Intact mAb Mass Check Standard

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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Milford, MA 01757 U.S.A. 
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©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