ADC 药物完整解决方案SCIEX 质谱 -- 超过 50 年的创新历程发布API III，是第一款SCIEX的商业化的
液质联用系统

发布5500系列，这是业界知名的
液相色谱三重四极杆质谱系统

SWATH™ Acquisition Compendium
2019

SCIEX 创立发布QTRAP® LC-MS/MS 系统，SCIEX创新的把三重四极杆和线性离子阱技术结合起来由 Barry French 博士和William Breukelman博士共同创立于多伦多大学

1970

SCIEX在这一年发布新产品
数量创新高，这其中包括
SWATH®采集技术

1981 1989 2001 2002 2005 2008 2009 2012 2013发布第一款SCIEX的商用三重四极杆(MS/MS)仪器

发布API 5000™系统，该系统是代表当时先进水平的
三重四极杆液质联用系统

发布API 4000™ LC-MS/MS 系统，成为了生物分析的标准手段

整合了Beckman
Coulter®公司
毛细管电泳业务

成为丹纳赫集团的一员，
并于2010年收购Eksigent
公司液相业务

The Power of Precision

发布Echo™ MS系统，这是一款基于声波激发耦合质谱(AEMS)技术
的超高检测样品通量的系统
发布第一款SCIEX的用于
台式精确质量质谱系统，
X500R QTOF 系统
发布SCIEX Now™
客户服务平台五十周年纪念发布BioPhase™ 8800 系统，一款可以同时检测8份样品的多通道毛细管电泳系统

发布专门控制三重四极杆质谱的
SCIEX OS 软件，是SCIEX创新的
核心动力

2015 2018 2020

2021

发布SCIEX Triple Quad™ 7500 系统，开启灵敏度的新时代

发布C100HT 生物制品分析系统,OptiFlow™ Turbo V 离子源, 和StatusScope® 远程监控服务发布了SCIEX OS软件3.0版本，
支持中文界面

2022

期待未来

发布ExionLC™ 2.0和ExionLC™ 2.0+ 系统，与SCIEX质谱系统兼容，实现一体化控制

发布ZenoTOF™ 7600系统，这是
一款高分辨精确质量质谱，是SCIEX跨跃式的质谱创新发展

发布ZenoTM SWATH® 数
据非依赖性采集(ZenoTM
SWATH DIA®)技术， 标
志着生物标志物发现及转化应用中又向前迈进
了重要一步

ADC 药物完整解决方案
抗 体 偶 联 药 物（antibody-drug conjugate，
ADC）是一类通过特定的连接子将靶向单克隆抗体
与高杀伤性的细胞毒性小分子药物偶联起来的生物
药，以单克隆抗体为载体将小分子细胞毒性药物高效地运输至目标肿瘤细胞中，起到治疗的目的。
近年来，随着新的连接子、小分子毒素、抗体以及靶点发现技术的不断更新，ADC 药物也发展迅速，仅 2019 年一年，FDA 就批准了三款 ADC 药
物，荣昌生物的国产 ADC 维迪西妥单抗也在 2021
年 6 月获 NMPA 批准上市。目前， 全球有 100 多项 ADC 药物相关的临床试验正在进行。

ADC 药物的结构和特点（Lancet Oncol. 2016 Jun; 17(6): e254–e262.）

小分子毒素抗体比 DAR（drug-to-antibody ratio）是 ADC 药物独特的重要质量属性，它代表抗体
偶联小分子毒性药物的平均数量，较低的 DAR 会降低 ADC 的效力，但过高的 DAR 又会影响 ADC 分析的药代动力学和毒性，普遍认为 DAR 在 2-4 之间是 ADC 药物的最优选。而偶联方式是影响 DAR 的重要因素，只有选择合适的偶联技术，才能让 ADC 的毒素均一稳定地连接在抗体上。
目前主要有三种偶联方式：1) 通过暴露在抗体表面的赖氨酸 , 2) 还原链间二硫键的半胱氨酸 , 3)
定点偶联技术来进行偶联。其中赖氨酸偶联的位点较多，每个抗体上可以挂 0~8 个小分子毒素，导
致每一批次的 ADC 产品异质性较大。而半胱氨酸偶联，由于二硫键的位置是固定的，所以偶联位置
也相对可控，但是其链接小分子毒素的个数取决于二硫键的还原程度。当 4 条链间二硫键均被完全
还原时，能携带 8 个毒素；当它被部分还原时，则可能生成 DAR 为 2、4、6、8 的产物，半胱氨酸偶联产物较赖氨酸，其均一性已经显著提高。定点偶联主要是使用生物技术人为地在单抗上引入链接位点。随着 ADC 偶联方式的进步，ADC 药物抗体比不均的问题也被逐步改善。
本文集提供 ADC 药物的解决方案，包括 ADC 药物的表征分析（分子量、DAR 值、肽图、偶连位点、
翻译后修饰），大小分子的绝对定量分析，小分子代谢物鉴定及电荷异质性分析等。运用三重四极杆液质联用系统、QTOF 高分辨液质联用系统，毛细管电泳技术，提供全面的 ADC 药物深度表征方案。

4 ADC 药物完整解决方案内容提要
应用电子活化解离（EAD）技术进行抗体 - 药物偶联物（ADC）表征 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Simple and fast-tracking of DAR distribution using intact multiple attribute methodology (Intact MAM) . . 12
Analysis of non-deglycosylated antibody-drug-conjugates by TripleTOF® high resolution
quadrupole-time-of-ﬂight instrument and eﬀective reconstruction software . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Native LC-MS Analysis of an Intact Cysteine-Linked Antibody-Drug-Conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Intact Analysis of Antibody Drug Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Structural Characterization of Antibody Drug Conjugates (ADCs) by a Combination of Intact,
Middle-up and Bottom-up Approaches using CESI-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Ultra-sensitive LC-MRM analysis for trastuzumab-emtansine quantiﬁcation in rat plasma . . . . . . . . . . . . . . . . 33
Sensitive and Accurate Quantitation of the Antibody-Drug Conjugate Ado-Trastuzumab
Emtansine in Rat Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Metabolite Identiﬁcation of Payload Species of Antibody Drug Conjugates with Noncleavable
Linkers using MetabolitePilot™ 2.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
CE-SDS 和 CIEF 方法对半胱氨酸偶联的抗体偶联药物的分析 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

ADC 药物完整解决方案 5

Characterization of an antibody-drug-conjugate (ADC) using
Enhancedsensitivity
for peptide (EAD)
quantification in a complex
electronactivateddissociation
应用电子活化解离（EAD）技术进行抗体-药物偶联物（ADC）表征matrixusing high-resolution
LC-MS/MS
Featuring the SCIEX ZenoTOF 7600 system with EAD and Protein Metrics Inc. software
Featuring
ZenoTOF
7600
LC-MS/MS7600
system
采用带有the
的SCIEX
系统和Protein Metrics公司软件
EAD
ZenoTOF™
Zoe Zhang1, Takashi Baba2, Pavel Ryumin2, Bill Loyd2, Jason Causon2, Kerstin Pohl1
1
2
SCIEX,Nandita,
US; SCIEX,
CanadaEshaniLei Xiong,
Elliott Jones, Zoe Zhang, Kerstin Pohl
SCIEX, USA

Characterization of an antibody-drug-conjugate (ADC) using

Here, the characterization of lysine-linked drugs in an ADC
collision-induced dissociation (CID) is able to provide amino acid
High-resolution
platforms, suchtime-of-flighttechnique
A significant
5-fold improvementin LLOQ
for peptide
sampleis demonstrated.
A bottom-up
approach
was chosen to
sequence confirmation,
but asthetraditional
harsh fragmentation
systems,
often lacksensitivity
due
to lossof ionThetransmissioninquantificationachieved
using
the ZenoTOF
7600 systemdetermine
thewaspositionof theconjugation.
Alternative
also breaks
the payload
into
small
pieces.
highly complex
betweenpulses.
The
Zeno
trap
controlscan
the beionvery
beam
from tofeaturing the1 Zeno
trap.
Comparedwith traditional
2
2 time-of-flight
21,2 was
2 TOF
1 suchfragmentationusingelectron-activateddissociation(EAD)
spectraderivedfromanapproachdifficult
Zoe Zhang , Takashi Baba , Pavel Ryumin , Bill Loyd , Jason Causon , Kerstin Pohl
theinterpret.
collision cellwhich facilitates
greatercan
ion provide
transmission
to insights
thesystems, thetrap structural
enables greater
MS/MS
byleveraged
forZenodetailed
information
onsensitivity
the drug andAlternativefragmentation
further
1
2
TOF
accelerator.
Therefore,
the duty
cycle is improved
to ≥90%,enhancingthewithduty
cycle.
In peptide
addition,backbone
the versatilityof TOFSCIEX,US;SCIEX,
Canadalinker,along
obtaining
information.
intosuch complex
samples,but previous
techniques
sufferedwhich
enhancesoveralltimes,
MS/MS
MS/MS data allows for the capability of post-acquisition
fromlong reaction
lowsensitivity.
sensitivity and lack ofdecisionsfor the selectionof fragmention(s) forantibodies
MRMHR. ForWiththe developmentin protein
engineering,andreproducibility.
The ZenoTOF 7600 system offers an exceptional combination of
cases
where
multipledominant
fragmentions
aregeneratedtheir related derivatives become the fastest growingclass ofmass
resolution, sensitivity, and acquisition speed for
3 peptide, the sum of XICs enabled greater常规方法，而对结合位点的深入表征，通常依赖于自下而上的方
from 本文对ADC样品中赖氨酸连接药物进行了表征，选择了一种
the target
therapeutics.
ADCs are one of those new modalities. ADCs a
A new, highly reproducible fragmentation type based on EAD1,2
quantitative
analysis. It also aids in the potential for: less ion path
1,2
sensitivity.
A 3-fold improvement
in monoclonal
LLOQ was observed
自下而上的方法来确定偶联的位置。利用电子活化解离（EAD)法。现在最广泛采用的碰撞诱导解离（CID）技术能够提供氨基酸areoften composedof a 150 kDa
antibodyfor(mAb)技was used to analyze the conjugated peptides from a commercial
tuning, increased sensitivity with the Zeno trap, ability to change
peptidesthat
leveraged
thesummingofmultipledominant
covalently
coupled with cytotoxic payloads, or other types of
ADC. The data were acquired with an untargeted 10 Hz rapid
术产生的离子碎片信息，可获取相关药物和连接子的详细结构信
序列确认，但是这种能量比较大的碎裂技术也将有效载荷分解为更
measured fragments post-acquisition and improved
4 ADCsfragment
ions whenMS/MS
ion current
wasdispersed.
drugs,
throughsynthetic
linkers.
show
a more complex
data-dependent acquisition (DDA) method and interpreted with息，同时获得多肽骨架信息。
小的片段，从这种方法获得的高度复杂的谱图可能很难解析。而能
reproducibility and accuracy.
structure and heterogeneity compared to unconjugated proteins,
Protein Metrics Inc. software. With this workflow, regular and
量更柔和的碎裂方法可以促进此类复杂样品的解析，而且CIQ技术
Traditional workflows for quantitative bioanalyses, such as
since随着蛋白质工程技术的进展，抗体及其相关衍生物成为治疗药the addition of a variable number of payload and linkers
advancedcharacterization
leveraging 7600
EAD-basedfragmentation
Keyfeaturesof the ZenoTOF
systemfor
immunological assays,
have been displaced by LC-MS/MS
可能需要更长的反应时间以及灵敏度低和缺乏可重复性的困扰。
3。ADCs是这些新的药物形式之一。ADCs通常
物中增长最快的一类
cansignificantly
themass
numberof proteoforms.5 To ensure highlyis achievable
in onepeptide
injection, enablinga streamlined
analysison triple enhance
quadrupole
spectrometers.
sensitive
quantification
1,2
由150safety
一种基于EAD 的创新、高度可重复的碎裂方法用于分析来自kDa单克隆抗体（mAb）与细胞毒性药物（有效载荷）通过
drug
andoftenefficacy,
in-depthcharacterization
ADCs
characterization
accessible to every user-level.
Immunoassays
lack anselectivity
andspecificity, andof
have
a is
4
• Demonstration of a 5-fold improvementin LLOQ for peptide
essential
duringtheirdevelopment.
This
includes
not
only
the
。与未偶联的蛋白相比，ADC
商业化ADC药物的偶联肽。使用10
特定偶联物（linker）共价偶联而成
Hz快速非靶向的数据依赖采集
limited linear dynamic range. While the triple quadrupole platformKeyfeatures
of
the
SCIEX
ZenoTOF
7600
quantification
using
the
Zeno
trap
to
accumulateions during
identification
andthe localizationof post-translational药物的结构复杂度和异质性更高，因为添加了多变的有效载荷和连（DDA）方法采集数据，并使用Protein Metrics 公司软件进行数据解
hasbeen a key
driverfor most bioanalytical
workflows, offering
each
TOF pulse for enhanced duty cycle
system5
modifications(PTMs)
on
the
mAb,
but
also
a
verification
of
the
great
sensitivity and quantitative performance,high-resolution。为确保药物安全性和有效接子，所以显著增加蛋白形式的数量
析。通过此工作流程，一次进样就可以应用基于EAD的碎片进行常• Reach enhanced sensitivity by summing of multiple highly
drug
conjugation.
Mass spectrometry
has become
the most 规和高级表征，从而获得可供每个用户使用的精简的表征方法。
accurate
mass spectrometry(HRAMS) (MS)has increasingly
been
性，ADC的深度表征在其开发过程中至关重要。这不仅包括对mAb• New depths
of ions
peptideEAD withabundant
fragment
with mapping
availabilityanalysis:of TOF MS/MS
data fast
1,2
widelyemployedmethodfor ADC characterization,owing to theadoptedfor quantitative
bioanalysis.
With the inherent
的翻译后修饰（PTMs）的鉴定和定位，还包括药物结合的验证。
DDA enables alternative fragmentation for routine, in-depth• Ensure exceptional accuracy and precision for quantitative
rapid
advancement
MS technologies.
Intactmass
analysis isadvantageof greaterofselectivity
with improved
mass
resolution,
analysis of next generation protein therapeutics and standard
由于质谱技术的飞速发展，质谱（MS）已经成为ADC表征中最广泛
SCIEX
ZenoTOF™
workflows
using the7600系统的主要功能
ZenoTOF 7600 system
as wellas themethod
flexibilityof TOFtoMS/MS
data,drug-to-antibody
the ZenoTOF 7600
theplatform
utilized
determine
ratio
mAbs
使用的方法。完整质量分析是用于确定药物与抗体比率（DAR）的
system whileprovidesexcellentquantitativein multiple(DAR),
deep
characterization
ofperformancethe sites of conjugation•
Easilyacquire,process,
and manage data on a single• •肽图分析的新深度：具有快速DDA的EAD技术可为下一代蛋白质Higher levels of structural information: Changing thedimensions.
usuallyrelies on bottom-up approaches. Most widely-adopted,
platform using the SCIEX OS Software治疗药物和标准单克隆抗体（mAb）的常规、深度分析提供可mechanism of fragmentation by tuning the electron energy选择的创新碎裂方法。
may provide a higher level of structural informationelectron activated dissociation (EAD)

Higher MS/MS sensitivity: Increased detection of fragments• •更高级别的结构信息：通过调节电子能量来改变碎裂模式,能会(5 to 10 fold) using the Zeno trap enables higher confidence in
提供更高级别的结构信息。
data assignment
更高的MS / MS灵敏度：使用Zeno™ trap(Zeno阱)技术，可产生•更多的碎片离子（5到10倍），从而提高了数据结果的置信度。
High reproducibility: Reproducible fragmentation with EAD
for singly, doubly, and multiply charged ions enables analysis• 高重现性：与其它碰撞碎裂技术和低重现性碎裂技术相比，使of more precursors than other alternative and low
用EAD进行单电荷、双电荷和多电荷离子碎裂时，具有更高的
reproducibility fragmentation techniques
重现性，可分析更多前体离子。
• Streamlined and easy-to-use: Fully automated data• 简单易用：使用带有SCIEXOS软件的EAD技术，以DDA模式进行
acquisition in DDA modeusing EAD with SCIEX OS Software,and automated data interpretation with Byos
software(Protein
全自动数据采集，并使用Byos软件（Protein
MetricsInc.）进行
Metrics Inc.) simplifies the entire user experience
自动数据解析，从而简化了分析流程、提升用户体验。
•

Figure 1.
1. Factor
Schematicsof the EADincell.
of improvement
LLOQ for peptide quantification with Zeno MRMHR compared to MRMHR. On average, a 5-fold improvement in图1. EAD碰撞池的示意图。
LLOQ
for peptide quantification was observed with Zeno MRMHR in comparison to standard MRMHR.
RUO-MKT-02-12834-ZH-A
RUO-MKT-02-12834-ZH-A

6 ADC 药物完整解决方案

p1

p1 p 1
本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。

Enhanced sensitivity for peptide quantification in a complex
表2. 质谱参数。
方法
matrix using
high-resolution
LC-MS/MS赖氨酸连接的ADC药物（曲妥珠单抗美坦新偶联样品制备：Featuring
the ZenoTOF
7600
LC-MS/MS system
物，T-DM1）用7.2
M盐酸胍和100mM Tris缓冲液（pH7.2）变性，参数

扫描模式(Scan mode)

MS

MS/MS

TOF-MS IDA dependent

然后加10 mM 二硫苏糖醇还原和30 mM碘乙酰胺进行烷基化。在极性(Polarity)
正离子模式
Eshani Nandita, Lei Xiong, Elliott Jones, Zoe Zhang, Kerstin
Pohl
37℃下用胰蛋白酶/ Lys-C酶消化16小时。
SCIEX, USA
喷雾气(Gas 1)
40 psi色谱条件： 4 μL（4 μg）的胰蛋白酶/ Lys-C消化液，采用ExionLC加热气(Gas 2)
40 psiHigh-resolution platforms, such as traditional time-of-flightAsignificant 5-fold improvementin LLOQ formm，1.7
peptide μm，130 Å，AD液相系统，通过CSH
C18色谱柱（2.1×100
气帘气(Curtaingas) sensitivity due to loss of ion transmission
30 psisystems, often lackinquantification was achieved using the ZenoTOF 7600 system
Waters）进行分离。流动相A为含0.1％甲酸的水溶液，流动相B是between
TOF pulses.
The Zeno trap controls the 350
ion ℃
beam fromfeaturing the Zeno trap. Compared with traditional time-of-flight
离子源温度(Source
temperature)
含0.1％甲酸的乙腈溶液。柱温保持在50 ℃，流速为300 μL/ min，
the collision cell which facilitates greater ion transmission to thesystems, the Zeno trap enables greater MS/MS sensitivity by离子喷雾电压(Ion spray voltage)
5200 V流动相梯度见表1。
TOF accelerator. Therefore, the duty cycle is improved to ≥90 %,enhancing the duty cycle. In addition, the versatility of TOF
去簇电压(Declustering
potential,DP)
20 V
which enhances overall MS/MS sensitivity.
MS/MS data allows for the capability of post-acquisition
表1. 肽图分析的色谱方法。
decisionsfor the selection of fragment ion(s) for MRMHR. For
碰撞能量(Collision energy)
8V
The ZenoTOF 7600 system offers an exceptional combination ofcases where multiple dominant fragment ions are generated
碰撞气(CADgas) sensitivity, and acquisition speed for7
时间（min）
流动相A[%]
流动相B[%]
mass
resolution,from the target peptide, the sum of XICs enabled greater
quantitative
analysis. It also
aids inion)
the potential for:5less ion path
最大候选离子(Maximum
初始
candidate
2.0 forsensitivity.
A 3-fold improvement98in LLOQ was observed
tuning, increased sensitivity with the Zeno trap, ability to change
peptides that leveraged the summing of multiple dominant强度阈值(Intensity threshold )100 cps
598
2.0
measuredfragments post-acquisition and improved
fragment ions when MS/MS ion current was dispersed.
离子电荷(Charge
states)2 to 10
reproducibility and accuracy.
690
10
Traditional workflows for quantitative bioanalyses, such as排除时间(Exclusion time )6 s after 2 occurrences
40
55
45
Key features of the ZenoTOF 7600 system for
immunological assays, have been displaced by LC-MS/MS
起始质荷比(Start mass)
100 m/z
150 m/z
44triple quadrupole mass
10 spectrometers. 90
analysis on
highlysensitive peptide quantification
终止质荷比(Stop
mass)
1,800
m/z
2,500
m/z
Immunoassays
often
lack
selectivity
andspecificity,
and
have
a
46
10
90
• Demonstration of a 5-fold improvement in LLOQ for peptide
limited linear dynamic range. While the triple quadrupole platform
电子KE(ElectronKE) the Zeno trap to accumulate
NA
eV
47
98
2.0
quantification using
ions7during
has been a key driver for most bioanalytical workflows, offering
each
TOF
pulse
for
enhanced
duty
cycle
电子束电流(Electron
beam current)NA4750 nA50
2.0
great sensitivity
and quantitative98performance, high-resolution
accurate mass
has increasingly
51 spectrometry (HRAMS)
1090 beenadopted for quantitative bioanalysis.1,2 With the inherent54
1090
advantage of greater selectivity with improved mass resolution,
98
2.0 7600
as well as55
the flexibility of TOF MS/MS
data, the ZenoTOF
system provides
excellent quantitative
performance in2.0
multiple
60.0
98
dimensions.

