1. No category

RECOMBINANT ANTIBODY GLYCOFORMS ASSAY BY LC AND LC/MS METHODS
Asish B. Chakraborty, Ying Qing Yu, Weibin Chen, Jeff Mazzeo
Biopharmaceutical Sciences, Waters Corporation, 34 Maple Street, Milford, MA 01757
30
23442 Da
Injectio n
Gradient:
28-32% B in 12.5 min
A: 0.02% TFA in Water
B: 0.018% TFA in ACN
LC-2Da
298,770 ± 5,988 (2%)
Normalized to G1F/G1F
Gradient: 23-29% B in 30 min
A: 0.02% TFA in water
B: 0.018% TFA in ACN
Man8
G2F
G2
Man7
Man6
G1F-GN
G1
G0
Man5
G0F-GN
G0-GN
%
Intensity
69+
39+
0
Xevo QTof MS
0.5 µg injected
46+
%
55+
59+
43+
65+
0
Batch 3
2000
2400
39+
2800
3200
m/z
3600
4000
4400
Figure 1.
Mass spectra of an intact antibody showing
enhanced sensitivity of Xevo QTof for intact protein analysis.
Figure 3.
MaxEnt1 deconvoluted mass spectra of
Trastuzumab Fc fragment (~50kDa) from three batches
displayed by BiopharmaLynxTM. The glycan profiles variations
from three production batches are illustrated. This approach
provides holistic view of the IgG1 glycoforms and paired
masses. Quantatitive information on individual glycoforms is
unavailable by this approach.
G1F-GN
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G2FS2
Man8
G2FS1
Man7
G2F
Table I. Quantitative comparison of Trastuzumab glycoforms with
different analytical methods. Glycoforms (G0, G1, G0F, G1F, G2F,
and Man5) detected by all techniques were quantitatively
compared. Isobaric G1 and G1F forms that were separated by
free glycan assay are combined for the comparison. Percent
were calculated from all glycoforms that were detected in a
specific batch.
CONCLUSIONS
Batch 2
• Several UPLC/ESI-QTof MS-based methods were
Batch 3
•
Figure 6. MaxEnt1 deconvoluted mass spectra of IgG HC
(~50kDa) generated from three different batches of
Trastuzumab. Detected glycoforms were labeled based on the
deconvoluted masses. Reduction of the antibody into
monomeric HC allowed quantitation of the individual
glycoform.
G1Fa
G1Fb
Man6
G2
G0F
Figure 10. Released N-glycan assay of the Trastuzumab from
three production batches. The figure shows the chromatograms
of 2-AB labeled glycans obtained in HILIC mode. The
identification of the 2-AB labeled glycans was achieved by online MS analysis (Xevo QTof).
Batch 1
50+
100
Intensity
Batch 3
46+
Man5
G1
Batch 3
•
GN=GlcNac
58+
G0F-GN
G0
G0-GN
Column:
ACQUITY UPLC BEH C4, 1.7 µm,
2.1 x 50 mm
Traditional Q-Tof
1.5 µg injected
51+
Detection: Fluorescence
λex=330 nm; λem=420 nm
Figure 8. UPLC/MS analysis of Lys-C digested (limited) and
partially
reduced
monoclonal
antibody.
Total
ion
chromatogram of Fc/2, LC, and Fd is shown.
Batch 2
Batch 2
Fd
Fc/2
Batch 1
Batch 1
LC
Fd
Man6
G1F/G2F
(G2F)2
G0F/G1F
G0F/G0F-GN
G0F/G0
(G1F)2
G0F/Man5
G0-GN
Man5/Man6
(Man5)2
Figure 2. MaxEnt1TM deconvoluted mass spectra of an intact
antibody showing mass accuracy, precision and intensity
variation for glycoforms on Xevo QTof over 28 injections.
G0F
160,527 ± 40,393 (20%)
G1F
192,990 ± 38,202 (19%)
63,309 ± 3724 (6%)
G0/G0F
(G0F)2
Figure 5. LC/MS (Acquity UPLC/Xevo QTof) analysis of reduced monoclonal antibody Trastuzumab (Batch 3).
LC
G0F
25
G1F-GN
20
G0
15
Man5
G0F-GN
10
Fc/2
Batc
Figure 7. Fragments generated by limited proteolysis of
monoclonal antibody with Lys-C followed by partial reduction.
G0-GN
5
G2FS1
0
Column:
ACQUITY UPLC BEH C4, 1.7 µm,
2.1 x 50 mm
Fc
+
G2FS1
148,056.7
Fab
Man8
G2F
HC
+
100
~ 6-fold improvement in sensitivity
Mass
Spectrum
G1F
LC
148,057.2
Partial
Reduction
+2
HC
Figure 4. Heavy chain (HC) and light chain (LC) fragments
generated by reduction of monoclonal antibody with DTT.
148,057.7
• This presentation compares several LC/MS assays with
LC/MS ANALYSIS OF INTACT IgG1 ANTIBODY
+
LC
G0F/G0F Mass Average mass precision: 1.1 ppm
carbohydrate moieties of therapeutic proteins.
LC System: Waters ACQUITY UPLC ®
Detector: ACQUITY UPLC FLR
2
G2
2
G1
Man7
8.6
8.0 ppm
• It is extremely important to accurately quantify the
MS Conditions
MS System: Waters XevoTM QTof MS
Ionization Mode: ESI Positive
Capillary Voltage: 3.0kV
Cone Voltage: 45V/25V
Source Temp: 100°C
Desolvation temp: 350°C
Desolvation Gas: 1000 L/h
Limited
Proteolysis
Lys-C
Digestion
314,856 ± 34,868 (11%)
quantifying
Batch 1
Average RMS mass
accuracy: 6.1 ppm
2.1
• Glycosylation
traditional released glycan assay for
glycosylation of a recombinant antibody.
Released N-Glycan Assays (2-AB Labeled)
4.3
growing therapeutics in the biopharmaceutical industry.
Recombinant antibodies contain carbohydrate moieties
(glycans) as a result of post-translational modifications.
The process of adding sugars to the protein at the
endoplasmic reticulum and the Golgi apparatus is
highly complex, resulting in variation of glycoforms.
plays
a
vital
role
in
stability,
biodisposition, in-vivo activity, solubility, serum halflife, and immunogenicity of an antibody drug and can
affect
efficacy,
target
binding,
folding
and
pharmacokinetic properties. Moreover, glycosylation of
recombinant antibodies differs with the cell culturing
parameters.
LC/MS ANALYSIS OF IgG1 Fc/2 FRAGMENT
28 injections of Antibody IgG1
6.7
• Recombinant monoclonal antibodies are the fastest
LC/MS ANALYSIS OF HC (REDUCED)
G0F
LC/MS ANALYSIS OF INTACT IgG1
OVERVIEW
Figure 9. Deconvoluted mass spectra of Trastuzumab, Fc/2
fragment from the three production batches. Lys-C digestion
followed by reduction resulted in monomeric Fc/2 (~25 kD).
Detected glycoforms were labeled based on the deconvoluted
masses.
compared for the identification and quantitation of
various glycoforms in a IgG1 antibody (Trastuzumab).
ESI-QTof MS analysis of both Fc/2 (from Lys-C
digestion and reduction) and HC (reduced) yielded
agreeable quantitation results on common glycoforms
in comparison with the N-release assays.
ACQUITY UPLC BEH Glycan columns offer superior
resolution in the separation of released glycans,
providing accurate measurement of the individual
glycoforms.
References:
1. Gadgil et.al J. Pharmaceutical Sciences 96, 2607–2621 (2007)
720003327EN