mabs and ADCs analysis by RP

mabs and ADCs analysis by RP Shanhua Lin, Ph.D. The world leader in serving science

Protein and mab Separation by HPLC Size difference? YES Size Exclusion Chromatography (SEC) MAbPac SEC-1 NO NO Charge difference? YES Ion Exchange Chromatography (IEX) MAbPac SCX-1 ProPac WCX-1 CX-1 ph gradient Buffer NO NO YES Reverse Phase Chromatography (RPC) MAbPac RP Hydrophobicity difference? YES Hydrophobic Interaction Chromatography (HIC) MAbPac HIC-1 MAbPac HIC-2 MAbPac HIC-Butyl ProPac HIC-1 2

Executive Summary Thermo Scientific MAbPac RP Column Designed for high-resolution separation of monoclonal antibodies (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 14 125 113 1 88 75 63 2 3 4 Column: MAbPac RP, 4 µm Format: 2.1 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B -1. 8 2. 8 2 25 2.5 5 5 2.7 5 5 2.8 8 2 3. 8 2 5 38 25 13 1 Temperature: 8 ºC Flow rate: 6mL/min.6 Inj. volume: 1 µl Detection: UV (28 nm) Sample: 1. Ribonuclease A (.5 mg/ml) 2. Cytochrome C (.5 mg/ml) 3. Lysozyme (.5 mg/ml) 4. mab (1 mg/ml) -2..5 1. 1.5 2. 2.5 3. Retention Time (min) 5

Reproducibility of MAbPac RP 12 1 8 6 4 2 #111 #91 #81-2 #71 #651 #51-4 #41 #31-6 #21 #11 1-8..5 1. 1.5 2. 2.5 3. 2 3 4 Column: MAbPac RP, 4 µm Format: 2.1 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B -1 8 2. 8 2 2.5 5 5 2.7 5 5 2.8 8 2 3. 8 2 Temperature: 8 ºC Flow rate:.6 ml/min Inj. volume: 1 µl Detection: UV (28 nm) Sample: 1. Ribonuclease A (.5 mg/ml) 2. Cytochrome Ct C(5 (.5 mg/ml) 3. Lysozyme (.5 mg/ml) 4. mab (1 mg/ml) Retention Time (min) 6

Loadability: Three Orders of Magnitude 2.5 1.25 -.5 1. (a).2 µg 18. (b).2 µg -2. 14 (c) 2 µg Column: MAbPac RP, 4 µm Format: 2.1 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B -1 85 15. 85 15 2.5 5 5 2.7 5 5 2.8 85 15 4. 85 15 Temperature: 8 ºC Flow rate:.6 ml/min Inj. volume: 1 µl Detection: UV (28 nm) Sample: mab -2 9 5-1 (d) 2 µg 1. 1.5 2. 2.5 3. 3.5 4. Retention Time (min) Area 5 45 4 35 3 25 2 15 1 5 y = 2.311x +.491 R² = 1 1 2 3 mab (µg) 7

Carryover 6 5 4 3 1: mab injection (peak area = 37.97) Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v) Gradient: Time (min) %A %B. 1 1. 1 11. 1 12. 1 14. 1 15. 1 2 1 Carryover <.62% 2: blank (Peak area =.233) Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 5 µl Detection: UV (28 nm) Sample: mab (5 mg/ml) -1. 1.3 2.5 3.8 5. 6.3 7.5 8.8 1. 11.3 12.5 13.8 15. Retention Time (min) 8

Stability at High ph Condition 14 125 113 1 88 75 63 5 38 25 13 After 6 hrs of.8 M NaOH wash at 8 C 1 Before NaOH wash 2 3 4 Column: MAbPac RP, 4 µm Format: 2.1 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B -1. 85 15. 85 15 2.5 5 5 2.7 5 5 2.8 85 15 4. 85 15 Temperature: 8 ºC Flow rate:.6 ml/min Inj. volume: 1µL Detection: UV (28 nm) Sample: 1. Ribonuclease A (.5 mg/ml) 2. Cytochrome C (.5 mg/ml) 3. Lysozyme (.5 mg/ml) 4. mab (1 mg/ml) -2.5 1. 1.5 2. 2.5 3. 3.5 Retention Time (min) 9