质谱条件： 采用SCIEX ZenoTOF™ 7600系统通过信息依赖采集• (ETC)
Reach enhanced sensitivity by summing NAof multiple highly
100abundant fragment ions with availability of TOF MS/MS data
Zeno 脉冲NA
ON• Ensure exceptional accuracy and precision for quantitative
累积时间
0.25
s
0.20
s
workflows using the ZenoTOF 7600 systembinsacquire,to sum process, and manage data4 on a single 4• TimeEasilyplatform using the SCIEX OS Software

数据处理： 采用Byos软件（Protein Metrics Inc.）进行数据解析。

（IDA）方法采集数据。EAD池的电子能量设置为7 eV。详细的方法
参数汇总在表2。

Figure 1. Factor of improvement in LLOQ for peptide quantification with Zeno MRMHR compared to MRMHR. On average, a 5-fold improvement in

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
RUO-MKT-02-12834-ZH-A
LLOQ for peptide quantification was observed with Zeno MRMHR incomparison to standard MRMHR.
RUO-MKT-02-12834-ZH-A

p1
p2

ADC 药物完整解决方案 7

The what, why and how

separation in combination with a fast scanning DDA method andEnhancedsensitivity for peptide
quantification
a complex
processing
using Protein Metrics Inc.in
software.
This
Ado-trastuzumab emtansine (T-DM1) was amongst the first ADC
是什么，为什么，怎么做偶联肽的分析
breakthroughtechnology realizes the dream of answering
therapeutics, receiving approval by the FDA in 2013 for thematrixusing high-resolution LC-MS/MS
complex questions in a routine and reproducible manner.
treatmentof human epidermal growth factor receptor 2 (HER2)曲妥珠单抗美坦新偶联物（T-DM1）是最早的ADC治疗药
该研究的重点是一个商品化的ADC药物：T-DM1的表征。DM1

positive, metastatic
breast cancer.
T-DM1
consists of asystem
Featuring
the ZenoTOF
7600
LC-MS/MS
物之一，于2013年获得FDA批准用于治疗人表皮生长因子受体2
和曲妥珠单抗之间的偶联反应具有靶向氨基的随机性。在曲妥珠单
Analysisof conjugated peptides

monoclonal antibody, trastuzumab, which is covalently
6，因此很容易结（HER2）阳性转移性乳腺癌。T-DM1是由单克隆抗体曲妥珠单抗 The
抗上的88个赖氨酸残基中，有40个是溶剂暴露的conjugatedto the cytotoxic
agent emtansine(DM1) Zoe
via a nonEshani
Nandita,
Lei Xiong,
Elliott Jones,
Zhang, Kerstinstudy
Pohlfocused on the characterization of a commercialized合。多种研究完整和亚基质量的方法已经应用于研究DAR，但是这和细胞毒药物美坦新（DM1）通过不可裂解连接子共价偶联而成ADC:
T-DM1.
The
conjugation
reaction
betweenDM1
andcleavable USA
linker (Figure 2). Combining target-specificity of a mAb
SCIEX,
trastuzumab
is of stochastic nature, targeting amines. Out of the些方法无法揭示精确的偶联位点。
（图2）。将单克隆抗体（mAb）的靶标特异性与细胞毒性药物
with the high potency of a cytotoxic drug takes advantage of the
88 lysine residues on trastuzumab, 40 are solvent exposed 6 andbest of two worlds, minimizing side effects.3 T-DM1 is linked to的高效率相结合，可充分利用两个方面的优势，最大限度地减少
High-resolution
platforms, suchas traditional time-of-flightAsignificant 5-fold improvement in LLOQ for peptide
本研究选择了与Zeno™
EAD相结合的DDA方法。采用这种
therefore
susceptible to conjugation.
Multiple approaches onamines,3 such as in the side chain of lysine residues of
副作用 。T-DM1是与氨基连接，如连接在曲妥珠单抗的赖氨酸残systems, often lack sensitivity due to loss of ion transmission inquantification
was achieved using the ZenoTOF 7600 system
方法，不仅可以执行常规的肽图分析，而且EAD可以在同一单
intactand subunit mass have been explored to study the DAR.
trastuzumab. Previous intact mass studies show that the
between TOF pulses. The Zeno trap controls the ion beam fromfeaturing
the Zeno trap. Compared with traditional time-of-flight
基的侧链中。先前的完整质量研究表明，T-DM1的平均DAR约为
These
approaches, however, cannot reveal theexact sites of theaverage DAR for T-DM1 is around 3.5.5,6 However there are 88
针分析中进行高级表征。此外，Zeno
EAD增强了碎片离子的5,6
the collision cell which facilitates greater ion transmission to thesystems,
the
Zeno
trap
enables
greater
MS/MSsensitivity
by
3.5.
conjugation.
lysine。但是曲妥珠单抗中有88个赖氨酸残基和4个N端基团，可residues and 4 N-terminal groups in trastuzumab, which
检
测能
力，从而正
确 鉴 定 了the低 duty丰 度 cycle
物质，
种方法允
使%,
用
TOF
accelerator.
Therefore,is 这
improved
to 许≥90
enhancing the duty cycle. In addition, theversatility of5 TOF
5。有效载荷的位点和结构
能会出现450万个以上的不同分子形式
could result in more than 4.5 million uniquemolecules. The site
公司.软件直接进行数据解析。这是针对ADC样品
Protein
MetricsHere,
a DDA
approachin combination
with Zeno EAD was
whichenhancesoverall
MS/MS sensitivity.
MS/MS data allows for the capability of post-acquisitionand structure of payload will directly affect drug efficacy and
将直接影响药物的功效和安全性，因此将其归类为关键质量属性
HR. Forchosen.
With
this approach, routine peptide mapping analyses [DM1]的SCIEX
decisionsfor theof fragment
MRMattributes
EAD技术的首次探索。图3展示了在偶联肽SCDK
safety, which
are selection
therefore classified
as ion(s)criticalfor
qualityThe
offers an
exceptional
combination of（CQA），并且需要在开发过程中进行全面表征和严格监控。当canbe ZenoTOF
performed,7600
whilesystem
EAD enables
advancedcharacterization
cases
multipledominant fragment
ions are generated
THTCPPCPAPELLGGPSVFLFPPKPK上观察到的碎裂模式的例子。在分(CQA)where
and requirecomprehensive
characterizationand tight
mass
resolution,
sensitivity,
and acquisition
speed for前，自下而上的方法是表征产品质量属性的选择方法，可同时进
in thesame,
single analysis.
Furthermore,
the detection
of
from
the
targetpeptide,
the
sum
of
XICs
enabledgreatermonitoring during development. Currently, bottom-up析中未观察到没有连接子和药物或其部分的肽，表明其完全偶联。
quantitative
analysis.
It alsoaidsidentificationin the potential
for:abundantless ion pathfragment
ions, andthus thecorrectof lowsensitivity.
A
3-foldimprovement
in
LLOQ
was
observedfor行鉴定和修饰位点的定位。LC-MS/
MS通常采用CID技术用于验证
approaches are the method of choice for the characterization of获得了此肽段高质量的MS/ EAD.
MS谱图，从而使该特定肽段的MS/ MS
tuning,increased
sensitivity
with
the
Zeno
trap,
ability
toforchange
species,isenhanced
by
Zeno
This
approachallowedpeptidesthat leveraged
summingof multiple
dominant
product quality
attributes,theenabling
the simultaneous偶联位点，因为每个DM1都会导大约957
amu的质量偏移。然而，
序列覆盖率达到96.6％。一个更占优势的碎片从m/z大于500的有效
fragments
andProtein
improved
themeasured
straight-forward
data post-acquisitioninterpretation using
Metrics Inc.
fragment
ionsandwhen
MS/MS ioncurrent was dispersed.
identification
localizationof modifications.
LC-MS/MS
采用CID碰撞技术除了导致肽段主链解离，还可能导致有效载荷药
reproducibility
accuracy. of SCIEX EAD technology forsoftware.
It is the andfirst exploration
载荷产生（请见图3中的标记）。观察到的有效载荷结构的主要裂
utilizing CID is commonly used to verify conjugation sites, as
物产生一系列小片段，例如m/z547.221、485.224和453.199离子，
an解位点是DM1的COO-C键，这种碎裂模式与先前利用CID技术产生的ADC sample. Figure 3 demonstrates an example of theTraditional
workflows for quantitative
bioanalyses, such aseach DM1 will lead to a ~957
amu mass shift. However, besides
5。虽然电子捕获解离（ECD）之类的碎裂这增加了谱图的复杂性fragmentation
pattern observed
on a conjugatedpeptide,Key
featuresof the ZenoTOF
7600
system for
immunologicalassays,have
beendisplacedby
LC-MS/MSthe dissociation of the peptide backbone, CID results in a series
一系列小碎片的数据不同5。较大分子量的药物碎片可以用作特征
SCDK[DM1]THTCPPCPAPELLGGPSVFLFPPKPK.
The peptide
analysison triple quadrupole
mass spectrometers.
highly sensitive peptide quantification
技术有望提供有关药物偶联肽的正交信息，但是其应用尚未得到
of small fragmentsfrom the payload
drug, such as m/z of碎片，以更具体地确认有效载荷的存在，并可以用来确认有效载荷without linker and drug or part of it was not observed in the
Immunoassays
often
selectivity
specificity,and have a547.221, 485.224,
andlack
453.199,
whichandincrease
the spectra
广泛探索。
• Demonstrationof aconjugation.
5-foldEAD技术用于增强的碎片离子检测，还
improvementin LLOQ
peptide的结构。此外，通过将Zeno
analysisindicating a fullHigh quality
MS/MSforspectra5 Although
limitedlineardynamicalternative
range. While
the triple quadrupole
complexity.
fragmentation
technology platformsuchquantification
using
the
Zeno
trap
to
accumulateions
duringwasachieved for the peptide, allowing for 96.6% MS/MS
可以很好地检测到来自肽主链的片段信息，从而提供有关肽段的分
SCIEXaZenoTOF™
7600系统，引入了一种强大的选择碎裂技术，has
been
key driverfor most bioanalytical
workflows,offering
as electron
capturedissociation(ECD) is expected
to provide
each
TOF
pulse
for
enhanced
duty
cycle
sequence
coverage for this particular peptide. One of the more
使科学家能够通过将液相分离技术结合DDA快速扫描方法获取的
子完整性的信息。由于酶的空间位阻，蛋白质上偶联药物的存在会
great
sensitivity
and quantitative
performance,high-resolution
orthogonal
information
on drug conjugatedpeptides,thedominant
fragments
wassensitivity
derived fromthe payloadwith an m/z• Reachenhancedby summingof multiple
highlyaccurate
mass(HRAMS)
has increasingly been
application
has spectrometry
not been
exploredextensively.
数据，且通过Protein
公司软件数据处理获得样品的深入图
导致样品制备酶解过程中的更多漏切位点。另外，赖氨酸残基和有Metrics
greaterthan 500fragment(see labelingin
Figure3). Theofmajorcleavageabundantionswithavailability
TOF
MS/MS
data
1,2
adoptedfor quantitative bioanalysis. With the inherent谱。这项突破性的技术实现了高重现性常规分析来解决复杂问题的效载荷之间的结合过程是随机反应。偶联的比率并不总是100％，site
for the payload structure was the COO-C bond ofWith the SCIEX
ZenoTOF
7600 system,a robustmass
alternative• observed
Ensure exceptional
accuracy and precision for quantitativeadvantageof greater
selectivity
with improved
resolution,
梦想。
the这导致了多样性和低丰度物质存在。当一个肽段中存在多个潜在连DM1. This fragmentation pattern is different from previous
fragmentation technique is introduced, enabling scientists to getworkflows using the ZenoTOF 7600 system
as well as the flexibility of TOF MS/MS data, the ZenoTOF 7600
publisheddata leveraging CID, which produced a series of small
接形式时，鉴定正确的连接位点可能是一个挑战。
an in-depth picture of their samples by using analytical-flow LC
system provides excellent quantitative performance in multiple•
Easily5 Larger
acquire,process,ofandon aassinglefragments.
fragments
themanage
drug candata
be useddimensions.
肽段ASQDVNTAVAWYQQKPGKAPK是这种具有挑战性的另一个
platform
using
the
SCIEX
OS
Softwaresignature fragments to confirm the existence of the payload
DM1linker例子（图4）。它包含一个漏切位点和一个脯氨酸相邻的N端赖氨
more
specifically, and can be leveraged to confirm the payload
ONH
O
structure.
Furthermore, fragments from the peptide backbone
酸，导致偶联位点的多种选择。但是，有了从EAD技术碎裂得到O
NH CH3
werealso well detected by applying Zeno EAD for enhancedHO丰富、高质量的MS/ MS质谱图，就可以实现药物定位的自动匹配HOOfragment ion detection, providing information on the molecular
CH3 CH3（图4A）。由于有效载荷靠近肽的C端，因此检测到的C离子比Z离
O
CH3
integrity of the peptide. The existence of conjugated drugs on the
O
H3C
N
O
子丰富（图4A），而未结合的肽显示出来自C端和N端的丰富片段
proteincan lead to more missed cleavages during enzymatic
CH3
N
S
CH3
O
N
O
CH3
（图4B）。众所周知因为电子捕获解离技术不会解离脯氨酸的N
samplepreparation, due to steric hinderance of the enzyme. In
addition,the conjugation process between lysine residues and 7。这
O端，我们还检测到了除了C15以外的从C3到C17的全系列C片段Cl
the提供了确凿的证据表明K15未与细胞毒药物偶联。此外，z4，z5和
payload is a stochastic reaction. The occupancy of theO
H3C
conjugation is not always 100%, which results in diverse and low
z7表明K18（而非K21）是药物偶联的正确位点。
abundant forms. When multiple potential positions exist in one
Figure 2. Schematics of cytotoxic payload and linker attached to mAb.
peptide, it can be a challenge to identify the correct site of
T-DM1 consists of DM1 (black) being attached to the mAb through an MCC
linker细胞毒药物有效载荷和连接子与mAb偶联的示意图。
(blue) targeting amine residues.
图2.
T-DM1由DM1 linkage. The peptide ASQDVNTAVAWYQQKPGKAPK is another（黑色），靶向连接氨基残基的MCC连接子（linker，蓝色）和单克隆抗体
HR
HR
Figure组成。 1. Factor of improvement in LLOQ for peptide quantification with Zeno MRM compared to MRM . On average, a 5-fold improvementp 3 in
LLOQ for peptide quantification was observed with Zeno MRMHR in comparison to standard MRMHR.

RUO-MKT-02-12834-ZH-A
RUO-MKT-02-12834-ZH-A

8 ADC 药物完整解决方案

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
p1
p3

Enhanced sensitivity for peptide quantification in a complex
matrix using high-resolution LC-MS/MS
Peptide

O

NH

+

H

HN

O

HO

Featuring the ZenoTOF 7600
LC-MS/MS system
O

CH3
CH3
N

NH

O

HO
Eshani Nandita, Lei Xiong,
Elliott
Jones, Zoe Zhang, Kerstin Pohl
H
O
CH CH
SCIEX, USA
3

H3C

Cl

3

O
CH

A significant 5-fold improvement3CHin3 LLOQ for peptide
Nquantification was achieved using the ZenoTOF 7600 systemOClfeaturing the Zeno trap. Comparedwith traditional time-of-flightO
systems, the Zeno trapHenables
greater MS/MS sensitivity by
3C
enhancing the duty cycle. In addition, the versatility of TOF
MS/MS data allows for the capability of post-acquisition
decisions for the selection of fragment ion(s) for MRMHR. For
cases where multiple dominant fragment ions are generated
from the target peptide, the sum of XICs enabled greater
sensitivity. A 3-fold improvement in LLOQ was observed for
peptides that leveraged the summing of multiple dominantfragment ions when MS/MS ion current was dispersed.

H3C

O

CH3 CH3

O

H3C

O

H

O

CH3

O

O

N
O

N
S
O

CH3

O

O

High-resolution platforms, such as traditional time-of-flightsystems, often lack sensitivity due to loss of ion transmission in
between TOF pulses. The Zeno trap controls the ion beam fromthe collision cell which facilitates greater
ion transmission to thePeptideO
HN
TOF accelerator. Therefore, theNHduty
cycle
isimproved
to
≥90
%,O
O
H
HO
H
which enhances overall MS/MS Osensitivity.
O+

CH3 CH3

O

H3C

CH3

O

N
The ZenoTOF 7600 system offers
anO exceptional
combination
of
N
S
CH
CH
O
CHspeed for O
mass resolution, sensitivity, andN acquisitionO
quantitative analysis. It also aidsin the potential for: less ion pathClO
tuning, increased sensitivity
H C with the Zeno trap, ability to change
measured fragments post-acquisition and improved
reproducibility and accuracy.
3

3

3

3

Traditional workflows for quantitative bioanalyses, such as
Key features of the ZenoTOF 7600 system for
immunological assays, have been displaced by LC-MS/MS
analysis on triple quadrupole mass spectrometers.
highly sensitive peptide quantification
Immunoassays often lack selectivity and specificity, and have a• Demonstration of a 5-fold improvement in LLOQ for peptide
limited 3.
lineardynamic range.
While
triple quadrupole
platform
FigureFragmentation
data fromthetheconjugatedpeptide SCDK[DM1]THTCPPCPAPELLGGPSVFLFPPKPK(z = +4) using Zeno EAD. Full scanquantification using the Zeno trap to accumulate ions during
MS/MS
data
conjugatedwith assigned workflows,ions from theoffering
peptide backbone, as well as the fragment from the payload.
has beena ofkey
driver forpeptide
most bioanalytical图3. 应用Zeno EAD得到的偶联肽SCDK [DM1] THTCPPCPAPELLGGPSVFLFPPKPK（z = + each4）的碎片数据。来自肽段主链指定偶联肽段离子的全扫描MS/ MS
TOF pulse for enhanced duty cycle
great
sensitivity and quantitative performance, high-resolution
数据，以及有效载荷中的碎离子信息。
• Reach enhanced
bywas
summingof multiple
highly4A).
examplethis spectrometry
type of challenging
peptide4). It been
contains
counterpart
linked tosensitivity
the peptide
also observed(Figureaccurate ofmass(HRAMS)has (Figure
increasingly
abundant
fragmentions
withavailability
of
TOF
MS/MS
data
a
miscleavage
site
and
a
lysineN-terminal
toaproline,leading
Unambiguous
characterization,
includingtheidentification
and
1,2
adopted for quantitative bioanalysis. With the inherent
to multiple options
forselectivity
the site ofwith
conjugation.
with thelocalizationof conjugatedpeptidesa single DDA
run, was• Ensure exceptional
accuracy
andinprecision
for quantitativeadvantageof greater
improvedHowever,mass resolution,
这个结果进一步证明了一个事实，即偶联的赖氨酸不能被胰结论rich,high-quality
MS/MSofspectra
derivedfromtheEAD,
anachieved
withusingthe SCIEX
ZenoTOF
7600
system and Zeno EAD.
workflows
the ZenoTOF
7600
systemaswell
as theflexibility
TOF MS/MS
data,
ZenoTOF7600
5，但K21作为连接位点除外。图4A谱图中最主要的离蛋白酶切割
automatic
assignment
of
the
drug
localization
wasachieved
This is an example of how ADC analysis, which was previously
system provides excellent quantitative performance in multiple
acquire, process, and manage data on a single
通过EAD的新型碎裂技术实现了具有多个潜在位点的多肽•• Easily(FigureBecause the payload is close to the C-terminus ofthought a challenge by LC-MS/MS, can be simplified by Zeno子（m /4A).
z 547.221）可以与有效载荷的解离联系在一起，这个同位
dimensions.
platformusing the SCIEX OS Software中药物偶联的准确定位thepeptide, more abundant c ions are detected than z ionsEAD.
素模式也支持这一发现，因为它适合包含卤素元素（Cl）的化合(Figure4A), while the unconjugated peptide shows abundant• 与传统的MS / MS分析相比，EAD技术获得更丰富的MS/MS物。同时还观察到与肽连接的相关对应物（图4A）。使用SCIEX
fragments from both the C-terminus and N-terminus (Figure 4B).
信息。应用Zeno EAD技术，即使对于中等强度或极低强度
ZenoTOF 7600系统中的Zeno EAD技术可以实现在单次DDA运行中鉴
A full series of c fragments from c3 to c17 was detected, except
的前体离子（例如低丰度的偶联肽），也能获得令人信服
定和定位偶联肽的精确表征。以前曾被认为ADC表征是LC-MS / MSfor c15, since it is well known that electron capture dissociation
的二级碎片和出色的数据质量
的一个挑战，这是Zeno
EAD如何简化ADC分析的示例。
techniques
do not dissociate
the N-terminal side of prolines.7• SCIEX ZenoTOF™ 7600系统强大、高重现性且易于使用的多This provides solid evidence that K15 is not linked to the drug. In
addition, z4, z5 and z7 show that K18, not K21, is the correct siteof drug conjugation.

