLC Ion-pair reversed phase HPLC with TEA: hexafluoroisopropanol (HFIP) ionpair (A) and methanol (B) 三 乙 胺 - 六 氟 异 丙 醇 离 子 对 Column temperature at >70 o C to denature ds-rna to its single anti-sense and sense strands 双 链 分 离 成 单 链 MS (SIM or MRM) ESI in negative ionization mood Deprotonated molecular ions with a multiplicity of different charge states 去 质 子 化 的 分 子 离 子 产 生 多 种 质 荷 比 离 子 14 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

15 Challenges of sirna LC MS assay development 难 点 讨 论 Incompatibility of chromatographic resolution and MS detection; limited ionization efficiency and signal suppression 色 谱 和 质 谱 不 兼 容, 有 限 的 离 子 化 和 信 号 抑 制 Sensitivity not high (typical LOQ is 1-10 ng/ml) Multiple charge states (ion dilution) Extensive adduction with Na+, K+, etc. Require ion-pair buffers Assay reproducibility Carryover Well controlled sample analytical procedures and dedicated LC-MS systems are required for sirna assay 15 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

16 Electronic Laboratory Notebooks (ELN) 电 子 实 验 室 笔 记 本 ELN is a computer program used to replace paper laboratory notebooks Benefits of ELN Use templates to capture all critical contents of experiments and data. Flag issues to avoid SOP deviations (e.g., use of expired reagent or instrument) Cross-link references Smooth data review processes Meet all CFR part 11 and GLP requirements. Provide audit trail to record the operator, time and date of data entry or change Improve laboratory efficiency and work productivity, reduce compliance risks and overall operational cost. 16 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

17 Transform from paper to paperless (ELN) 17 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

18 Typical Bioanalytical templates in ELN 生 物 样 品 分 析 实 验 室 常 用 模 板 18 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

19 Factors to consider in implementing ELN in BA lab 实 施 ELN 前 考 虑 事 项 Interface with existing instruments, Wason LIMS and other databases Establishment of electronic archiving and linking with ELN Development of various BA templates to cover entire BA workflow Identify ELN vendors 19 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

20 Conclusions LC-MS has become a fundamental tool for Bioanalysis in every stage of drug discovery and development As demonstrated in the four highlighted areas in this presentation, many new techniques, procedures, and processes have been developed in recent years toward faster, cheaper, and better ways of providing quality bioanalytical results 20 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only

21 Acknowledgements Francis Tse Jimmy Flarakos Wenkui Li Annamaria Jakab Bin Yang Jennifer Davis 21 Nanjing Pharma conference Jie Zhang 28-Jun-2014 Business Use Only