The Power of Precision
ZenoTOF 7600 system
SCIEX 7500 system
SCIEX Triple Quad 6500+ system
SCIEX 5500+ system
QTRAP 6500+ system
QTRAP 4500 system
View all mass
Intabio ZT system
Echo MS system
2.0 High Throughput Multiplexing system
View all integrated
BioPhase 8800 system
8000 Plus system
800 Plus system
Advance your research with front-end
instruments designed to help you realize the full power of your mass
spectrometer. SCIEX has the broadest portfolio of ESI-MS front-ends
can facilitate various flow rates, sample requirements and
sensitivities. No one else offers the entire range
flow, microflow, nanoflow LC-MS and even ultra-low flow
ExionLC AE system
ExionLC 2.0 series
View all front-end HPLC MS
Differential mobility spectrometry (DMS) and
ion mobility spectrometry are analytical techniques used to separate
ions based on their gas phase mobility. Multiple types of ion
devices exist, such as drift tubes, traveling wave, and high-field
asymmetric waveform devices. Learn how you can separate yourself
Differential Ion Mobility.
The latest ion sources from SCIEX enable
enhanced sensitivity and robustness with greater desolvation range
across all MS platforms, from Triple Quad to QTRAP and QTOF.
Turbo V ion
OptiFlow Turbo V ion
View all ion sources
vMethod applications are pre-configured and verified LC-MS/MS
that reduce the need for method development – significantly cutting
time, effort and money to deploy a new assay. Every vMethod provides
method conditions, recommended sample prep, LC and MS conditions,
details for applicable MS/MS library databases for key applications.
and personal care products (PPCPs)
and protein bioanalysis
Explore vMethod applications
feature request portal
Biologics Explorer software
Molecule Profiler software
In addition to being your trusted partner for the precise detection and quantitation of molecules, SCIEX offers accessories for your lab to support your work.
Mass spectrometry accessories
High resolution and QTRAP
libraries can dramatically enhance the quality of your analysis,
you much improved confidence in your data. With a comprehensive
at your fingertips, you can easily create methods and process
and non-targeted screening data on your complex samples, faster and
easier than ever before.
All in one
HR-MS/MS library with NIST 2017
Explore the library
Boost the performance of your mass
spectrometer and improve sensitivity, productivity, and data
iChemistry Solutions are the world's only reagents and
that are custom designed with your success in mind.
RNA 9000 Purity & Integrity kit
kit for amino acid analysis of hydrolysates
kit for amino acid analysis of physiological fluids
Protein CE-SDS Purity Analysis kit
MS calibration kits
CZE rapid charge variant analysis kit
BioPhase Fast Glycan Labeling and Analysis kit
View all consumables
QTOF – Quadrupole
Time of Flight
QTRAP® – Triple Quad Linear
SWATH® – Data Independent
SelexION® – Differential
MicroLC – Microflow
View All Technology
ADME and DMPK
Viral vector characterization
High-throughput mass spectrometry
Targeted protein degraders and PROTACs
Extractables and leachables
Residual nucleic acid analysis
Lipid nanoparticles and non-viral carrier
Messenger RNA analysis
Plasmid and DNA analysis
Synthetic oligonucleotide analysis
Cell line analysis
Charge heterogeneity analysis
Intact protein analysis
Peptide mapping analysis
Subunit mass analysis and middle-down
View all Biopharma and pharma
Clinical Mass Spec Operators
Clinical Method Developers
Clinical Lab Managers
View All Clinical
Pesticides & herbicides
Soil and biota
View All Environmental
How do you protect your reputation and meet today’s global food
standards? Whether you are a commercial lab or a food manufacturer,
quality of your food testing data is vital to your business. SCIEX
solutions help you meet maximum residue limits (MRLs) with
data that you can genuinely count upon. With a portfolio of
applications, your lab can quickly and easily react to diverse
Authenticity & Profiling Analysis
& Food Contact Substance Analysis
Food and Beverage Testing
How do you ensure the integrity of your
results in an industry that is never constant? By accurately
even the smallest compound angles you can deliver evidence that
SCIEX forensic analysis solutions deliver fast, highly accurate
data across a multitude of compounds and biomarkers, from the known
the new and novel.
