Intact Monoclonal Antibody
Characterization Using A Bench-Top
Orbitrap Mass Spectrometer
Zhiqi Hao Thermo Fisher Scientific
San Jose, CA
2
Image from Kozlowski et al. n engl j med 365;5 nejm.386 org august 4, 2011
Aspirin (180 Da)
MAb (150,000 Da)
Monoclonal Antibody (mAb) – an Analytical Challenge
3
mAb Characterization – HR/AM Mass Spectrometry a Natural Fit
• Some of the analytical challenges:
Deamidation (+1 Da)
Oxidation (+16 Da)
Disulfide scrambling
Sequence truncation
Change in glycosylation
Aggregation
etc…
• Accurate characterization requires high resolution and accurate mass MS
• Characterizations are performed at intact protein, sub-structure and peptide levels
• Mass ~150 kDa • 2 light chains (~20 kDa each) • 2 heavy chains (~50 kDa each) • 16 disulfide bonds • Glycans
4
Outline
• The new generation OrbitrapTM mass analyzer • Intact antibody characterization using the Q ExactiveTM
quadrupole Orbitrap mass spectrometer • Introducing Q Exactive quadrupole Orbitrap mass spectrometer
• Intact mAb analysis • Mass measurement
• Relative quantification of glycoforms
• Structure characterization
• Fab
• Summary
5
• The new generation OrbitrapTM mass analyzer
Exactive Plus
Q Exactive
Orbitrap Elite
6
Intact Protein Detection in Orbitrap Analyzer
0 1000 2000 3000 t, ms
Myoglobin (16.9 kD)
z: +16
0 1000 2000 3000 t, ms
0 1000 2000 3000 t, ms
0
Transferrin (78kD)
z: +40
Enolase (46kD)
z: +24
Solutions: • Higher Vacuum • Hardware Innovation • Software Innovation
Fast Signal Decay: • Collision of ions • Fragmentation of ions • De-phasing of ion packets
7
#29216 IT: 500.000 ST: 1.05 uS: 1 NL: 7.15F: FTMS + p ESI Full ms [0.00-384.00]Info: Ufill=0.01 MCal=24h RF=1810V TmpDiff
0 1 2 3 4 5 6 7 8 9 10m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive
Inte
nsity
6.56290.3585
6.4464
6.40610.50106.34466.3241
6.2629
6.24326.10036.0396
5.83635.77555.63274.96222.14822.0607 4.51544.08912.18082.0204
3.68333.52083.2571 9.90649.55888.95501.1884 8.84477.86337.2506
#27955 IT: 10.000 ST: 0.28 uS: 1 NL: 7.15E2F: FTMS + p ESI Full ms [0.00-96.00]Info: Ufill=0.01 MCal=9h RF=1430V Stable=2min
0 1 2 3 4 5 6 7 8 9 10 11m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive In
tensit
y
0.1758
0.3435
0.4006
0.5435 4.7990 7.63040.6147 4.32664.0796 7.36875.5774 10.38089.58811.0611 10.63228.87952.9427 6.8835 7.7499 10.9545
ms ms
8.9 ms < 0.6 ms
Earlier Orbitrap Instruments
Q Exactive Orbitrap Elite Exactive Plus
Reduced Detection Delay Improves Intact Protein Measurement
0 1000 2000 3000 t, ms
0
Transferrin (78kD)
z: +40
<10ms Efficient Capture of Initial Transient
Stronger Signal
Better Mass Accuracy
8
0
20000
40000
60000
80000
100000
120000
140000
160000
0 5 10 15
Re
solu
tio
n
Scan Speed [Hz]
Exactive
Q-Exactive Resolution
at m/z 200
Resolution
at m/z 400
Max. scan
speed (Hz)
17,500 12,500 12
35,000 25,000 7
70,000 50,000 3
140,000 100,000 1.5
Resolution increased by 2X
Speed increased by 2X
Improved Resolution and Scan Speed
9
• Intact antibody characterization using the Q ExactiveTM
quadrupole Orbitrap mass spectrometer
• Introducing Q Exactive quadrupole Orbitrap mass spectrometer
• Intact mAb analysis • Mass measurement
