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Application Note
Biotherapeutics and Biosimilars
AuthorSuresh Babu C.V. Agilent Technologies, Inc.
AbstractThe development and manufacturing of biosimilars require comparability studies with innovator biotherapeutic products. This Application Note presents a peptide mapping analysis of monoclonal antibody (mAb) biologics to assess the similarities and differences between innovator and two biosimilars of rituximab using an Agilent 6545XT AdvanceBio LC/Q-TOF system. The sequence coverage map of tryptic digested mAbs was >97%. Liquid chromatography/mass spectrometry (LC/MS) peptide separation profiles show differences in peptide abundances between innovator and biosimilar mAbs, revealing different extents of post-translational modifications (PTMs) such as deamidation and oxidation.
LC/MS/MS Peptide Mapping Comparison of Innovator and Biosimilars of Rituximab
2
IntroductionThe production of mAb-based therapeutics is growing rapidly.1 Patents covering innovator mAbs that entered the market approximately two decades ago are expiring, opening opportunities for the development of cost-effective biosimilars. Biosimilars are close copies of their reference products, but are not identical due to inevitable differences in their manufacturing processes. These processes are highly complex, and slight deviations may introduce variability and intrinsic molecular heterogeneity to the product. Regulatory agencies require thorough characterization of biosimilars to prove they are comparable to innovator products in terms of safety, purity, and efficacy. The goal of
biosimilar product development is to elucidate the similarities and differences between innovator and biosimilar drugs. Analytical comparability is a fundamental requirement to show biosimilarity.
Critical quality attributes (CQAs) of biologics define their efficacy and safety. PTMs such as glycosylation, oxidation, and deamidation variants are amongst the CQAs that can significantly impact biological function. In the biopharmaceutical industry, LC/MS-based peptide mapping serves as a primary tool for protein identification as well as monitoring PTMs. High resolution, reliable chromatographic separations, and accurate mass measurements are essential for the success of peptide mapping experiments.
In this study, peptide mapping of an innovator and two biosimilar mAbs of rituximab was performed using an integrated workflow consisting of an Agilent AssayMAP Bravo liquid-handling platform, an Agilent 1290 Infinity II LC system, an Agilent 6545XT AdvanceBio LC/Q-TOF, and data analysis through Agilent MassHunter BioConfirm software (Figure 1). The percentage sequence similarity of the heavy chain, light chain, and intact mAbs were compared to determine the amino acid coverage. The results of the study demonstrate the suitability of Agilent’s peptide mapping workflow for identifying and monitoring important PTMs such as deamidation and oxidation.
Sample preparation Separation Detection Data processing and report
Agilent AdvanceBio Peptide Mapping column
Agilent AssayMAP Bravo Agilent 1290 Infinity II LC
Agilent 6545XT AdvanceBio
LC/Q-TOF with Iterative MS/MS
Agilent MassHunter
BioConfirm B.10
Figure 1. Agilent peptide mapping workflow.
3
Experimental
MaterialsInnovator and biosimilar monoclonal antibodies were purchased from a local distributor (Singapore), and stored according to the manufacturers’ instructions. Trizma base, guanidine-hydrochloride, tris(2-carboxyethyl)phosphine (TCEP), iodoacetamide (IAA), formic acid, trifluoroacetic acid, and LC/MS grade solvents were purchased from Sigma-Aldrich. High-quality sequence grade trypsin was obtained from Agilent Technologies, Inc.