重碎裂技术，使用户能够以简单的方式解决具有挑战性的分析问题

• 使用Protein Metrics 公司的软件进行自动数据处理，可实This is further supported by the fact that conjugated lysines are
not cleavable with trypsin5, excluding K21 as the site of
现复杂生物治疗药物的可重现、常规和高级表征
attachment. The most dominant species in the spectrum in
本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
RUO-MKT-02-12834-ZH-A
Figure4A (m/z 547.221) can be linked to the dissociation of the
payload.
The
isotopepatternsupports
thisfindingsince
it fits a with Zeno MRMHR compared to MRMHR. On average, a 5-fold improvement in
Figure 1. Factor of improvement in LLOQ for peptide quantificationHR
LLOQ
for peptide
quantification
was observedwith Zeno
in comparison to standard MRMHR.
compoundcontaining
a halogenelement (Cl).
The MRM
associated
RUO-MKT-02-12834-ZH-A

p1
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4

ADC 药物完整解决方案 9

Enhanced sensitivity for peptide quantification in a complex
matrix using high-resolution LC-MS/MS
O

NH

O

NH

O
Featuring the ZenoTOF
7600
LC-MS/MS system
HO
O

H

O

O

HO

H

O

CH3 CH3O

H C

3CH3 CH3 Jones, Zoe Zhang, Kerstin Pohl O
Eshani Nandita, Lei Xiong, Elliott
CH3
CH3
O
SCIEX, USAH3C
N
O

CH3
CH3
N in LLOQ for peptide
A significant 5-fold improvement

quantification was achieved usingO the ZenoTOF 7600 system H3C
Clfeaturing the Zeno trap. Comparedwith traditional time-of-flightO
H3C
systems, the Zeno trap
enables greater MS/MS sensitivity byenhancing the duty cycle. In addition, the versatility of TOF
MS/MS data allows for the capability of post-acquisition
decisions for the selection of fragment ion(s) for MRMHR. For
cases where multiple dominant fragment ions are generated
from the target peptide, the sum of XICs enabled greater
sensitivity. A 3-fold improvement in LLOQ was observed for
peptides that leveraged the summing of multiple dominantfragment ions when MS/MS ion current was dispersed.

A

Traditional workflows for quantitative bioanalyses, such as
immunological assays, have been displaced by LC-MS/MS
analysis on triple quadrupole mass spectrometers.
Immunoassays often lack selectivity and specificity, and have alimited linear dynamic range. While the triple quadrupole platform
has been a key driver for most bioanalytical workflows, offering
great sensitivity and quantitative performance, high-resolution
accurate mass spectrometry (HRAMS) has increasingly been
adopted for quantitative bioanalysis.1,2 With the inherent
advantage of greater selectivity with improved mass resolution,
as well as the flexibility of TOF MS/MS data, the ZenoTOF 7600
system provides excellent quantitative performance in multiple
dimensions.

B

Peptide

NH

O
CH3

O

N
CH3

N
S
O

O

O

Cl
High-resolution
platforms, such as traditional time-of-flightsystems, often lack sensitivity due to loss of ion transmission in
between TOF pulses. The Zeno trap controls the ion beam fromthe collision cell which facilitates greater ion transmission to the
TOF accelerator. Therefore, the duty cycle is
O improved to ≥90 %,which enhances overall MS/MS sensitivity.
NH

O

PeptideThe ZenoTOF 7600 system offers an exceptional combination ofCH3Omass resolution,
sensitivity, and acquisition
speed forN
HO
N
quantitative analysis.
It also Saids in the potential for: less ion pathOO
CH3tuning, increased
sensitivity with the Zeno trap, ability to change
measured fragments post-acquisition and improved
reproducibility and accuracy.

Key features of the ZenoTOF 7600 system forhighly sensitive peptide quantification• Demonstration of a 5-fold improvement in LLOQ for peptide
quantification using the Zeno trap to accumulate ions during
each TOF pulse for enhanced duty cycle• Reach enhanced sensitivity by summing of multiple highly
abundant fragment ions with availability of TOF MS/MS data
• Ensure exceptional accuracy and precision for quantitative
workflows using the ZenoTOF 7600 system• Easily acquire, process, and manage data on a single
platform using the SCIEX OS Software

Figure 4. Fragmentation data from the conjugated/naked peptide ASQDVNTAVAWYQQKPGK[DM1]APK (z = +3) using Zeno EAD. A: Full scan
MS/MS data of conjugated peptide with assigned fragment ions from the peptide backbone, as well as the fragments from the payload. B: Full scan
应用Zeno图4. data
EAD得到的来自偶联/非偶联肽ASQDVNTAVAWYQQKPGK
[DM1]
APK（z
3）的碎片的数据。
A：来自肽段主链指定偶联肽段离子的全扫MS/MSof nakedpeptide with assigned ions from the peptide backbone.
Thelinker =is +depictedin blue, the DM1
drug in black
描MS / MS数据，以及有效载荷中的碎离子信息。 B：来自肽段主链指定非偶联肽的全扫描MS / MS数据。 连接子显示为蓝色，DM1药物显示为黑色

Figure 1. Factor of improvement in LLOQ for peptide quantification with Zeno MRMHR compared to MRMHR. On average, a 5-fold improvement
inp5

RUO-MKT-02-12834-ZH-A
LLOQ for peptide quantification was observed with Zeno MRMHR本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
in comparison to standard MRMHR.
RUO-MKT-02-12834-ZH-A

10 ADC 药物完整解决方案p1p5Enhanced sensitivity for peptide quantification in a complex
参考文献
matrix using high-resolution LC-MS/MS
1. Baba T et al. (2015) Electron capture dissociation in a branched

Featuring
the ZenoTOF
LC-MS/MS
systemradio-frequency
ion trap, Anal7600
Chem, 87,785-792.
2. Baba TNandita,
et al. (2020)Lei
Dissociation
biomolecules
an intense
EshaniXiong, ofElliott
Jones,byZoe
Zhang, Kerstin Pohl
low-energy
SCIEX,
USAelectron beam in a high sensitivity time-of-flight mass
spectrometer. Submitted to JASMS.
A significant 5-fold improvement in LLOQ for peptide
3. Beck, Alain was
et al. achieved(2010) Strategies
thesystem
next
quantification
using andthe challenges
ZenoTOF for
7600

generationof therapeutic
antibodies.
Nattraditional
Rev Immunol
10(5):345-52.
featuring
the Zeno
trap. Comparedwithtime-of-flightsystems, the Zeno trap enables greater MS/MS sensitivity by
4. Botzanowski, Thomas et al. (2017) Insights from native
enhancing the duty cycle. In addition, the versatility of TOF
mass spectrometry approaches for top- and middle- level
MS/MS data allows for the capability of post-acquisition
characterizationof site-specificantibody-drug
Mabs,
decisionsfor the selectionof fragmention(s) forconjugates.
MRMHR. For
9(5):801-811.
cases where multiple dominant fragment ions are generated
from the target peptide, the sum of XICs enabled greater
5. Chen, Liuxi et al. (2016) In-depth structural characterization ofsensitivity. A 3-fold improvement in LLOQ was observed for
Kadcyla (ado-trastuzumab emtansine) and its biosimilar candidate.
peptides that leveraged the summing of multiple dominantMabs, ions8(7): 1210-1223.
fragment
when MS/MS ion current was dispersed.
6. Harris Reedet al. (2015)
Native mass bioanalyses,
spectrometry andTraditional
workflowsfor quantitative
suchionasmobility
characterizationof
trastuzumab
emtansine,alysine-linked
immunological assays, have been displaced by LC-MS/MS
analysison triple
quadrupole
massSci
spectrometers.
antibody
drug conjugate
Protein24(8):1210-1223.
Immunoassays often lack selectivity and specificity, and have a
7. Fort, Kyle et al (2018) Exploring ECD on a benchtop Q Exactive
limited linear dynamic range. While the triple quadrupole platform
spectrometer.
Proteome Resworkflows,
17(2) :926-933.
has orbitrap
been a mass
key driverfor mostJbioanalyticaloffering
great
sensitivity
andquantitative
performance,high-resolution
The SCIEX clinical diagnostic portfolio is For In Vitro Diagnostic Use.
accurate mass spectrometry (HRAMS) has increasingly been
Rx Only. Product(s) not available in all countries. For information
adopted for quantitative bioanalysis.1,2 With the inherent
on availability, please contact your local sales representative or
advantage of greater selectivity with improved mass resolution,referasto thehttps://sciex.com/diagnostics.
All other
are7600
For
as well
flexibility of TOF MS/MS data,
theproducts
ZenoTOFResearch
Use
Only.
Not
for
use
in
Diagnostic
Procedures.
system provides excellent quantitative performance in multiple
dimensions.

High-resolution platforms, such as traditional time-of-flightsystems, often lack sensitivity due to loss of ion transmission in
between TOF pulses. The Zeno trap controls the ion beam fromthe collision cell which facilitates greater ion transmission to the
TOF accelerator. Therefore, the duty cycle is improved to ≥90 %,which enhances overall MS/MS sensitivity.
The ZenoTOF 7600 system offers an exceptional combination ofmass resolution, sensitivity, and acquisition speed forquantitative analysis. It also aids in the potential for: less ion pathtuning, increased sensitivity with the Zeno trap, ability to change
measured fragments post-acquisition and improved
reproducibility and accuracy.

Key features of the ZenoTOF 7600 system forhighly sensitive peptide quantification• Demonstration of a 5-fold improvement in LLOQ for peptide
quantification using the Zeno trap to accumulate ions during
each TOF pulse for enhanced duty cycle• Reach enhanced sensitivity by summing of multiple highly
abundant fragment ions with availability of TOF MS/MS data
• Ensure exceptional accuracy and precision for quantitative
workflows using the ZenoTOF 7600 system• Easily acquire, process, and manage data on a single
platform using the SCIEX OS Software

SCIEX临床诊断产品线仅用于体外诊断。仅凭处方销售。这些产品并非在所有国家地区都提供销售。获取有关具体可用信息，请联系当地销售代表或查阅https://sciex.com.cn/diagnostics 。
所有其他产品仅用于研究。不用于临床诊断。本文提及的商标和/或注册商标，也包括相关的标识、标志的所有权，归属于AB Sciex Pte. Ltd. 或在美国和/或某些其他国家地区的各权利
所有人。
© 2021 DH Tech. Dev. Pte. Ltd. RUO-MKT-02-12834-ZH-A

SCIEX中国
北京分公司

上海公司及中国区应用支持中心

广州分公司

北京市朝阳区酒仙桥中路24号院
上海市长宁区福泉北路518号
Figure 1. Factor of improvement in LLOQ
for peptide quantification with Zeno
MRMHR compared to MRMHR. 广州市天河区珠江西路15号
On average, a 5-fold improvement in
珠江城1907室
1号楼5层 with Zeno MRMHR in comparison
1座502室LLOQ for peptide quantification was observed
to standard MRMHR.

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ADC 药物完整解决方案 11

For research use only. Not for use in diagnostics procedures.

Simple and fast-tracking of DAR distribution using intact
multiple attribute methodology (Intact MAM)
DAR determination for cystine-linked ADC (Vorsetuzumab) featuring SCIEX OS software
Kazuko Inoue1, Ryo Yokoyama2, Chie Inagaki2, Greg Roman3, and Zoe Zhang3
1

Eisai Co.,Ltd., Japan 2SCIEX Japan, 3SCIEX USAThis technical note highlights the utility of SCIEX's flexible
solution for intact MAM within SCIEX OS software. This example
shows the drug antibody ratio (DAR) monitoring for antibodydrug conjugates (ADCs). The streamlined MAM data flow fromdata acquisition to data processing in compliance-ready SCIEXOS software will also be demonstrated.
ADCs are biotherapeutics composed of small molecule drugs
conjugated to an antibody scaffold. An ADC molecule consists ofthree components: monoclonal antibody, linker, and cytotoxic
drug. As a result, ADCs have a very complex molecular structurewith macromolecules and small molecule characteristics. The
information about the average DAR is essential for assessing
ADC qualities. For instance, a low DAR could reduce the efficacy
of a drug. Therefore, DAR values are one of the critical quality
attributes (CQAs) for the therapeutic index of ADCs.

monkey and mouse plasma. A time course study was
performed to showcase the streamlined LC-MS based
workflow, combining SCIEX high-resolution QTOF mass
spectrometry with user-friendly data analysis software.

Key features of SCIEX's intact MAM solution
for DAR monitoring• High throughput: DAR monitoring for a large sample set• Intuitive software: Powerful product attribute definition,
tracking, and quantification with flexible custom calculations
for CQAs based on specific user needs.
• Streamlined solution: Complete software solution for
acquisition and quantitative tracking of DAR changes.
• Compliance-ready: DAR quantification and monitoring are
performed in compliance-ready SCIEX OS software.

In this technical note, we demonstrate SCIEX's intact MAM
workflow in DAR determination of a cystine-linked ADC from

Figure 1. A streamlined workflow for DAR monitoring using SCIEX intact MAM

RUO-MKT-02-15063

12 ADC 药物完整解决方案

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
p1

For research use only. Not for use in diagnostics procedures.

Methods

B consisting of 0.1% formic acid in water and 0.1% FA in
acetonitrile, respectively.

Sample preparation:
Vorsetuzumab Mc-VC-PAB-MMAE ADC was purchased from
Creative Biolab Inc., (Shirley, NY). Human, monkey, mouseplasma, and PBS samples were incubated with vorsetuzumab
MMAE ADC (30 µL, 0.1 mg/mL) for time intervals of 0, 24, 48,
and 72 hrs. These samples were diluted with HBS-EP buffer(260 µL), followed by the addition of biotinylated human CD27
ligand as antigen of vorsetuzumab (10µL of 0.2 mg/mL aqueous
solution). The mixture was incubated at room temperature for 15
minutes to generate conjugates with biotinylated human CD27
and vorsetuzumab MMAE ADC. Subsequently, streptavidin
immobilized magnetic beads (Dynabeads M-280 streptavidin)
were suspended into the resulting mixture and incubated at room
temperature for 15 minutes with continuous mixing. Afterincubation, the resulting magnetic beads collected from the
mixture were washed with HBS-EP buffer twice.
Table 2. Chromatography for intact and subunit analysis.
Time [min]Mobile Phase A [%]Mobile Phase B [%]Mass spectrometry:
LC-MS data were acquired using the TripleTOF 6600+ system.
The key TOF parameters are listed in Table 2.
Table 2. MS parameters for subunit and intact mass analysis.
Parameter

Setting

Scan mode

TOF-MS

Polarity

Positive

Intact protein mode

ON

Gas 1

60 psiGas 2

60 psi

Curtain gas

30 psi

Temperature

450 oCIon spray voltage

5500 VCAD gas680

Initial80

20Time bins to sum

8

50

50

Accumulation time

9

10

90

Start mass

500 m/z

12

10

90

Stop mass

4,000 m/z
120 V
10 V

12.1

80

20

Declustering potential

17

80

20

Collision energy

The biotinylated vorsetuzumab MMAE ADC was released fromthe streptavidin magnetic beads using a pre-mixed PNGaseF
reaction solution (1uL of rapid PNGaseF diluted with 37°C
prewarmed MilliQ water). Then it was incubated at 37°C for 60
minutes to conduct on-bead deglycosylation. After collecting themagnetic beads from the rapid PNGaseF reaction mixture, thebeads were washed with HBS-EP buffer. The vorsetuzumab
MMAE ADC captured on the beads was eluted with 50 uL ofacetonitrile/MilliQ water/formic acid (10/90/0.01 V/V/V). The
eluates were treated with 10 mM TCEP at a final concentrationfor 30 minutes at 37°C to reduce the intra-chain disulfide bond of
the ADC. The reduced ADC sample was injected into a
TripleTOF 6600+ system with a UPLC system.

0.5 sData processing:
Reconstruction and average DAR calculation of cystine-linked
ADCs were performed in compliance-ready SCIEX OS software,version 2.2.

Chromatography:
Subunits were separated using an ACQUITY BEH protein C4 1.7
um, 2.1 x 50 mm analytical column (Waters), which was kept at
80°C in the column oven of a Nexera 30A (Shimadzu) UHPLC
system. Table 1 shows the LC gradient used for subunit
separation at a flow rate of 0.3 mL/min with mobile phases A andRUO-MKT-02-15063

p2
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ADC 药物完整解决方案 13

For research use only. Not for use in diagnostics procedures.

Human_0h

Human_24h

Human_48h

Human_72h

Human_24h

Human_48h

Human_72h'UXJB'UXJB'UXJB'UXJB

Human_0h'UXJB'UXJB

Figure 2. Reconstructed spectrum from Cys-linked ADC incubated in human plasma. Top: Heavy chain. Bottom: light chain. Peaks corresponding
to the molecule carrying a higher number of drug payloads decreased throughout incubation time.

Data collection for DAR calculations
Compared to traditional antibodies, ADCs go through morepotential biotransformations with a payload in vivo and in vitro,
which can change DAR distribution significantly. 1 The change ofin vivo DAR distribution of trastuzumab emtansine was recently

RUO-MKT-02-15063

14 ADC 药物完整解决方案reported in literature .2,3 DAR is one of the CQAs that must be
closely monitored during ADC manufacture and storage as it canaffect drug efficacy and safety. In this work, a time course
stability study was designed to investigate the changes of DAR
distribution from incubation of vorsetuzumab in different plasma.
Data were collected, evaluated, and reconstructed using SCIEX

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
p3

For research use only. Not for use in diagnostics procedures.

Figure 3. Parameter definition for each subunit of vorsetuzumab MMAE ADC in SCIEX OS software. SCIEX OS software allows
for convenient deconvolution of the respective subunits related to the ADC.
OS software over the time course study for vorsetuzumab MMAE
ADC in human plasma, as shown in Figure 2. The ADC peaks
with a different number of drug payloads were labeled in thereconstructed spectra. The dominant species observed for theheavy chain carried 0-3 drug molecules, while the light chain was
mainly conjugated with 0 or 1 drug payload. The DAR
distributions of both chains were shifted toward the lower
payloads throughout incubation.

automated DAR calculation, as shown in Figure 4. The
percentage of peak area for each payload from each subunit wascalculated using the formula displayed in Figure 4A. As
vorsetuzumab MMAE ADC has two heavy chains and two lightchains, the total ratio of each payload needs to be multiplied by
2, as shown in Figure 4B. Finally, the average DAR was
calculated by adding all the ratios from each payload (Figure
4C).

Intact MAM for automated DAR monitoring
Method generation for DAR calculation
SCIEX OS software offers a simple and streamlined intact MAM
solution4. Attributes can be easily defined, and integration
parameters for individual components can be adjusted toachieve accurate quantitation in SCIEX OS software, as shownin the previous technical note. 4 Figure 3 displays reconstruction
and peak integration parameters for developing intact MAM.
Using the advanced reconstruction parameters, the user candefine the number of spectra to average, S/N threshold,resolution, and mass range for the assay. The attribute level canbe calculated within each attribute group using flexible custom
calculations. In this technical note, a method was built for

RUO-MKT-02-15063

Vorsetuzumab MMAE ADC samples incubated with plasma were
purified through immunocapture. On-bead reduction anddeglycosylation were performed to reduce sample complexity,
and a TripleTOF 6600+ system was employed to measure the
ADC's heavy chain and light chain. Molecular mass andquantitative information of each subunit were obtained using
SCIEX's intact MAM solution. The average DAR of the entire
ADC can be calculated by building a custom formula within
SCIEX OS software, as illustrated in Figure 4. Detailed resulttables can be obtained and customized for accelerating data
review (Figure 5). The table includes all the quantification resultsfrom intact MAM workflow, such as the ADC's absolute and本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
p4

ADC 药物完整解决方案 15

For research use only. Not for use in diagnostics procedures.

(B)

(A)

Drug_0
Drug_1
Drug_2
Drug_3(C)

Figure 4. Custom formula for DAR calculation. (A) The calculation for the % area of each payload. Drug_1 is shown as an example. (B) The
calculation for the DAR of each payload. Drug_0, Drug_1, Drug_2, and Drug_3 on the heavy chain are shown as an example. (C) The calculation for
the average DAR of the entire ADC.

(A) Result table

PBS
Mouse (Control)
Human

Monkey(B) Metric plot

TIC

MS

Deconvolution

Figure 5. Result table and metric plot of DAR in SCIEX OS software. (A) Result table showing reconstructed peak area and DAR calculated
customized formula. TIC, MS, and reconstructed spectrum were displayed on the same page. (B) Metric plots allow for easy visualization of DAR
distribution changes.

RUO-MKT-02-15063

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
p5

16 ADC 药物完整解决方案For research use only. Not for use in diagnostics procedures.

percentage peak areas with a different number of drugs andConclusions• SCIEX's flexible solution for intact MAM within SCIEX OS
software offers a powerful workflow for ADC analysis byproviding a streamlined and compliant software package,from data acquisition through data analysis

Average DAR (Intact ADC)
4.5
4.0
3.5

• The combination of SCIEX high resolution mass
spectrometry and streamlined software presents a cuttingedge solution for attribute monitoring in processdevelopment, enabling faster decision making

3.0
2.5
2.0
1.5

• The TripleTOF 6600+ system and SCIEX OS software
provide an excellent DAR monitoring tool.

1.0
0.5
0.0

1

2

3

4

5

6

7

8

9

References

10 11 12 13 14 15 161; Human_0hr、2; Human_24hr 3; Human_48hr 4; Human_72hr
5; Monkey_0hr、6; Monkey _24hr 、 7; Monkey _48hr 、 8; Monkey _72hr
9; Mouse_0hr、10; Mouse _24hr 、 11; Mouse _48hr 、 12; Mouse _72hr
13; PBS_0hr、14; PBS _24hr 、 15; PBS _48hr 、 16; PBS _72hr

1.
Simone, chadt et al. (2019) Are Biotransformation
Studies of therapeutic proteins needed? Scientificconsiderations and technical challenges. Drug Metab
Dispos. 2019 Dec;47(12):1443-1456.
2.
Keyang, Xu et al (2013) Characterization of the drug-toantibody ratio distribution for antibody–drug conjugates inplasma/serum. Bioanalysis. 2013 May;5(9):1057-71.