Homeland security (coming soon!)
Cannabis and hemp potency testing
View All Forensic
Metabolic Flux Analysis
and SNP Analysis
View All Life
The SCIEX Now Learning Hub offers the most diverse and flexible learning options available, with best-inclass content that helps you to get the most out of your instrument and take your lab to the next level. Available personalized learning paths based on the latest memory science ensure better knowledge retention, and automated onboarding and enrollment means you’ll get up and running faster.
SCIEX Now Learning Hub offers the most diverse and
flexible learning options available, with best-in-class content that
helps you to get the most out of your instrument and take your lab
the next level. Available personalized learning paths based on the
latest memory science ensure better knowledge retention, and
onboarding and enrollment means you’ll get up and running faster.
SCIEX Learning Manager provides you with the infrastructure to assign, monitor and report on your staff's
competency through a single digital platform. Effectively manage the training process for new hires, ensure
continuous staff development and access information with a single log-in to your SCIEX account.
You can browse, filter, or search our
extensive list of training offerings. Choose from over 100
eLearnings or search for an instructor-led course near you. Once you
select the course you want to take, you will be directed to
Learning Hub for enrollment (login required).
Login to SCIEX Now Learning Hub
Success Programs at Your
Scientist Training at Your Site
Visit all Training
SCIEX Now Learning Hub
Manage my instruments
SCIEX Now New Feature Request
Visit your SCIEX
Declaration of conformance
Safety data sheets
Certificates of analysis
View All regulatory documents
Software and IT services
LC-MS service plans
Protect Plus Suite for your new LC-MS
CE service plans
Clinical service plans
StatusScope remote monitoring
Software accelerator program
Software support plans
Software support policy
Premium access content
Academic partnership program
View all partnership programs
Join the SCIEX community today to
interact with your peers, share and exchange ideas, develop
your knowledge, stay up-to-date with the latest products,
post insights and questions, comment on others and receive
support. This community is designed to help you, our
customers, move science forward and get the answers you
need. We’re committed to engaging with and listening to you,
to create the best customer experience possible and to
contribute to the success of your work.
Environmental / Industrial
Food and Beverage
Life Science Research
Now Feature Requests
View All Community
Echo® MS Center Of Excellence
Our favorite papers
Meet our executives
In the news
You've got questions. We've got experts who
can help. Contact us to find out more, talk to a specialist, explore
solutions or get expert support.
Request a quote
SCIEX success network
Request hosted catalog
Global public relations
Bosnia and Herzegovina
United Arab Emirates
United Republic of Tanzania
AB Sciex is doing business as SCIEX. © 2010-2018 AB Sciex. The trademarks mentioned herein are the property
of the AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license. Beckman
Coulter® is being used under license. Product(s) may not be available in all countries. For
information on availability, please contact your local representative. For
research use only. Not for use in diagnostic procedures.
Download Tech Note (PDF)
Featuring an icIEF-UV/MS workflow using the Intabio ZT system from SCIEX
Rashmi Madda, Rita Nichiporuk, Mariam ElNaggar, Scott Mack, Maggie Ostrowski and Zoe Zhang
This technical note highlights the reproducibility of a streamlined icIEF-UV/MS workflow for charge variant separation, UV quantitation, and peak identification of monoclonal antibodies (mAbs) by Intabio ZT system coupled to ZenoTOF 7600 system. The icIEF-UV/MS measurements of NISTmAb were performed across 45 injections with 3 cartridges using the Intabio ZT system, resulting in high inter- and intra-cartridge reproducibility (%CVs <5%).