• Relative quantification of glycoforms
• Structure characterization
• Fab
10
Q ExactiveTM - Innovations and Features
The first benchtop Quadrupole-Orbitrap mass spectrometer
ORBITRAP
Resolution: 140,000
Mass Accuracy: < 3 ppm
QUADRUPOLE MASS FILTER
Mass Range: 50 – 6000 amu Prec Isolation: 0.4 – 10 amu
HCD cell and C-Trap
ION SOURCE
S-Lens
11
AntiHer2_2ug_5000 4/23/2012 4:35:25 PM
RT: 0.00 - 20.02
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
5.89
0.37 6.71 7.23 7.96 15.518.35 15.949.08 11.069.55 11.45 16.6212.10 13.07 13.4110.00 14.990.85 17.215.713.743.15 5.071.70 18.762.53 4.35 19.77
NL:2.79E9TIC MS AntiHer2_2ug_5000
AntiHer2_2ug_5000 #439-512 RT: 5.73-6.07 AV: 74 NL: 3.04E7T: FTMS + p ESI sid=45.00 Full ms [1800.00-5000.00]
1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
2907.25
2797.59 3025.87
3088.892745.82
2695.90
2968.59
3154.57
2647.78 3223.152601.35
3294.722556.54
2513.21 3369.59
2430.84 3447.922353.71
3529.932246.72 3616.002149.04 3801.46 3901.522003.93 4006.911899.12 4122.56 4235.94 4365.32
AntiHer2_2ug_5000 #439-512 RT: 5.73-6.07 AV: 74 NL: 3.04E7T: FTMS + p ESI sid=45.00 Full ms [1800.00-5000.00]
2940 2960 2980 3000 3020 3040 3060 3080 3100 3120m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
2965.3810
3025.8740
3088.8882
2971.84453032.5158
3095.5851
3019.5958
2959.2434
3082.4699
2977.68572996.3881
3038.23963055.3179
3101.77532933.2498 2953.0596
3013.3330 3075.9493
AntiHer2_2ug_5000 4/23/2012 4:35:25 PM
RT: 0.00 - 20.02
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
5.89
0.37 6.71 7.23 7.96 15.518.35 15.949.08 11.069.55 11.45 16.6212.10 13.07 13.4110.00 14.990.85 17.215.713.743.15 5.071.70 18.762.53 4.35 19.77
NL:2.79E9TIC MS AntiHer2_2ug_5000
AntiHer2_2ug_5000 #439-512 RT: 5.73-6.07 AV: 74 NL: 3.04E7T: FTMS + p ESI sid=45.00 Full ms [1800.00-5000.00]
1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
2907.25
2797.59 3025.87
3088.892745.82
2695.90
2968.59
3154.57
2647.78 3223.152601.35
3294.722556.54
2513.21 3369.59
2430.84 3447.922353.71
3529.932246.72 3616.002149.04 3801.46 3901.522003.93 4006.911899.12 4122.56 4235.94 4365.32
Total Ion Chromatogram
Averaged full MS spectrum across 1 min elution peak
Intact Monoclonal Antibody Mass Measurement
G0F+G1F
G1F+G2F
G0F+G0F
G0+G0F
G0F+G2F
• 2 ug mAb on column • 5 min gradient • Instrument resolution setting = 17.5K
12
Deconvoluted Spectrum
-7 ppm G0F+G1F
G0F+G0F G0F+G2F (or 2G1F)
G1F+G2F
G2F+G2F G0+G0F
G0+G0 2xMan5
G1F+G2F+SA
5.0 ppm
-0.7 ppm -8.5 ppm
-0.9 ppm
• 9 glycoforms identified, all within ~12 ppm of theoretical average mass (+/-2 Da mass accuracy)
• Deglycosylated form detected with high mass accuracy at low relative abundance
• Deconvoluted spectrum is free of artifacts and the various forms of the protein are well resolved
13
Reproducible Mass Measurement for IgG Glycoforms
• -6.9 +/- 6.4 ppm mass error across all measurements
• Mass measurement is highly reproducible, even across instruments
RAW file Q Exactive G0+G0F G0F+G0F G0F+G1F G0F+G2F G1F+G2F
1 1 -10.5 0.7 -10.5 -13.8 -18.0
2 1 -11.6 -1.1 -8.8 -11.2 -12.0
3 1 5.1 -5.0 -2.6 5.1 5.6
4 2 -14.3 3.0 -6.9 -5.4 -5.9
5 2 -8.6 -2.2 -12.2 -12.5 -12.9