Figure 6. Average DAR of Intact ADC and DAR of each payload results. A;
Average DAR monitoring from each sample. B; DAR monitoring of eachpayload result.

3.
Kaur, Surinder (2013) Bioanalytical assay strategies forthe development of antibody–drug conjugate biotherapeutics
Bioanalysis. 2013 Jan;5(2):201-26

average DAR (Figure 5A). The results can be sorted by sample,
targeted attribute, or modification event.
A metric plot can be created to visualize the change of each
attribute (Figure 5B), offering a quick way of detecting changes
in the molecule. The metric plot in Figure 5B clearly shows thatthe DAR distribution decreased throughout incubation ofvorsetuzumab MMAE ADC in different plasma (human, monkey,
and mouse). By comparison, the control samples incubated with
PBS did not show this trend, as the DAR remained consistentover 3 days of incubation. The change in DAR distribution canalso be visualized using a bar graph, as displayed in Figure 6.
These results suggested that vorsetuzumab MMAE ADC
underwent a potential biotransformation process of the payload
in plasma, leading to a decrease in the average DAR value.

4.

Compliant Attribute Monitoring for Biopharmaceutical ProductQuality Attributes Employing Intact Mass Analysis. SCIEX
technical note, RUO-MKT-02-11314-A.

The SCIEX clinical diagnostic portfolio is For In Vitro Diagnostic Use. Rx Only. Product(s) not available in all countries. For information on availability, please contact your local sales
representative or refer to https://sciex.com/diagnostics. All other products are For Research Use Only. Not for use in Diagnostic Procedures.
Trademarks and/or registered trademarks mentioned herein, including associated logos, are the property of AB Sciex Pte. Ltd. or their respective owners in the United States and/or certain
other countries (see www.sciex.com/trademarks).
© 2022 DH Tech. Dev. Pte. Ltd. RUO-MKT-02-15063

RUO-MKT-02-15063

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
p6

ADC 药物完整解决方案 17

Analysis of non-deglycosylated antibody-drug-conjugates by
TripleTOF® high resolution quadrupole-time-of-flight
instrument and effective reconstruction software
1

2

2

2

1

3

Milla Neffling , Bruno Genet , David Bugnazet , Eric Lacassie , Jason Causon , Justin Blethrow , Eric Johansen
13

2

SCIEX, Warrington, UK; Sanofi-Vitry Biologics SCP/Analytics/Characterization Sanofi-Aventis recherché &development Centre de3

Recherche de Vitry-Alfortville, Vitry-Sur-Seine, France; SCIEX, Redwood Shores, CA

Introduction
Antibody drug conjugates (ADC’s) are an important class ofbiotherapeutic compounds delivering a targeted, usuallycytotoxic drug selectively to the target cell. Lysine –linked
ADC’s can be very complex with multiple payloads conjugatedto the same biotherapeutic protein. The drug-antibody-ratio(DAR) needs to be characterized and monitored from batch-tobatch, to ensure safety and efficacy of the biotherapeutic.
Analysis time bottleneck in a conventional set-up would be thedeglycosylation step, which is usually required to eliminate
complexity in the sample. Here we present data acquired froma non-treated ADC with multiple payloads, and N-glycosylation
intact. Despite the complexity, reproducible analysis andprocessing was achieved. Differential ion mobility was used tofurther reduce the complexity and the backgroundinterferences.
Intact protein analysis for antibodies and ADCs is a rapid
method for global observations of changes to product from lotto-lot. Intact protein analysis is used to control mass ofcomplete protein product, (i.e. to discover possible clipping or
truncation), glycosylation heterogeneity, and to assess the
DAR of ADCs. These aspects should be vigorously controlled,
as biological therapeutics are produced in cells, andglycosylation patterns may vary due to changes in production.
The drug conjugation efficiency should also be assessed toensure safety and efficacy.
Reproducible, reliable and trustworthy data and processing are
the most important factors to assess the information from
intact protein analysis.
Lysine linked ADC’s are a common class of ADCs, with a highnumber of possible conjugation sites in the structure (IgG
amino acids sequence typically has more than 70 Lys –residues). Monitoring and characterization of the DAR calls forstrong data quality and confidence in data processing, with
automated calculations to allow fast decision making on thequality of the product. The tools allowing for good quality
spectraandautomatedbatch
processinginthebiopharmaceutical industry allow not only for the sample

RUO-MKT-02-2814-A

18 ADC 药物完整解决方案

preparation time to be reduced, but also to allow the responsetime to the synthetic chemist department to be reduced. The
speed of the analysis would also allow for the opportunity to lookat more synthesis options in the same timeframe, and therefore
faster synthesis method optimization.
Total analysis time depends on the sample preparation. The
simpler this can be made, the more samples can be analyzed in areasonable time frame. To reduce the sample preparation timehigh data quality, and orthogonal separation methods can beused to evaluate product complexity. Differential ion mobility has
been shown to effectively reduce the data complexity and to
separate features of different sizes and mobility. Here, theinterferences in the spectra were separated from the ADC ofinterest.

Figure 1 (A) comparison of raw spectra from the chromatographic system
1: reverse phase separation. In Blue: spectra with SelexION® technology
cell optimized for the transmission of ADC ions. In Pink: no differential
mobility separation applied. (B) Reconstructed spectrum of the SelexION
technology-acquired data.

Experimental
Lysine-linked ADC was kindly provided by Sanofi-Vitry (France).
The amino acid sequence and drug moiety structure areproprietary information.
The ADC sample was analyzed using TripleTOF® 5600 coupled
to Shimadzu Nexera UHPLC, by two different chromatographic
set-ups:p1

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1.

Agilent SB C-8 (5μm 1mmx75mm). Gradient with 20 min
run time, 200 μL/min flow rate. Solvents A: 0.1% formic
acid (FA) water; B: 0.1% FA Acetonitrile.

iterations employed (Figure 4). Batch submission in
BioPharmaView
softwaredemonstrated
high
level
of
reproducibility in both, analysis and reconstruction processing.

2.

Waters Acquity UPLC BEH 200 SEC (1,7μm
4,6mmx300mm). Gradient with 35 min run time, 300-400
μL/min flow rate. Solvents A: Ammonium formate 25mM,
1% FA; B: Acetonitrile. Column temperature 60 ºC in bothcases. 5-10μg of ADC loaded on column per run.

Non-deglycosylated ADC was identified to carry up to 7 payloads
per antibody (Figures 4 and 5). The glycosylation pattern showed
three glycan structures to be the most significant ones
(combinations of G0F-G0F, GOF-G1F, G1F-G1F, G1F-G2F, G2FG2F; Figure 4.). The raw data was complex, carrying
heterogeneity from both, the drug conjugation and theglycosylation. The charge state envelope showed highest
intensity signal for charge states from about +40 to +65 inchromatographic system 1, and +35 to +60 in thechromatographic system 2.

SelexION DMS technology was used in chromatographic
system 1. Data was acquired with a TOF-MS scan over themass range (m/z = 400-4500).Peak reconstruction was
performed using BioPharmaView™ software.

Figure 2 Optimization of the SelexION technology compensation
voltages (CoV) on a monoclonal antibody (mAb). Panes have beenmarked with the applied CoV (here shown from -6 to 9). The separation
voltage was optimized to 3500V. For large proteins such as mAbs, thetransmission optimum was found with negative CoVs; for the smaller
proteins (Ab light chain) and interferences, the transmission optimum
was found with positive CoVs.

Figure 3 Chromatographic system 2 (SEC separation) Total ion
Chromatogram (3A) and the obtained ADC raw spectrum (3B) and azoom-in of the spectrum (3C) with the charge states highlighted

Results
In the initial (higher through-put) RP (C-8) set-up with SelexION
differential mobility separation technology was used to reduce
background interference to allow for cleaner spectra and better
processing (Figure 1). The signal from the light chain (MWaround 25kDa) and the formulation background interference
was separated from the signal of the ADC (MW around 150kDa) by separation voltage (SV) of 3500 V and compensation
voltage (CoV) of 3. The SelexION parameters were optimized
by multiple injections to ramp through compensation voltages
from -12V to +12V, with the lower molecular weight features
having transmission optimum at the higher end (6-12V) and thehigher molecular weight features having their transmission
optimum at lower CoV values (-3 - 0) (Figure 2).
The second chromatographic set-up, with size exclusion typeseparation, allowed for reconstruction quality spectra to beacquired in normal mode with the TripleTOF system (Figure 3).
Data processing was achieved in BioPharmaView™ software
which employs maximum entropy reconstruction, here 100

RUO-MKT-02-2814-A

Figure 4 BioPharmaView software reconstruction of the data presented in
Figure 3 Drug load was up to 7 per antibody moiety, with all the different
drug conjugations demonstrating the same glycosylation pattern of
combinations of G0Fs to G2Fs.

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ADC 药物完整解决方案 19

In addition to reconstruction, BioPharmaView software performs
automated DAR calculations. The calculations for the samples
shown in Figure 4 are shown in Figure 5. The batch processingof six injections of the same sample are shown in Figure 6.

Figure 5 Automated DAR calculations for the non-deglycosylated ADC.
The reconstruction area is given as a value, and also as a percentageof the total area per each multiplicity of conjugation. The multiplicity isrepresented as columns and the final DAR ratio is calculated.

Figure 6 Reconstruction of analysis of six injections of the nondeglycosylated ADC: (6A) the overlay of six reconstructed spectra, (6B)
the DAR ratios and (6C) the multiplicity represented as columns for the six
injections.

Conclusions
Complex ADC’s can be reproducibly analyzed without time
consuming sample preparation to deglycosylate the protein. The
reconstruction with BioPharmaView software enabled both the
DAR to be calculated and the glycosylation pattern to bemonitored within a single sample and single analysis for thiscomplex biotherapeutic. The sample analysis time was reduced toobtain higher through-put by excluding sample pretreatment time,
combining shorter analysis time with SelexION technology
separation, and by enabling fast data-analysis batch-processing
by BioPharmaView software
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© 2015 AB Sciex. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners.
AB SCIEX™ is being used under license.
Document number: RUO-MKT-02-2814-A

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20 ADC 药物完整解决方案Native LC-MS Analysis of an Intact Cysteine-Linked
Antibody-Drug-Conjugate
Featuring SCIEX TripleTOF® 6600 system with IonDrive™ Turbo V source and BioPharmaView™
Software 3.0.1
Jennifer Schanz1, Jason Tonillo1, Nicolas Rasche1, Kerstin Pohl2, Denise Scherbl3, Roland Kellner1, Sean
McCarthy2
1
Merck KGaA, Darmstadt, Germany; 2SCIEX Framingham, MA, USA; 3SCIEX, Villebon sur Yvette, France
The concept of antibody-drug-conjugates (ADC) delivering acytotoxic compound specifically to malignant target cells with thehelp of a (at that time called) “heptophore” was described by
Paul Ehrlich in 1913. This very reasonable concept increases
selectivity for delivery and minimizes off-target toxicity. Morethan 100 years later, much development focusing amongst
others on the protein itself, cytotoxic drugs, linkers, andconjugation chemistry have been developed. Usually,
conjugation of payloads are obtained via lysine-linkage orcysteine-linkage using native or engineered residues. Linkers
binding to the protein through thiols from cysteines offer anadvantage of less heterogeneity compared to lysine-linked ADCs
and the potential to be reduced in cellular compartments
releasing the drugs inside the cells while keeping the conjugates
intact during circulation.
With heterogeneity being a major challenge in the developmentof ADCs, a variety of analytical tools are applied throughout
characterization. While conjugated light and heavy chains can beanalyzed separately after reduction of remaining inter chain
disulfide bonds using reversed-phase LC-MS detection. To
monitor the intact ADC, native LC-MS analysis provides a nondenaturing approach to analyze intact ADCs. For native analysis,
size exclusion chromatography (SEC) with volatile, aqueous
buffers enables scientists to determine the drug-antibody-ratio(DAR) on intact level for ADCs. The DAR is a critical quality
attribute (CQA) of ADCs which needs to be assessed during thecharacterization of biotherapeutics. Furthermore, proteins takeup fewer charges during native analysis compared to reversed
phase conditions, resulting in less distribution of the signal andan increased spacing of different features of the sample on them/z-axis. Finally, it is less unlikely to induce artefacts during theanalysis.

assessed on intact level. In addition, the heterogeneity of thesample was investigated and reconstructed masses wereassigned to different protein features.

Key Feature of TripleTOF 6600 System with
IonDrive Turbo V source and BioPharmaView
Software• High resolution and high sensitivity for native MS analysis ofproteins and protein complexes• Efficient desolvation of native eluents without disruption ofprotein complexes• Automatic matching or protein forms and DAR calculation of
ADCs

Here, the advantages applying native conditions for thecharacterization of ADCs using a TripleTOF 6600 system aredescribed. A cysteine-linked ADC sample obtained from Merck
KGaA, Darmstadt, Germany was used as a model molecule forwhich MS parameters were optimized and the DAR was

RUO-MKT-02-9757-A

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ADC 药物完整解决方案 21

Materials and Methods
SampleTable 2. MS Parameters.

A cysteine-linked ADC sample (see Figure 1) based on a mousemonoclonal antibody (mAb) against hen egg lysozyme (HEL)

Parameter

Setting

Scan Mode

TOF-MS positiveGS1

60 psiGS2

60 psi

Curtain Gas

35 psi

Source Temperature

250 °CIon Spray Voltage

5000 V

Figure 1. Conjugation example via cysteines for MC vc-PABMMAE.

Time Bins to Sum

80

Accumulation Time

1 seccoupled with maleimidio caproyl (MC) valine-citrulline (vc) paminobenzyl oxycarbonyl (PAB) monomethyl auristatin E (MMAE)(anti-HELxMC-vc-PAB-MMAE) obtained from Merck KGaA,
Darmstadt, Germany (c = 4.09 mg/mL).

TOF Start

1,000 m/z

TOF Stop

7,000 m/z

Declustering Potential

100 V

Collision Energy

7 eV

Chromatography and Mass Spectrometry
An ExionLC™ system coupled to a SCIEX accurate mass
spectrometer TripleTOF 6600 system equipped with an IonDrive
Turbo V source were used for data acquisition. An isocratic
chromatography was used with conditions as shown in Table 1.
The MS parameters used are shown in Table 2.

Table 1. Chromatographic Conditions.
Parameter

Value

ColumnPhenomenex bioZen SEC-3 150×4.6 mm, 1.8 µm

Mobile Phase A

50 mM ammonium acetate, pH 7.2

Mobile Phase B

50 mM ammonium acetate, pH 7.2

Flow Rate

150 µL/min

ColumnTemperature

26 °C

Injection Amount

25 µl / 100 µg

Data processing:
Data was processed using BioPharmaView Software 3.0.1.

RUO-MKT-02-9757-A

Results
Analyzing the ADC sample under native conditions using the
TripleTOF 6600 system fitted with the IonDrive Turbo V source
resulted in a charge state envelope at higher m/z values
compared to denaturing analysis as expected (see Figure 2).
Using a source temperature of 250°C, reduced ion spray voltage
of 5000 V, and a low declustering potential, the non-covalentsample was efficiently ionized, desolvated, and detected in its
intact state. Under native-like conditions the protein maintains a
3-D structure and therefore does not take up as many charges as
when being analyzed under denaturating conditions (envelope
between 5000-6700 m/z, Figure 2). Even though soft source
conditions are needed for keeping the molecule intact, thedesolvation of the molecules with the IonDrive Turbo V source
could be performed successfully. The source ensures highefficiency in transferring heat to the molecule via two efficientheaters, so that even when using low temperatures desolvation,
nicely separated protein features on an m/z scale (Figure 2, upper
right-hand corner), are obtained. The high quality raw spectrum
was reconstructed to a “zero charge state” using BioPharmaView
software allowing to assign the major proteoforms (Figure 3) withtheoretically calculated masses based on the sequence andknown modifications such as glycosylations and linker/drug
information. The protein was detected without any drug load(naked mAb), with two drugs and with four drugs attached. For

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22 ADC 药物完整解决方案each drug load, different glycoforms were observed as expected(see Figure 3).
By processing with BioPharmaView software the main
proteoforms were automatically assigned and used to calculate a
DAR value of 2.78 based on the reconstructed peak areas (Figure4). Furthermore, the drug load was visualized within the software
as a bar graph for easy and quick comparison of different samples
or batches (Figure 4, upper right hand corner).
Additional peaks were found in the reconstructed data which wereconfirmed by the high quality spectral raw data using the help of
the so called hypermass tool: Any given peak in the reconstructeddata can be calculated back to the potential charge states in theraw data helping the user to discriminate between real peaks
being supported by raw data and potential artefacts (see figure 5).
These peaks could be linked back to partial loss of the MC-vcPAB-MMAE load (delta mass of 760 Da) and to the loss of
glycosylation with a delta mass of 1445 Da (see green and orange
labelling in Figure 3). It is not clear if the losses are induced during
sample preparation, storage, or from the analysis. Interestingly,
loss of glycosylation has not been observed in native studies of
non-ADC molecules.1

Figure 2. TOF-MS raw data for intact ADC sample. Upper rightcorner shows zoomed view of charge state z-28.

Δ~760 Da

Δ~760 Da

Δ~760 DaG1F 2xΔ~760 Da

Δ~760 Da

Δ~760 Da

Δ~1445 Da

Δ~1445 Da
Δ~1445 Da

G1F/G2FG1F/G2FG1F/G2FG1F/G2FG0F 2xG0F/G1FG1F 2xnaked mAb
G0F 2xG0F/G1FG1F 2xG1F/G2F

Δ~1445 Da

G0F 2xG0F/G1FG1F 2x1445 Da ≙ G0F

G1F 2x760 Da ≙ part of linker and drugmAb + 4 drugs
G0F 2xG0F/G1FG0F 2xG0F/G1F

mAb + 2 drugs

Figure 3: Reconstructed TOF-MS data. Main glycoforms of naked mAb, mAb with drug load two and four can be found. In addition deltamasses of 760 Da and 1445 Da being linked to the loss of the drug and a part of the linker (orange arrows) and a loss of one G0F (green
arrows) could be assigned.

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ADC 药物完整解决方案 23

Figure 4: Automatic processing in BioPharmaView software. Automatic assignment of reconstructed peaks, DAR calculation and graphicaldisplay of drug load revealing a DAR of 2.78.

TOF-MS raw data

Reconstructed data

Figure 5: Hypermass feature in BioPharmaView software. Highlighting a peak in the reconstructed data (right hand side) results in a
calculation of all potential charge states in the raw data (red highlights, left hand side) enabling a user to discriminate between real
information and artefacts.

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24 ADC 药物完整解决方案Conclusions
Native SEC-MS provides orthogonal information to otheranalytical tools for ADC samples during characterization. The
TripleTOF 6600 system and IonDrive Turbo V source resulted inhigh quality data for accurate measurement of non-covalently
bound samples such as cysteine-linked ADCs in an intact state
under native conditions with minimal optimization. The source
technology of the IonDrive Turbo V source combines an excellent
desolvation of the molecule with soft ionization. The processing of
the raw data with BioPharmaView software enables users to get
access to critical quality attributes such as DAR with minimalhands-on time. The high quality raw data provided additional
information on less abundant protein features giving further
insights into the complex molecular details of ADC samples.

References
1.

SCIEX Technical Note. Native Analysis of Intact Monoclonal
Antibody Aggregates Using the TripleTOF® 6600 System. RUOMKT-02-8791-A.

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ADC 药物完整解决方案 25

,Intact Analysis of Antibody Drug Conjugates
Trastuzumab Emtansine Analysis using Benchtop X500B QTOF Mass Spectrometer
Wen Jin1, Suya Liu1, Doug Simmons1, Ian Moore1, Sean McCarthy2 and Sibylle Heidelberger1
71 Four Valley Dr. Concord, ON L4K 4V8, Canada
500 Old Connecticut Path, Framingham, MA, 01701, USA
Introduction

Experimental

Antibody-drug conjugates (ADCs) are an emerging class ofbiotherapeutics. They offer the specificity of monoclonal
antibodies while incorporating cytotoxic payloads to efficiently
target and kill infected cells. By their nature, ADCs are highly
complex as they use an antibody backbone which must be wellcharacterized. Adding to this complexity is conjugation of thecytotoxic payloads or drugs to the antibody. The number of drugs
attached to the antibody has been shown to impact the safety
and efficacy of the resulting ADC, and as such, must be
characterized and monitored through development. [1-3]Samples were prepared either neat for the glycosylated form or
using PNGase F (New England BioLabs (Ipswich, MA, USA) using
their standard protocol.

Presented here is a streamlined approach for the analysis of
ADCs to rapidly and accurately calculate the drug-to-antibody
ratio (DAR). We will discuss the use of the new, compact X500B
QToF, powered by SCIEX OS, with data processing using
BioPharmaView™ 2.0.1 for routine characterization of ADCs andcalculation of DAR using both the glycosylated anddeglycosylated forms.

Figure 1: Trastuzumab emtasine. T-DM1 consists of a trastuzumab
backbone with MCC linker binding DM1, a cytotoxic drug to the antibody
via lysine residues.