Recombinant mAbs are widely used in biotherapeutic applications due to their high specificity and efficacy. One challenge associated with the characterization of antibody-based biotherapeutics is sample heterogeneity caused by physiochemical transformations and post-translational modifications (PTMs) that might occur during mAb manufacturing.1 Monitoring and characterizing the heterogeneity of mAbs is important to assess critical quality attributes (CQA) of biotherapeutics to ensure drug safety and efficacy.
The Intabio ZT system offers direct chip-based integration of icIEF-UV with ZenoTOF 7600 system that can deliver rapid and reproducible separation and characterization of intact mAb charge variants, and assessment of proteoforms. 2,3 The Intabio ZT system coupled with ZenoTOF 7600 system offers a streamlined workflow for reproducible separation, identification and relative quantitation of mAb charge variants in a highthroughput manner. 3, 4
In this technical note, the icIEF-UV/MS workflow was employed to provide reproducible separation of the main, acidic and basic variants of NISTmAb across 45 injections using 3 different cartridges (Figure 1).
Figure 1. Reproducible separation and detection across 45 runs on 3 cartridges using the Intabio ZT system coupled to the ZenoTOF 7600 system. Comparable NISTmAb charge variant patterns were observed between the icIEF-UV profile (left) and the MS base peak electropherograms (BPEs, right). Consistent separation of main, acidic and basic charge variant peaks was achieved across 45 injections using 3 cartridges.
Equipment: Intabio ZT system (SCIEX) and Intabio ZT cartridge (SCIEX, P/N 5088248) were used for the separation of NISTmAb and its charge variants. MS detection was performed on the ZenoTOF 7600 system (SCIEX, P/N 5080337) equipped with OptiFlow interface components (SCIEX, P/N 5084645).
Chemicals and reagents: The Intabio system – Electrolyte and Mobilizer kit (P/N 5088205) was used for anolyte, catholyte and mobilizer. Anolyte and mobilizer were used undiluted. The stock catholyte solution was 1% and diluted to 0.25% for use in the reagent drawer. The stock anolyte is 1% formic acid and catholyte is 1% diethylamine. The mobilizer is composed of 25% acetic acid25% acetonitrile and 50% water.
A 500mM cathodic spacer solution containing free base Larginine (Arg) (purity ≥ 98.5%, Sigma-Aldrich, P/N A8094-25G) was prepared by dissolving 0.870 mg of Arg powder into 10 mL of Milli-Q water. The electrolytes and cathodic spacer solutions were stored at room temperature. pI markers (CanPeptide) were individually dissolved in Milli-Q water at 5 mg/mL.
Prior to icIEF-UV/MS analysis, NISTmAb was desalted with a Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL (Thermo Fisher Scientific, P/N 89882).
Samples containing 400 µg/mL NISTmAb, 10mM Arg, 1 % Pharmalyte 3 to 10 (Cytiva P/N 17045601), 2.5% Pharmalyte 8 to 10.5 (Cytiva, P/N 17045501) and 6.0 µg/mL peptide pI markers were vortexed and then degassed by centrifugation at 3900 cf.
icIEF-UV/MS analysis: For the reproducibility test, 400 ug/mL NISTmAb solution was mixed with carrier ampholytes, and internal pI markers. The sample was separately analyzed with 3 different Intabio ZT cartridges.
The icIEF separation was achieved using the parameters shown in Table 1. UV absorbance measurements were collected at 1 Hz during the focusing and mobilization steps. The samples were introduced into the ZenoTOF 7600 system with a metered 2 µL/min flow of chemical mobilizer. The TOF MS data were acquired using the parameters shown in Table 2.
Table 1. icIEF separation parameters.
Data processing: UV profiles and mass spectra from the icIEFUV/MS analysis of NISTmAb were analyzed using the Biologics Explorer software. Each peak in the icIEF-UV profile was integrated to determine peak area and percent composition. Intact masses were estimated from the raw mass spectrum under each peak of the icIEF-MS profile utilizing a charge deconvolution algorithm with a mass range setting between 145,000 and 150,000 Da.
Table 2. TOF MS parameters.
