mabs and adcs analysis by reversed phasetools.thermofisher.com/...adc-reversed-phase...en.pdf ·...

Shanhua Lin, Ph.D.

mAbs and ADCs analysis by RP

The world leader in serving science

Protein and mAb Separation by HPLC

Size Exclusion Chromatography (SEC)Size difference?

MAbPac SEC-1YES

Ion Exchange Chromatography (IEX)Charge difference?

YES

NO

MAbPac SCX-10ProPac WCX-10C ff

NO

Chromatography (IEX)

R PhNO

CX-1 pH gradient Buffer

NO

Reverse Phase Chromatography (RPC)

Hydrophobicity difference?MAbPac HIC 10

YES MAbPac RP

Hydrophobic Interaction Chromatography (HIC)

MAbPac HIC-10MAbPac HIC-20MAbPac HIC-ButylProPac HIC-10

YES

2

Executive Summary

• Designed for high-resolution separation of monoclonal antibodies (mAbs) and mAb fragments (including LC HC Fc Fab scFc

Thermo Scientific™ MAbPac™ RP Column

(mAbs) and mAb fragments (including LC, HC, Fc, Fab, scFc, and F(ab)2) by reverse phase chromatography

• Developed for analytical chemists who need High Resolution Accurate Mass determination of monoclonal antibody variants, antibody drug conjugates (ADC), and proteins

3

RP Columns Are Important for mAb Analysis

• mAbs are highly heterogeneous and are susceptible to degradation• Glycosylation• Modifications on the termini• Oxidation• Deamidation• Isomerization• Reduction of disulfide bond• Aggregation

• LC/MS analysis of mAb and mAb fragments can reveal these modifications without complete digestion and peptide mapping.

4

Fast Separation of Proteins

Column: MAbPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: H2O/TFA (99.9 : 0.1 v/v)Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1113

125

140

3Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1

v/v/v/)Gradient:

Time (min) %A %B-1.0 80 200.0 80 202 5 50 5075

88

100

2.5 50 502.7 50 502.8 80 203.0 80 20

Temperature: 80 ºCFlow rate: 0 6 mL/min

50

63

75

24

Flow rate: 0.6 mL/minInj. volume: 1 µL Detection: UV (280 nm)Sample: 1. Ribonuclease A (0.5 mg/mL)

2. Cytochrome C (0.5 mg/mL)3. Lysozyme (0.5 mg/mL)4 Ab (1 / L)

13

25

38

1

4. mAb (1 mg/mL)

0.00 0.50 1.00 1.50 2.00 2.50 3.00-20

0

5

Retention Time (min)

Reproducibility of MAbPac RP

Column: MAbPac RP, 4 µmFormat: 2.1 ×50 mm

1203

Format: 2.1 50 mmMobile phase A: H2O/TFA (99.9 : 0.1 v/v)Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1

v/v/v/)Gradient:

Time (min) %A %B-1 80 20

60

80

100

24

-1 80 200.0 80 202.5 50 502.7 50 502.8 80 203.0 80 20

0

20

40

#1101

#901

1

Temperature: 80 ºCFlow rate: 0.6 mL/minInj. volume: 1 µL Detection: UV (280 nm)Sample: 1. Ribonuclease A (0.5 mg/mL)

2 C t h C (0 5 / L)60

-40

-20

#901#801#701#651#501#401#301

2. Cytochrome C (0.5 mg/mL)3. Lysozyme (0.5 mg/mL)4. mAb (1 mg/mL)

0.00 0.50 1.00 1.50 2.00 2.50 3.00

-80

-60 #201#101


6

Loadability: Three Orders of Magnitude

1.25

2.50 (a) 0.02 µg Column: MAbPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: H2O/TFA (99.9 : 0.1 v/v)Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1

v/v/v/)Gradient: Time (min) %A %B

-0.50

10.0

18.0 (b) 0.2 µg

( )-1 85 150.0 85 152.5 50 502.7 50 502.8 85 154.0 85 15

-2.0140 (c) 2 µg

4.0 85 15Temperature: 80 ºCFlow rate: 0.6 mL/minInj. volume: 1 µL Detection: UV (280 nm)Sample: mAb

-20900 (d) 20 µg

y = 2.3101x + 0.0491R² = 1

20

25

30

35

40

45

50

Area

1.00 1.50 2.00 2.50 3.00 3.50 4.00

-100

500

0

5

10

15

20

0 10 20 30mAb (µg)

7


Carryover

Column: MAbPac RP, 4 µmFormat: 3 ×50 mmMobile phase A: H2O/TFA (99.9 : 0.1 v/v)Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1

1: mAb injection (peak area = 37.97)500

600

v/v/v)Gradient:

Time (min) %A %B0.0 100 01.0 100 011.0 0 100

400

12.0 0 10014.0 100 015.0 0 100

Temperature: 80 ºCFlow rate: 0.5 mL/min

200

300

Inj. volume: 5 µL Detection: UV (280 nm)Sample: mAb (5 mg/mL)2: blank (Peak area = 0.233)Carryover

Stability at High pH Condition

140

Column: MAbPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: H2O/TFA (99.9 : 0.1 v/v)Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1

v/v/v/)113

125

140

After 6 hrs of 0.8 M NaOH wash at 80 C

3

Gradient:Time (min) %A %B-1.0 85 150.0 85 152.5 50 502.7 50 50

75

88

1002

4

2.8 85 154.0 85 15

Temperature: 80 ºCFlow rate: 0.6 mL/minInj volume: 1 µL38

50

63

1

Inj. volume: 1 µL Detection: UV (280 nm)Sample: 1. Ribonuclease A (0.5 mg/mL)

2. Cytochrome C (0.5 mg/mL)3. Lysozyme (0.5 mg/mL)4. mAb (1 mg/mL)

0

13

25 Before NaOH wash

0.50 1.00 1.50 2.00 2.50 3.00 3.50-20

0


9

( )

Separation of mAb Fragments

10

Structure of IgG and Typical Forms of Heterogeneity

11

mAb Fragments

(a)

(b)

(c)(c)

12

mAb and mAb Fragments Analysis

mAb

Column: MAbPac RP, 4 µmFormat: 3 ×50 mmMobile phase A: H2O/FA/TFA (99.88 : 0.1:0.02 v/v/v)Mobile phase B: MeCN/ H2O/FA/TFA (90: 9.88

140 mAb(a)

:0.1:0.02 v/v/v/v)Gradient:

Time (min) %A %B0.0 80 201.0 80 2011.0 55 45

-20

50.0

80.0

LC

HC(b)

12.0 55 4514.0 80 2015.0 80 20


-10.0

50

90

Fc

Fab(c)

Inj. volume: 5 µL Detection: UV (280 nm)Sample: (a) trastuzumab (5 mg/mL)

(b) trastuzumab + DTT (4 mg/mL)(c) trastuzumab + Papain (2 mg/mL)(d) trastuzumab + IdeS (2 mg/mL)

-10100 F(ab)2(d)

(d) trastuzumab + IdeS (2 mg/mL)

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

-10

50 scFc

13


LC/MS Analysis of Intact mAb and Glycosylation Profile

RT: 4.44 - 13.13

1008.26 NL:

5 59E8 Column: MAbPac RP 4 µm

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

Relative Abundance

8 44

5.59E8TIC MS herceptin_5mgperml

Column: MAbPac RP, 4 µmFormat: 3 ×50 mmMobile phase A: H2O/FA/TFA (99.88 : 0.1:0.02

v/v/v)Mobile phase B: MeCN/ H2O/FA/TFA (90: 9.88


TIC

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0Time (min)

0

5

10

15

20 8.44

12.748.6310.5310.339.888.97 9.729.377.395.935.685.43 6.095.134.47 6.996.74 7.64

herceptin_5mgperml #167-173 RT: 8.22-8.33 AV: 7 NL: 8.14E6T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

75

80

85

90

95

1003223.20

3088.923294.80 3369.64

3154.64

3025.91

2965.42 3447.98

Gradient:Time (min) %A %B0.0 80 201.0 80 2011.0 55 4512.0 55 4514 0 80 20

MS spectrum

10

15

20

25

30

35

40

45

50

55

60

65

70

75

Relative Abu

ndan

ce

2907.27

3530.09

3616.14

2851.393706.50

2797.55

3801.532745.82

2695.91

2647.763901.54

2601.37

2513.182471.32

2430.80

2394.25

3917.63

14.0 80 2015.0 80 20

Temperature: 80 ºCFlow rate: 0.5 mL/minInj. volume: 1 µL

2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

52283.77

2183.132057.49 3994.21 MS Detection: positive-ion mode

Mass Spec: Q Exactive™ PlusSample: trastuzumab (5 mg/mL)

herceptin_5mgperml #167-173 RT: 8.22-8.33 AV: 7 NL: 6.26E6T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

70

75

80

85

90

95

100 2965.42

2968.61m/z at 50+

15

20

25

30

35

40

45

50

55

60

65

Relative Abu

ndan

ce 2962.22

2971.87

2959.34

14

2930 2935 2940 2945 2950 2955 2960 2965 2970 2975 2980 2985 2990 2995 3000m/z

0

5

102974.96

2996.302953.05 2993.072936.54 2977.492956.45

2933.53 2980.99 2986.822939.75 3002.662946.14

LC/MS Analysis of Reduced mAb

Column: MAbPac RP 4 µmLC HCRT: 3.00 - 11.00

20

30

40

50

60

70

80

90

100

Rel

ative

Abu

ndan

ce

8.287.43

NL:2.54E8TIC MS Herceptin_HC+LC_1ul



:0.1:0.02 v/v/v/v)G di t

TICLC HC

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

uAU

0

10 8.58 8.86 10.7710.373.943.63 9.114.793.23 10.024.34 9.765.20 5.50 5.95 6.10 7.956.41 6.968.28

