7
The initial calculation based on the sequence published in
the DrugBank database
2
resulted in a mass that did not
match the masses obtained in our experiments. Comparison
of the DrugBank sequence with a previously published
sequence
3
revealed a difference in one amino acid at position
219, located in the conserved region of the heavy chain, Ala
versus Val. The sequence containing the Ala at position 219
did match well with the results obtained from intact mass
measurements as well as with previously reported results.
4
To further verify this, a series of additional experiments
was performed.
After reducing the antibody (without alkylation), the
analysis of separated light and heavy chain was performed
applying different resolution settings to account for whether
or not isotopic resolution can be achieved based on
molecular weight. Due to the smaller molecular weight of
the light chain, it is possible to obtain an isotopically
resolved spectrum, whereas for the heavy chain this is not
possible since it is about twice as large as the light chain.
To apply different resolution settings, the method was set up
in two segments (140k resolution for the scans acquired
from 0 to 15.8 min, and 17.5k resolution from 15.8 to
30 min) and the gradient was optimized to achieve well-
separated peaks of the light and heavy chain.
On both monolithic columns evaluated in this study
(PepSwift 250 mm x 0.2 mm i.d. and ProSwift RP-10R
50 mm x 0.1 mm i.d.), the separation of the two peaks by
more than 2 min was equally possible (Figure 6). The mass
spectra obtained from the light chain and from the heavy
chain (Figures 6b and 6c) were submitted for deconvolution.
The isotopically resolved light chain spectrum was
deconvoluted using the Xtract algorithm, resulting in a
monoisotopic molecular weight of 23,025.3758 Da, which
matches the calculated mass by 2.5 ppm. The heavy chain
was deconvoluted using the ReSpect algorithm, resulting in
three peaks, each of which represents one of the major
glycoforms, G0F, G1F, and G2F (Figure 7).
Table 5. Chemical composition and masses of monosaccharides
Table 6. Monoisotopic and average atomic masses of the elements used to calculate the molecular masses in Tables 4 and 5
Elemental compositions
C
H
N
O
S
MW
(monoisotopic)
MW
(average)
G1F/G2FSA2 (HexNAc)8 (Hex)9 (Fuc)2 (SA)2
152
248
10
109
0
3957.41704
3959.6
G2F/G2FSA (HexNAc)8 (Hex)10 (Fuc)2 SA
147
241
9
106
0
3828.37445
3830.5
G2F/G2FSA2 (HexNAc)8 (Hex)10 (Fuc)2 (SA)2
158
258
10
114
0
4119.46986
4121.7
Sum 2 x HC +2 x LC (4 x pyroGlu, -2K)
6414
9896 1692 2006 44 144,127.20750 144,216.6
minus 32 S-S bond protons
6414
9864 1692 2006 44 144,094.95710 144,184.3
2HC + 2LC - 16 S-S bonds + (Man5)2
6506 10016 1696 2076 44 146,527.80282 146,618.5
2HC + 2LC - 16 S-S bonds + (G0F)2
6526 10048 1700 2084 44 146,984.02484 147,075.0
2HC + 2LC - 16 S-S bonds + G0F/G1F
6532 10058 1700 2089 44 147,146.07766 147,237.1
2HC + 2LC - 16 S-S bonds + G0F/G2F or (G1F)2
6538 10068 1700 2094 44 147,308.13049 147,399.3
2HC + 2LC - 16 S-S bonds + G1F/G2F
6544 10078 1700 2099 44 147,470.18331 147,561.4
2HC + 2LC - 16 S-S bonds + G2F/G2F
6550 10088 1700 2104 44 147,632.23613 147,723.5
2HC + 2LC - 16 S-S bonds + G1F/G2F SA
6555 10095 1701 2107 44 147,761.27872 147,852.7
2HC + 2LC - 16 S-S bonds + G1F/G2F (SA)2
6566
10112 1702 2115 44 148,052.37414 148,143.9
2HC + 2LC - 16 S-S bonds + G2F/G2F SA
6561
10105 1701 2112 44 147,923.33155 148,014.8
2HC + 2LC - 16 S-S bonds + G2F/G2F (SA)2
6572
10122 1702 2120 44 148,214.42696 148,306.1
Sum
Formula
Monoisotopic
Mass
Average
Mass
C O N H
Sialic Acid
C
11
O
8
NH
17
291.09542
291.3
11 8
1 17
Galactose
C
6
O
5
H
10
162.05282
162.1
6
5
0 10
N-Acetylglucosamine C
8
O
5
NH
13
203.07937
203.2
8
5
1 13
Mannose
C
6
O
5
H
10
162.05282
162.1
6
5
0 10
Fucose
C
6
O
4
H
10
146.05791
146.1
6
4
0 10
Monoisotopic Mass
Average Mass
C
12.0000000
12.01074
H
1.00782503
1.00794
N
14.0030740
14.00674
O
15.9949146
15.99940
S
31.9720707
32.06608