6 2 -14.3 -6.6 -12.3 -14.8 -10.1
-7 ppm
G0F+G1F
G0F+G0FG0F+G2F (or 2G1F)
G1F+G2F
G2F+G2FG0+G0F
G0+G02xMan5
G1F+G2F+SA
5.0 ppm
-0.7 ppm -8.5 ppm
-0.9 ppmIgG glycoform mass measurement accuracy (error in ppm)
14
• Relative abundances are highly reproducible across runs
Reproducible Relative Abundances for IgG Glycoforms
IgG glycoform relative abundances (%)
RAW file Q Exactive G0+G0F G0F+G0F G0F+G1F G0F+G2F G1F+G2F
1 1 12.9 74.1 100.0 67.0 23.4
2 1 12.0 72.8 100.0 66.2 22.0
3 1 12.2 75.0 100.0 67.0 23.6
4 2 12.7 75.7 100.0 63.6 21.6
5 2 13.2 75.4 100.0 64.8 21.0
6 2 12.9 76.6 100.0 64.7 21.6
CV 3.4% 1.6% N.A. 3.9% 4.4%
-7 ppm
G0F+G1F
G0F+G0FG0F+G2F (or 2G1F)
G1F+G2F
G2F+G2FG0+G0F
G0+G02xMan5
G1F+G2F+SA
5.0 ppm
-0.7 ppm -8.5 ppm
-0.9 ppm
15
Characterization of Fab
Papain_digest_no_cys_nonlinear-2ul #525-556 RT: 18.97-19.51 AV: 32 NL: 1.86E6T: FTMS + p ESI Full ms [800.00-3500.00]
1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive A
bund
ance
1475.30
1430.59 1522.86
1348.93
1573.57
1311.46
1627.82
1276.061685.91
1748.311242.55
1815.511888.07
1966.741210.68
2052.18
1180.472145.45
2247.55
2008.66 2097.89
1900.37
2160.00 2318.22
intact Fab Fab light Fab heavy
Reduction
Intact Fab
Fab light
LC MS
Fab heavy
47177.6 Da 23038.8 Da 24148.3 Da
PPN_red_60C_02 #1157-1169 RT: 20.34-20.47 AV: 13 NL: 4.38E4T: FTMS + p ESI Full ms [1000.00-2700.00]
1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive A
bund
ance
1510.311421.47
1610.80
1342.58
1725.83
1272.02
1208.29
1689.821440.80
1619.461487.38
1579.531547.13 1661.981466.57
1714.22
1760.17
1733.09
1385.811356.36
1323.971281.05
1412.83
1263.861222.44
PPN_red_60C_02 #1138-1144 RT: 20.13-20.18 AV: 7 NL: 1.30E5T: FTMS + p ESI Full ms [1000.00-2700.00]
1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive A
bund
ance
1440.89
1356.12
1537.021280.95
1646.581213.53
1153.05 1773.26
1328.25 1689.951475.811388.321272.79 1413.63
1562.471162.23 1613.361507.74
1288.45
1657.88
1256.64 1723.071748.38
RT: 7.95 - 20.71
8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relati
ve Abu
ndance
13.95
14.18
16.78 17.64 18.18 18.71 19.36 20.4310.72 11.14 12.118.25 9.32 9.648.89
NL:2.04E8TIC MS PPN_noCys_Red_140K
Fc
Preparation and identification of Fab light and heavy chains
• The 10 Da mass difference between the intact and the sum of the reduced Fab suggests 5 disulfide bonds, just as expected
16
High Resolution Measurement of Fab on LC Time Scale
20111109_Reduced_140k_Dionex_03 #893-919 RT: 36.12-36.86 AV: 27 NL: 5.71E5T: FTMS + p ESI Full ms [600.00-3000.00]
1440.2 1440.4 1440.6 1440.8 1441.0 1441.2 1441.4 1441.6 1441.8m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive
Abun
danc
e
1440.85 1440.97
1441.101440.78
1441.16
1440.72
1441.22
1440.661441.29
1440.60
1441.411441.481440.53
1440.091440.23 1440.48 1441.54
1441.661441.78
20111109_Reduced_140k_Dionex_03 #894-919 RT: 36.15-36.86 AV: 26 NL: 7.12E5T: FTMS + p ESI Full ms [600.00-3000.00]
800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relativ
e Abun
dance
1213.61z=19 1281.03
z=181098.03
z=211048.16z=22 1356.21
z=171152.93
z=20 1440.85z=16
1536.97z=15
1002.68z=23
960.98z=24
1646.75z=14
922.62z=? 1773.28
z=13
887.06z=?