7.42

NL:6.06E5UV_VIS_1 UV Herceptin_HC+LC_1ul

Gradient:Time (min) %A %B0.0 80 201.0 80 2011.0 55 4512.0 55 45

UV

70

80

90

1001954.16

1803.88

2131.63

1675 14

NL: 2.52E7Herceptin_HC+LC_1ul#152 RT: 7.42 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0Time (min)

0

50000

1000008.85 9.107.736.806.636.35

14.0 80 2015.0 80 20


LC

1000

10

20

30

40

50

60

70

Rel

ativ

e A

bund

ance

1675.14

1563.47 2344.74

1465.80

1379.68

1303.031967.87 2146.62

1234.471687.01 2361.04

2233.121575.621819.28

1478.76

1762.96

2095.001392.86

1638.291539.03 NL: 6.19E6Herceptin_HC+LC 1 l#178 RT 8 28

UV Detection: 280 nmMS Detection: positive-ion modeMass Spec: Q Exactive™ PlusSample: reduced trastuzumab (4 mg/mL)

Herceptin_HC+LC_1ul #178 RT: 8.28 AV: 1 NL: 6.19E6

20

30

40

50

60

70

80

901692.83

1493.851751.18

1446.471813.69

1410.901880.81

1953.111368.36

2031.28

2115.811336.67

2200.692308.14 2418.021269.96

1180.58

LC_1ul#178 RT: 8.28 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00] HC

T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

1638.29

1633.01

1643.60

31+

15

1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400m/z

0

10 1238.962472.972253.37 2358.10

2158.66

1600 1605 1610 1615 1620 1625 1630 1635 1640 1645 1650 1655 1660 1665 1670m/z

0

5

10

15

20

1631.64 1640.081634.91

1628.281625.611646.93

1671.371624.16 1661.331605.32 1608.89 1666.591653.791620.161600.00 1615.28

S ti f Ab F t ith Ch V i tSeparation of mAb Fragments with Charge Variant

16

DTT Reduction and IdeS Digest: scFc, LC, and Fd

17

Separation of mAb Fragments Containing Lys Variant

20 0

Column: MAbPac RP, 4 µmFormat: 3.0 ×50 mmMobile phase A: H2O/TFA (99.9 : 0.1 v/v)Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1 16.0

18.0

20.0

Fd´

v/v/v/)Gradient:

Time (min) %A %B0.0 70 301.0 70 3011.0 60 40

12.0

14.0

scFc,0 Lys

scFc

LC

12.0 60 4014.0 70 3015.0 70 30

Temperature: 80 ºCFlow rate: 0.5 mL/min6.0

8.0

10.0scFc,1 Lys

Inj. volume: 2 µL Detection: UV (280 nm)Sample: Infliximab+IdeS+DTT (2 mg/mL)

2.0

4.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

-2.0

0.0

18


LC/MS Analysis: scFc, LC, and Fd´

remicade+ides+dtt_2mgperml 11/12/2014 1:27:41 PM

RT: 5.00 - 15.00

60

80

100

ndan

ce

8.68 NL:7.27E8TIC MS Remicade+IDES_2mgperml

IDESscFc F(ab)2

Lysine variants

60

80

1000

20

40

60

Rel

ativ

e A

bu

7.016.92

8.91 12.8112.6110.809.39 9.59 9.998.358.007.39 7.796.075.76 6.225.31 6.627.89

8.327.006.93

ml

NL:1.65E8TIC MS remicade+ides+dtt_2mgpermlIDES +DTTFd

LC

Lysine variants

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0Time (min)

0

20

4012.7712.44

11.049.938.97 9.588.57 9.33 14.866.02 6.225.825.21 7.336.62

1949 211810 10 NL: 7.93E6scFc, 1 Lys +10

50

100

Abu

ndan

ce 0

50

1000

50

1001949.211810.10 2111.62

2303.361583.92 2533.73 2815.06 3166.901407.93 3619.141267.41 2789.992272.03 2342.391939.301800.90 2100.94

2291.841575.83 2801.002521.06 3150.871400.83 3600.831261.03 2141.30 3371.812975.16 3877.912517.64 2653.632394.88 2805.21