1 Separation of mab Fragments

Structure of IgG and Typical Forms of Heterogeneity 11

mab Fragments (a) (b) (c) 12

mab and mab Fragments Analysis 14 mab -2 8. 5. -1. 9 5-1 (a) (b) (c) LC Fc HC Fab 1 F(ab ) (d) 2 5 scfc Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B. 8 2 1. 8 2 11. 55 45 12. 55 45 14. 8 2 15. 8 2 Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 5 µl Detection: UV (28 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) -1 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 11. 12. 13. 14. 15. Retention Time (min) 13

LC/MS Analysis of Intact mab and Glycosylation Profile RT: 4.44-13.13 RelativeAbundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 TIC 8.26 NL: 559E8 5.59E8 TIC MS Column: herceptin_5 mgperml 2 844 8.44 15 1 5 8.63 12.74 4.47 5.13 5.43 5.68 5.93 6.9 6.74 6.99 7.39 7.64 8.97 9.37 9.72 9.88 1.33 1.53 4.5 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 11. 11.5 12. 12.5 13. Time (min) herceptin_5mgperml #167-173 RT: 8.22-8.33 AV: 7 NL: 8.14E6 T: FTMS + p ESI sid=4. Full ms [1.-4.] 3223.2 1 MS spectrum 388.92 95 3294.8 3369.64 3154.64 9 325.91 85 8 2965.42 3447.98 75 Relative Abundance 7 65 6 55 5 45 4 35 3 25 2 15 1 5 261.37 2695.91 2647.76 2745.82 2797.55 2851.39 297.27 353.9 3616.14 376.5 381.53 391.54 3917.63 MS 2513.18 2471.32 243.8 2394.25 2283.77 2183.13 257.49 3994.21 2 22 24 26 28 3 32 34 36 38 4 m/z herceptin_5mgperml #167-173 RT: 8.22-8.33 AV: 7 NL: 6.26E6 T: FTMS + p ESI sid=4. Full ms [1.-4.] 1 95 9 85 8 75 m/z at 5+ 2965.42 2968.61 Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B. 8 2 1. 8 2 11. 55 45 12. 55 45 14. 8 2 15. 8 2 Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 1 µl Detection: positive-ion mode Mass Spec: Q Exactive Plus Sample: trastuzumab (5 mg/ml) 7 65 Relative Abundance 6 55 5 45 4 35 3 2962.22 2971.87 25 2 15 2959.34 1 2974.96 2956.45 5 2933.53 2936.54 2953.5 2993.7 2996.3 2939.75 2977.49 2946.14 298.99 2986.82 32.66 293 2935 294 2945 295 2955 296 2965 297 2975 298 2985 299 2995 3 m/z 14

