安捷伦生物药物分析解决方案

Biologic Characterization PK/PD/Tox Efficacy Assay Development Human Biomarker Identification
Cell line development Fermentation Optimization Impurity Profiling Purification process
通过MS/MS分析修饰位点
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不同样品肽谱结果Mirror比较
（可进行>=3个样品的比较）
比较结果： R+S:表示在两个样品都存在 R: 只在样品R中存在 S: 只在样品S中存在
样品R化合物列表
样品S化合物列表
MS图 UV和TIC图Mirror MS/MS图
Glycosylation site Fc
采
1260/90 LC, Chip-LC Q-TOF/TOF
1260/90 LC, Chip-LC Q-TOF
mAb-Glyco-Chip LC Q-TOF/TOF
硬 件 软 件
集
分 析
Deconvolution Match Sequence
Molecular Feature Extraction Match Sequence
Biologic Discovery
Preclinical Development
Bioprocess Development
Clinical Trials
Regulatory Approval & Manufacturing
Genetics Research
Biologic Expression Binding Assay development Biologic Structural Analysis Hybridoma/DNA Library screening
日程安排
12.19日
上午
1. 安捷伦单克隆抗体分析完整解决方案（周志贵）
2. 样品前处理注意事项（史律） 下午 1. 数据采集方法 2. 数据分析方法 12.20日 1. 上机练习
2. 问题讨论
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Agilent Restricted December 19, 2012
安捷伦生物药物分析 解决方案
MassHunter Software
MassHunter BioConfirm
周志贵 安捷伦应用工程师 zhi-gui_zhou@
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Agilent’s Analytical Tools in Biologics Development
Disease Research
Worklist (Sequence)
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AutoTune + Calibration
Calibration Report
One “Click”

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Qualitative + BioConfirm Automated workflow + “File open”action
Glycan G2F G1F G0F G0
Structure
Average mass 1769.6 1607.5 1445.4 1299.3
完整抗体表征 肽谱和翻译后修饰分析 糖基分析
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December 19, 2012
安捷伦单克隆抗体分析完整解决方案
Heavy Chain Disulfide shuffling Pyroglutamate Light Chain Fab Deamidation/oxidation
氨基酸序列 二硫键位置
Light Chain
Fab Deamidation/oxidation Hinge
修
饰
糖型和糖基化
Fc Truncation (lysine) Glycosylation site
Fucose – 146 Da Mannose – 162 Da N-Acetylglucosamine – 203 Da Galactose – 162 Da
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Agilent Restricted December 19, 2012
“MFE” + “BioConfirm”
安捷伦独有:分子特征提取（MFE） 通过精确质量数和MS/MS分析氨基 酸序列覆盖率 综合考虑同位素质量精度及同位素 丰度比
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Agilent Restricted December 19, 2012
Molecular Feature Extraction Search by glycan library
完整单抗分析 特 点
两种去卷积方法计算分子 量 精确质量数分析单抗修饰
肽谱分析
安捷伦独有:分子特征提取 精确质量数和MS/MS分析 氨基酸序列和修饰位点
糖基分析
完整单抗直接进样，在线 酶解 12分钟完成单抗糖型分析
BioConfirm Report
Molecular Weight Potential Modifications
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两种去卷积算法
单抗原始质谱图 两种去卷积算法（Maximum Entropy 和 Peak Modeling ）
得到不同糖型的单抗的精确分子量
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pMod Resolves Overlapped Peaks
IgG 1
IgG2
These two peaks are 39 Da apart
IgG1 and IgG2 mix Not resolved by Maximum Entropy IgG1 and IgG2 mix Resolved by pMod
糖型
Agilent Restricted December 19, 2012
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LC/MS 肽谱分析工作流程
Acquisition Qual BioConfirm Comparison
MassHunter Acquisition Define Target Sequence
Define Digest Enzymes and Potential Modifications
Agilent’s Analytical Tools in Biologics Development
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Confidentiality Label December 19, 2012
单克隆抗体的表征
Heavy Chain Antigen binding Disulfide shuffling Pyroglutamate
same or not
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80多种常见修饰
通过精确分子量可以对单抗修饰进行分析
• More than 80 predefined modifications in the chemical data dictionary • Allows user defined modifications • Allows C13, N15, O18, D labeled amino acid 修饰
All at a glance
BioConfirm has the ability to see the MS, MS/MS, and EIC/ECC on a per compound basis all at the same time:
ECC
MS MS/MS
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Compare Protein Digest module allows comparison of biosimilars and to monitor batch-tobatch variation
Match?
BioConfirm Report
Sequence Coverage %
Potential Modifications and their Locations
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DA Reprocessor for Batch Analysis
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LC/MS 完整单抗分析工作流程
Acquisition Qual/ BioConfirm BioConfirm Comparison
Human Biomarker and PK Analysis
Product-Related Quality Control Process-Related Quality Control
Disease Pathways
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Confidentiality Label December 19, 2012
pMod
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Mirror Plots of Deconvoluted Spectra
Allow fast comparison of two samples Differential plot (dotted line) tells you if biosimilars are the
Agilent Restricted December 19, 2012
Molecular Feature Extractor (MFE): TOF MS-only or QTOF MS and MS/MS data for: --Protein Digest (peptide mapping) --Filter features found by quality using Q- score NEW!
MassHunter Acquisition Integrate Chromatogram Define Target Sequence NEW! Extract Spectra Define Potential Modifications Mirror Plot allows comparison of biosimilars and to monitor batch-tobatch variation
特 点
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Agilent Confidential December 19, 2012
Agilent’s 1290 HPLC and 6530/40/50 QTOF
Agilent 6540 Ultra High Definition QTOF
低延迟体积 高精密度
分析速度快
高质量准确度 宽动态范围
Agilent 1290 Infinity LC
高采集速度下 高分辨率
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December 19, 2012
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Instrument Actuals/Status (configurable)
System Diagram
Real-Time Spec/Chro Display
Method Editor LC + MS !
Deconvolute Spectra: TOF or Q-TOF MS-only data for: --Intact Protein --Synthetic Peptide --Oligonucleotide --Maximum Entropy --Peak Modeling NEW!
Match?
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样品R MS列表
样品S MS列表
样品S
Agilent Restricted December 19, 2012
样品R
糖谱分析工作流程
糖谱分析：安捷伦从硬件到软件，从样品处理到数据分析有着完整的解决方案
应用文集：
LC, CE, Chip-LC, LC-MS, CE-MS，色谱柱方案
详情参见：/en-US/Solutions/Biotherapeutics-Biosimilars/Glycan-Analysis/Pages/default.aspx