Sample preparation: trypsin digestionInnovator and biosimilar mAbs were reduced/alkylated and trypsin-digested followed by desalting using the Agilent AssayMAP Bravo liquid-handling platform. Briefly, sample plates containing mAbs (10 µg/µL) were reconstituted in denaturation buffer (8 M guanidine-HCl with 5 mM TCEP and 150 mM Tris, pH 8) and incubated at 60 °C for 60 minutes. After denaturation and reduction of disulfide bonds, alkylation of free cysteines was carried out by adding 133 mM iodoacetamide (40 minutes at room temperature, protected from light). Guanidine-HCl concentration was reduced, and the pH
of the solution was adjusted to pH 7 to 8 with 150 mM Tris-HCl before trypsin digestion. Trypsin digestion (20:1, protein to protease w/w) was performed overnight at 37 °C. The samples were later acidified with 0.1% formic acid to halt the action of trypsin using the AssayMAP reagent transfer utility. AssayMAP Bravo Peptide Cleanup protocol (desalting) was performed using C18 reversed-phase cartridges. The cartridges were primed with 100 µL of 60 % acetonitrile 0.1% trifluoroacetic acid equilibrated with 50 µL of 0.1% trifluoroacetic acid, loaded with 50 µL of mAb tryptic digested samples at a flow rate of 5 µL/min, washed with 50 µL of 0.1% trifluoroacetic acid, and eluted with 15 µL of 60 % acetonitrile, 0.1% trifluoroacetic acid at a flow rate of 5 µL/min and mixed into 160 µL of 0.1% formic acid.
InstrumentationLC system
Agilent 1290 Infinity II LC System including:
• Agilent 1290 Infinity II High Speed Pump (G7120A)
• Agilent 1290 Infinity II Multicolumn Thermostat (G7116B)
• Agilent 1290 Infinity II Multisampler (G7167B)
MS systemAgilent 6545XT AdvanceBio LC/Q-TOF
LC/MS analysisLC/MS peptide map separation was performed on an Agilent AdvanceBio Peptide Mapping column (2.1 × 150 mm, 2.7 µm) using a 30-minute gradient. The data were acquired in iterative MS/MS mode (Table 1).
Data analysisThe data were processed using the peptide mapping workflow in Agilent MassHunter BioConfirm 10. Briefly, molecular features were extracted and matched against the innovator mAb’s sequence with fixed modifications (alkylation: C) and variable modifications (oxidation: M, deamidation: N, Q, lysine loss: K, and pyroglutamate: E). The mass tolerance was 10 ppm for MS, and 30 ppm for MS/MS. Sequence coverage was calculated based on peptide spectrum matches to MS/MS features and were filtered by a 1% false discovery rate (FDR).
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Table 1. LC/MS conditions.
Parameter Agilent 1290 Infinity II LC System
Column Agilent AdvanceBio Peptide Mapping 2.1 × 150 mm, 2.7 µm, 120Å
Injection Volume 2 µL (2 µg/µL)
Sample Thermostat 5 °C
Mobile Phase A 0.1% formic acid in water
Mobile Phase B 0.1% formic acid in acetonitrile
Gradient
At 0 minutes & 3% B At 30 minutes & 40% B At 33 minutes & 90% B At 35 minutes & 90% B At 37 minutes & 3% B At 40 minutes & 3% B
Stop Time 40.1 minutes
Column Temperature 60 °C
Flow Rate 0.4 mL/min
Parameter Agilent 6545XT AdvanceBio LC/Q-TOF
Ion Mode Positive ion mode, dual AJS ESI
Drying Gas Temperature 325 °C
Drying Gas Flow 13 L/min
Sheath Gas Temperature 275 °C
Sheath Gas Flow 12 L/min
Nebulizer 35 psi
Capillary Voltage 4,000 V
Nozzle Voltage 0 V
Fragmentor Voltage 175 V
Skimmer Voltage 65 V
Oct RF Vpp 750 V
Reference Mass 121.050873, 922.009798
Acquisition Mode Data were acquired in Extended Dynamic Range (2 GHz)
Ms Mass Range 110 to 1,700 m/z
Acquisition Rate 8 spectra/sec
Auto Ms/Ms Range 50 to 1,700 m/z
Ms/Ms Acquisition Rate 3 spectra/sec
Isolation Width Narrow (~1.3 m/z)
Precursors/Cycle Top 10
Collision EnergyCharge state Slope Offset 2 3.1 1 3 and >3 3.6 –4.8
Threshold for MS/MS 1,000 counts and 0.001%
Dynamic Exclusion On One repeat, then exclude for 0.1 or 0.2 minutes
Precursor Abundance Based Scan Speed Yes
Target 25,000 counts/spectrum
Use MS/MS Accumulation Time Limit Yes
Purity 100% stringency, 30% cutoff
Isotope Model Peptides
Sort Precursors By charge state then abundance; +2, +3, >+3
5
Results and discussionMassHunter BioConfirm is a comprehensive and user-friendly data analysis tool for protein analysis. Figure 2 shows the BioConfirm 10.0 software peptide mapping workflow layout. For peptide mapping experiments, the software automatically extracts
peptide characteristic features with a peptide find algorithm, and matches MS/MS spectra with possible PTMs to confirm and validate the peptide sequence.2 In addition, the software performs relative quantification of PTMs automatically, and displays the information as histograms for quick data review, enabling faster decisions.
