Waters Intact mAb Mass Check Standard User Manual
[ CARE AND USE MANUAL ]
Intact mAb Mass Check Standard
CONTENTS
I. INTRODUCTION
II. STORAGE AND STABILITY
III. RECONSTITUTION OF THE INTACT
mAB MASS CHECK STANDARD FOR
MS OPTIMIZATION
IV. MOLECULAR WEIGHT INFORMATION
V. EXAMPLES OF USING THE INTACT
mAB MASS CHECK STANDARD FOR
MS OPTIMIZATION WITH Q-Tof,
Tof AND SQD
VI. ORDERING INFORMATION
I. INTRODUCTION
A monoclonal antibody is a glycosylated protein with a molecular
weight of about 150 kDa. Intact mass analysis of mAb by LC/MS is a
routine analytical task performed by many different biopharmaceutical
organizations, and provides a rapid method to investigate the integrity
of the biological sample. Low molecular weight protein standards that
are commonly available for instrument tuning or performance checking
(e.g. myoglobin) are not well suited to check the instrument performance
when the analysis of monoclonal antibody is needed because analytical
(instrument) conditions may greatly deviate from that for mAb analysis
due to molecular weight and structure difference ( e.g. glycosylation).
The Intact mAb Mass Check Standard is a LC/MS standard used as a
qualitative tool for intact mass analysis. It is an intact mouse protein
purified by Protein-A with a known molecular weight, ideal for higher
molecular weight intact mass measurements. It is provided in a Waters Max
Recovery Vial (PN 18600327c).
Upon a simple reconstitution step at a customer site, the mAb sample
is ready for injection/analysis as a true intact mAb reference standard.
Because the mAb sample has a similar MW range to conventional
therapeutic monoclonal antibodies, it serves a convenient reference
sample for MS instrument tuning and calibration check for intact mass
analysis of high molecular weight proteins.
[ CARE AND USE MANUAL ]
II. STORAGE AND STABILITY
The lyophilized powder is recommended to be stored long term
at -20 °C until the expiration date printed on the label. Once
reconstituted, the solution is stable for one week when stored at 8 °C.
III. RECONSTITUTION OF THE INTACT mAB MASS
CHECK STANDARD FOR MS OPTIMIZATION
The Intact mAb Mass Check Standard is 1 mg per vial of a
lyophilized solid. Depending on system requirements please
follow the below solubilization instructions:
SYNAPT G1/G2 and Xevo G1/G2
Add 100 µL of DI water to the Max Recovery Vial. Sonicate for 5
minutes. The mAb concentration of this solution will be 10 mg/
mL or 67 µM. Dilute this solution 20 fold with Eluent A of the LC
mobile phase to prepare a solution containing 3.3 µM mAb and
inject 5-20 µL for testing the LC/MS system.
SYNAPT/Xevo G2-S
Add 100 µL of DI water to the Max Recovery Vial. Sonicate for
5 minutes. The mAb concentration of this solution will be 10 mg/mL
or 67 µM. Dilute this solution 200 fold with Eluent A of the LC
mobile phase to prepare a solution containing 0.33 µM mAb
and inject 5-20 µL for testing the LC/MS system.
SQD
Add 200 µL of DI water to the Max Recovery Vial. Sonicate for
5 minutes. The mAb concentration of this solution will be 5 mg/
mL or 33 µM. Inject 5-10 µL for testing the LC/MS system.
V. EXAMPLES OF USING THE INTACT mAB
MASS CHECK STANDARD FOR MS
OPTIMIZATION WITH Q-Tof, Tof AND SQD
Table 1: Deconvoluted MW of the Major Glycoform of the mAb
Peak Number mAb Glycoform Expected MW
1
M + G0F + G0F
2 M + G0F + G1F 148,383
3 M + G1F + G1F 14 8, 545
4
M + G1F + G2F
5 M + G2F + G2F 148,868
*aglycosylated mAb
ESI-TOF
MS spectrum
2200
Deconvoluted
TOF spectrum
2600 3000 3400 3800
+50
+53
+47
2
3
1
4
5
148,220
148,707
m/z
IV. MOLECULAR WEIGHT INFORMATION
Light Chain Formula: Avg. MW =
C
106 6 H164 9 N287 O343 S7
Heavy Chain Formula: Avg. MW =
C
2170 H3338 N562 O661 S19
Intact Protein Formula: Average MW =
C
6472 H9940 N169 8 O2008 S52
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.
2
Intact mAb Mass Check Standard
2419 8 Da
484848 Da
145,330 Da
147600 148000 148400 148800 149200 149600
Mass (Da)
Figure 1: Example Data obtained with ESI-TOF Instrumentation.
mass
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