854.32z=?
823.77z=?
798.98z=?
1707.55z=?
1403.22z=?
1467.62z=?
1836.75z=?
1587.56z=?
1784.51z=?
Monoisotopic mass measured at 140 K
Fab Measured Expected Error (ppm)
H 24133.8700 24133.7937 2.7
L 23025.3342 23025.3125 0.9
Baseline resolved isotopic peaks of light chain
+15 charge
17
Identification and Relative Quantification of Impurities
Papain_digest_no_cys_nonlinear-2ul #428-465 RT: 17.41-18.00 AV: 38 NL: 7.39E6T: FTMS + p ESI Full ms [800.00-3500.00]
1100 1200 1300 1400 1500 1600 1700 1800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1430.63
1475.31
1388.57
1522.871348.92
1573.56
1311.491627.82
1685.911276.09
1242.53 1748.291815.54
1210.67
1180.47
1151.64
1609.46
1124.301654.55
1716.84 1780.78 1831.26
Papain_digest_no_cys_nonlinear-2ul #428-465 RT: 17.41-18.00 AV: 38 NL: 5.97E6T: FTMS + p ESI Full ms [800.00-3500.00]
1516 1518 1520 1522 1524 1526 1528 1530 1532m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1522.87
1523.49
1521.461517.50
1520.54 1526.181519.51 1524.86 1528.041516.941518.56
1530.381529.26 1532.30
Sub-structure Resolution Impurity Delta Mass (Da) Relative intensity (%)
Fab 17.5K 16.3 18.1
Fab heavy 140K 16.0158 21.9
PPN_red_37C_140K_120504084725_XT_00001_M_ #1 RT: 1.00 AV: 1 NL: 3.57E4T: FTMS + p ESI Full ms [1000.00-2700.00]
24120 24130 24140 24150 24160 24170 24180m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
24147.93
24146.94 24149.94
24145.91
24150.94
24151.9424144.89
24152.9524143.90
24153.93
24154.9724156.0024141.95
24163.9024166.90
24140.89 24169.9224129.93 24138.85 24173.0624123.9724116.87
Oxidized Species of Fab
Intact Fab Fab heavy Resolution =140 K Resolution =17.5 K
Average mass Monoisotopic mass
18
20111109_Reduced_tHCD20light_Dionex03 #94-111 RT: 36.20-36.80 AV: 18 NL: 6.63E4T: FTMS + p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [ ...
200 400 600 800 1000 1200 1400 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive A
bund
ance
324.19z=1
437.28z=1
739.40z=1159.09
z=1 524.31z=1
216.10z=1
812.34z=1 1175.91
z=9409.28
z=11322.90
z=8974.97
z=6303.13
z=11076.53
z=4839.48
z=3703.74z=3 1435.71
z=6619.30
z=21512.03
z=7
1377.71z=3
1751.88z=5
Targeted HCD on six precursors of different charge states
Top-down Sequencing Using Unique Spectrum Multiplexing Mode
Six Individual isolation and fragmentation events
One Orbitrap detection event
High resolution top-down MS/MS
Full MS of light chain 20111109_Reduced_140k_Dionex_03 #894-919 RT: 36.15-36.86 AV: 26 NL: 7.12E5T: FTMS + p ESI Full ms [600.00-3000.00]
800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relative
Abundan
ce
1213.61z=19 1281.03
z=181098.03
z=211048.16z=22 1356.21
z=171152.93
z=20 1440.85z=16
1536.97z=15
1002.68z=23
960.98z=24
1646.75z=14
922.62z=? 1773.28
z=13
887.06z=?
854.32z=?
823.77z=?
798.98z=?
1707.55z=?
1403.22z=?
1467.62z=?
1836.75z=?
1587.56z=?
1784.51z=?