Multiple peptide mapping files can be processed and compared across different protein digest samples. With these powerful and user-friendly features, MassHunter BioConfirm software is well suited for comparative assessment of innovator and biosimilar peptide mapping experiments.
Figure 2. Screenshot of Agilent MassHunter BioConfirm 10.0 software for peptide mapping analysis.
Method setup Sample table
Chromatograms
Sequence coverage
MS spectrum MS/MS spectrum
Peptide list
Innovator
Biosimilar 1
Biosimilar 2
Innovator
Biosimilar 1
Biosimilar 2
Innovator
Biosimilar 2
Biosimilar 1
6
Peptide mapping is routinely used to identify proteins by confirming the amino acid sequence. It is also the preferred approach for the characterization and monitoring of PTMs. This study chose an innovator and two biosimilar mAbs for comparability assessment. Figure 3 shows the LC/MS of tryptic peptide map
of innovator and biosimilar mAbs. The 30-minute gradient on the AdvanceBio Peptide Mapping column produced a high-resolution peptide separation. Using the automatic peptide mapping workflow in MassHunter BioConfirm software, peptide masses from the LC/MS/MS run were matched with the theoretical digest
with preferred modifications included in the mAb sequence. The BioConfirm software automatically identified and assigned the peptides for the MS/MS data. The result of iterative MS/MS gives >97% sequence coverages for heavy chain and light chain sequences (Table 2).
Figure 3. Comparison of tryptic digested mAbs. TIC of (A) Innovator, (B) Biosimilar 1, and (C) Biosimilar 2. The arrows highlight the difference in TIC profiles, and the below ECCs represent the corresponding peptides.
00.20.40.60.81.01.21.41.61.82.02.2
10.3 10.6 10.8
0
2
4
6
8
0
2
4
6
8
0
2
4
6
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
16.7 16.8 16.90
0.10.20.30.40.50.60.70.80.91.01.1
20.2 20.40
0.5
1.0
1.5
2.0
2.5
3.0
21.2 21.4 21.6
QTPGRGLEWIGAIYPGNGDTSYNQK VVSVLTVLHQDWLNGKEY TTPPVLDSDGSFFLYSKLSLSLSPG
Innovator
Biosimilar 1
Biosimilar 2
Biosimilar 2
Biosimilar 1
Innovator
Innovator
Biosimilar 1
Biosimilar 2 Biosimilar 2
Biosimilar 1
Innovator
SL
SL
SP
GK
SL
SL
SP
G
QT
PG
RG
LE
WIG
AIY
PG
NG
DT
SY
NQ
K
VV
SV
LT
VL
HQ
DW
LN
GK
EY
TT
PP
VL
DS
DG
SF
FL
YS
KL
×107
×107
×107
×106 ×105 ×105×104
A
B
C
Acquisition time (min)
Acquisition time (min) Acquisition time (min) Acquisition time (min) Acquisition time (min)
Co
un
ts
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
8.0 8.2
SLSLSPGK
Innovator
Biosimilar 1
Biosimilar 2×106
Acquisition time (min)
Co
un
ts
Co
un
ts
Co
un
ts
Co
un
ts
Co
un
ts
Co
un
tsC
ou
nts
7
The total ion chromatogram (TIC) of tryptic digest obtained from auto MS/MS was compared to find the similarity/differences between the innovator and biosimilar mAbs. Figure 3 shows that inspection of the ion chromatograms reveal differences in the expression levels of peptides. The C-terminal sequence (SLSLSPGK) is enriched in biosimilar 2 (~eight minutes) compared to innovator and biosimilar 1. Conversely, the lysine-truncated peptide form (SLSLSPG) was enriched in
the innovator mAb (~10.4 minutes). The three other peptides, QTPGRGLEWIGAIYPGNGDTSYNQK, VVSVLTVLHQDWLNGKEY, and TTPPVLDSDGSFFLYSKL show different intensity levels between innovator and biosimilar mAbs.