LC/MS Analysis of Reduced mab RT: 3. - 11. TIC 1 9 8 7 6 5 4 Relative Abundance uau LC HC 8.28 NL: 7.43 2.54E8 TIC MS Herceptin_ HC+LC_1ul 3 2 1 8.58 8.86 3.94 4.79 6.41 6.96 5.2 5.5 5.95 6.1 7.95 3.63 9.11 4.34 9.76 3.23 1.2 1.37 1.77 8.28 NL: 6 6.6E5 UV UV_VIS_1 55 UV Herceptin_ 5 HC+LC_1ul 45 4 35 7.42 3 25 2 15 1 8.85 9.1 5 7.73 6.35 6.63 6.8 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 11. Time (min) Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B. 8 2 1. 8 2 11. 55 45 12. 55 45 14. 8 2 15. 8 2 Relative Abundance 1 9 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 3 LC 1234.47 HC 1954.16 183.88 2131.63 1675.1414 1563.47 2344.74 1465.8 1379.68 1762.96 1819.28 1967.87 2146.62 133.3 1687.1 1392.86 2361.4 1478.76 1575.62 295. 2233.12 1638.29 1539.3 1692.83 1493.85 1751.18 1446.47 1813.69 141.9 188.81 1953.11 1368.36 231.28 1336.67 2115.81 NL: 2.52E7 Herceptin_HC+ LC_1ul#152 RT: 7.42 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 6.19E6 Herceptin_HC+ LC_1ul#178 RT: 8.28 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 1 µl UV Detection: 28 nm MS Detection: positive-ion mode Mass Spec: Q Exactive Plus Sample: reduced trastuzumab (4 mg/ml) Herceptin_HC+LC_1ul #178 RT: 8.28 AV: 1 NL: 6.19E6 T: FTMS + p ESI sid=4. Full ms [1.-4.] 1 95 9 85 8 75 7 65 6 55 5 45 Relative Abundance 4 35 31+ 1633.1 1638.29 2 118.58 22.69 1269.96 238.14 2418.2 1 1238.96 2158.66 2253.37 2358.1 2472.97 12 13 14 15 16 17 18 19 2 21 22 23 24 m/z 3 1643.6 25 2 15 1631.64 1634.91 164.8 1 1625.61 1628.28 5 1646.93 16. 165.32 168.89 1615.28 162.16 1624.16 1653.79 1661.33 1666.59 1671.37 16 165 161 1615 162 1625 163 1635 164 1645 165 1655 166 1665 167 m/z 15

16 Separation of mab Fragments with Charge Variant

DTT Reduction and IdeS Digest: scfc, LC, and Fd 17

Separation of mab Fragments Containing Lys Variant 2. 18. 16. 14. 12. scfc, 1. 1 Lys 8. scfc, Lys LC Fd Column: MAbPac RP, 4 µm Format: 3. 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B. 7 3 1. 7 3 11. 6 4 12. 6 4 14. 7 3 15. 7 3 6. 4. Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 2 µl Detection: UV (28 nm) Sample: Infliximab+IdeS+DTT (2 mg/ml) 2.. -2. 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 11. Retention Time (min) 18

LC/MS Analysis: scfc, LC, and Fd remicade+ides+dtt_2mgperml 11/12/214 1:27:41 PM RT: 5. - 15. ndance Relative Abu Abundance Relative 1 8 6 4 2 1 8 6 4 scfc F(ab ) 2 Lysine variants 6.92 5.31 5.76 6.7 6.22 6.62 7.1 6.93 7. 7.39 7.79 8. 8.35 7.89 8.32 LC Fd 8.68 NL: 7.27E8 TIC MS Remicade+ IDES IDES_2mgper ml 8.91 9.39 9.59 9.99 1.8 12.61 12.81 IDES +DTT 2 12.44 12.77 11.4 5.21 5.82 6.2 6.22 6.62 7.33 8.57 8.97 9.33 9.58 9.93 14.86 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 11. 11.5 12. 12.5 13. 13.5 14. 14.5 15. Time (min) 1 5 1 scfc, 1Lys +1 1949.21 NL: 7.93E6 181.1 2111.62 1583.92 233.36 2533.73 2815.6 3166.9 1267.41 147.93 2272.3 2342.39 2789.99 3619.14 18.9 1939.3 21.94 scfc, Lys 2291.84 +1 5 1575.83 2521.6 281. 315.87 1261.3 14.83 2141.3 2975.16 3371.81 36.83 3877.91 2517.64 1 2394.88 2653.63 285.21 F(ab ) 2283.56 2975.14 5 2 2134.7 3167.1 3385.33 149.67 1443.87 1558.97 1753.58 1963.9 356.18 3775.9 3926.93 1953.79 1 183.55 2131.3 +1 LC 1674.78 2344.28 5 264.75 1465.66 293.28 1172.8 132.8 1984.84 2232.89 2467.57 2757.96 3125.63 3348.71 366.34 396.77 171.46 1832.57 1 1973.51 Fd 159.38 2137.84 +1 5 2332.4 2565.3 285.7 326.39 1222.6 135.79 2162.31 317.95 342.18 3664.33 3946.31 1 12 14 16 18 2 22 24 26 28 3 32 34 36 38 4 m/z NL: 1.65E8 TIC MS remicade+ ides+ dtt_2mgperml Remicade+IDES_2mgperml#144 RT: 6.92 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 9.43E6 Remicade+IDES_2mgperml#149 RT: 7.1 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 5.76E7 Remicade+IDES_2mgperml#19 RT: 8.68 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 2.6E7 remicade+ides+dtt_2mgperml#173 RT: 7.89 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 1.49E7 remicade+ides+dtt_2mgperml#187 RT: 8.32 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] 19