2975.142283.56 3167.012134.70 3385 33 3506 18

NL: 7.93E6Remicade+IDES_2mgperml#144 RT: 6.92 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00] NL: 9.43E6Remicade+IDES_2mgperml#149 RT: 7.01 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00] NL: 5.76E7Remicade+IDES_2mgperml#190 RT: 8.68 AV: 1 T: FTMS + p ESI sid=40.00 Full ms

scFc, 1 Lys

F(ab)2

10

+10scFc, 0 Lys

1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 40000

50

1000

50

1000

Rel

ativ

e

2134.70 3385.33 3506.181963.90 3775.90 3926.931753.581558.971443.871049.671953.79 2131.301803.55

2344.281674.78 2604.75 2930.281465.66 3348.71 3606.341302.80 3125.63 3906.772757.962467.571172.80 2232.891984.841832.571710.46 1973.51

2137.841509.38 2332.04 2565.30 2850.07 3206.391350.79 3664.331222.06 3420.183017.95 3946.312162.31

S p S s d 0 00 u s[1000.00-4000.00] NL: 2.06E7remicade+ides+dtt_2mgperml#173 RT: 7.89 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00] NL: 1.49E7remicade+ides+dtt_2mgperml#187 RT: 8.32 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

LC

Fd

+10

+10

19

m/z

LC/MS Analysis: scFc

F:\Xcalibur\...\Remicade+IDES_2mgperml 11/12/2014 12:23:15 PM

RT: 5.00 - 15.00

50

1008.68

7.018.91 12.8112.6110.809.39 9.59 9.998 358 007 39 7 796 075 76 6 225 31 6 62

NL: 7.27E8TIC MS Remicade+IDES_2mgperml

0

50

1000

50

100

Rel

ativ

e A

bund

ance 0

10.809.39 9.59 9.998.358.007.39 7.796.075.76 6.225.31 6.627.89 8.327.006.93

12.7712.4411.049.938.97 9.588.57 9.33 14.866.02 6.225.825.21 7.336.626.92

8.617.10 8.86 9.44 12.6812.377.39 7.64 11.748.00 8.25 10.30 10.909.64 14.745.71 14.446.326.025.06 5.36

NL: 1.65E8TIC MS remicade+ides+dtt_2mgperml

NL: 3.11E6m/z= 2533.21-2534.16 MS Remicade+IDES 2 l

scFc, 1 Lys

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0Time (min)

0

50

1000 7.02

8.797.396.83 7.54 12.7812.08 13.0410.607.89 8.20 11.8711.469.64 13.88 14.34 14.946.175.975.21 6.325.41

IDES_2mgpermlNL: 3.41E6m/z= 2520.32-2521.26 MS Remicade+IDES_2mgperml

2533.73 NL: 3.11E6

scFc, 0 Lys

+10 1 Lys2533 73

1000

20

40

60

80

100

Rel

ativ

e A

bund

ance

2533.73

2549.84

2535.882520.742511.22

2529.78

2566.402552.102546.072499.01

2521.06

3 6Remicade+IDES_2mgperml#144 RT: 6.92 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

NL: 2.92E6

scFc, 1 Lys

10 0 L

+10, 1 Lys2533.73

2521.06

2460 2470 2480 2490 2500 2510 2520 2530 2540 2550 2560 2570 2580 2590 2600 2610 2620 26300

20

40

60

80

100

2537.08

2523.01 2553.562500.86 2539.562517.112533.60

2565.67 2584.182486.30

Remicade+IDES_2mgperml#149 RT: 7.01 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

scFc, 0 Lys +10, 0 Lys

20

m/z

S ti f Ab F t ith O id ti V i tSeparation of mAb Fragments with Oxidation Variant

21

Oxidized mAbs

Methionine and Tryptophans are usually oxidized.

22

Separation of mAb Fragments Containing Oxidation Variant

7 5

12.0 Column: MAbPac RP, 4 µmFormat: 3 ×50 mmMobile phase A: H2O/TFA (99.9 : 0.1 v/v)Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1 LC

HCOxidized HC(a)

2.5

5.0

7.5 v/v/v)Gradient:

Time (min) %A %B0.0 70 301.0 70 3011.0 60 40

-2.0

9.0

12.0 60 4014.0 70 3015.0 70 30


Fd(b)

2.0

4.0

6.0 Inj. volume: 2 µL Detection: UV (280 nm)Sample: (a) trastuzumab + DTT (2 mg/mL)

(b) trastuzumab + DTT + IdeS (1 mg/mL)

LC

scFcOxidized scFc

1.0 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0

-1.0


23


LC/MS Analysis: Oxidized HC

LCRT: 2.00 - 12.00

50

55

60

65

70

75

80

85

90

95

100

ve A

bund

ance

8.33

10.2710.21

NL:9.66E7TIC MS 112714_Herceptin+H2O2+DTT_2mgperml



:0.1:0.02 v/v/v/v)