RUO-MKT-02-5468-C

LCMS analyses were conducted using a benchtop X500B QTOF
mass spectrometer using SCIEX OS equipped with an Exion LC™
system. Table 1 lists the LCMS conditions used in these analyses.
Data was processed using BioPharmaView™ for reconstruction of
the intact protein and calculation of DAR.
Table 1 Exion LC™ conditions
ColumnMobile phase A
Mobile Phase B
Flow rate
Column Temperature

Agilent Poroshell 300SB-C8 1.0 x 75mm 5 µm
0.1 % Formic acid in water
0.1% Formic acid in acetonitrile
0.2 mL/mino
75 C

Table 2 X500B mass spectrometry conditions
Source parameters:

CUR
GS1
GS2
Ion Spray Voltage
Source Temperature
TOFMS mass rangeDP
Accumulation time
Time bins to sum
Intact Protein Mode (IPM)
Large Proteins (>70kDa)
Decrease Detector Voltage
DP

30
50
50
5000 Vo

400 C

900 – 4000 m/z
250 V
0.5 s
80
On
On
On
250 V

Results and Discussion
Glycosylated T-DM1
For this study, we used trastuzumab emtansine (T-DM1), a lysine
conjugated ADC for the treatment of HER-2 positive metastatic
breast cancer. T-DM1 is comprised of an antibody, trastuzumab,
covalently linked via lysine residues to cytotoxic drug molecules
which are liberated following internalization by target cells (Figure1). As drug molecules are attached to the antibody following
expression, assessment of the drug-to-antibody ratio (DAR) must
be determined regularly as part of the drug development process.

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26 ADC 药物完整解决方案We began our study by determining the DAR of the intact ADC.
As shown in Figure 2A, the resulting raw spectrum is highly
complex. Using the BioPharmaView reconstruction algorithm,
we generated high quality reconstructed spectra clearly showing
the different DAR species as well as the glycoprofile for each
DAR. The resulting reconstruction of the raw data gives us arange of between 0-8 drugs attached to the trastuzumab (Figure2B). As expected, the glycoprofile for each DAR is consistentacross each of the DAR species.

A
Figure 3: Trastuzumab emtansine with 3 drugs attached. A closer
look at the reconstructed DAR shows evidence for the main glycoforms:
1 G0F/G0F, 2: G0F/G1F, 3:G1F/G1F and 4: G1F/G2F withcorresponding additions of one MCC linker without DM1.

Using BioPharmaView, (DAR) can be calculated based on thereconstructed peak area distribution of the different drug
conjugated antibodies. DAR was calculated to be 3.49 (Figure.
5), which agrees well with previously reported value of 3.5 [1-3].

B

Figure 2: T-DM1 raw (A) and reconstructed (B) data. Raw data (2A)
includes glycosylations and ADC which increases the normal complexityof an antibody spectrum while 2B shows the reconstruction using
BioPharmaView and calculating 0-8 drugs attached to the trastuzumab.

Looking closely at each reconstructed DAR separately, weclearly see evidence for each of the main glycoforms as well as acorresponding set of species with a mass shift of 221 Da and not
219 Da which would be the mass of the linker itself. (Figure 3).
This has previously been reported to be due to a reaction withlysine residues, which results in chemical crosslinking. The initial
reaction of trastuzumab to the MCC linker produces anintermediate which, in some cases, due to the proximal locationof lysines involves a second reaction causing the inter-chain
cross linking and a linker species of 221 Da without the DM1. [2]

RUO-MKT-02-5468-C

Figure 4: Calculation of DAR for trastuzumab emtasine using
BioPharmaView™. Calculation of DAR using the software gives a
simple table and graph, showing the median multiplicity of the ratio.
Highlighted in the top right is the ratio calculated which was 3.49.

Deglycosylated T-DM1
We then removed the N-linked glycans using PNGaseF toprovide a spectrum with reduced complexity as the peaks
attributed to the glycoforms would be removed. As shown in
Figure 5A, we can clearly see that the glycoform complexity has
been reduced. In addition, the presence of the species with a
221 Da higher than each corresponding DAR species is evident
confirming our previous findings.

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ADC 药物完整解决方案 27

Figure 6: DAR calculation for deglycosylated trastuzumab
emtansine. The 8 drug conjugates (highlighted bottom left) are thenused to calculate the DAR ratio (highlighted top left) to give a DAR ratioof 3.26.

Conclusion

Figure 5: Deglycosylated T-DM1 raw (A) and reconstructed (B)spectrum. Raw data (5A) from the deglycosylated is used to reconstruct
the intact protein mass (5B).

The reconstructed spectrum for the deglycosylated trastuzumab
emtasine confirms the results observed in figure 2 of theglycosylated form. Both confirm from 0 – 8 drugs attached to the
trastuzumab backbone. DAR calculations were performed using
the software, and the resulting DAR value is consistent withpreviously reported values. [2]We have shown that the benchtop X500B QTOF systemproduces data with such quality that it can be used for routine
analysis of such complex biologics such as ADCs which require
high resolution data to identify glycosylations and the number ofdrugs bound to the antibody. We have shown that the massaccuracy is such that we are able to identify a prominent 221 Da
mass shift that has been reported by Jacobson et al as aconjugation of the trastuzumab-MCC linker intermediate with aproximal lysine residue. Processing of the data andreconstruction was performed using BioPharmaView resulting infast reconstruction and accurate DAR calculations. The resulting
software was able to calculate the DAR ratio to be 3.46 on theglycosylated form compared to the 3.5 listed in literature. [2]References[1] Michael T. Kim, Yan Chen, Joseph Marhoul and Fred Jacobson. Statistical Modeling of the
Drug Load Distribution on Trastuzumab Emtansine (Kadcyla), a Lysine-Linked Antibody Drug
Conjugate. Bioconjugate Chem. 2014, 25, 1223−1232[2] Yan Chen, Michael T. Kim, Laura Zheng, Galahad Deperalta, and Fred Jacobson. Structural
Characterization of Cross-Linked Species in Trastuzumab Emtansine (Kadcyla). Bioconjugate
Chem. 2016, 27, 2037−2047[3] Liuxi Chen, Lan Wang, Henry Shion, Chuanfei Yu, Ying Qing Yu, Lei Zhu, Meng Li, Weibin
Chen, and Kai Gao. In-depth structural characterization of Kadcyla (ado-trastuzumab emtansine)and its biosimilar candidate. MABS, 2016, VOL. 8, NO. 7, 1210–1223

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© 2018 AB Sciex. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners.
AB SCIEX™ is being used under license.
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28 ADC 药物完整解决方案Structural Characterization of Antibody Drug Conjugates(ADCs) by a Combination of Intact, Middle-up and Bottom-up
Approaches using CESI-MS
Stephen Lock,1 Nassur Said,2 Rabah Gahoual,2 Alain Beck,3
Yannis-Nicolas François,2 and Emmanuelle Leize-Wagner2
1 SCIEX,UK
2 Laboratoire de Spectrométrie de Masses des Interactions et des Systèmes (LSMIS),
Université de Strasbourg, Strasbourg, France
3 Centre d’Immunologie Pierre Fabre, Saint-Julien-en-Genevois, France

Introduction
Antibody drug conjugates (ADCs) represent a rapidly growingclass of biopharmaceuticals. ADCs are formed by the conjugationof an active drug species to a monoclonal antibody,1 and theyoften result in a distribution of products containing varying
numbers of active drugs bound at different locations aroundthe antibody.
Capillary electrospray ionization (CESI) is the integration ofcapillary electrophoresis (CE) and electrospray ionization (ESI)
into a single process in a single device (Figure 1).2 CESI-MS

Figure 1. OptiMS - Ultra low flow ESI Interface.

operates at low nL/min flow rates and offers several advantages,

Materials and methods

including increased ionization efficiency and a reduction in

Chemicals: All chemicals were reagent grade and were

ion suppression. CESI-MS separates analytes by their charge
and size, and it is therefore a separation mechanism thatpurchased from Sigma Aldrich or alternative suppliers.3
Brentuximab vedotin was produced by Millennium

is complementary to more traditional techniques, such as

Pharmaceuticals/Takeda.

reversed-phase LC.
This document summarizes the work recently published by the
research group at LSMIS at the Université de Strasbourg.3 In
this application note, we will show how CESI-MS can be used
to characterize a gold standard ADC, brentuximab vedotin(BV). BV is a cysteine-linked mono-methyl auristatin E (MMAE)
monoclonal antibody (mAb), which was attached without
disrupting the heavy and light chain linkages of the mAb.3 We will

Sample Preparation: BV was buffer exchanged with 200 mM
ammonium acetate buffer (pH 7.0) using Amicon filters before
intact analysis.3
For middle-up analysis, BV was cleaved at the hinge region
by proteolysis using IdeS (FabriCATOR, Genovis) to obtain
Fc/2 fragments and an F(ab’)2 fragment. After digestion wascompleted, the sample was again buffer exchanged into 200 mM

show how CESI-MS is used as a nano-spray infusion device toammonium acetate buffer (pH 7.0) before CESI-MS analysis.

identify the drug antibody ratio of the sample. We will also show

For bottom-up analysis, a sample of desalted IdeS cleaved

how CESI-MS can be used to study the “middle-up” structure forthis protein, for example, by analyzing the light chain, Fab andF(ab’)2 subunits. Finally, a bottom-up analysis of a tryptic digest
will be performed by CESI-MS to fully sequence the ADC.

ADC, which had undergone buffer exchange and was diluted
with RapiGest (Waters) and reduced with dithiothreitol (DTT).
Before enzymatic digestion, 10% of acetonitrile was added tothe sample. The reduced protein was then digested with trypsin
overnight, isopropanol (40%) and formic acid (1% v/v) wereadded and the final solution diluted using ammonium acetate(50 mM, pH 4.0) to produce a final concentration of 2.2 μM.3

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ADC 药物完整解决方案 29

CESI-MS method: For the analysis of intact and digested
samples, a bare fused silica OptiMS cartridge (30 μm ID x 91 cm)
from SCIEX was used and was thermostatted using recirculating
liquid coolant regulated at 20°C used. For the analysis of tryptic
digests, the sample was injected hydrodynamically (10 psi,
60 s) and peptides were separated using conditions shown in
Table 1 with a background electrolyte of 10% acetic acid. For MS

Important:• A separation current above 5 μA might cause permanent
damage to the separation capillary.
• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

analysis of tryptic digests, a SCIEX TripleTOF® 5600+ LC-MS/

Results

MS System was fitted with the NanoSpray® III Ion Source. Gas

When CESI is used as a nano-spray infusion device, the sample

1 and 2 were not used, the curtain gas was set to 5 psi andthe temperature of the interface was set at 150 °C. Ionization
at these very low-flow rates occurred by simply applying theionspray voltage (1,450 V). MS data was acquired using a TOF
survey scan (m/z 100–2,000 amu), which triggered MS/MS
acquisition (m/z 100–2,000 amu).

ActionTime(min)

PressureDirection
(psi)

Rinse

3.5

100

Rinse

1.5

Rinse

3

Rinse

is simply pushed to the MS detector at low nL/min flow rates.4
Desalted intact BV was analyzed in this mode to confirm themolecular weight of this ADC and measure the drug to antibody
ratio (DAR), which is the drug loading on the antibody andtypically ranges from 0 to 8. An example of the data achieved
is shown in Figure 2, which gives an overview of the analysis
of BV by CESI-MS.

Voltage(kV)

Solution

Forward0

0.1 M NaOH

100

Forward

0

Water

100

Forward0

0.1 M HCl

5100

Forward

0

10% Acetic acid

Rinse

2

75

Reverse

0

10% Acetic acid

Rinse

60s

10

Forward0

Sample Vial

Injection

10s

5Forward

0

10% Acetic acid

Separation

35

0.1

Forward20

10% Acetic acid

Voltage2

10Forward1

10% Acetic acid

Table 1. CESI separation conditions used for the analysis of tryptic digests.

For nano-spray infusion, the CESI capillary was flushed with
10% acetic acid (5 min, 50 psi) and then with sample (5 min,
50 psi), and MS data was acquired at 5 psi. After each analysis,
the capillary was flushed with 10% acetic acid (10 min, 50 psi) toprevent carryover. For these nano-spray infusion experiments,
MS data was acquired using a maXis 4G system (Bruker).
The maXis system was optimized for each analysis using theactual sample and ion funnels with values that ranged from
300–400 Vpp. The electrospray voltage ranged from 1,200–
1,800 V, drying gas was set at 1.5 L/min and the source
temperature was set at 150 °C.

Figure 2. Overview of brentuximab vedotin structural characterization
using sheathless CE-MS. (a) Native MS infusion for average DAR
determination and drug loading distribution assessment. (b) Middle-up
and bottom-up analysis.

Based on the charge state of the deconvoluted mass spectrumof the intact BV shown in Figure 2, the average DAR value was
between 3.8 and 3.9. A distribution of baseline resolved species
of BV was observed with masses that corresponded to theintact mAb linked with 0 to 8 payloads of the drug. The massaccuracies of intact BV were in total agreement with the results
reported in the literature.3

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30 ADC 药物完整解决方案Using the same experimental conditions as the intact analysis, a

CESI-MS has been previously used for the analysis of tryptic

middle-up sample was analyzed to elucidate the location of thedigests of mAbs.5 The next set of experiments performed on

drug loading on the mAb, which had been cleaved at the hinge

BV were the bottom-up analysis of a tryptic digest to determine

region (Figure 2). The raw data highlighted multiply charged

the location of post-translational modification (PTM) sites,

protein peaks in 3 separate regions across the mass range ofthe location of the drug linked to the mAb and the amino acid

2,000–6,000 amu (Figure 3). When deconvoluted, the multiply

sequence. The tryptic digestion protocol had been adapted fromcharged protein envelopes depicted in Figure 3 showed that thethe classical approach to improve the overall digestion, which

free light chain (LC) protein subunit (MW = 25040.1 ± 0.1 Da) had

was affected by the presence of the drug molecules bound to

1 drug molecule linked to it as well as 2 glycation modifications.

the mAb.3 CESI-MS analysis took less than 35 min. Due to

The smaller Fab subunit (approximately 48–55 kDa) had 0–4

the sensitivity and efficiency of the CESI-MS analysis, 100%molecules of drug linked to the protein and was present as asequence coverage could be obtained for BV in a single injection

dimer. The F(ab’)2 subunit (approximately 97–108 kDa) had 0–8

with the identification of the peptides based on their accurate

drug molecules attached to the protein subunit. As the drug units

molecular weight as well as sequence data from MS/MS analysis.

were spread across multiple regions of the mAb, the IdeS middle-

The presence of the organic solvent in the sample preparation

up sample could not be used to calculate the DAR for ADCs, but

did not seem to have an effect on the separation of the tryptic

does give information about the location of the drug molecules

peptides demonstrated by the detection of small (3 amino acids)

within BV.

to large (63 amino acid) peptides.
Modified peptides (including N-glycopeptides) were detected in
the analysis, for example, TKYPREEQYN297STYR was observed
to have 11 glycoforms. Regarding drug-loaded-peptides, 4 weredetected (Figure 4), which was aided by the presence of organic
solvent in the sample preparation to prevent the loss of thesehydrophobic modified peptides.

Figure 3. (a) MS spectra corresponding to native MS nano ESI infusionof middle-up BV. (b) Charge state deconvoluted mass spectra of(1) LC- drug conjugated subunit, (2) Fab subunits with the incorporationof 0 to 4 drug molecules and Fc/2 homodimers and (3) F(ab’)2 subunits
with the incorporation of 0 to 8 drug molecules.

Figure 3. MS and MS/MS spectra of drug-loaded peptides. (a) [GEC]- 1 drug, (b) [SCDK] -1 drug, (c) [THTCPPCPAPELLG] - 1 drug and(d) [THTCPPCPAPELLG] - 2 drug molecules

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ADC 药物完整解决方案 31

Identification of these drug containing peptides was confirmed
by MS/MS data analysis, which highlighted the presence ofseveral diagnostic drug fragment ions. A single drug loaded
peptide was located on the light chain of the mAb (GEC),
and the other peptides were present on the heavy chain. The
THTCPPCPAPELLG peptide actually had the potential ofcontaining 2 drug molecules, and these 2 different peptides havebeen separated by CESI-MS.

Conclusions
A CESI-MS protocol for structural characterization of ADC
molecules has been developed. By using CESI-MS in 2 different
modes, several properties of an ADC could be confirmed,
including:• DAR ratio calculation using native conditions• Drug distribution on the F(ab’)2 and Fc/2 using nano-spray
infusion CESI-MS analysis of an IdeS digested middle-up
sample using native conditions• 100% sequence coverage of the ADC and identification of
the drug location as well as the location of other PTMs
• Characterization of drug loaded peptides by analysis of
MS/MS spectra

References
1. Chari, R.V.J. Targeted cancer therapy: conferring specificity
to cytotoxic drugs. Accounts Chem. Res. (2008), 41: 98–107.
2. Busnel, J.-M. et al. High Capacity Capillary ElectrophoresisElectrospray Ionization Mass Spectrometry: Coupling a
Porous Sheath-less Interface with Transient-Isotachophoresis.
Anal. Chem. (2010), 82: 9476–9483.
3. Said, N., Gahoual, R., Kuhn, L., Beck, A., François, Y.-N.,
Leize-Wagner, E. Structural characterization of antibody
drug conjugate by a combination of intact, middle-up andbottom-up techniques using sheathless capillary electrosprayTandem mass spectrometry as nanoESI infusion platform
and separation method. Analytica Chimica, Acta (2016).
http://dx.doi.org/10.1016/j.aca.2016.03.006.
4. Gahoual, R., Busnel, J.M., Wolff, P., Francois, Y.-N.,
Leize-Wagner, E. Novel sheathless CE-MS interface as an
original and powerful infusion platform for nanoESI study:
from intact proteins to high molecular mass noncovalent
complexes. Anal. Bioanal. Chem. (2014), 406: 1029–1038.
5. Gahoual, R., Busnel, J.M., Beck, A., Francois, Y.-N.,
Leize-Wagner, E. Full antibody primary structure andmicrovariant characterization in a single injection usingtransient isotachophoresis and sheathless capillaryelectrophoresis-tandem mass spectrometry. Anal. Chem.
(2014), 86: 9074–9081.

For further information on this topic we would like to referreaders to the full scientific publication on which this applicationnote is based.3

The SCIEX clinical diagnostic portfolio is For In Vitro Diagnostic Use. Rx Only. Product(s) not available in all countries. For information on availability, please contact your local sales
representative or refer to https://sciex.com/diagnostics. All other products are For Research Use Only. Not for use in Diagnostic Procedures. Trademarks and/or registered trademarks
mentioned herein, including associated logos, are the property of AB Sciex Pte. Ltd. or their respective owners in the United States and/or certain other countries.
© 2020 DH Tech. Dev. Pte. Ltd. RUO-MKT-02-4400-B
AB SCIEX™ is being used under license. Beckman Coulter® is being used under license.

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Ultra-sensitive LC-MRM analysis for trastuzumab-emtansine
quantification in rat plasma
Featuring the SCIEX Triple Quad™ 7500 LC-MS/MS System – QTRAP® Ready, powered by SCIEX OS
SoftwareZuzana Demianova and Lei Xiong
SCIEX, USA
With the adoption of immunoaffinity based sample preparation,
an ultra-low LLOQ of 1 ng/mL was achieved. The assay showshigh precision, accuracy, and good linearity, demonstrating therobustness and performance of the developed method.
Quantification of protein therapeutics in biological matrices, playa critical role in the drug discovery and development process.
LC-MS/MS has been routinely adopted for quantification of thismolecular class in bioanalytical laboratories, serving as analternative technology to the traditional ligand binding assays(LBAs). Although mass spectrometry offers higher specificity
than LBA, there still remains a need for improved sensitivity for
accurate quantification of low concentration analytes in complex
matrices while still maintaining high analysis throughput,
instrument easy-of-use, and robustness.
In this work, the SCIEX Triple Quad 7500 System coupled withan analytical flow HPLC system is employed to quantify
trastuzumab-emtansine in rat plasma. Multiple hardware
improvements on the ion source and the front end of the mass
analyzer significantly boost the system sensitivity.1

Key features of the peptide quantification
workflow• Immunoaffinity-LC-MRM workflow2 offers solid quantificationof trastuzumab-emtansine in rat plasma at 1 ng/mL, with highprecision, accuracy, and linearity• Hardware improvements on the SCIEX 7500 System provide
significant gains in sensitivity for peptide quantification: theOptiFlow® Pro Ion Source with E Lens™ Technology provides
improvements in ion generation and the D Jet™ Ion Guide
improves ion sampling• SCIEX OS Software—an easy to use, compliance ready,single platform for acquisition, processing and data
management

Figure 1. Extracted ion chromatograms (XICs) of selected MRM for trastuzumab-emtansine in rat plasma. From left to right are blank, 0.5 ng/mL(LOD) and 1 ng/mL (LLOQ).