LC/MS Analysis: scfc F:\Xcalibur\...\Remicade+IDES_2mgperml 11/12/214 12:23:15 PM RT: 5. - 15. 8.68 NL: 7.27E8 1 TIC MS 5 7.1 Remicade+ IDES_2mgperml 531 5.31 576 5.76 67 6.7 622 6.22 662 6.62 739 7.39 779 7.79 8 8. 835 8.35 8.91 9.39 9.59 9.99 1.8 12.61 12.81 7.89 1 8.32 NL: 1.65E8 6.93 7. TIC MS remicade+ides+ 5 12.44 12.77 dtt_2mgperml 5.21 5.82 6.2 6.22 6.62 7.33 8.57 8.97 9.33 9.58 9.93 11.4 14.86 6.92 NL: 3.11E6 1 m/z= 2533.21-2534.16 5 scfc, 1 Lys MS 5.6 5.36 5.71 6.2 6.32 7.1 7.39 7.64 8. 8.25 8.61 8.86 9.44 9.64 1.3 1.9 11.74 12.37 12.68 14.44 14.74 Remicade+ 7.2 IDES_2mgperml 1 NL: 3.41E6 scfc, Lys m/z= 5 252.32-2521.26 5.21 5.41 5.97 6.17 6.32 6.83 7.39 7.54 7.89 8.2 8.79 9.64 1.6 11.46 11.87 12.8 12.78 13.4 13.88 14.34 14.94 MS Remicade+ 5. 5.5 6. 6.5 7. 7.5 8. 8.5 9. 9.5 1. 1.5 11. 11.5 12. 12.5 13. 13.5 14. 14.5 15. IDES_2mgperml Time (min) Relative Abundance Relative Abundance 1 8 6 4 2 1 8 6 4 2 scfc, 1 Lys scfc, Lys 2486.3 2533.73 2529.78 2511.22 252.74 2499.1 2521.6 2521.6 +1, 1 Lys 2533.73 NL: 3.11E6 Remicade+ IDES_2mgperml#144 RT: 6.92 AV: 1 T: FTMS + p ESI sid=4. Full ms 2549.84 [1.-4.] 2535.88 2546.7 2552.1 +1, Lys 2537.8 2533.6 25.86 2517.11 2523.1 2539.56 2553.56 246 247 248 249 25 251 252 253 254 255 256 257 258 259 26 261 262 263 m/z 2566.4 2565.67 2584.18 NL: 2.92E6 Remicade+ IDES_2mgperml#149 RT: 7.1 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] 2

Separation of mab Fragments with Oxidation Variant 21

Oxidized mabs Methionine and Tryptophans are usually oxidized. 22

Separation of mab Fragments Containing Oxidation Variant 12. Column: MAbPac RP, 4 µm (a) Oxidized HC HC Format: 3 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) LC Mobile phase B: MeCN/ H 75 2 O/TFA (9: 9.9 :.1 7.5 v/v/v) Gradient: 5. Time (min) %A %B. 7 3 2.5 1. 7 3 11. 6 4 12. 6 4 14. 7 3-2. 15. 7 3 9. (b) Fd Temperature: 8 ºC Flow rate:.5 ml/min 6. LC Inj. volume: 2 µl Detection: UV (28 nm) Sample: (a) trastuzumab + DTT (2 mg/ml) 4. scfc (b) trastuzumab + DTT + IdeS (1 mg/ml) 2. Oxidized scfc -1. 1. 2.5 3.8 5. 6.3 7.5 8.8 1. 11.3 12.5 13.8 15. Retention Time (min) 23

