Monoclonal antibodies comprise a significant proportion of biotechnology-driven
molecules used for diagnostic and therapeutic applications. The inherent heterogeneity
of such products has dictated the need for thorough analytical characterization
methodologies so that safe, effective and reproducible products can be produced.
In addition, while antibodies can have vastly differing binding selectivity, the overall
structure is highly conserved between antibodies of the same class, thus standard
analytical methods can often be used as a starting point for developing an optimized
analytical strategy for an individual molecule1. LC and High Resolution MS have become
a powerful tool as part of the standard analysis package to:
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Characterize these important biomolecules
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Assess batch-to-batch variation
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Study antibody structure
Intact mAb Mass Check Standard
IgG1: Critical in Many Therapeutic and Diagnostic Applications
Figure 1. Structure of the Intact mAb Mass Check Standard. This particular IgG was found to contain
N-linked biantennary carbohydrates linked to each of the heavy chains. The major product variants
that were observed correspond to terminal pyroglutamic acids and glycoform heterogeneity (galactose
additions to the fucosylated biantennary core).
S S
S S
S S
S S
S S
S S
S
S
S
S
S
S
S
S
S
S
S
S
S S
S S
S S
S S
Gal
Fuc
Man
GlcNAc
2
INTACT mAb MASS CHECK STANDARD
Intact mass analysis of mAbs by LC/MS is a routine analytical
task performed by many different biopharmaceutical laboratories,
and provides a rapid approach for confirming antibody mass and
glycosylation profile. Low molecular weight protein standards that
are commonly available for instrument tuning or performance
checks (e.g. myoglobin) are not well suited to check mAb
instrument performance, as optimal instrument settings may differ
for the smaller nonglycosylated protein.
The Intact mAb Mass Check Standard is a LC/MS standard can be
used as a qualitative tool for confirming LCMS system operation.
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Known molecular weight for multiple glycoforms, ideal for
higher molecular weight intact mass measurements.
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Provided in convenient Waters Max Recovery Vial
(P/N 18600327c) for direct solubilization and injection.
Molecular Weight Information
Light Chain Formula:
Avg. Mw =
C
1066 H1649 N287 O343 S7 24197.7
Heavy Chain Formula:
Avg. Mw =
C
2170 H3338 N562 O661 S19
48484.3 Da
Intact Protein Formula:
Average Mw =
C
6472 H9940 N1698 O2008 S52
145,329.7 Da
Total number of Disulfide Bond = 17
Fixed Modification on 2 x Pyroglutamic Acid Q
For sequence information to cut/paste please go to:http://www.waters.com/
waters/nav.htm?cid=134634380 and Click on Intact mAb Mass Check Standard
Protein Sequence
The figure below contains the protein structure information.
For Waters Mass Spectrometry users who wish to process the
intact protein LC/MS data using Waters Informatics software such
as BiopharmaLynx or UNIFI, the sequence can be incorporated into
data processing methods (this is also located on the website
for easy cut/paste).
Modifications on the Protein:
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17 Disulfide Bonds
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N-terminus of each heavy chain has fixed Pyroglutamic
acid from Q
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One N-linked glycosylation on each heavy chain
Figure 2. Protein Sequence Information
3
Chain 1: Light Chain
Chain 2: Heavy Chain
Because this mAb sample has a similar MW range to conventional
therapeutic monoclonal antibodies, the Waters Intact mAb Mass
Check Standard serves as a convenient reference sample for mass
analysis of large biopolymers. Figures 4 and 5 show examples
on various mass spectrometry systems.
Examples of Using the Intact mAb Mass Check Standard for
MS Optimization with Q-TOF, TOF and SQD
Peak Number
mAb Glycoform
Expected MW
1
M* + G0F + G0F
148,220.4
2
M* + G0F + G1F
148,382.5
3
M* + G1F + G1F
148,544.6
4
M* + G1F + G2F
148,706.7
5
M* + G2F + G2F
148,868.8
*aglycosylated mAb
Table 1: Deconvoluted MW of the Major Glycoform of the mAb.
Figure 3. Combined mass spectrum (inset) and deconvoluted mass spectrum of
the light chain.
Light Chain Formula: C
1066H1649N287O343S7
Avg. MW = 24197.7
24000
24100
24200
24300
24400
Da
24500
24600
24700
24800
mass
24198 Da
Figure 4. Combined mass spectrum (inset) and deconvoluted mass spectrum of
the glycosylated heavy chain.
Heavy Chain Formula: C
2170H3338N562O661S19
Avg. MW = 48484.3 Da
Da
49800 49900 50000 50100 50200 50300 50400 50500 50600 50700
mass
48484 Da
Gal
Fuc
Figure 5. Example data obtained with ESI-TOF instrumentation.
1
2
3
4
5
ESI-TOF
MS spectrum
Deconvoluted
TOF spectrum
+50
+47
+53
Mass (Da)
2200
147600
148000
148400
148800
149200
149600
2600
3000
3400
3800
m/z
mass
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F: 1 508 872 1990
www.waters.com
Waters Corporation and the Science of W hat’s Possible are registered trademarks of Waters Corporation.
All other trademarks are the property of their respective owners.
©2014 Waters Corporation. Produced in the U.S.A. July 2014 720004610EN TC-PDF
References:
1. Chakraborty, A., Berger S., Gebler J., Characetrization of an IgG1 Monoclonal
Antobody and Related Sub-Strcutures by LC/ESI-TOF/MS. Waters Application
Note, March 2007 PN 720002107EN.
2. Rapid Commun. Mass Spectrom. 2008; 22: 29–40
ORDERING INFORMATION
Description
Part No.
Intact mAb Mass Check Standard
186006552
Figure 6. Example data obtained with ESI-Quad instrumentation.
G0/G0F
G1F/G1F + G0F/G2F
G1F/G2F
G2F/G2F
G0F/G0F
Mass (Da)
147800
148000
148200
148400
148600
148800
149000
mass
ESI-Quad MS spectrum
Deconvoluted Quad spectrum
p
50+
70+
60+
58+
57+
56+
m/z
2000
2100
2200
2300
2400
2500
2600
2700
2800
2900