PTMs are CQAs that may be linked to drug efficacy and safety. Therefore, it is important to identify and compare the levels of PTMs between the biosimilar and innovator mAb pairs. Data on two commonly found modifications, oxidation and deamidation, between innovator and biosimilar mAbs were presented.
OxidationOxidation is a common PTM of mAbs, and can occur at various stages of bioprocessing development such as purification, formulation, and storage. To
compare the oxidation levels of innovator and biosimilar mAbs, a representative heavy chain DLTMISR peptide was selected. Figure 4 compares MS/MS spectra, separation of oxidized and unmodified DLTMISR peptides, and the relative quantification of M256 oxidation. Inspection of y4 and y5 ion masses shows an increase of 16 Da in oxidized MS/MS spectra, suggesting oxidation of the Met residue. Figure 4C shows the triplicate measurements of relative oxidation levels of M256 as a histogram, a useful feature of MassHunter BioConfirm software, for all the mAbs. The examination of histograms for M256 oxidation levels for innovator and biosimilar 1 mAbs were comparable (1.48% and 1.27% respectively), whereas for biosimilar 2, a slightly higher level of oxidation (2.5%) was observed.
Table 2. Average sequence coverage.
mAb Samples
Sequence Coverage
Heavy Chain Light Chain
Innovator 98.23 100
Biosimilar 1 98.10 99.06
Biosimilar 2 97.59 100
Figure 4. Oxidation analysis of DTLMISR peptide. (A) MS/MS spectrum of unmodified and oxidized peptide, (B) ECC overlay, and (C) relative quantification of M256 (three replicate measurements).
0
0.5
1.0
20 60 100 140 180 220 260 300 340 380 420 460 500 540 580 620 660 700
L
86.0965b
2
b2
b2
21
7.0
81
7
y1
y1
y1
17
5.1
19
0M
10
4.0
53
6
y2
y2
y2
26
2.1
50
8
y3
375.2355
y4
506.2759
y5
y3
y4
y5
y3
y4
y5
619.3605
b3
b3
b3
33
0.1
66
5
y5
2+
b2
y1
y2
y3
y4 y
5b4
b4
y5
2+
b2
b2
y1
y1
y2
y2
y3
y4
y5
y3
y4
y5
b3
b3
y5
2+
y5
2+
y5
2+
31
0.1
83
0
b4
461.2067
14
4.0
65
31
29
.06
52
18
9.0
87
1
24
5.1
31
2
28
4.1
60
1
31
2.1
55
6
35
8.2
13
4
40
9.2
17
4
53
2.2
21
4
60
2.3
30
4
64
4.3
13
7
0
0.5
1.0
1.5
2.0
2.5
20 60 100 140 180 220 260 300 340 380 420 460 500 540 580 620 660 700
L86.0969
21
7.0
82
7
17
5.1
18
9M
12
0.0
46
1
14
4.0
64
9
26
2.1
51
1
318.1822
37
5.2
34
3
522.2705635.3572
47
7.1
97
6
56
.04
87
18
9.0
87
9
24
6.1
34
0
28
4.1
59
8
34
6.1
82
9
39
4.2
06
94
17
.21
51
50
7.2
50
3
54
6.3
48
9
57
1.3
56
65
88
.28
59
66
3.3
27
56
82
.38
84
0
0.5
1.0
Time (min)
7.5 8.5 9.5
Co
un
ts
Co
un
ts
Oxidized
Unmodified
0
1
2
3
4
5
6
7
8
9