LC/MS Analysis: Oxidized HC RT: 2. - 12. 1 Relative Abundance 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 1 9 8 7 6 5 4 3 2 1 1 9 8 7 Relativ ve Abundance (a) TIC LC 8.33 oxidized HC HC NL: 9.66E7 TIC MS 112714_He rceptin+ H2O2+ DTT_2mgp erml 1.27 1.21 1.78 11.44 3.87 5.13 4.93 5.49 6.9 6.39 3.2 6.75 7.15 7.35 3.37 4.7 5.69 8.93 2.51 7.7 2.81 9.53 2 3 4 5 6 7 8 9 1 11 12 Time (min) 1638.76 NL: 1.6E6 112714_Herceptin+H2O2+ DTT_2mgperml#221 RT: 1.17 AV: 1 T: FTMS + p (b) Oxidized HC +31 ESI sid=4. Full ms [1.-4.] 1633.54 1632.23 1636.88 164.11 1644. 1628.94 1649.17 1652.8 1626.28 161.4 1612.88 1622.36 165.43 166.83 1617.35 1665.64 (c) HC 1633.5 NL: 9.61E5 1638.26 112714_Herceptin+H2O2+ DTT_2mgperml#227 RT: 1.29 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B. 75 25 1. 75 25 11. 63 37 12. 63 37 14. 75 25 15. 75 25 Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 2µL MS Detection: positive-ion mode Mass spec: Q Exactive Plus Sample: oxidized trastuzumab, reduced by DTT (2 mg/ml) 6 5 4 3 2 1 1643.51 1631.54 1636.69 1639.65 1647.53 161.59 1614.85 1625.56 1653.75 167.87 1622.58 1657.35 1664.3 165 161 1615 162 1625 163 1635 164 1645 165 1655 166 1665 m/z 24

LC/MS Analysis: Oxidized scfc RT: 2. - 12. RelativeAbundance ve Abundance Relativ 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1 5 1 9 8 7 6 5 4 3 2 1 1 9 8 7 (a) TIC Oxidizied scfc scfc 7.37 7.16 LC 2.5 2.85 3.25 3.45 3.91 4.21 4.87 5.7 5.52 5.82 6.28 6.48 7.79 8.92 9.38 9.63 8.35 Fd 2 3 4 5 6 7 8 9 1 11 12 Time (min) 2525.6 (b) Oxidized scfc (c) scfc 2524.8 2525.6 2541.99 +1 2557.97 2529.45 256.15 251.98 2545.66 252.76 2521.75 2566.26 2524.8 254.38 1.1 1.42 1.79 11.9 NL: 7.51E7 TIC MS 112714_He rceptin+ H2O2+ DTT+ IDES_1mgp erml NL: 5.9E5 112714_Herceptin+H2O2+ DTT+IDES_1mgperml#149 RT: 7.16 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] NL: 4.26E5 112714_Herceptin+H2O2+ DTT+IDES_1mgperml#16 RT: 7.42 AV: 1 T: FTMS + p ESI sid=4. Full ms [1.-4.] Column: MAbPac RP, 4 µm Format: 3 5 mm Mobile phase A: H 2 O/FA/TFA (99.88 :.1:.2 v/v/v) Mobile phase B: MeCN/ H 2 O/FA/TFA (9: 9.88 :.1:.2 v/v/v/v) Gradient: Time (min) %A %B. 75 25 1. 75 25 11. 63 37 12. 63 37 14. 75 25 15. 75 25 Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 2µL MS Detection: positive-ion mode Mass spec: Q Exactive Plus Sample: Oxidized trastuzumab, reduced by DTT and digested by IdeS (1 mg/ml) 6 5 4 3 2 1 259.45 2527.98 2544.22 2556.93 251.48 252.11 256.78 2585.84 246 248 25 252 254 256 258 26 262 m/z 25