(a) TICoxidized HC

HC

2 3 4 5 6 7 8 9 10 11 12Time (min)

0

5

10

15

20

25

30

35

40

45

Relativ

10.78 11.445.134.93 5.493.873.37 4.07 5.69 8.936.09 6.393.02 6.75 7.15 7.352.81 9.537.702.51


Time (min) %A %B0.0 75 251.0 75 2511.0 63 3712 0 63 37

50

60

70

80

90

100

Abu

ndan

ce

1638.76

1633.54

NL: 1.06E6112714_Herceptin+H2O2+DTT_2mgperml#221 RT: 10.17 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

12.0 63 3714.0 75 2515.0 75 25

Temperature: 80 ºCFlow rate: 0.5 mL/minInj volume: 2 µL

(b) Oxidized HC +31

80

90

1000

10

20

30

40

50

Rel

ativ

e A

1644.001628.94

1636.88 1640.111632.23

1649.17 1652.801626.281612.881610.041605.43 1660.831617.35 1665.641622.36

1633.051638.26

NL: 9.61E5112714_Herceptin+H2O2+DTT_2mgperml#227 RT: 10.29 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

Inj. volume: 2 µL MS Detection: positive-ion modeMass spec: Q Exactive™ PlusSample: oxidized trastuzumab, reduced

by DTT (2 mg/mL)(c) HC

1605 1610 1615 1620 1625 1630 1635 1640 1645 1650 1655 1660 16650

10

20

30

40

50

60

70

1643.51

1631.541636.69

1639.651610.59 1614.85 1625.56 1653.751647.531607.87 1622.58 1657.35 1664.30

24

m/z

LC/MS Analysis: Oxidized scFc

FdRT: 2.00 - 12.00

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

10.42

8.35

7.37

7.16

NL:7.51E7TIC MS 112714_Herceptin+H2O2+DTT+IDES_1mgperml



:0.1:0.02 v/v/v/v)

(a) TICLC

Fd

Oxidizied scFcscFc

2 3 4 5 6 7 8 9 10 11 12Time (min)

0

5

10

15

20

25

30

35

40

10.10

10.79 11.905.074.87 6.285.825.524.213.91 9.633.45 6.483.25 9.388.922.852.50 7.79


Time (min) %A %B0.0 75 251.0 75 2511.0 63 3712 0 63 372525 60

50

60

70

80

90

100

ve A

bund

ance

2541.99

2525.60

NL: 5.09E5112714_Herceptin+H2O2+DTT+IDES_1mgperml#149 RT: 7.16 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

12.0 63 3714.0 75 2515.0 75 25

Temperature: 80 ºCFlow rate: 0.5 mL/minInj volume: 2 µL

(b) Oxidized scFc+10

2525.60

70

80

90

1000

10

20

30

40

Rel

ativ

2557.972529.45

2510.98 2545.662560.15

2521.752502.76 2566.26

2524.08

2540.38

NL: 4.26E5112714_Herceptin+H2O2+DTT+IDES_1mgperml#160 RT: 7.42 AV: 1 T: FTMS + p ESI sid=40.00 Full ms [1000.00-4000.00]

Inj. volume: 2 µL MS Detection: positive-ion modeMass spec: Q Exactive™ PlusSample: Oxidized trastuzumab, reduced

by DTT and digested by IdeS (1 mg/mL)(c) scFc

2524.08

2460 2480 2500 2520 2540 2560 2580 2600 26200

10

20

30

40

50

60

70

2509.45 2527.982544.22 2556.93

2501.48 2520.11 2560.78 2585.84

25

m/z

High Resolution MS

+16

70

80

90

100

ance

1578.97

1579.10

1578.84

1579.22

NL: 7.70E5112714_Herceptin+H2O2+DTT+IDES_1mgperml_141126204149#560-577 RT: 7.06-7.22 AV: 18 T: FTMS + p ESI Full ms [1000.00-4000.00]

(a) Oxidized scFc

+16

20

30

40

50

60

Rel

ativ

e A

bund

a

1579.35

1578.661580.09

1579.78

1578.531580.22

1580.35

80

90

1000

101578.34

1580.841576.35 1577.531577.15 1581.281575.22 1582.28 1583.47 1583.851582.84

1577.971577.85

1578.16

1577.72

NL: 8.74E5112714_Herceptin+H2O2+DTT+IDES_1mgperml_141126204149#583-601 RT: 7.28-7.45 AV: 19 T: FTMS + p ESI Full ms [1000.00-4000.00]

(b) scFc

30

40

50

60

70 1578.28

1578.911577.60

1578.41

1579 16

1575 1576 1577 1578 1579 1580 1581 1582 1583 1584m/z

0

10

20

30 1579.16

1579.351579.47

1577.47

1576.781575.34 1580.221576.16 1580.72 1581.97 1582.53 1583.791583.34

26

Separation of ADC DAR forms

27

Site-selective Antibody-drug Conjugates (ADCs)