Intra- and inter-cartridge relative abundance reproducibility
The combination of icIEF separation with UV quantitation and MS identification provides a high-resolution separation and comprehensive characterization of intact mAbs and their charge variants, as well as proteoform identification. The charge variants of NISTmAb were monitored across 45 injections using 3 different cartridges to evaluate the reproducibility of the icIEFUV/MS workflow, Figure 1.
The intra- and inter-cartridge reproducibility of NISTmAb separation and quantitation were assessed based on 3 metrics. First, the relative percent abundances of the main, acidic 1, and basic 1 and basic 2 variants were determined using each of 3 different cartridges. Additionally, the icIEF-UV and icIEF-MS profiles acquired from the 3 cartridges were correlated and compared. Finally, the pI and peak separation were compared across multiple runs on the same cartridge.
Figure 2. Relative abundances of the main, acidic 1 and basic 1 peaks of NISTmAb based on icIEF-UV/MS analysis of 45 injections on 3 different cartridges. The %CVs for the relative abundance of each charge variant were <5% based on the icIEFUV profiles.
The icIEF-UV/MS analyses of NISTmAb using 3 different cartridges provided consistent pI values and relative abundances of the main, acidic1, basic 1 and basic 2 charge variants (Figure 2 and Table 3). The %CVs of the pI of the main peaks were <0.1% and the %CVs of the relative abundances of all variants based on the icIEF-UV profiles were <5% (Table 3). These results demonstrate the high inter-cartridge reproducibility of the icIEF-UV/MS workflow.
Intra-cartridge pI value and resolution reproducibility
The icIEF-UV profiles were compared to evaluate the reproducibility of charge variant separation and detection across 15 injections on the same cartridge. Figure 3 shows the overlay of icIEF-UV profiles from 15 injections (blank injections are not shown).
Table 3. Separation reproducibility and relative peak area results for multiple runs on 3 cartridges.
These data demonstrate the ability of a single cartridge to reliably separate intact NISTmAb and its charge variants. High intra-cartridge separation reproducibility was measured based on the resolution between the main peak and the variant containing 1 C-terminal lysine across 45 injections on 3 different cartridges. This measurement was obtained directly from the software. A resolution of 1.5 is often indicative of baseline separation in icIEF analysis. Here, a separation resolution of 2-2.5 was consistently obtained for the icIEF-UV profiles of these 2 species across all runs (Figure 4). These results demonstrate the separation power and reproducibility of analysis on the Intabio ZT system.
Figure 3. Intra-cartridge reproducibility. The icIEF-UV profiles showing charge variant separation from 15 sequential injections demonstrate the reproducibility of analysis on the Intabio ZT system. Consistent separation of main, acidic 1, basic 1 and basic 2 variants was observed from 15 sequential injections.
Figure 4. Consistent resolution was observed across 45 injections on 3 different cartridge. The scatter plot shows a 2-2.5 separation resolution in the icIEF-UV profile between the main and basic 1 (1 C-terminal Lys) variant peaks based on 45 consecutive injections. A %CV of 6.3% was observed. These results demonstrate the consistency between technical replicates on the Intabio ZT system.
Evaluation of carryover between runs
To assess the carryover between runs for the charge variant analysis, an alternating sequence of blank and highly concentrated NISTmAb (up to 1 mg/mL) samples was injected into the Intabio ZT system. The icIEF-UV profiles of the alternating blank and NISTmAb injections show that carryover was not detected in the blank runs between sample injections (Figure 5). These results demonstrate the absence of carry over in the described analysis on the Intabio ZT system.
Figure 5. icIEF-UV profile of blank runs followed by injections of highly concentrated NISTmAb (1 mg/mL) sample using the Intabio ZT system. No carryover was detected in the blank injections between sample runs.
In summary, this technical note demonstrates the high inter- and intra-cartridge reproducibility of the Intabio ZT system and the power of this system for separating and characterizing different proteoforms of mAbs.
The Destination for All Your Support Needs