High Resolution MS Relative Abunda ance 1 9 8 7 6 5 4 3 2 1 1 9 8 7 6 (a) Oxidized scfc 1578.84 1578.66 1578.53 +16 1578.97 1579.1 1579.22 1579.35 1579.78 158.9 158.22 158.35 1578.34 1575.22 1576.35 1577.15 1577.53 158.84 1581.28 1582.28 1582.84 1583.47 1583.85 (b) scfc 1577.97 1577.85 1578.16 1577.72 1578.28 NL: 7.7E5 112714_Herceptin+H2O2+DTT+ IDES_1mgperml_14112624149 #56-577 RT: 7.6-7.22 AV: 18 T: FTMS + p ESI Full ms [1.-4.] NL: 8.74E5 112714_Herceptin+H2O2+DTT+ IDES_1mgperml_14112624149 #583-61 RT: 7.28-7.45 AV: 19 T: FTMS + p ESI Full ms [1.-4.] 5 4 1577.6 1578.41 1578.91 3 1579.1616 2 1 1577.47 1579.35 1576.78 1579.47 1575.34 158.22 1576.16 158.72 1581.97 1582.53 1583.34 1583.79 1575 1576 1577 1578 1579 158 1581 1582 1583 1584 m/z 26

27 Separation of ADC DAR forms

Site-selective Antibody-drug Conjugates (ADCs) β-1,4-galactosidase Add in GalT(Y289L) UDP-GalNAz N 3 DIBO-MMAE MMAE MMAE N 3 3 N 3 MMAE MMAE 37 C, 5 hr 37 C, ON N 3 25 C, ON Unlabeled Ab Cleave Terminal Gal Azide-Activated Ab (stable for long-term storage) Antibody drug conjugate (ADC) 28

MMAE Modified Trastuzumab ADC 25. 2. 15. 1. 5. Column: MAbPac RP, 4 µm Format: 2.1 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v) Gradient: Time (min) %A %B. 65 35.5 65 35 4.5 45 55 5. 45 55 5.5 65 35 6. 65 35 Flow rate:.6 ml/min Temperature: 8 ºC Inj. volume: 2 µl Detection: UV (28 nm) Sample: trastuzumab-mmae. -5. 1. 1.5 2. 2.5 3. 3.5 4. Retention Time (min) 29

3 Separation of PEGylated Protein

PEGylated Protein Analysis 9. 6. 4. 2. -1. 14. 1. (a) (b) Column: MAbPac RP, 4 µm Format: 3. 5 mm Mobile phase A: H 2 O/TFA (99.9 :.1 v/v) Mobile phase B: MeCN/ H 2 O/TFA (9: 9.9 :.1 v/v/v/) Gradient: Time (min) %A %B. 55 45 1. 55 45 11. 25 75 12. 25 75 14. 55 45 15. 55 45 Temperature: 8 ºC Flow rate:.5 ml/min Inj. volume: 1 µl Detection: UV (28 nm) Sample: (a) PEGylated protein (11 mg/ml) (b)de-pegylated protein (1.24 mg/ml) 5. -2. 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 11. Retention Time (min) 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: 7 C to 8 C. To remove carryover, washing column with high h organic and 1 mm NaOH. For better separation resolution, use.1% TFA in mobile phase. For LC/MS analysis, use.1% FA and.2% TFA in mobile phase. 32

mabs and ADCs analysis by HIC Shanhua Lin, Ph.D. 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 Gradient elution: High to Low salt concentration Relatively mild conditions are used; so biomolecules do not become denatured during the separation 35