Parallel Filling and Detection Spectrum Multiplexing
19
30% backbone fragments
Sequence Coverage Achieved for Fully Reduced Fab
Light chain Fab heavy chain
34% backbone fragments P Score = 2.2E-34
• ‘Signal protein’ mode • 5 ppm fragment ion tolerance
P Score = 4.9E-19
20
Top-down Approach Can Provide Structural Information
Mapping disulfide linkage in partially reduced light chain PPN_noCys_Red_140K #129-133 RT: 13.79-14.10 AV: 5 NL: 8.83E4
T: FTMS + p ESI Full ms [1000.00-2700.00]
1151.5 1152.0 1152.5 1153.0 1153.5 1154.0m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
1152.73z=20
1152.78z=20
1152.63z=20 1152.83
z=20
1152.58z=20
1152.93z=20
1152.98z=20
1152.53z=20
1153.03z=201152.48
z=201153.13
z=201151.98z=201151.48
z=?1152.38
z=201151.77
z=20 1153.53z=?1153.18
z=201153.68
z=? 1153.98z=? 1154.32
z=?
• Deconvoluted monoisotopic mass was 23021.4185 Da, 4 Da less than expected.
• Top-down sequencing mapped two internal disulfide bonds
• Further sample processing produced completely reduced light chain with monoisotopic mass of 23025.3342 Da, only 0.9 ppm off expected.
+20 charged
partially reduced light chain completely reduced light chain
21
•Summary
22
Summary
• Accurate, reproducible and fast mass measurement of intact mAb was achieved using the Q Exactive MS and Protein Deconvolution software.
• The Q Exactive high resolution top-down analysis and ProSight PC sequence assignment generated extensive, accurate sequence information for Fab light chain and heavy chain, offering a fast way to confirm amino acid sequence and detect unexpected modifications.
• Both, precise mass measurements and extensive, high confident sequence and structural information can be obtained for intact mAb using this workflow solution that combines fast chromatography, high resolution MS, high throughput msx HCD and high fidelity data analysis.
23
Intact Monoclonal Antibody Characterization Workflow
Cell culture
Affinity purification
Charge separation
Desalting /
H/L chain separation
AntiHer2_2ug_5000 4/23/2012 4:35:25 PM
RT: 0.00 - 20.02
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
5.89
0.37 6.71 7.23 7.96 15.518.35 15.949.08 11.069.55 11.45 16.6212.10 13.07 13.4110.00 14.990.85 17.215.713.743.15 5.071.70 18.762.53 4.35 19.77
NL:2.79E9TIC MS AntiHer2_2ug_5000
AntiHer2_2ug_5000 #439-512 RT: 5.73-6.07 AV: 74 NL: 3.04E7T: FTMS + p ESI sid=45.00 Full ms [1800.00-5000.00]
1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
2907.25
2797.59 3025.87
3088.892745.82
2695.90
2968.59
3154.57
2647.78 3223.152601.35
3294.722556.54
2513.21 3369.59
2430.84 3447.922353.71
3529.932246.72 3616.002149.04 3801.46 3901.522003.93 4006.911899.12 4122.56 4235.94 4365.32
20111109_Reduced_tHCD20light_Dionex03 #94-111 RT: 36.20-36.80 AV: 18 NL: 6.63E4T: FTMS + p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [ ...
200 400 600 800 1000 1200 1400 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive A
bund
ance
324.19z=1
437.28z=1
739.40z=1159.09
z=1 524.31z=1
216.10z=1
812.34z=1 1175.91
z=9409.28
z=11322.90
z=8974.97
z=6303.13
z=11076.53
z=4839.48
z=3703.74z=3 1435.71
z=6619.30
z=21512.03
z=7
1377.71z=3
1751.88z=5
MS of intact antibody
Top-down HCD of light chain
-7 ppm
G0F+G1F
G0F+G0FG0F+G2F (or 2G1F)
G1F+G2F
G2F+G2FG0+G0F
G0+G02xMan5
G1F+G2F+SA
5.0 ppm
-0.7 ppm -8.5 ppm
-0.9 ppm
from crude cell culture to HR/AM characterization
• Intact Mass
• Relative quan of glycoforms
• Sequence confirmation
• Structural information
Poster : ThP15,
Shanhua Lin et. al
http://portal.thermo-brims.com
24
Acknowledgement
• Thermo Fisher Scientific, San Jose and Bremen Yi Zhang David Horn Seema Sharma Xiaoyue Jiang Andreas FR Huhmer Patrick K Bennett Markus Kellmann Thomas Moehring
• Barnett Institute, Northeastern University, Boston Shiaw-Lin Wu
• CGMP BPPF, Development Center for Biotechnology, Taipei Irene, Ae-Ning Lin
Yi-Hsuan Pan Ya-Fen Yang