RUO-MKT-02-12237-A

p1

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ADC 药物完整解决方案 33

Methods
Immunocapture: A streptavidin coated immunoaffinity magnetic
bead slurry was aliquoted and washed with PBS buffer (1x) threetimes. Biotinylated goat anti-human IgG antibody was added tothe beads and incubated at room temperature for 1 hour withshaking. The conjugated beads were washed three times and resuspended in PBS buffer. Calibration standard samples wereprepared by spiking trastuzumab-emtansine and SILuMab(internal standard) into rat plasma and performing serial dilution.
The concentrations of trastuzumab-emtansine in plasma ranged
from 0.5 - 20,000 ng/mL. To each calibration standard sample,
250 µL of PBS Buffer (1×) and 50 µL conjugated bead slurry
were added and the mixtures were incubated at room
temperature for 1 hour with shaking. The beads wereaccumulated by magnetic stand and washed sequentially with
PBS buffer and 10 mM ammonium bicarbonate. The target
proteins were eluted by incubating the beads with 0.1% TFA inwater and vortexing for 10 min.
Protease digestion of immuno-enriched eluents: The eluentswere neutralized with 500 mM ammonium bicarbonate in waterand incubated at 95 °C for 10 mins with shaking. The samples
were cooled to room temperature and incubated with 1 µg oftrypsin/Lys-C mix per sample overnight at 37 °C. The digestion
was aborted by adding formic acid. The samples were
centrifuged at 15,000×g for 5 min. The supernatants werecollected and subjected to LC-MS/MS analysis.
LC-MS/MS conditions: Samples were analyzed in triplicate by a
SCIEX Triple Quad 7500 System, coupled with an ExionLC
System. The method details are summarized in Tables 1, 2 and3. The data were processed using the Analytics module in
SCIEX OS Software 2.0.
Table 1. Chromatographic conditions.

Table 2. Gradient information.
Time [min]Mobile phase A [%]Mobile phase B [%]Initial

95

5.0

0.7

95

5.0

0.8

90

10

3.5

75

25

4.0

60

40

4.5

10

90

6.0

10

90

6.1

95

5.0

7.7

95

5.0

Table 2. Mass spectrometric conditions.
Parameter

Value

Parameter

Value

Curtain gas

40 psi

Source temperature

500 °CIon source gas 1

50 psiIon source gas 2

50 psiCAD gas11

Ion spray voltage

Name

Q1

Q3

CE

CXP

IYPTNGYTR1

542.8

808.4

23

13

IYPTNGYTR2

542.8

405.1

23

13

FTISADTSK1*

485.2

721.3

23

15

FTISADTSK2

485.2

608.2

22

15

GPSVFPLAPSSK1

1700 V

593.8

699.4

31

15

Parameter

Value

GPSVFPLAPSSK2

593.8

846.5

28

15

ColumnPhenomenex bioZen Peptide XB-C18 50×2.1
mm; 2.6 µm

FNWYVDGVEVHNAK[H]

562.9

713.3

23

15

GPSVFPLAPSSK[H]

597.8

707.4

28

15

Mobile phase A

Water with 0.1 % formic acid

Mobile phase B

Acetonitrile with 0.1 % formic acid

Flow rate

500 µL/min

Column temperature

40 °C

Injection volume

30 µL

RUO-MKT-02-12237-A* MRM transition for the best sensitivity[H] MRM transitions of internal standard peptides from SILuMab

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34 ADC 药物完整解决方案Quantification results

Conclusions• A highly sensitive immunoaffinity-LC-MRM workflow forquantifying trastuzumab emtansine in rat plasma wasdemonstrated• By using the SCIEX Triple Quad 7500 System coupled withanalytical flow HPLC, trastuzumab-emtansine was solidly
quantified at 1 ng/mL level• Good reproducibility, accuracy and wide linear dynamic range
as 4.5 orders of magnitude were achieved at the same time

Figure 2. Quantification result summary.

The SCIEX Triple Quad 7500 System integrates innovations thatprovide improvements in both ion generation and ion sampling.
The OptiFlow Pro Ion Source with E Lens Technology provides
improvement in ion generation and the D Jet Ion Guide efficiently
captures and transmits the ions in the high gas flow behind theorifice plate.1

References
1.

Enabling new levels of quantification. SCIEX technical note
RUO-MKT-02-11886-A.

2.

Quantification of trastuzumab in rat plasma using an
improved immunoaffinity-LC-MS/MS method. SCIEX
technical note RUO-MKT-02-7597-A.

With the optimized method, the presented immunocapture-LCMRM assay demonstrated ultra-high sensitivity to quantify
trastuzumab-emtansine in rat plasma, with the LLOQ at 1 ng/mL
and the LOD at 0.5 ng/mL (Figure 1). As summarized in Figure
2, the assay accuracy is 86-106% and %CVs are below 10% for
all tested samples. The calibration curve covered 4.5 orders ofmagnitude (1-20000 ng/mL) (Figure 3) and displayed aregression coefficient (r) of 0.996 using a weighting of 1/x2.

Figure 3. Calibration curve for trastuzumab-emtansine in rat plasma displayed with log-log format. Concentrations range from 1 ng/mL to
20,000 ng/mL.

The SCIEX clinical diagnostic portfolio is For In Vitro Diagnostic Use. Rx Only. Product(s) not available in all countries. For information on availability, please contact your local sales
representative or refer to https://sciex.com/diagnostics. All other products are For Research Use Only. Not for use in Diagnostic Procedures.
Trademarks and/or registered trademarks mentioned herein, including associated logos, are the property of AB Sciex Pte. Ltd. or their respective owners in the United States and/or certain
other countries.
© 2020 DH Tech. Dev. Pte. Ltd. RUO-MKT-02-12237-A. AB SCIEX™ is being used under license.

RUO-MKT-02-12237-A

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ADC 药物完整解决方案 35

For Research Use Only. Not For Use In Diagnostic Procedures

Drug Discovery and Development

Sensitive and Accurate Quantitation of the Antibody-Drug
Conjugate Ado-Trastuzumab Emtansine in Rat Plasma
High-Sensitivity Bioanalysis of ADC using BioBA with M3 MicroLC and QTRAP® 6500+ LC-MS/MS system
Khatereh Motamedchaboki,1 Remco van Soest1 and Ian Moore2
1
SCIEX, USA, 2SCIEX, Canada

Overview
Who Should Read This: Senior Scientists, Lab Directors
Focus: Improved quantitation of total antibody-drug
conjugates (ADCs) using BioBA solution and microflow
liquid chromatography-mass spectrometry (LC-MS/MS).
Goal: Develop an accurate and sensitive trap-and-elute
microflow LC-MS/MS method to improve sensitivity andlinear dynamic range of the BioBA High Capacity Sample
Enrichment method for quantitation of antibody-drug
conjugates in complex matrices.
Problem: Traditional LC-MS/MS methods for the quantitationof antibody-drug conjugates and similar biotherapeutics often

Key Features:deliver insufficient sensitivity. While we have seen improvement• The SCIEX BioBA Kit includes a generic method and all
the reagents necessary for immunocapture and digestion
to quantitate ADCs in plasma

in sensitivity for the antibody-drug conjugate ado-trastuzumab
emtansine quantitation in rat plasma using direct injection
microflow LC-MS/MS,1 this approach results in long sample
loading times, and MS contamination as a divert valve could
not be used.
Results: The SCIEX M3 MicroLC with a QTRAP 6500+ mass
spectrometer provides up to 5X lower limit of quantitation for
the antibody-drug conjugate ado-trastuzumab emtansine in
rat plasma. Quantitation over 5 orders of magnitude linear
dynamic range was achieved with r value of 0.995.

• Microflow LC-MS/MS allows quantitation over a wide linear
dynamic range of 5 orders of magnitude (1ng/ml-100ug/ml)• Up to 4X improvement in s/n ratio enables quantitation at
5x lower concentration than can be achieved with traditional
flow LC-MS/MS• Robust on-line trap-and-elute sample loading and desalting
increases column lifetime and reduces solvent consumption
and costs
Fast-Growing Field: Protein biotherapeutics like immunoglobulin

Key Challenges:

G (IgG)-derived monoclonal antibodies and antibody-drug• Ligand binding assays (LBAs) like ELISA have been utilized
in the past for the quantitation of biotherapeutics suchas monocolonal antibodies (mAbs) and ADCs. Thesemethods have limited linear dynamic range, cross reactivity
and poor reproducibility• Quantitation of mAbs over a wide dynamic range at low
nanogram levels often requires greater sensitivity than canbe achieved with LC-MS/MS with traditional LC flow rates• Scientists who are familiar with quantitation of traditional
small molecule therapeutics and new to analysis of theselarge and heterogeneous proteins conjugates require arobust and sensitive work flow which is easy to implement
and reproducible in different laboratories

RUO-MKT-02-5037-A

36 ADC 药物完整解决方案

conjugates occupy a rapidly increasing share of thepharmaceutical industry due to their lower toxicity, higher potency
and target specificity. Ado-trastuzumab emtansine (Kadcyla)
is the first HER2-targeted treatment for metastatic breast cancer.
It is made of two cancer-fighting drugs, a monoclonal antibody
trastuzumab (Herceptin) which targets HER2 and a lysine
conjugated chemotherapy drug. It is made to bring chemotherapy
inside HER2-positive cancer cells and kill them with less harm
to normal cells.
Due to the heterogeneous nature of lysine conjugatedadotrastuzumab emtansine, several bioanalytical assays,
p1

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including ligand binding assays (LBAs), are required during theemtansine in rat plasma using a hybrid LBA LC-MS/MS

drug development process. Ligand binding assays have limited

approach. The BioBA sample preparation kit and BioBA genericdynamic range, which makes hybrid LBA LC-MS/MS assays

immunocapture strategy is employed, and then followed bya more attractive choice for quantitation of the total antibody.

analysis of signature peptides on a M3 MicroLC coupled to a

These hybrid assays are based on quantitation of signature

QTRAP 6500 mass spectrometer. This method takes advantage

peptides which provide a wide linear dynamic range.

of a trap-and-elute strategy for fast sample injection and desalting

The BioBA High Capacity Sample Enrichment Kit provides a
hybrid LBA LC-MS/MS workflow with a sample preparation
protocol, and a generic method for pharmacokinetic studies ofbiotherapeutics during pre-clinical or phase I-IV studies. The
BioBA workflow (Figure 1) includes the enrichment of mAbs
from plasma using magnetic streptavidin beads conjugated withan anti-Human IgG for enrichment and quantitation of the total
antibody with a generic or specific signature peptide. A target
specific immunocapture strategy can also be employed witha recombinant target protein or an anti-idiotype antibody.

to increase throughput of lower flow rate microflow LC analysis.

Experimental Design
Sample Preparation: 10x spiking solutions of ado-trastuzumab
emtansine were first prepared in 1X BioBA bind/wash buffer
containing 0.01% BSA (bovine serum albumin), then spiked into
Sprague-Dawley rat plasma, K2EDTA (BioreclamationIVT) at
the final concentrations of 0.5-100000 ng/ml. SILuMab (SigmaAldrich), was used as internal standard (IS) and was added tothe plasma samples prior to BioBA immunocapture processing.
A blank and double blank sample was also prepared. The
double blank only had 1X BioBA bind/wash buffer containing
0.01% BSA and the blank sample had rat plasma with additional
internal standard. Spiked plasma samples (50 µL) were mixed
with internal standard and processed based on the BioBA genericmethod2 with some modified steps to reduce background signal
and improve signal to noise (s/n) ratio in order to utilize theadvantage of higher sensitivity provided by microflow LC. The
modified steps include; an extra hour of incubation of conjugatedbeads with sample (2 hrs vs 1 hr in generic method) followedwith two wash steps with 500 µL of BioBA bind/wash buffer and a
500 µL of 50 mM ammonium bicarbonate instead of the two wash
steps with 200 µL of BioBA bind/wash buffer in generic method.

Figure 1. BioBA Immuno-Enrichment and Sample Processing
Workflow. BioBA workflow includes immuno-enrichment ofmAbs and ADCs, reduction, alkylation, digestion and a generictraditional flow LC-MS/MS method for the quantitation of themAbs and ADCs in plasma.

BioBA magnetic beads offer several advantages including:ease of handling, scalability, improved sample recovery, parallel

Also a 1:10 dilution of stock BioBA digestion buffer (500 mM
ammonium Bicarbonate) was used for preparing iodoacetamide,
Trypsin/Lys-C and a mass spec compatible surfactant was used,with 1ug total Trypsin/Lys-C for each sample digestion.
Traditional Flow LC

Microflow LC

Time (min)%BTime (min)%Bprocessing of samples using a variety of magnetic stands

0

5

0

3

and use in high-throughput formats with robotics.3 Following

0.7

5

0.7

5

enrichment, the proteins are eluted, reduced, alkylated, and

0.8

10

0.8

10

3.5

25

3.5

25

5

40

5

40

5.1

95

5.1

95

5.9

95

10.0

95

6

3

10.1

3

7

3

15

3

digested before detection using MRM based LC-MS/MS analysis
of generic or signature peptides of mAbs of interest. The trypsin/lys-C enzyme mixture allows for the most efficient digestion.
Digestion can be completed within three and half hours,
enabling the workflow to be completed in a single day.1
In this application note we describe a robust and sensitive
workflow for total antibody quantitation of ado-trastuzumab
p2

RUO-MKT-02-5037-A

Table1. Gradients used for Traditional and Microflow LC-MS/MS.

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ADC 药物完整解决方案 37

Traditional Flow Liquid Chromatography: A Shimadzu

Traditional Flow LC

Microflow LC

Electrode ID

100 µm25 µm

Curtain Gas

30

20

Collision GAS

High

High

IonSpray Voltage

5500

5000

at a flow rate of 0.5 ml/min. Wash solvent for the autosampler

Temperature

600

250

was 20/20/60 methanol/acetonitrile/IPA. Injection volume was

Ion Source Gas 1

50

20

25 µL, and the column was kept at 40° C.The gradient method

Ion Source Gas 2

65

20

Prominence HPLC system with two LC-20AD pumps, CTO-20A
column oven and a SIL-20AC autosampler was used. The
column was a 100 x 2.1 mm Kinetex C18 2.6 µm 100 Å column(Phenomenex). Mobile phase A, water with 0.1% formic acid,and mobile phase B, acetonitrile with 0.1% formic acid was used

used is listed in Table 1.

MS Parameters

Table3. Source Parameters

Microflow Liquid Chromatography: A SCIEX M3 MicroLC-TE
system, with two microLC gradient pumps and an integrated

with a 25 µm ID electrode (SCIEX). The transitions and MS

autosampler was used in combination with a source mounted

parameters were optimized using DiscoveryQuant software

column oven (SCIEX). A 10 x 0.3 mm trap column packed with(SCIEX) and kept constant for both the traditional flow and5 µm 120 Å ChromXP C18 CL and an analytical column 50 x

microflow LC experiments. The source and gas parameters are

0.3 mm HALO Peptide ES-C18 2.7 µm 160 Å column was used

listed in table 3. The MS parameters are listed in table 4, and(SCIEX). Mobile phase A in the analytical gradient was water

MultiQuant™ 3.0.2 software (SCIEX) was used for data analysis.

with 0.1% formic acid, mobile phase B was acetonitrile with 0.1%

Sample for both microflow and traditional flow LC-MS/MS

formic acid (Table 1) with flowrate of 10 µL/min. The column

analysis was prepared on the same day to exclude variations intemperature was set to 40° C. Injection volume was 25 µL, andresponse due to sample preparation. Three replicate LC-MS/MS

the autosampler needle and valve wash consisted of two cycles

injections were acquired for both the traditional flow and trap-and-using mobile phase B, followed by one cycle using mobile phase

elute microflow LC analysis.

A. For trapping conditions, mobile phase A in the loading gradient
was water with 0.1% formic acid, mobile phase B was acetonitrile

Results and Discussion

with 0.1% formic acid. Sample was loaded from the injection loop

Improved Sensitivity: The calibration curve of Ado- trastuzumab

onto the trap column using 100% A for two and half minute at

Emtansine standards in rat plasma matrix (0.5-100,000 ng/

50 µL/min flow rate. The trap was then washed with 95% Bml) was generated using MultiQuant Software (Table 5, 6) for

followed by 100% A each at 50 µL/min for 5 minutes after every

data acquired by traditional and microflow LC using signature

injection (Table 2).

tryptic peptides IYPTNGYTR and FTISADTSK. Figure 2 shows

Mass Spectrometry and Data Processing: A SCIEX 6500
QTRAP with IonDrive™ Turbo V source was used. For the
microflow LC experiments, the standard electrode was replaced

the calibration curve for signature peptide IYPTNGYTR using
Microflow LC. Figure 3 shows the extracted ion chromatograms(XIC’s) of the signature peptide (FTISADTSK) used forquantitation of trastuzumab in both methods at the 5 ng/ml
and 10 µg/ml level. S/N ratio was improved by 4 fold for bothTime (min)

%B

0

0

were determined using the requirements of precision < 20% and10accuracy between 80 and 120% at LLOQ, and at any higher

2.50

concentration a precision <15% and accuracy between 85% and

2.6

95

115%. LLOQ improved by a factor of 5 using the microflow LC

895

trap-and-elute method using both signature peptides and the

8.1

0

15

0

Table2. Gradient used for the Trap Wash Workflow.

RUO-MKT-02-5037-A

peptides using microflow LC. The LLOQ’s for both methods

generic peptide DTLMISR. For the traditional LC method thelimit of quantification (LOQ) was 5 ng/ml where the LOQ of 1 ng/
ml was achieved by microflow LC. Both the traditional flow andmicroflow LC methods showed good linearity with r >0.99.

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
p3

38 ADC 药物完整解决方案Transitions

Q1 Mass (Da)Q3 Mass (Da)Time (msec)

Signature Peptide Sequences

DP (volts)

CE (volts)

CXP (volts)

1

423.2

629.4

20

S-DTLMIS[R].heavy 1

40

24

18

2

423.2

516.3

20

S-DTLMIS[R].heavy 2

60

22

17

3

418.2

619.3

20

G-DTLMISR.1

60

22

15

4

418.2

506.2

20

G-DTLMISR.2

40

20

18

5

542.8

405.8

20

S-IYPTNGYTR.1

120

23

10

6

542.8

808.2

20

S-IYPTNGYTR.2

60

16

11

7

485.2

608.2

20

S-FTISADTSK.1

50

25

25

8

485.2

721.3

20

S-FTISADTSK.2

90

20

15

Table 4. Transitions and MS Parameters for Signature Peptides of Ado-Trastuzumab Emtansine and Internal Standard. Peptide transitions
in bold were used for quantification and the 2nd peptide transitions were used for confirmation with S-DTLMIS[R].heavy 1 transition used
for internal calibration.
Microflow LC
ActualConcentration

Traditional Flow LC

Mean Measured
Concentration (ng/ml)

Accuracy (%)

1

1.00

100.42

3.24

-

-

-

5

4.87

96.92

7.61

4.63

92.63

7.48

CV (%)

Mean Measured
Concentration

Accuracy (%)

CV (%)

10

10.43

104.29

9.37

11.50

115.00

7.30

50

46.94

93.89

6.35

49.65

99.29

8.05

100

89.38

89.38

2.41

99.52

99.52

1.62

1000

921.6

92.16

4.38

921.6

92.16

0.71

10000

10930

109.27

0.56

10120

101.24

4.00

25000

27720

110.86

3.04

26270

105.06

2.04

50000

54780

109.57

3.04

52720

105.441.41

100000

93250

93.25

4.35

89650

89.65

2.93

Table 5. Quantitation Curve for Standard Ado-Trastuzumab Emtansine using Traditional Flow and Microflow LC-MS/MS. MultiQuant analysis
based on peptide IYPTNGYTR resulted in an accurate quantitation with single digit CV% and r value of 0.995. Micro LC-MS/MS provides 5X
increased in sensitivity with wider linear dynamic range as compared to Traditional LC-MS/MS.
Microflow LC

Traditional Flow LC

Actual Concentration (ng/ml)

Mean Measured
Concentration (ng/ml)

10.9694

96.94

8.04

-

-

-

2

2.109

105.43

6.80

-

-

-

2.5

2.552

102.10

4.65

-

-

-

5

4.73

94.71

8.04

4.15

83.12

19.83

Accuracy (%)

CV (%)

Mean Measured
Concentration (ng/ml)

Accuracy (%)

CV (%)

10

10.44

104.35

3.27

10.44

104.42

15.65

50

49.91

99.82

2.14

52.25

104.49

2.23

100

112.6

112.59

6.81

122.4

122.41

4.11

1000

1052

105.181.18

1017

101.67

0.91

10000

10150

101.49

3.68

10430

104.25

4.00

25000

23040

92.15

3.59

26230

104.93

3.63

50000

42620

85.24

3.94

48300

96.60

2.68

Table 6. Quantitation Curve for Standard Ado-Trastuzumab Emtansine using Traditional Flow and Microflow LC-MS/MS. MultiQuant analysis
based on peptide FTISADTSK resulted in an accurate quantitation with single digit CV% for micro LC data and CV of 20% at LLOQ for Traditional LC data with r value of 0.995 and 0.998 for Microflow and traditional flow LC. Micro LC-MS/MS provides 5X increased in sensitivity withwider linear dynamic range as compared to Traditional LC-MS/MS.
p4

RUO-MKT-02-5037-A

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ADC 药物完整解决方案 39

Microflow LC

Calibration for IYPTNGYTR_2: y = 0.00142x + 0.00518(r = 0.99551) (weighting 1/x^2) 1 ng/ml-100 µg/ml

130
120
110
100

r=0.995

Area Ratio

90
80
70
60
50
40
30
20
10
0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

Concentration Ratio
Figure 2. Quantitation Curve for Standard Ado-Trastuzumab Emtansine using Signature Peptide IYPTNGYTR. MultiQuant quantitation
curve using peptide IYPTNGYTR resulted in an accurate quantitation with single digit CV% and r value of 0.995 using peak area and1/x^2 weighting.