MAbPac HIC family MAbPac HIC-1 MAbPac HIC-2 MAbPac HIC-Butyl Chemistry Proprietary polyamide Proprietary alkylamide Butyl Substrate Spherical, high purity Spherical, high purity Hydrophilic polymer silica particles silica particles Particle Size 5 µm 5 µm 5 µm Pore size 1, Å 1, 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-1 MAbPac HIC-2 MAbPac HIC-Butyl ph Range 2.-8. 2.-9. 2.-12. Temperature Limit 6 6 6 ( C) Organic Compatibility -1% -1% -5% Flow Rate.5-1. ml/min (recommended) 1.5 ml/min (max) Maximum Pressure 6, psi (4.6 1 mm) 8, psi (4.6 25 mm) 6, psi (4.6 1 mm) 8, psi (4.6 25 mm) 4, psi (4.6 1 mm) 37

Separation of Aggregates on MAbPac HIC-1 Absorbance (ma AU) 22 2 15 1 5 mab variants Monomer Aggregate Column: MAbPac HIC-1, 5 µm Format: 4.6 1 mm Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7 7. Mobile phase B: 1 mm sodium phosphate, ph 7. Gradient: Time (min) %A %B -5. 6 4. 6 4 1. 6 4 29. 1 34. 1 Temperature: 3 ºC Flow rate: 5.5 ml/min Inj. volume: 1 µl (4 mg/ml) Detection: UV (28 nm) Sample: Monoclonal antibody 5 1 15 2 25 3 Retention Time (min) 38

Oxidized mabs Methionine and Tryptophans are usually oxidized. 39

Separation of Oxidized mab on MAbPac HIC-2 Absorbance (ma AU) 6 Native mab Column: MAbPac HIC-2, 5 µm Format: 4.6 25 mm 5 Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, ph 7. Gradient: 4 Time (min) %A %B -6. 5 5. 5 5 2. 5 5 3 3. 1 35. 1 2 1 Oxidized variants Temperature: 3 ºC Flow rate:.5 5mL/min Inj. volume: Untreated mab: 2 µl (1.25 mg/ml) Oxidized mab: 2 µl (1.25 mg/ml) Detection: UV (28 nm) Sample: Untreated mab H 2 O 2 oxidized mab 1 14 18 22 26 3 Retention Time (min) 4

Antibody-Drug Conjugates (ADCs) 41

Heterogeneity of Cys-Linked ADC 42

Separation of Cys-Linked ADC on MAbPac HIC-Butyl 4 Absorbance (mau) 3 2 DAR 2 DAR 4 Column: MAbPacHIC-Butyl, 5 µm Format: 4.6 1 mm Mobile phase A: 1.5 M ammonium sulfate, 5 mm sodium phosphate, ph 7. / isopropanol (95:5 v/v) Mobile phase B: 5 mm sodium phosphate, ph 7. / isopropanol (8:2 v/v) Gradient: Time (min) %A %B -5. 5 1. 1 1. 1 15. 1 2. 1 1 DAR DAR 6 DAR 8 Temperature: 25 ºC Flow rate: 1. ml/min Inj. volume: 5 µl (5 mg/ml) Detection: UV (28 nm) Sample: Cys-conjugated ADC mimic 4 8 12 16 2 Retention Time (min) 43

Separation of Bispecific mab 1 88 75 Absorbance (mau) 63 5 38 4 25 3 132 1 Column: MAbPac HIC-2, 5 µm Format: 4.6 1 mm Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, p ph 7. Gradient: Time (min) %A %B -5. 6 4. 6 4 1 1. 6 4 15. 1 2. 1 Temperature: 3 ºC Flow rate: 1. ml/min Inj. volume: A+B: 2 µl A+D: 12 µl C+B: 1 µl C+D: 2 µl Detection: UV (28 nm) Sample: A+B (.87 mg/ml) A+D (1.4 mg/ml) C+B (1.72 mg/ml) C+D (1. mg/ml) -1 2 2. 38 3.8 5 5. 63 6.3 75 7.5 88 8.8 1. 11.3 12.5 13.8 15. 16.3 18. Retention Time (min) 44