20 60 100 140 180 240 280 320 360 400 440 480 520 560 600 640 680
L
86.0954
21
7.0
80
5
17
5.1
18
6
26
2.1
50
2
375.2347
33
0.1
62
4
506.2752
619.3595
10
4.0
51
6
14
4.0
64
1
18
9.0
85
8
24
5.1
26
6
28
4.1
58
7
31
2.1
54
7
35
8.2
07
0
41
8.2
23
1
46
1.2
06
0
58
9.3
32
6
66
6.3
68
9
0
0.5
1.0
1.5
2.0
20 60 100 140 180 220 260 300 340 380 420 460 500 540 580 620 660 700
L86.0970
21
7.0
83
4
17
5.1
18
5 26
2.1
51
8
31
8.1
80
7
375.2363
33
0.1
66
6
522.2709
635.3553
56
.05
08
70
.02
83
11
7.9
21
7
14
4.0
66
5
24
4.0
98
6
28
6.1
81
1
33
6.7
05
2
39
4.2
23
8
41
7.2
12
9
45
8.2
73
4
48
7.2
72
0
54
6.3
36
9
57
1.3
55
95
90
.28
97
0
0.5
1.0
Time (min)
7.5 8.5 9.5
Oxidized
Unmodified
Co
un
tsCo
un
ts
L
86
.09
65
21
7.0
81
9
17
5.1
18
0
60
.05
42
0
0.5
1.0
20 60 100 140 180 220 260 300 340 380 420 460 500 540 580 620 660 700
M
10
4.0
53
0
26
2.1
50
4
33
0.1
65
6
31
0.1
83
2
375.2350
506.2759
619.3609
14
4.0
65
51
69
.13
20
18
9.0
86
7
24
5.1
31
3
28
4.1
60
02
31
2.1
54
5
35
7.2
22
7
40
9.2
15
7
44
6.2
41
6
48
8.2
67
9
52
8.2
85
0
58
5.3
19
8
64
5.3
35
9
67
5.3
78
6
0
1
2
3
20 60 100 140 180 220 260 300 340 380 420 460 500 540 580 620 660 700
L86.0963
T
74
.05
95
17
5.1
17
7 21
7.0
81
2
26
2.1
44
8
31
8.1
79
4
33
0.1
67
8
37
5.2
36
4
522.2686
47
7.2
02
4
63
5.3
54
4
56
.04
89
11
3.0
71
1
14
4.0
65
3
17
0.0
92
4
18
9.0
87
0
24
5.1
22
3
28
6.1
81
7
35
2.1
84
0
41
7.2
11
8
45
8.2
71
3
49
5.7
46
9
54
4.3
32
4
57
1.3
57
4
61
7.3
29
2
68
3.4
00
7
Co
un
ts
0
0.5
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Time (min)
7.5 8.5 9.5
Co
un
ts
Oxidized
Unmodified
Inn
ova
tor
Bio
sim
ilar
1
Bio
sim
ilar
2
Unmodified Oxidized×104
×104
×103 ×103
×103
×103 ×106
×106
×106
A B C
Mass-to-charge (m/z) Mass-to-charge (m/z)
Mass-to-charge (m/z) Mass-to-charge (m/z)
Mass-to-charge (m/z) Mass-to-charge (m/z)
8
DeamidationDeamidation is another commonly found modification that occurs primarily at asparagine (N) and glutamine (Q) residues. Deamidation on N residues occurs more rapidly than on Q residues. To compare the deamidation levels of innovator and biosimilar mAbs, the heavy
chain GFYPSDIAVEWESNGQPENNYK peptide sequence (375–396) was used. Figure 5 shows the GFYPSDIAVEWESNGQPENNYK analysis results. Analysis of MS/MS spectra showed a +1 Da mass shift of y9 product ions onwards, while y ions from y8 downwards are the same, indicating
Asn388 deamidation. The analysis of the results of relative quantification showed higher Asn388 deamidation levels in biosimilars 1 and 2 (19.99% and 19.83% respectively) compared to the innovator mAb (10.92%).