2.00e51.50e51.00e59.50e49.00e4

2.3e6
2.2e6

Microflow LC

2.0e6

10000 ng/ml

1.9e6
1.8e6

8.50e4

1.7e6

8.00e4

1.6e6

7.50e4

1.5e6

7.00e4

1.4e6

Intensity

6.50e4

Intensity

Traditional Flow LC

2.1e6

10000 ng/ml

6.00e45.50e45.00e44.50e4

1.3e6
1.2e6
1.1e6
1.0e6
9.0e5

4.00e48.0e5

3.50e47.0e5

3.00e46.0e5

2.50e45.0e5

2.00e44.0e5

1.50e4

3.0e5

1.00e42.0e5

0.00e4

1.0e5

00
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

0.0

0.5

1.0

1.5

Time (min)7000

Intensity

6000

2.045

Microflow LC

2.0

2.5

3.0

3.5

4.0

Time (min)900

Traditional Flow LC

5 ng/ml

800

5 ng/ml

S/N=20

700

S/N=5

5000

600

4000

500

2.229

400

3000

300
2000

200

1000

100

0

0
1.8

1.9

2.0

Time (min)

2.1

1.8

2.0

2.2

Time (min)Figure 3. Signal Intensity Improvement using M3 MicroLC. A 4 fold improvement ins/n ratio was achieved using micro flow LC. XIC data for micro flow and traditional
flow LC for 10 µg/ml (Top) and 5ng/ml (Bottom) is shown here with improved s/n.

RUO-MKT-02-5037-A

40 ADC 药物完整解决方案

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
p5

Column Carryover: Carryover was determined by injecting thedigest of the immunocaptured extract from a blank+IS plasma
sample after an injection of the ULOQ of 100,000 ng/ml using
traditional flow LC-MS/MS, and The trap-and-elute microflow LC
method. Observed carryover was 0.0075 and 0.007 respectively.
The carryover was 1/3 of the response at the LLOQ (1 ng/ml)
which is slightly higher than required 20%. Additional washingof trap and column is required to reduce the carryover if wider

References
1. Antibody Drug Conjugate Bioanalysis using the BioBA
Solution. SCIEX Technical Note, RUO-MKT-02-4864-A
2. BioBA High Capacity Sample Enrichment Kit- Protocol,
RUO-IDV-05-3429-A | 5046523 A
3. Automating Biologics Signature Peptide Quantitation,
SCIEX Technical Note, RUO-MKT-02-4029-B

dynamic range is required.

4. Improving Sensitivity in Bioanalysis using Trap-and-EluteMicroLC-MS/MS. SCIEX Technical Note, RUO-MKT-02-2849

Conclusion

5. Improving Sensitivity for an Immunocapture LC-MS/MS Assayof Infliximab in Rat Plasma Using Trap-and-Elute Micro
LC-MS/MS, SCIEX Technical Note, RUO-MKT-02-3413

Up to 5x lower LLOQ was achieved using a trap-and-elute
microflow LC-MS/MS method at 10 μL/min, compared to using
a direct inject traditional flow LC-MS/MS method at 500 μL/
min for the quantitation of ado-trastuzumab emtansine using
the signature tryptic peptides IYPTNGYTR and FTISADTSK
in samples prepared using BioBA magnetic bead based
immunocapture.
The trap-and-elute method ensures similar throughput while
injecting the same 25 μL of extracted sample, while protecting
the column and MS from contamination. Similar results wereobserved for infliximab using M3 MicroLC with SCIEX 6500
QTRAP.4,5

Who is SCIEX? SCIEX company’s global leadership and worldclass service and support in the capillary electrophoresis andliquid chromatography-mass spectrometry industry have madeOptimized BioBA sample preparation protocol in combination

it a trusted partner to thousands of the scientists and lab analysts

with microflow LC provides wider linear dynamic range of 4.5 forworldwide who are focused on basic research, drug discovery

traditional flow and linear dynamic range of 5 for microflow as

and development, food and environmental testing, forensics

compared to the data obtained from generic method optimized

and clinical research.

for traditional LC using IYPTNGYTR peptide transitions.1
This workflow offers a solution for applications where mAb’s
need to be quantified in low concentrations and/or when

Contact Us: sciex.com/contact-us

sample volumes are limited.

AB Sciex is doing business as SCIEX.
© 2017 AB Sciex. For research use only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of the AB Sciex Pte. Ltd. or their respective owners.
AB SCIEX™ is being used under license.
RUO-MKT-02-5037-A 01/2017

RUO-MKT-02-5037-A

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International Sales
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For our office locations please call the division
Phone 508-383-7800
headquarters or refer to our website atsciex.com
sciex.com/offices

ADC 药物完整解决方案 41

Metabolite Identification of Payload Species of Antibody Drug
Conjugates with Noncleavable Linkers using MetabolitePilot™ 2.0
Software and TripleTOF® 6600 System
Ian Moore and YuanQing Xia
, Concord, ON, Canada


Metabolism studies of ADCs must consider manydifferent species: payload, payload plus linker,
and payload plus linker conjugated an amino acidor peptidePotential biotransformations of all the above
species must also be considered

Missing, lowlevel drug metabolites in complex
biological matrices



Incomplete metabolite information leading torepeated sample analysis and decreased
productivity



Maintaining data quality for both quantitative andqualitative
analysisinahighthroughput
environmentThe SCIEX Advanced Biotransform Solution featuring the new
MetabolitePilot 2.0 software, TripleTOF® 6600 and Exion AD LC system.











A dedicated ADC processing workflow that
performs targeted searching for all components of
the ADC: payload, linker and antibody

Dedicated ADC structure assignment workspaceand MS/MS fragment interpretation workspace
where both payload and peptide are considered

Integrated correlation function allows comparisonof metabolism across multiple samples for timecourse studies or interspecies comparison, using
both MS and analog data.

Antibody drug conjugates (ADC) are designed for targeted
delivery of a cytotoxic molecule to a diseased orcancerous tissue. ADCs are composed of three

42 ADC 药物完整解决方案


















































RUO-MKT-02-5472-A



Multiple peak finding strategies utilizing MS and
MS/MS information for targeted and untargeted
searching for drug related metabolites







The structures of the linker plus payload of
trastuzumab emtansine (TE) and SigmaMAb Antibody Drug Conjugate
mimic (MSQC8), sites of conjugation to the mAb are highlighted in red.

p1
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components: the antibody used for selective targeting, thecytotoxic drug and a linker to connect the two. The goal indesign and development of ADCs is to maximize delivery
to the targeted tissue and minimize delivery to untargetedtissues. There are currently two FDA approved ADC
therapies on the market for the treatment of cancer andresearch efforts in ADC technology have grown across thepharmaceutical industry to encompass therapeutic areas
other than cancer.
There are two classes of antibody drug conjugate (ADC)
linkers: cleavable and noncleavable. Cleavable linkers arelabile and are designed to release the unmodified
cytotoxin upon internalization by the target cell due to achange in chemical environment (e.g. pH) or a specificprotease inside the cell. A noncleavable linker does notcontain any mechanism for payload release and relies on
antibody degradation through cellular processing forrelease of the active species. In these ADCs the active
species is the cytotoxic payload with the linker and one ormore amino acids from the antibody.
In addition to studying the usual small molecule
metabolism of the payload molecule separate from theantibody, a necessary step in the development of ADCs is
the identification of the active payloads of both cleavableand noncleavable linkers from the ADC and any
metabolism products involving active forms of likely
payload products. In addition to plasma or serum
incubations,   systems of purified cellular fractions(lysosomes or S9) or purified enzymes are often used tocharacterize active payloads released from the ADC.
The number of possible species that can be generated
when considering the metabolic/catabolic fate of an ADC
is vast. One must consider payloadlinker species with
one or more amino acids attached plus potential
biotransformations of the linker and/or payload plus any
cleavage or degradation products. To address thischallenging task SCIEX has introduced a specific ADC
TM
workflow in the new MetabolitePilot 2.0 software. In thistech note the ADC specific workflow features of the new
MetabolitePilot 2.0 were used to identify payload products
and metabolites from two noncleavable linker ADCs after
incubation with human liver lysosomes and S9 fraction.



Trastuzumab
emtansine(TE)
waspurchased
commercially. SigmaMAb Antibody Drug Conjugate mimic(MSQC8) was provided by SigmaAldrich. Human liver
lysosomes (HLL), S9 fraction and catabolism buffer werekindly provided by Xenotech.

RUO-MKT-02-5472-A

ADCs were incubated with either HLL (0.25 mg/mL) or S9
fraction (1 mg/mL). ADC concentration was 50 g/mL ineach incubation. Control samples included ADC without
HLL or S9 and HLL or S9 without ADC. Total reaction
volume was 200 l, consisting of 70 L water, 20 L
catabolism buffer (10x), 100 L HLL or S9 and 10 L
analyte stock solutions.
At different time points (1, 2, 4 and 26 hours) a 50 Laliquot was removed and processed. Sample processing
involved: adding 250 L of cold ACN, then centrifuging at
14 x  for 15 minutes. The supernatant was removed to aclean tube and dried under vacuum at 37 °C. Dried
samples were then reconstituted in 50 L 5% mobile
phase B for injection.


Data was collected on a SCIEX TripleTOF® 6600 Systemusing SWATH® acquisition. TOF MS acquisition coveredm/z 300 to 2000. MS/MS data were collected using 25
variable SWATH windows focused around the m/z of thelinker payload plus the amino acid of conjugation. Five 13
Da windows centered around the parent mass (1103 for T
E and 789 for MSQC8) and expanding to windows of 50,
100 and 200 to cover a mass range of 50 to 2000 Da. A
collision energy of 40 with collision energy spread of 15
was used. Total scan time for the SWATH method was
850 ms.


Samples were chromatographed on a SCIEX Exion AD
system using a Phenomenex Aeris C18 Peptide column(2.0 x 150 mm), 1.7 m. Elution was performed using alinear gradient from 5% to 80% B over 26 mins. Mobile
phase A was water with 0.1% formic acid and mobile
phase B was acetonitrile with 0.1% formic acid.


An ADC specific processing method was created in
MetabolitePilot 2.0 software for both TE and MSQC8.
Briefly, the first step is to provide the chemical structure of
the payload and linker, designate the amino acid of
attachment, the type of conjugation chemistry involved
and then enter the antibody sequence. Next the size andidentity of protein/peptide sequence fragments from theantibody linked to the payload were chosen. Nonspecificcleavage of two peptide bonds plus disulfide bondcleavage was used and up to five amino acids wereconsidered. Then a biotransformation list and set ofcleavage metabolites of the payload was chosen. Since
the incubation in HLL and S9 occurs at ~pH 5 in a
reducing environment the biotransformation list contained

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p2

ADC 药物完整解决方案 43

only: loss of water, demethylation, internal hydrolysis andthe parent (payload plus linker). Phase I and II metabolismof the parent was not considered for this  system. A
reference MS/MS spectrum was available for DM1+MCC
and used to designate characteristic product ions andneutral losses. A reference spectrum for the MSQC8
payload was not available.
Peak finding for both ADCs included TOF MS with bothpredicted and generic peak finding algorithms. The TOF
MSMS peak finding algorithm was used for TE since a
reference MS/MS spectrum was available, at least 2
characteristic product ions (485.22, 547.22) and 1 neutral
loss were selected and the advanced MS/MS filter was set
to 1 unit below confident. A retention time window of 3 to
29 minutes was used to search for metabolites products.
A sample to control comparison ratio of >3 was used andmetabolites up to a charge state of 3 were considered.


TM

The MetabolitePilot 2.0 batch workspace was used tosetup and process the SWATH data collected fromincubations of both ADCs with HLL and S9 incubations at
all time points using the data processing parametersdescribed above. Both control samples were used during
processing of each time point. The batch processing tableof MetabolitePilot 2.0 has been expanded from 30 to 200
rows to increase throughput and the results from eachsample may be viewed individually in the results
workspace as they are completed before the whole batch
is finished.
A total of 59 potential metabolites of TE were found in the
26 hour time point of the incubation with HLL within theretention time window of 3 to 29 minutes. As TE contains
a noncleavable linker the released payload species
consists of DM1MCC plus a lysine residue. Using theresults workspace of MetabolitePilot 2.0 Software thisreleased product was found at retention times 15.01 and15.27 minutes as the released payload is a pair ofdiastereomers (Figure 3). The released payload wasfound in the TOF MS spectra as singly charged anddoubly charged protonated species and as the singly
charged sodium adduct all within ±2.0 ppm (m/z1103.4763, 552.2145 and 1125.4573).

  The results workspace of MetabolitePilot 2.0 software
displaying results from the incubation of TE with human liver lysosomes.
The released payload species (payload+linker+lysine) was found at
retention times 15.01 and 15.27 minutes as a pair of diastereomer peaks.
The structure of the payload and linker species is displayed on the left
with the amino acid of conjugation below. Also displayed is the MS and
MS/MS spectra of the released payload species.

  The interpretation results workspace of MetabolitePilot 2.0
software displaying results of the released payload species(payload+linker+lysine) from the incubation of TE with human liver
lysosomes. In this workspace product ions in the MS/MS spectra areassigned to the identified metabolite. There are separate assignments forboth the small molecule payload and sequences of the mAb.

After identification of the released payload species in the
results workspace further confirmation was performed
using MS/MS in the interpretation workspace (Figure 4).
Once a putative metabolite is identified the structure of the

RUO-MKT-02-5472-A

44 ADC 药物完整解决方案

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
p3

payload and linker is loaded into the fragmentinterpretation workspace and the putative peptide
sequence is loaded into the sequence workspace and theresidue of conjugation is assigned. Next the assign
fragments feature is used to calculate potential structures
for the fragments and neutral losses in the MS/MS
spectrum. For the singly charged released payload
species 4 common product ions (indicated in orange) were
found in the MS/MS at 140.0701, 467.2086, 485.2209 and
547.2208 within ±4.0 mDa. Three of the four common
product ions had structures proposed as indicated in boldon the chemical structure.

fold between the 4 and 26 hour time points. A similar foldincrease was seen for the S9 incubations. The dipeptide
payload species were seen predominantly in the 4 hourand 26 hour samples and did not increase in peak area as
rapidly as the released payload plus lysine. The rate ofappearance of released payload species is a complex
proposition due to the heterogeneous nature of the TE
molecule with DAR species from 0 to 8 and different lysine
residues involved in conjugation within each DAR species.

A similar workflow was followed to identify and confirm
other released payload species containing larger portionsof the antibody sequence in the 26 hour sample. Fourother catabolites containing the unmodified DM1MCC
conjugated to a dipeptide sequence were found that had
at least two product ions in common and appeared as a  List of released payload plus linker species from the
incubation of TE with HLL and S9.





Parent17.6

Parent+YK

15.17

15.40

Parent+K

15.01

15.27

Parent+KV

15.1

15.4

Parent+KS

15.0

15.3

Parent+KA

14.8

15.1





  The correlation workspace of MetabolitePilot 2.0 software
displaying results of the released payload species (payload+linker+lysine)from the incubation of TE with human liver lysosomes. In this workspace
overlaid XICs from the LC chromatogram, MS and MS/MS spectra areoverlaid. The most abundant species found was payload+linker+lysine,
payload+linker plus dipeptide fragments containing lysine were also
observed.

pairs of diastereomeric peaks. Table 1 lists their identity
and sequence. No catabolites conjugated to anythinglarger than a dipeptide were confirmed in the sample, thislikely due to a combination of the length of the incubationand the sample workup which involved an organic
precipitation.
TM

2.0
The correlation workspace of MetabolitePilot
software was next used to confirm the presence of theidentified metabolites across the assay time points andbetween liver fraction experiments. The correlation
workspace has an interactive graph window where results
can be displayed as an x, y plot, bar graph or table. In
addition to the correlation workspace overlaid XIC’s, TOF
MS and TOF MS/MS spectra are also displayed for the
chosen metabolites/catabolites. Figure 5 displays thecorrelation plot of the released payload plus lysine fromthe HLL incubation. It was also observed in the 2 and 4
hour samples but not in the 1 hour time point. The peakarea of the compound (15.01 and 15.27 peaks) increasedby 4fold between the 2 and 4 hour time points and by 5

RUO-MKT-02-5472-A

MSQC8 is a cysteine linked ADC that is not a therapeutic
molecule but an ADC mimic. A reference MS/MS
spectrum was not available for the dansyl fluorophore plus
linker so peak finding was performed using predicted
metabolites and generic peak finding. A total of 119
potential metabolites and catabolites were found within theretention time window of 3 to 25 minutes and ±5.0 ppm.
The linker in MSQC8 is also a noncleavable linker andthe released payload species for this ADC mimic would be
the dansyl fluorophore plus linker and cysteine. This
molecule is found in both single and doubly charged forms
at 14.92 minutes as a single peak. The interpretation
workspace (Figure 6) was then used to assign peaks in
the MS/MS, this was a critical step as no reference
spectra of the payload and linker was available. The
structural interpretation tool assigned structures to four of
the ions in the MS/MS all within ±2.0 mDa and three of thefour ions contained the dansyl ring.

p4
本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。

ADC 药物完整解决方案 45

  List of ions with m/z shifts of 14 Da found in the MS and
MS/MS spectra of MSQC8 after incubation with HLL and S9.








TOF MS789.3662

775.3502

TOF MS/MS 1

449.2599

435.2390

TOF MS/MS 2

432.2329

418.2311

TOF MS/MS 3

336.1750

322.1596  The interpretation results workspace of MetabolitePilotTM 2.0
software displaying results of the released payload species(payload+linker+cysteine) from the incubation of MSQC8 with humanliver lysosomes. There are 3 product ions with proposed structures
assigned to the payload species that include the dansyl fluorophore:
449.2599, 432.2329 and 336.1750.

Among the list of potential catabolites/metabolites aputative
demethylated
metaboliteofthepayload+linker+cysteine was identified in the TOF MS at
13.05 minutes. Again the interpretation workspace wasused to assign ions in the MS/MS to the metabolite
structure for confident ID. Three ions in the MS/MS
spectra were found with a mass difference of 14 Da whencompared with the ions assigned in the released payload
species. Based on the assigned fragment structures from
TM
MetabolitePilot 2.0 software and their mass accuracies;
the site of demethylation was assigned to thedimethylamino group of the naphthalene ring of the
payload (Figure 6).
The correlation workspace was used to visualize theappearance of both of these metabolites across all time
points and sample sets. The payload+linker+cysteine wasfound in both HLL and S9 fraction and at all time points
andincreased
over
time.
The
demethylated
payload+linker+cysteine were observed in only the 4 and26 hour time points in both the HLL and S9 incubations.



























  Structural assignment of the demethylated MSQC8 released
payload species.

The SCIEX Advanced Biotrans solution with the
TripleTOF™ 6600 system and new MetabolitePilot 2.0
software effectively addresses the complex task of
performing metabolite and catabolite ID on ADCs fromcomprehensive peak detection with SWATH® acquisition
to metabolite finding. MetabolitePilot 2.0 software features
a dedicated ADC processing workflow that performs
targeted searching for all components of the ADC:
payload, linker and antibody plus cleavage metabolites
and biotransformations. This comprehensive knowledge
base combined with untargeted searching for drug related
metabolites and peak finding strategies utilizing MS and
MS/MS information ensures success for scientists
studying ADC metabolism.


Thanks to Chris Bohl of Xenotech for helpful discussions.



AB Sciex is doing business as SCIEX.
© 2017 AB Sciex. For Research Use Only. Not for use in diagnostic procedures.The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners.
AB SCIEX™ is being used under license. 
Document number: RUOMKT025472A

RUO-MKT-02-5472-A

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。

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46 ADC 药物完整解决方案For Research Use Only. Not for use in Diagnostic Procedures.

Technology

The Power of Precision

CE-SDS 和 CIEF 方法对半胱氨酸偶联的抗体偶联药物的分析
任挺钧、陈泓序

CE-SDS和CIEF方法对半胱氨酸偶联的抗体偶联药物的分析
CE & Biopharma，Sciex，China

1、前言
Heterogeneity analysis of cysteine-linked
antibody-drug conjugate by抗体偶联药物（）是一种将化学药物通过连接物（linker）与抗体偶
CE method联形成的药物。它既有抗体的靶向性又有化学药物的强杀伤力，成为肿瘤治疗用药的研究与发展热Antibody-drug conjugates, ADC

点。2011 年 8 月和 2013 年 2 月，ADC 药物 brentuximabvedotin（商品名 Adcetris）和 ado-

任挺钧、陈泓序
Tingjun Ren, Hongxu Chen

trastuzumabemtansine（商品名 Kadcyla）分别获 FDA 批准上市。该两种 ADC 药物上市仅仅是 ADC
研发热潮的缩影，目前，世界范围内有约 60 种 ADC 药物处于临床阶段[1-2]。

CE & Biopharma，Sciex，China

ADC 常见的偶联方式有赖氨酸偶联和半胱氨酸偶联。赖氨酸是抗体中最常见的连接位，其氨
基可以与连接物的羧基反应，得到较为稳定的酰胺键。一般的，抗体中约有 80 个赖氨酸，其中 10个赖氨酸可作为连接位点。对于半胱氨酸偶联的方式，由于抗体表面没有游离的巯基，可以通过对

打断抗体链间已有的二硫键，形成游离的巯基，实现偶联[3]。图1
断裂重链或者轻链间的二硫键，在抗体编码中加入半胱氨酸，另一方面，可以通过用还原剂打断抗Key Words: Antibody-drug conjugate, Cysteine-linked, CE-SDS,[4][3]
体链间已有的二硫键，形成游离的巯基，实现偶联。图 1 简单描述了赖氨酸和半胱氨酸可能的位。
简单描述了赖氨酸和半胱氨酸可能的位点的结构示意图
cIEF, Heterogenecity analysis
点的结构示意图[4]。

Abstract: Antibody drug conjugates (ADCs) are innovative

biopharmaceutical emerging in recent years. And this kind of

biotherapeutics show unprecedented intrinsic heterogeneity, so
separation technologies play a critical role in their characterization.
In this tech note, heterogeneity of a Cys-linked ADC is analyzed byusing CE-SDS and cIEF. In CE-SDS, we established the reduced andnon-reduced method to evaluate the purity of the ADC and explain

图 1 半胱氨酸和赖氨酸偶联位点及连接方式示意图the meaning of each peak in CE-SDS. In cIEF method, we compared
the change of charge variant distribution between ADC and themonoclonal antibodies. Both two method can give us the referenceof quality control assessment to the ADCs.