DIBO-MMAE

Add inGalT(Y289L)UDP-GalNAzβ-1,4-Galactosidase N3

N3N3N3

MMAEMMAE

MMAEMMAE

25 C, ON37 C, 5 hr

Azide-Activated Ab(stable for long-term storage)

Antibody drug conjugate (ADC)Unlabeled Ab Cleave Terminal Gal

37 C, ONN3 N3 MMAE MMAE

28

MMAE Modified Trastuzumab ADC

Column: MAbPac RP, 4 µmFormat: 2.1 ×50 mmMobile phase A: H2O/TFA (99.9 : 0.1 v/v)Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1 20.0

25.0

v/v/v)Gradient:

Time (min) %A %B0.0 65 350.5 65 354.5 45 55

15.0

5.0 45 555.5 65 356.0 65 35

Flow rate: 0.6 mL/minTemperature: 80 ºCInj. volume: 2 µL

5 0

10.0

j µDetection: UV (280 nm)Sample: trastuzumab-MMAE

0.0

5.0

1.00 1.50 2.00 2.50 3.00 3.50 4.00

-5.0


29

( )

Separation of PEGylated Protein

30

PEGylated Protein Analysis

Column: MAbPac RP, 4 µmFormat: 3.0 ×50 mmMobile phase A: H2O/TFA (99.9 : 0.1 v/v)Mobile phase B: MeCN/ H2O/TFA (90: 9.9 :0.1 v/v/v/)Gradient:

Ti ( i ) %A %B

6.0

9.0

(a)

Time (min) %A %B0.0 55 451.0 55 4511.0 25 7512.0 25 7514.0 55 45

2.0

4.0

15.0 55 45

Temperature: 80 ºCFlow rate: 0.5 mL/minInj. volume: 10 µL Detection: UV (280 nm)

-1.0

10 0

14.0

(b)( )

Sample: (a) PEGylated protein (11 mg/mL)(b)de-PEGylated protein (1.24 mg/mL)

5.0

10.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

-2.0

31


Tips and Tricks for RP LC/UV and LC/MS Analysis

• System QC mix: Ribo A, Cytochrome C, Lysozyme, BSA.• Most mAb should be analyzed at higher temperature: 70 C to 80 C.

T hi l ith hi h i d 100 M• To remove carryover, washing column with high organic and 100 mMNaOH.

• For better separation resolution, use 0.1% TFA in mobile phase.• For LC/MS analysis, use 0.1% FA and 0.02% TFA in mobile phase.

32

Shanhua Lin, Ph.D.

mAbs and ADCs analysis by HIC

The world leader in serving science

What is Hydrophobic Interaction Chromatography?

34

What is Hydrophobic Interaction Chromatography?

• Chromatographic techniques that separates biomolecules according to differences in their surface hydrophobicity

• Orthogonal to IEX and SEC• Orthogonal to IEX and SEC

• Gradient elution: High to Low salt concentration

• Relatively mild conditions are used; so biomolecules do not becomeRelatively mild conditions are used; so biomolecules do not become denatured during the separation

35

MAbPac HIC family

MAbPac HIC-10 MAbPac HIC-20 MAbPac HIC-ButylChemistry Proprietary polyamide Proprietary alkylamide Butyl

Substrate Spherical, high purity silica particles

Spherical, high purity silica particles

Hydrophilic polymer

Particle Size 5 µm 5 µm 5 µm

P i 1 000 Å 1 000 Å NPore size 1,000 Å 1,000 Å Non-porous

*Capacity 28 mg/mL 24 mg/mL 9 mg/mL

*Capacity : Amount of Lysozyme / column volume (mg/mL)

36

MAbPac HIC Family

MAbPac HIC-10 MAbPac HIC-20 MAbPac HIC-ButylpH Range 2.0-8.0 2.0-9.0 2.0-12.0

Temperature Limit 60 60 60Temperature Limit (°C)

60 60 60

Organic Compatibility

0-100% 0-100% 0-50%

Flow Rate 0.5-1.0 mL/min (recommended)1.5 mL/min (max)

Maximum Pressure 6,000 psi (4.6 × 100 mm)8,000 psi (4.6 × 250 mm)

6,000 psi (4.6 × 100 mm)8,000 psi (4.6 × 250 mm)

4,000 psi (4.6 × 100 mm)

37

, p ( ) , p ( )

Separation of Aggregates on MAbPac HIC-10

200

220

Column: MAbPac HIC-10, 5 µmFormat: 4.6 ×100 mmMobile phase A: 2 M ammonium sulfate, 100 mM

sodium phosphate pH 7 0

Monomer

150

AU

)

sodium phosphate, pH 7.0Mobile phase B: 100 mM sodium phosphate, pH

7.0Gradient:

Time (min) %A %B-5.0 60 400 0 60 40

100

Abs

orba

nce

(mA 0.0 60 40

1.0 60 4029.0 0 10034.0 0 100

Temperature: 30 ºCFl t 0 5 L/ i

mAbvariants

50

Flow rate: 0.5 mL/minInj. volume: 10 µL (4 mg/mL)Detection: UV (280 nm)Sample: Monoclonal antibody

Aggregate

0 5 10 15 20 25 30

0

38

0 5 10 15 20 25 30Retention Time (min)

Oxidized mAbs

Methionine and Tryptophans are usually oxidized.

39

Separation of Oxidized mAb on MAbPac HIC-20

50

60


Native mAb

40

50 Mobile phase A: 2 M ammonium sulfate, 100 mMsodium phosphate, pH 7.0

Mobile phase B: 100 mM sodium phosphate, pH 7.0Gradient:

Time (min) %A %B-6.0 50 500 0 50 50A

U)

30

0.0 50 502.0 50 50

30.0 0 10035.0 0 100

Temperature: 30 ºCFlow rate: 0 5 mL/minA

bsor

banc

e (m

A

Oxidized variants

10

20 Flow rate: 0.5 mL/minInj. volume: Untreated mAb: 20 µL (1.25 mg/mL)Oxidized mAb: 20 µL (1.25 mg/mL)

Detection: UV (280 nm)Sample: Untreated mAb

H2O2 oxidized mAb

10 14 18 22 26 30

0

40

10 14 18 22 26 30Retention Time (min)

Antibody-Drug Conjugates (ADCs)

41

Heterogeneity of Cys-Linked ADC

42

Separation of Cys-Linked ADC on MAbPac HIC-Butyl

Column: MAbPacHIC-Butyl, 5 µmFormat: 4.6 ×100 mmMobile phase A: 1.5 M ammonium sulfate, 50

mM sodium phosphate pH 7 0 /

40

DAR 4

mM sodium phosphate, pH 7.0 / isopropanol (95:5 v/v)

Mobile phase B: 50 mM sodium phosphate, pH 7.0 / isopropanol (80:20 v/v)

Gradient:Time (min) %A %B

5 0 100 0

30

(mA

U)

-5.0 100 00.0 100 01.0 100 0

15.0 0 10020.0 0 100

T t 25 ºC

20

Abs

orba

nce DAR 2

Temperature: 25 ºCFlow rate: 1.0 mL/minInj. volume: 5 µL (5 mg/mL)Detection: UV (280 nm)Sample: Cys-conjugated ADC mimic

10

DAR 0

DAR 6

DAR 8

0 4 8 12 16 20

0

43


Separation of Bispecific mAb



88

100

p p p , p7.0

Gradient:Time (min) %A %B-5.0 60 400.0 60 401 0 60 40

63

75

e (m

AU

)

1.0 60 4015.0 0 10020.0 0 100

Temperature: 30 ºCFlow rate: 1.0 mL/minInj volume: A+B: 20 µL

38

50

Abs

orba

nce

4Inj. volume: A+B: 20 µL

A+D: 12 µLC+B: 10 µLC+D: 20 µL

Detection: UV (280 nm)Sample: A+B (0.87 mg/mL)

A+D (1 40 mg/mL)13

253

2A+D (1.40 mg/mL)C+B (1.72 mg/mL)C+D (1.00 mg/mL)

2 0 3 8 5 0 6 3 7 5 8 8 10 0 11 3 12 5 13 8 15 0 16 3 18 0-10

1

44

2.0 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 18.0Retention Time (min)

Method Development

• Type of salt (lyotropic salt, salting-out agents)• Ammonium sulfate• Sodium chloride• Sodium chloride

• pH of the mobile phase• pH 7: sodium phosphate• pH 5: sodium acetate

• Starting salt concentration• Addition of organic solvent (acetonitrile or isopropanol)g ( p p )• Sample matrix• Flow rate

T t• Temperature

45

Mobile Phase Recommendation

• Mobile phase formula 1• Mobile phase A: 2 M ammonium sulfate, 100 mM sodium phosphate, pH 7.0• Mobile phase B: 100 mM sodium phosphate, pH 7.0p p p p

• Mobile phase formula 2• Mobile phase A: 1 5 M ammonium sulfate 50 mM sodium phosphate pH 7 0 /• Mobile phase A: 1.5 M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 /

isopropanol (95:5 v/v)• Mobile phase B: 50 mM sodium phosphate, pH 7.0 / isopropanol (80:20 v/v)