Co
un
tsC
ou
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Co
un
ts
Co
un
tsC
ou
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Co
un
ts
Inn
ova
tor
Bio
sim
ilar
1
Bio
sim
ilar
2
Unmodified OxidizedA B C
Mass-to-charge (m/z) Mass-to-charge (m/z)
Mass-to-charge (m/z) Mass-to-charge (m/z)
Mass-to-charge (m/z) Mass-to-charge (m/z)
0
1
2
3
4
5
6
7
100 300 500 700 900 1100 1300 1500 1700
P
70
.06
54
Y
13
6.0
75
7
y1
y1
y2
y2
y2
y3
y3
y4
y4
y7
y6
y6
y9
y10
y10
y11
y11
y12
y13
y14
y12
y13
y14
y19
2+
y19
2+
y19
2+
y20
2+
y20
2+
y8
14
7.1
14
b2
20
5.0
98
2 30
0.1
19
33
10
.17
77
b3
b3
y3
y4
b3
b6
b6
b7
b8
y7
y8
b7
b8
y6
y8
b7
b8
b10
y9
b10
y9
b10
b11
b11
y10 y
202+
b11
b12
b12
y11 y
12y
13
y14
b12
b15
36
8.1
58
6
42
4.2
18
8
48
4.2
39
3
53
8.2
63
3
64
9.2
63
16
67
.28
54
71
2.3
59
4
76
4.3
56
67
80
.35
61
85
1.3
90
58
92
.41
95 9
49
.43
80
10
63
.48
32
10
79
.50
51
08
8.9
94
7
11
50
.51
25
11
70
.52
51
12
65
.58
17
Precursor2+
Precursor2+
Precursor2+
1272.5714
12
79
.55
89
13
94
.63
57
14
65
.63
71
15
94
.67
90
16
93
.74
89
16
52
.72
08
0
1.0
2.0
100 300 500 700 900 1100 1300 1500 1700
0.5
1.5
P
70
.06
49
12
10
.54
06
84
.08
14
Y
13
6.0
75
91
47
.11
18
18
5.0
93
22
05
.09
35 3
00
.11
92
31
0.1
77
5
36
8.1
64
7
42
4.2
22
1
53
8.2
61
4
58
3.3
05
9
71
2.3
45
3
76
4.3
58
97
80
.35
68
85
1.3
93
1
89
2.4
11
8
94
9.4
39
2
10
64
.46
53
10
79
.50
91
10
89
.48
59
11
51
.49
66
11
71
.02
07
12
66
.58
31
1273.0619
12
80
.53
56
14
66
.61
10
15
95
.66
18
16
94
.72
47
10
27
.49
06
13
21
.49
66
0
0.5
1.0
Time (min)17.2 17.6
0
0.5
1.0
1.5
2.0
2.5
3.0
100 300 500 700 900 1100 1300 1500 1700
Y
13
6.0
75
4
P
y1
y2 y
3
y4
b3
b4
b2
b2
b6
y6
y7
y8
y9
b7
b8 b
10
y10
y20
2+
y19
2+
y11
y13
y12
y14
b13
Precursor2+
y2
y3
y4
b3
b6
Y
P y1
b2
Y
P
y6
y6
y7
y7
y8
y8
y9
y9
b7
b7
b8
b8
b10
b10
y10
y20
2+
y19
2+
y19
2+
y10
2+
y11
y11
y13
y12
y14
y13
y12
y14
b12
b11
y10
y20
2+
b11
Precursor2+
Precursor2+
y4
b6
y1
y1
70
.06
62
14
7.1
12
2
36
8.1
62
2
17
7.1
01
9
23
8.1
23
4
30
0.1
19
13
10
.17
42
41
3.2
04
24
24
.21
94
48
4.2
36
2
53
8.2
59
4
66
7.2
81
5
71
2.3
49
3
76
4.3
56
47
80
.35
41
85
1.3
90
1
89
2.4
05
7
94
9.4
36
3
10
15
.93
71
10
63
.46
65
10
80
.49
24
10
88
.98
68
11
50
.50
66
11
70
.52
19
12
65
.58
59
1272.5636
12
79
.54
21
13
34
.52
80
13
94
.62
11
14
65
.62
99
15
94
.66
29
16
93
.73
98
0
0.5
1.0
1.5
100 300 500 700 900 1100 1300 1500 1700
Y
13
6.0
75
8
F
12
0.0
80
6
15
26
.60
34
84
.08
09
17
7.1
03
01
47
.11
12
25
9.1
36
8
30
3.1
53
7
34
1.1
40
1
38
7.2
00
7
53
8.2
61
1
48
5.2
42
5
58
3.3
08
65
76
.26
57
66
7.2
80
4
76
4.3
54
67
80
.35
37
85
1.4
00
8
89
2.4
10
09
49
.44
26
10
64
.47
25 10
79
.50
47
10
89
.48
64
11
71
.51
85
12
66
.58
73
1273.0616
12
80
.55
01
13
94
.62
13 1
46
6.6
18
9
15
95
.66
82
16
94
.72
04
15
14
.66
99
0
0.5
1.0
Time (min)17.2 17.6
0
1
2
3
4
100 300 500 700 900 1100 1300 1500 1700
13
6.0
75
21
47
.11
30
20
5.0
95
4
70