图1. 半胱氨酸和赖氨酸偶联位点及连接方式示意图。
ADC 药物的质量控制与单克隆抗体类药物分析类似，主要针对其异质性进行分析，包括大小异质性及电荷异质性分析。而毛细管电泳技术近年来以其分离度高、分析速度快及自动化程度高成

ADC药物的质量控制与单克隆抗体类药物分析类似，主要针对为了生物制药质控中不可或缺的分析技术，其中主要包括毛细管凝胶电泳（CE-SDS）进行纯度分

其异质性进行分析，包括大小异质性及电荷异质性分析。而毛细

析（大小异质性）和毛细管等电聚焦（cIEF）进行等电点和电荷异质性分析。

前言管电泳技术近年来以其分离度高、分析速度快及自动化程度高成

本研究采用 Sciex 公司的 PA800 Plus 生物制药分析系统，以通过还原剂打开二硫键后通过半胱

为了生物制药质控中不可或缺的分析技术，其中主要包括毛细管氨酸偶联的 ADC 为例，建立和完善了 CE-SDS 和 cIEF 对该种 ADC 药物的分析方法，对其大小异

抗体偶联药物（Antibody-drug conjugates, ADC）是一种将化凝胶电泳（CE-SDS）进行纯度分析（大小异质性）和毛细管等电质性和电荷异质性进行了分析，为 ADC 药物的质量控制提供参考。

学药物通过连接物（linker）与抗体偶联形成的药物。它既有抗体

的靶向性又有化学药物的强杀伤力，成为肿瘤治疗用药的研究与发展热点。2011年8月和2013年2月，ADC药物brentuximabvedotin（商品名Adcetris）和ado-trastuzumabemtansine（商品名
Kadcyla）分别获FDA批准上市。该两种ADC药物上市仅仅是ADC研发热潮的缩影，目前，世界范围内有约60种ADC药物处于临床阶
段[1-2]。
ADC常见的偶联方式有赖氨酸偶联和半胱氨酸偶联。赖氨酸
是抗体中最常见的连接位点，其氨基可以与连接物的羧基反应，

聚焦（cIEF）进行等电点和电荷异质性分析。

本研究采用Sciex公司的PA800 Plus生物制药分析系统，以通过还原剂打开二硫键后通过半胱氨酸偶联的ADC为例，建立和完善了
CE-SDS和cIEF对该种ADC药物的分析方法，对其大小异质性和电荷异质性进行了分析，为ADC药物的质量控制提供参考。

2、仪器和试剂
2.1 仪器得到较为稳定的酰胺键。一般的，抗体中约有80个赖氨酸，其中毛细管电泳仪（PA800 Plus生物制药分析系统，PDA和UV10个赖氨酸可作为连接位点。对于半胱氨酸偶联的方式，由于抗检测器，Sciex）；5430型台式高速冷冻离心机（Eppendorf，

体表面没有游离的巯基，可以通过对断裂重链或者轻链间的二硫

Germany）；MA3涡旋混合器（IKA，Germany）；Thermomixer

键，在抗体编码中加入半胱氨酸，另一方面，可以通过用还原剂

compact型恒温混匀仪（Eppendorf，Germany）。

RUO-MKT-02-10284-ZH-A
RUO-MKT-02-10284-ZH-A

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
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ADC 药物完整解决方案 47

The Power of Precision

2.2 试剂

表1. 分析多个样品时配制的 cIEF Master Mix。

IgG Purity/Heterogeneity Assay Kit（PN A10663）和Advanced
试剂

每个样品
需要的量
（µL）

样品数量

M6250）、碘乙酰胺（PN I-1149）、磷酸（PN 345245）、氢氧化

3 M 尿素-cIEF 胶

180

×（____ + 1）

钠（PN 221465）、氢氧化铵溶液（PN 221228）、亚氨基二乙酸

cIEF Kit（PN A80976）采购自Sciex。两性电解质（PN 17-0456-01）
采购自GE Healthcare。其余所有试剂均为分析纯。巯基乙醇（PN

3-10 两性电解质

12

×（____ + 1）

（PN 220000）、精氨酸（PN A5006）冰醋酸（PN 537020）和尿

阴极稳定剂

20

×（____ + 1）

素（U0631）均采购自Sigma-Aldrich。实验所需用水均采用二次去

阳极稳定剂

2

×（____ + 1）

pI 标准品12

×（____ + 1）

pI 标准品22

×（____ + 1）

pI 标准品32

×（____ + 1）

离子水（Double Deionized Water, DDI Water）。Ultracel YM-10 滤膜（Millipore PN A11530）。
1）CE-SDS：

总体积
（µL）

碘乙酰胺溶液：取46 mg 碘乙酰胺与1.5 mL EP管中，加入1 mL
DDI Water溶解混匀。现用现配，避光保存。

3、方法

2）cIEF：阳极液、 阴极液、化学迁移剂、酸性冲洗液、4.3 M尿素溶
液、阳极稳定液、阴极稳定液、3 M Urea-cIEF Gel溶液的配制方法

3.1 CE-SDS：
分离条件：毛细管：非涂层毛细管、50 µm内径、30 /20 cm
总/有效长度；进样：-5 kV，20 s；分离：-15 kV， 40 min；数据采[5]

参照Sciex cIEF试剂盒说明书 。

集频率：4 Hz；样品室温度：25 ℃；毛细管温度：25 ℃；窗口狭
缝：2号（100×200 µm）；PDA检测器，检测波长：220 nm；

2.3 样品及前处理
裸抗和其通过半胱氨酸偶联的ADC样品均来自某合作单位。
取 400 µL 样品入超滤管（截留分子量10 kD）中，14000 rpm，
离心10 分钟；加入300 µL DDI water，14000 rpm，离心10 分钟；再加入300 µL DDI water，14000 rpm，离心10 分钟。调整样品最终浓度为5.0 mg/mL，盐离子浓度低于50 mM。

新毛细管的预平衡：碱性冲洗液，20 psi 压力冲洗10分钟；酸性冲洗液，20 psi 压力冲洗5分钟；DDI Water，20 psi 压力冲洗2分
钟；Gel Buffer，70 psi 压力冲洗10分钟。最后，以-15 kV电压平衡
10 分钟。
每针之间冲洗：碱性冲洗液，70 psi 压力冲洗3分钟；酸性冲
洗液，70 psi 压力冲洗1分钟；DDI Water，70 psi 压力冲洗1分钟；1）CE-SDS：

Gel Buﬀer，70 psi 压力冲洗10分钟。

取两支1.5 mL EP管，分别取20 μL样品于两支EP管中，然后各

3.2 cIEF：

加入75 μL的Sample buffer使得体积为95 μL。然后再向两支EP管
中分别加入5 μ L 2-巯基乙醇（还原法）和碘乙酰胺溶液（非还原法）。盖上瓶盖，充分混合后离心1 分钟。然后置于70 ℃水浴10分钟。随后离心去除气泡，吸取100 μL置于样品管中。

分离条件：毛细管：中性涂层毛细管、50 μm内径、30 /20 cm
总/有效长度；进样：25 psi，99 s；聚焦：采用200 mM磷酸和300
mM氢氧化钠作为阳极液和阴极液，25 kV，15分钟；化学迁移：
用350 mM醋酸来代替阳极液，30 kV，30分钟。数据采集频率：2）cIEF：

4 Hz；样品室温度：10 ℃；毛细管温度：25 ℃；窗口狭缝：2 号

分析多个样品的时候，推荐预先配制样品混合物母液（Master（100×200 µm）；UV检测器，检测波长：280 nm。

Mix），如表1。Master Mix 可简化样品配制并且使误差最小化。在
表格中输入样品的数量，然后样品数量加1 计算，按照计算的各个
溶液的体积进行配制Master Mix。

新毛细管的预平衡：酸性冲洗液，50 psi 压力冲洗5分钟；DDI
Water，50 psi压力冲洗2分钟；cIEF Gel，50 psi压力冲洗5分钟。
每针之间冲洗：4.3 M尿素溶液，50 psi压力冲洗3分钟；DDI

随后，取10 µL样品与240 µL上述溶液进行混合，涡旋混匀。

Water，50 psi压力冲洗2分钟。

取出200 µL置于样品管中。

RUO-MKT-02-10284-ZH-A
RUO-MKT-02-10284-ZH-A

48 ADC 药物完整解决方案

本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
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The Power of Precision

4、结果与讨论

非还原CE-SDS分析：
由于该ADC药物是通过半胱氨酸偶联的，在进行偶联时，需利

4.1 CE-SDS法进行纯度（大小异质性）分析

用还原剂打开抗体结构中的二硫键。因此，ADC 药物会解离成多

CE-SDS因其分析速度快，重现性好，分辨率高，稳定性强种碎片组分，且碎片类型与小分子药物的偶联位点相关。其非还

等特点，已经成为检测蛋白类药物分析大小变异体的首选方法。

原CE-SDS分析谱图见图3。根据分子量的估算，1-5号峰分别为经过图 2 某 ADC 药物的还原法 CE-SDS 电泳谱图。电泳条件见第 3 节。

药典中要求对单抗药物的还原（Reduced, R）和非还原（Non-偶联的轻链( LC)，重链( HC)，重轻链（HL）、重重链（HH）、重[6]
Reduced, NR）样品进行分别检测 。两种分析方法中样品均需要[1]。
重轻链（HHL），6号峰为完整的裸抗
非还原 CE-SDS 分析：

与十二烷基硫酸钠（sodium dodecyl sulfate, SDS）络合，但还原由于该 ADC 药物是通过半胱氨酸偶联的，在进行偶联时，需利用还原剂打开抗体结构中的对还原和非还原谱图的峰进行分析，不仅可监测各个成分的单抗纯度分析时需要使用巯基乙醇等还原剂将单抗结构中的二 二硫键。因此，ADC药物会解离成多种碎片组分，且碎片类型与小分子药物的偶联位点相关。其[11]。如，在非还原CE-SDS中纯度，还可反映出药物的偶联效率
硫键断裂，形成轻链（Light Chain，LC）和重链（Heavy Chain， 非还原
CE-SDS 分析谱图见图 3。根据分子量的估算，1-5 号峰分别为经过偶联的轻链( LC)，重链（图3），1号轻链峰为单峰，说明在非还原条件下，游离的轻链
、重重链（HH）、重重轻链（HHL）,6 号峰为完整的裸抗[1]。对还原和非还原HC）。而非还原单抗纯度分析则需要保持二硫键稳定不断裂，故 ( HC)，重轻链（HL）
已全部偶联，还原谱图中的未偶联轻链（图2中峰1）来源于HL、不仅可监测各个成分的纯度，还可反映出药物的偶联效率[11]。如，在非还原 CE需加入巯基封闭剂对裸露的巯基进行封闭[7]，防止游离巯基介导的 谱图的峰进行分析，链间二硫键断裂而产生碎片[8,9]。

SDS HHL和完整裸抗的还原产物（图3中的3、5和6号峰）。
中（图 3），1 号轻链峰为单峰，说明在非还原条件下，游离的轻链已全部偶联，还原谱图中的未偶联轻链（图 2 中峰 1）来源于 HL、HHL 和完整裸抗的还原产物（图 3 中的 3、5 和 6 号峰）。

对于ADC药物的纯度分析，采用同药典中对单抗分析相同的分
析策略，即对ADC进行还原和非还原法的纯度进行测定[3]。
还原CE-SDS分析：
通过CE-SDS还原法谱图（图2），可推断1号峰为未偶联的轻链（含量8.03%），2号峰为偶联的轻链（含量23.13%），3号峰为偶联和未偶联的重链之和（含量 64.73%），4号峰在此条件下，视
为杂质（含量4.12%）。由结果可知，通过还原CE-SDS分析，能够将偶联和未偶联药物小分子的轻链实现分离[10]。而重链上可能会
偶联1至4个药物小分子，而在还原法CE-SDS谱图中并未发现偶联和未偶联重链的分离[11]。通过1和2号峰含量的变化，可以在一定

图 3 某 ADC 药物的非还原法 CE-SDS 电泳谱图。电泳条件见第 3 节。

图3. 某ADC药物的非还原法CE-SDS电泳谱图。电泳条件见第3节。

程度上反应出轻链的偶联效率，并对二者进行准确的定量。通过
方法的结果我们可知，1和2号峰所代表的片段之所以能够实现分
离，正是源于其偶联药物分子后分子量的差异，二者的分离度达
到了1.4，能够实现对1和2号峰的准确定量。而并未发现偶联和未
偶联的重链得以分离，可能是由于重链分子量较大，偶联前后分子量变化在重链中体现不明显所致。

4.2 cIEF法进行等电点及电荷异质性分析
如图4所示，（A）和（B）分别代表了裸抗及其对应的ADC药
物。采用3个pI Marker（10.0、9.5、5.5）进行等电点的线性计算（R2>0.999），获得裸抗主峰的实测值为8.67，与根据氨基酸序列计算的理论值8.73十分接近。当通过半胱氨酸偶联药物小分子后，
由于药物分子的引入以及偶联后分子结构发生了变化[8]，所以ADC
药物的等电点和电荷变异体相对于裸抗也发生了变化。以峰面积
百分比含量最高的峰为主峰，则该ADC药物主峰的等电点由裸抗的8.67变化至8.10，电荷变异体的pI值范围整体向酸性移动（裸抗：8.12-8.76；ADC药物：7.48-8.89）。

图 2 某 ADC 药物的还原法 CE-SDS 电泳谱图。电泳条件见第 3 节。

图2. 某ADC药物的还原法CE-SDS电泳谱图。电泳条件见第3节。
非还原 CE-SDS 分析：

由于该 ADC 药物是通过半胱氨酸偶联的，在进行偶联时，需利用还原剂打开抗体结构中的
本内容仅为专业人士提供学术交流与参考，不用于商业营销及其他目的。
RUO-MKT-02-10284-ZH-A二硫键。因此，ADC 药物会解离成多种碎片组分，且碎片类型与小分子药物的偶联位点相关。其RUO-MKT-02-10284-ZH-A

非还原 CE-SDS 分析谱图见图 3。根据分子量的估算，1-5 号峰分别为经过偶联的轻链( LC)，重链p2( HC)，重轻链（HL）、重重链（HH）、重重轻链（HHL）,6 号峰为完整的裸抗[1]。对还原和非还原谱图的峰进行分析，不仅可监测各个成分的纯度，还可反映出药物的偶联效率[11]。如，在非还原 CESDS 中（图 3），1 号轻链峰为单峰，说明在非还原条件下，游离的轻链已全部偶联，还原谱图中的未偶联轻链（图 2 中峰 1）来源于 HL、HHL 和完整裸抗的还原产物（图 3 中的 3、5 和 6 号峰）。

ADC 药物完整解决方案 49

4.2 cIEF 法进行等电点及电荷异质性分析如图 4 所示，（A）和（B）分别代表了裸抗及其对应的 ADC 药物。采用 3 个 pI Marker（10.0、
9.5、5.5）进行等电点的线性计算（R2>0.999），获得裸抗主峰的实测值为 8.57，与根据氨基酸序列计算的理论值 8.63 十分接近。当通过半胱氨酸偶联药物小分子后，由于药物分子的引入以及偶联

The Power of Precision

后分子结构发生了复杂的变化[8]，所以 ADC 药物的等电点和电荷变异体相对于裸抗也发生了复杂的变化。以峰面积百分比含量最高的峰为主峰，则该 ADC 药物主峰的等电点由裸抗的 8.57 变化至
8.00，电荷变异体的 pI 值范围整体向酸性移动（裸抗：8.02-8.66；ADC 药物：7.38-8.79）。

参考文献[1] Chen T, Chen Y, Stella, C, Dedley C, Gruenhagen J, Antibody-Drugconjugate characterization by chromatographic and electrophoretic
techniques, Journal of Chromatography B, 1032 (2016) 39-50.
[2] Tsuchikama, K, An ZQ, Antibody-drug conjugates: recent advancesin conjugation and linker chemistries, Protein&Cell, 9 (2018) 33-46.
图 4 某 ADC 药物偶联前后的 cIEF 电泳谱图。（A）裸抗；（B）ADC 药物。电泳条件见第 3 节。

图4. 某ADC药物偶联前后的cIEF电泳谱图。（A）裸抗；（B）ADC药物。电
泳条件见第3节。
5、结论

[3] 王兰，夏懋，高凯，抗体偶联药物的研究进展与质量控制，中国生物工程杂质，34 (2019) 85-94。
[4] Rowan E. Moore, Kelly Broster, Ken Cook, Kyle D’Silva, Eric

本研究建立了基于半胱氨酸偶联的 ADC 药物的异质性分析方法。使用 CE-SDS 和 cIEF 方法

Niederkofler, Aaron O. Bailey, Jonathan Bones, and Michael W. Dong,

分别对其大小异质性和电荷异质性进行分析。通过还原和非还原 CE-SDS 的方法，对该样品进行了

Antibody–Drug Conjugates: Perspectives and Characterization, LCGC纯度的评价。1）在还原条件下，偶联和未偶联的轻链可以实现高分离度的分离，并可以对这两个

North America, 36(2018) 362-374.

5、结论

本研究建立了基于半胱氨酸偶联的ADC药物的异质性分析方片段进行准确的定量分析。2）在非还原条件下，对该类半胱氨酸偶联的
ADC 样品，可实现六种片[5] 等电点分析试剂盒说明书，Sciex，2019。
段的分离和定量。3）通过还原和非还原 CE-SDS 的对比分析，能够在一定程度上反映出药物的偶法。使用CE-SDS和cIEF方法分别对其大小异质性和电荷异质性进联效率。4）在裸抗及其对应的 ADC 药物的 cIEF 方法分析中，通过其主峰的等电点及其电荷变异[6] 单抗分析大小变异体测定法(3127)，中国药典，2015版。
行分析。通过还原和非还原CE-SDS的方法，对该样品进行了纯度构体的变化，能够有效的监控其等电点的变化并分离 ADC 药物的酸碱修饰。

的评价。1）在还原条件下，偶联和未偶联的轻链可以实现高分离由于 ADC 药物高度复杂的异质性，我们更需要组合多种理化分析技术对其进行检测。本研究[7] Vithayathil P J, Richards F M. Modification of the Methionine
度的分离，并可以对这两个片段进行准确的定量分析。2）在非还
通过毛细管电泳方法的建立，实现了 ADC 类药物大小异质性和电荷异质性分析，对该类生物技术
Residue in the Peptide Component of Ribonuclease-S, J Biol Chem,
原条件下，对该类半胱氨酸偶联的ADC样品，可实现六种片段的分
药物的研究和质量控制提供了参考。
1960, 235 (8) 2343-2351。

离和定量。3）通过还原和非还原CE-SDS的对比分析，能够在一定

程度上反映出药物的偶联效率。4）在裸抗及其对应的ADC药物的cIEF方法分析中，通过其主峰的等电点及其电荷变异构体的变化，能够有效的监控其等电点的变化并分离ADC药物的酸碱修饰。
由于ADC药物高度复杂的异质性，我们更需要组合多种理化分析技术对其进行检测。本研究通过毛细管电泳方法的建立，实现
了ADC类药物大小异质性和电荷异质性分析，对该类生物技术药物

[8] 刘振东, 高铁, 徐玲丽, 杨勇, 王庆民, 陈泓序，十二烷基硫酸钠
毛细管电泳法分析非还原单克隆抗体药物纯度的前处理条件优
化，色谱，37 (2019) 666-670。
[9] 齐连权，韦薇，常翠云，白玉，罗建辉，抗体偶联药物及其质
量研究与质量控制，中国新药杂质，28 (2019) 1428-1432。
[10] 李萌，朱磊，武刚，崔永霏，于传飞，刘春雨，张峰，王文
波，陈伟，高凯，王兰, 抗体联药物抗CD30-VC-MMAE的异质性

的研究和质量控制提供了参考。

分析，中国药学杂质，51(2016) 1091-1095。
[11] Michael M. C. Sun, Kevin S. Beam, Charles G. Cerveny, Kevin J.
Hamblett, Richard S. Blackmore, Michael Y. Torgov, Felicia G. M.
Handley, Nathan C. Ihle, Peter D. Senter, and Stephen C. Alley,
Bioconjugate Chen., 16(2015)1282-1290.

For Research Use Only. Not for use in Diagnostic Procedures.
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AB SCIEX™ is being used under license.
© 2019 DH Tech. Dev. Pte. Ltd.

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