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Innovation with IntegrityBioPharma
mAb Development made easy
maXis II for Biopharma Analysis
Determining the monoisotopic mass for large molecules (> 25 kDa) and related variants is essential for mAb developability assessments during early stage development.
The maXis II UHR-QTOF mass spectrometer together with the SNAP-II deconvolution algorithm possesses an intrinsic edge for the rapid profiling of large proteins and their heterogeneities.
The maXis II acquires True Isotopic Pattern (TIP) raw data that allows for rapid determination of modifications such as deamidation of the light chain (LC). Coupled to SNAP-II algorithm, these subtle modifications can be routinely characterized at the sub-unit level.
Sub-unit characterization (e.g. de-amidation) using the power of True Isotopic Pattern (TIP) raw data
maXis II: Unmatched Performance
True Isotopic Pattern (TIP)
23435 23440 23445 23450 23455
L Chain
Asn 30
MWth = 23428.524MWob = 23428.530
Asp30 orisoAsp30
Asn 30
MWth = 23429.508MWob = 23429.522∆mass = +0.992Da
Asp30 orisoAsp30
The ‘de facto’ standard for biologics
Intact Analysis
MaxEnt deconvolution spectrum measured in blue. Simulated mass spectrum in black.
Sub-unit Analysis
F(ab')2
Fc/2
Released Glycan Analysis
Glycopeptide Analysis
Higher Order Analysis
Increase speed to clinic with the maXis II:
The maXis II comes with electron transfer dissociation (ETD) capability as well as a high-mass option (HMO) for native MS applications.
• Understand and predict PK properties of antibodies by thorough characterization of glycans• Accurately determine deamidation• Rapid drug-antibody ratio (DAR) analysis• Reduce downstream manufacturing risks• Monitor batch to batch variability
Mass spectrum of a heavy chain with resolved isotopes
~80,000 resolution
Mr mono (meas) = 50613.041 DaMr mono (expected) = 50613.004 Da
∆Mr/Mr = 0.73 ppm
50625 50630 50635 50640 50645 50650 50655 50660 50665 m/z
Glycosylation is a crucial quality attribute for many biopharmaceuticals, as small changes in glycosylation profiles can have significant impact on stability, efficacy and/or safety.
Identifying, monitoring and controlling the glycosylation levels is key for successful biopharmaceutical development and the maXis II confidently addresses these key challenges in many pharmaceutical labs today at the intact, subunit, glycopeptide and released glycan level.
Superior Glycan Profiling Performance
Confidently determine:• Relative abundances (The most glycan IDs and
1-sigma groups in NISTmAb round robin study)*
• Composition (glycopeptide or released)**
• Localization within protein sequence
(glycopeptide)
Profiling at the intact level usually has a mass accuracy of around 10 ppm accuracy, as illustrated above. With resolved isotopes (< 50 kDa) it drops under 1 ppm. The maXis II UHR-QTOF sets the performance standard, with an unrivaled dynamic range for large proteins and glycoforms.
146500 146600 146600 146800 146900 147000 m/z
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Inte
nsi
ty
x103
1465
93.8
58G
2F/a
gly
con
1467
54.8
17G
0F/a
gly
con
1469
18.6
84G
2F/a
gly
con
146900 147400 147900 148400 148900 m/z
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Inte
nsi
ty
x104
1465
93.8
58G
0F/a
gly
con
1476
28.7
60G
0F/G
0F-G
lcN
Ac
(1) MaxEnt Spectrum
1467
54.8
17G
1F/a
gly
con
1465
93.8
58G
2F/a
gly
con
1478
36.1
48
G0F
/G0F
-Glc
NA
c
1480
38.5
46G
0F/G
0F
1479
98.8
76G
0F/G
1F-G
lcN
Ac
1481
63.5
35G
0F/G
0F +
K14
8200
.633
G0F
/G1F
1483
62.8
59G
1F/G
1F
1483
27.3
77G
0F/G
1F +
K
1485
25.1
37G
1F/G
2F
1484
89.7
36G
1F/G
1F +
K
1485
25.1
37G
2F/G
2F
1488
48.