.06
51
30
0.1
20
33
10
.17
46
36
8.1
60
8
41
3.2
03
64
24
.22
30
46
5.2
12
04
84
.23
84
53
8.2
60
8
58
3.3
05
9
63
9.2
82
76
67
.28
51
76
4.3
57
17
80
.36
05
85
1.3
94
8
94
9.4
40
5
10
63
.47
97
10
79
.50
46
10
88
.99
35
11
50
.51
33
11
70
.52
86
12
66
.58
16
1272.5170
12
79
.55
54
13
94
.62
68
14
65
.63
99
15
94
.67
87
16
93
.76
06
0
0.5
1.0
100 300 500 700 900 1100 1300 1500 1700
13
6.0
74
11
47
.11
08
20
6.1
00
4
70
.06
49
18
5.0
90
2
22
9.1
17
6
31
0.1
75
4
36
8.1
59
0
42
4.2
18
7
53
8.2
63
2
61
8.2
44
6
66
7.2
72
7
76
4.3
60
07
81
.36
32
85
1.3
96
7
89
2.4
24
8 94
9.4
44
4
10
64
.45
35
10
79
.50
51
10
89
.99
18
11
71
.52
05
11
51
.50
47
1273.0659
12
80
.53
89
14
66
.62
15
14
81
.66
33
15
95
.67
39
16
94
.72
87
0
1
Time (min)17.2 17.6
Unmodified
Unmodified
Unmodified
Deamidated
Deamidated
Deamidated
11
51
.49
27
×103
×103
×103
×103
×103
×103
Figure 5. Deamidation analysis of GFYPSDIAVEWESNGQPENNYK peptide sequence (375–396). (A) MS/MS spectrum of unmodified and deamidated peptide, (B) ECC overlay, and (C) relative quantification of N388 (three replicate measurements).
9
PTMs on other peptides were also analyzed using the histogram feature of BioConfirm. Figure 6 shows the representative relative quantification for other modified residues of innovator and biosimilar mAbs. The histogram comparison display provides an easy and quick way of monitoring the levels of quality attributes across a series of samples.
Figure 6. Representative histograms showing relative quantitation of PTMs (oxidation, deamidation, Lys truncation, and N-terminal cyclizations of Gln to pyroGlu).
Heavy Chain
M432 - Oxidation M21 - Oxidation W90 - Oxidation
Light Chain Light Chain
Heavy Chain
Q366 - Deamidation
Light Chain
N137 - Deamidation
Heavy Chain
N290 - Deamidation
Heavy Chain
N33 - Deamidation
Heavy Chain
K451 - Lys loss Heavy Chain
Q1 - PyroGlu
Light Chain
Q1 - PyroGlu
Innovator
Biosimilar 1
Biosimilar 2
www.agilent.com/chem
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019 Printed in the USA, December 18, 2019 5994-1495EN DE.0897222222
ConclusionThis Application Note demonstrates an integrated peptide mapping workflow to study innovator and biosimilar mAbs using an Agilent AssayMAP Bravo platform, Agilent Accurate Mass 6545XT AdvanceBio LC/Q-TOF, and easy-to-use Agilent MassHunter BioConfirm software for data analysis. The results obtained on peptide mapping provides excellent sequence coverage for innovator and biosimilar mAbs. Precise characterization of PTMs was achieved due to high-efficiency chromatographic separation and high-quality MS/MS spectra. This integrated approach also allowed relative quantification of various PTMs between innovator and biosimilar mAbs.
References1. Ecker DM1, Jones SD, Levine HL. The
Therapeutic Monoclonal Antibody Market. MAbs. 7(1):9-14 2015
2. Agilent Technologies, publication number 5991-8552EN.