• Mobile phase formula 3• Mobile phase A: 2 M ammonium sulfate, 20 mM sodium acetate, pH 5.0• Mobile phase B: 20 mM sodium acetate, pH 5.0

46

Optimization of the Starting Salt Concentration

Column: MAbPac HIC-20, 5 µm

25

30Dimension: 4.6 ×100 mmMobile phase A: 2 M ammonium sulfate, 100 mM


7.0Gradient 1:

15

20

Time (min) %A %B-5.0 100 00.0 100 01.0 100 0

15.0 0 10020.0 0 100m

AU

)

10

5 0 0 0 00Gradient 2:

Time (min) %A %B-5.0 80 200.0 80 201.0 80 20

15 0 0 100

Abs

orba

nce

(m

0

515.0 0 10020.0 0 100

Gradient 3:Time (min) %A %B-5.0 60 400.0 60 401 0 60 401 6 M

1.2 M

0 2 4 6 8 10 12 14 16 18 20

-51.0 60 40

15.0 0 10020.0 0 100

Temperature: 30 ºCFlow rate: 1.0 mL/minI j l 10 L

2.0 M

1.6 M

47

0 2 4 6 8 10 12 14 16 18 20 Inj. volume: 10 µL Detection: UV (280 nm)Sample: Trypsin inhibitor (5 mg/mL)


Addition of Organic Solvent in Mobile Phase B

Column: MAbPac HIC-20, 5 µm

45

, µDimension: 4.6 ×100 mm

1)Mobile phase A: 2 M ammonium sulfate, 100 mM

sodium phosphate, pH 7.0Mobile phase B: 100 mM sodium phosphate pH

30

Mobile phase B: 100 mM sodium phosphate, pH 7.0

2)Mobile phase A: 2 M ammonium sulfate, 100 mM


7 0 / isopropanol (90:10 v/v)mA

U)

20

7.0 / isopropanol (90:10 v/v)3)Mobile phase A: 2 M ammonium sulfate, 100 mM


7.0 / isopropanol (80:20 v/v)Abs

orba

nce

(m

10 3

2

Gradient:Time (min) %A %B-5.0 100 00.0 100 01.0 100 0

1 0 0 100

10% Isopropanol

20% Isopropanol

0 2 4 6 8 10 12 14 16 18 20-5

115.0 0 10020.0 0 100


No solvent

48

0 2 4 6 8 10 12 14 16 18 20Detection: UV (280 nm)Sample: Trypsin inhibitor (5 mg/mL)Retention Time (min)

Effect of Matrix on Peak Shape

40

60Column: MAbPac HIC-20, 5 µmFormat: 4.6 ×100 mmMobile phase A: 2 M ammonium sulfate, 100 mM

sodium phosphate, pH 7.0Mobile phase B: 100 mM sodium phosphate, pH 2

34a

20

ob e p ase 00 sod u p osp a e, p7.0

Gradient:Time (min) %A %B-5.0 100 00.0 100 01 0 100 0

1

2

AU

)

60

01.0 100 0

15.0 0 10020.0 0 100

Temperature: 30 ºCFlow rate: 1.0 mL/minInj volume: 10 µL2

3 4b

Abs

orba

nce

(mA

20

40

Inj. volume: 10 µL Detection: UV (280 nm)Sample: Protein mixture

Sample matrix: a) Waterb) 1 M ammonium sulfate, 50

M di h h t H 7 0

1

0

0 4 8 12 16 20

mM sodium phosphate, pH 7.0

Peaks: 1) Myoglobin2) Ribonuclease A 3) Lysozyme4) α-Chymotrypsinogen A

49


Tips and Tricks for HIC Analysis

• Wash the system thoroughly before and after analysis using DI water.

• Make sure the ammonium sulfate does not cause high• Make sure the ammonium sulfate does not cause high background signal.

• If the protein is less hydrophobic (indicated by broader p y p ( yfronting peak and short retention time), increase the salt concentration (lyotropic agent) of mobile phase A or you will need a more hydrophobic columnneed a more hydrophobic column.

• If the protein is less hydrophobic try increasing the run temperature.

• If the protein is very hydrophobic, try adding organic solvent to obtain better efficiency and resolution.

50

MAbPac HIC-20 Conditioning

• This column requires conditioning by injecting ≥750 µg of protein.• Ovalbumin or BSA can be used for this purpose.

Aft t i th l t l ti d t diti• After storing the column storage solution, you may need to re-condition the column.

51

Protein Standard

• Myoglobin, RNase A, Lysozyme, α-Chymotrypsinogen A• Make 10 mg/mL of each protein

Th i th t i ith th f ll i ti• Then mix these proteins with the following ratio• Myoglobin : RNase A : Lysozyme : α-Chymotrypsinogen A = 2:4:1:2

52

Thank you!

53

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