683
G2F
/G2F
+ H
ex
*Maria Lorna A. De Leoz et al: NIST Interlaboratory Study on the Glycosylation of NISTmAb, NIST Pubs(2017), DOI: 10.6028/NIST.IR.8186
**H. Hinneburg et al.: The Art of Glycopeptide Destruction, J. Am. Soc. Mass Spectrom. (2015), DOI: 10.1007/s13361-015-1308-6
Increased Confidence in Released Glycan Analysis• Empirical spectra library comprising accurate mass and MS/MS
spectra of 2AB and RapiFluor® labeled glycans (25 glycoproteins
including most IgG subtypes and over 15 cell lines)
• Optimized LC and MS acquisition method
• Data processing with built-in expert knowledge. Scoring based on
fragment intensities and quantitation of coeluting glycans
• Report generation including MS and fluorescence based data with
minimized user intervention
• Workflow can be applied to regulated environments
GlycoFilerTM ResultsSample Preparation
2-aminobenzoic acid
RapiFluor
Compound ID
20
Main peaksassigned withglycan structures
GlycoFilerTM Workflow
• Enzymatic release of glycans from
the protein
• Fluorescence labeling
• HILIC-UPLC-FLR-CID-MS/MS
MeasurementHILIC-UPLC-FLR-CID-MS-MS
FLR Detection MS DetectionESI-QTOF-MS/MS
Data ProcessingAutomated
Integrationof FLR Peaks
IdentificationMatch with Glycan
Spectra Library
FLR-MS Data Hybridization
Table of glycan structures correlated to relative fluorescence peak areas
Data ProcessingAutomated
Reporting
Certificate of Analysis
Calculation of biologically relevant parameters
Dr. Sven Bahrke, Glycotype GmbH, Germany
“ GlycoFiler TM improved our lab productivity by reducing the time spent on evaluating data, especially in the case of complex glycan mixtures with coeluting peaks. In addition the ability to unambigu-ously identify and precisely quantify even low abundant structures reduces the non-annotated peak area to a minimum as well as the risk of false identifications. Integrated and automated quality control of the analyses as well as straightforward comparison of results between products round out the GlycoFiler TM workflow.“
EGFR (8 glycosylation sites)
Extract from GlycoFiler reports: Fluorescence chromatograms for an IgG1 and EGFR released glycans annotated with the main identified structures.
The maXis II easily detects and quantifies micro-heterogeneities directly on intact antibodies including under native conditions. This makes the maXis II the perfect platform to rapidly develop assays for drug distribution and average drug loading (DAR) without waiting for HPLC methods to be established.
Comparability with orthogonal methods and robustness makes it possible to validate these methods for GLP operations.
Rapidly Characterize Drug-Antibody Conjugates under GLP
• Interday• Intersite• Under GLP
Validation of drug distribution and DAR assay under native conditions on the maXis II UHR-QTOF
Method Reproducibility Assessment
%Peak Area
Prep 1Prep 2Prep 3Prep 4Prep 5Prep6
MeanSD
%RSD
0.0000.0000.0000.0000.0000.0000.0000.0000.000
0-Drug% 1-Drug% 2-Drug% DAR
3.8893.6194.1774.1993.8204.1923.9830.2065.162
96.11196.38195.82395.80196.18095.80896.0170.2060.214
1.961.961.961.961.961.961.960.000.00
%Peak AreaAnalyst
Analyst 1 - Day 1
Analyst 2 - Day 1
Analyst 1 - Day 2
Mean
SD
RSD
% 0-Drug
% 1-Drug
% 2-Drug DAR
0.000
0.000
0.000
0.000
0.000
0.000
4.177
3.500
3.883
3.853
0.277
7.193
95.823
96.500
96.117
96.147
0.277
0.288
1.96
1.96
1.96
1.96
0.00
0.00
Top-Down sequencing using ETD
One software platform including features such as intact protein and sub-unit analysis or peptide screening and quantitation workflows, which routinely provide artifact free quantification with highest dynamic range, fully integrating LC-UV, MS and MS/MS data.
Integrated data acquisition. Simple GUI for starting and monitoring acquisition runs.
Predefined, customizable report formats, supports 21 CFR Part 11 requirements.
BioPharma Compass: Designed in collaboration with the pharmaceutical industry
Automatic result assessment, • Algorithm based similarity scoring
Butterfly plots for easy comparison of chromatograms and spectra
Released glycans and glycopeptides MS/MS based identification
Protein Peaks Expected Peaks Confirmed Cosine Similarity Accepted
LC 2 2 1.0000 Yes
Fc2 9 9 1.0000 Yes
Fd 2 2 1.0000 Yes
BioPharma Compass
Bru
ker
Dal
toni
cs is
con
tinua
lly im
prov
ing
its p
rodu
cts
and
rese
rves
the
rig
ht
to c
hang
e sp
ecifi
catio
ns w
ithou
t no
tice.
© B
DA
L 05
-201
8,18
5970
0
Bruker Daltonik GmbH
Bremen · GermanyPhone +49 (0)421-2205-0
Bruker Scientific LLC
Billerica, MA · USA Phone +1 (978) 663-3660
[email protected] - www.bruker.com
For research use only. Not for use in diagnostic procedures.
Scan the QR-Code for more Details
7 Reasons the maXis II makes mAb development easy
• 80,000 FSR (Full Sensitivity Resolution) with high intra-scan dynamic range• True Isotopic Pattern (TIP) not limited by space charge effects for accurate DAR analysis• SNAP-II algorithm for accurate monoisotopic mass determination for large biologics• Supports 21 CFR Part 11• Electron Transfer Dissociation• Native mass spectrometry enabled• Glycan analysis with Glycofiler library
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