Method Development Type of salt (lyotropic salt, salting-out agents) Ammonium sulfate 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) p Sample matrix Flow rate Temperature 45

Mobile Phase Recommendation Mobile phase formula 1 Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, p ph 7. Mobile phase formula 2 Mobile phase A: 1.5 M ammonium sulfate, 5 mm sodium phosphate, ph 7. / isopropanol (95:5 v/v) Mobile phase B: 5 mm sodium phosphate, ph 7. / isopropanol (8:2 v/v) Mobile phase formula 3 Mobile phase A: 2 M ammonium sulfate, 2 mm sodium acetate, ph 5. Mobile phase B: 2 mm sodium acetate, ph 5. 46

Optimization of the Starting Salt Concentration Absorbance (m mau) Column: MAbPac HIC-2, 5 µm Dimension: 4.6 1 mm 3 Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, ph 25 7. Gradient 1: Time (min) %A %B 2-5. 1. 1 1. 1 15. 1 15 2. 1 Gradient 2: Time (min) %A %B 1-5. 8 2. 8 2 1. 8 2 5 15. 1 2. 1 Gradient 3: Time (min) %A %B 1.2 M -5. 6 4 16M 1.6. 6 4 1 1. 6 4-5 15. 1 2. 1 2. M Temperature: 3 ºC 2 4 6 8 1 12 14 16 18 2 Flow rate: 1. ml/min Inj. volume: 1 µl Retention Time (min) Detection: UV (28 nm) Sample: Trypsin inhibitor (5 mg/ml) 47

Addition of Organic Solvent in Mobile Phase B Absorbance (m mau) 45 3 2 Column: MAbPac HIC-2, 5 µm Dimension: 4.6 1 mm 1) Mobile phase A: Mobile phase B: 2) Mobile phase A: Mobile phase B: 3) Mobile phase A: Mobile phase B: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. 1 mm sodium phosphate, ph 7. 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. 1 mm sodium phosphate, ph 7./isopropanol (9:1 v/v) 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. 1 mm sodium phosphate, ph 7. / isopropanol (8:2 v/v) 1 3 2 1 2% Isopropanol 1% Isopropanol No solvent Gradient: Time (min) %A %B -5. 1. 1 1. 1 15. 1 2. 1-5 Temperature: 3 ºC Flow rate: 1. ml/min 2 4 6 8 1 12 14 16 18 2 Inj. volume: 5 µl Detection: UV (28 nm) Retention Time (min) Sample: Trypsin inhibitor (5 mg/ml) 48

Effect of Matrix on Peak Shape Absorbance (ma AU) 6 4 2 6 4 a b 1 1 2 2 3 3 4 4 Column: MAbPac HIC-2, 5 µm Format: 4.6 1 mm Mobile phase A: 2 M ammonium sulfate, 1 mm sodium phosphate, ph 7. Mobile phase B: 1 mm sodium phosphate, ph 7. Gradient: Time (min) %A %B -5. 1. 1 1 1. 1 15. 1 2. 1 Temperature: 3 ºC Flow rate: 1. ml/min Inj. volume: 1 µl Detection: UV (28 nm) Sample: Protein mixture 2 4 8 12 16 2 Retention Time (min) Sample matrix: Peaks: a) Water b) 1 M ammonium sulfate, 5 mm sodium phosphate, h ph 7. 1) Myoglobin 2) Ribonuclease A 3) Lysozyme 4) 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 background signal. If the protein is less hydrophobic (indicated by broader fronting peak and short retention time), increase the salt concentration (lyotropic agent) of mobile phase A or you will need 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. 5

MAbPac HIC-2 Conditioning This column requires conditioning by injecting 75 µg of protein. Ovalbumin or BSA can be used for this purpose. 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 1 mg/ml of each protein Then mix these proteins with the following ratio Myoglobin : RNase A : Lysozyme : α-chymotrypsinogen A = 2:4:1:2 52

Thank you! 53