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ADC Drug to Antibody Ratio (DAR)

HIC

Proteomix RP

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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.

• Proteomix HICBu NP5, 4.6x35mm, P/N: 431NP5-4603

(polymer based)

• BioHIC 100A, 3um, 4.6x100mm, P/N: 313100-4610 (silica based)

• BioHIC 300A, 3um, 4.6x100mm, P/N: 313300-4610 (silica based)

Sepax HIC HIC hydrophobic interaction separation

min 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

0

200

400

600

800

1000

1200

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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.

Column: Proteomix® HICBu-NP5 (5 mm, 4.6 x 35 mm, Development lot) Mobile phase: A: 2 M ammonium sulfate in 0.025 M sodium phosphate, pH 7.0, B: 0.025 M sodium phosphate pH 7.0, C : 100% IPA Flow rate: 0.8 mL/min, Detector: UV 214 nm, Column temperature: 25 ℃ Sample: Herceptin/ADC1/ADC2, 1 mg/mL in 25 mM sodium phosphate, Injection: 10 mL,

T (min)

%B %C

0 25 0

15 75 25

20 75 25

21 25 0

31 25 0

Herceptin

Cysteine ADC 1

Herceptin and its ADCs separation on Proteomix ® HIC

Cysteine ADC 2

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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc. 4

Proteomix® RP-1000 • With the high cross-linking structure, Proteomix® RP-1000 resins

provide excellent chemical and physical stability, as well as high rigidity to tolerate high pressure, high temperature (80 degrees).

• MAb Herceptin and its corresponding ADCs are successfully separated on the Proteomix® RP-1000, 5um, 4.6x100mm with protein peaks indicating different hydrophobicity with minimum sample carry over. Ideal tool for DAR caculation.

• Proteomix® RP-1000, 5um 2.1x50mm or 4.6x50mm provides high throughput and fast analysis of mAb, ADC and other protein therapeutics with online mass spec determination capability.

MAb Herceptin and its ADCs Separation- 4.6 x 100 mm

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Column: Proteomix® RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN; Flow rate: 1.0 mL/min; Detector: UV 210 nm; Column temperature: 80 °C; Sample: Herceptin and ADCs 1 mg/mL diluted in water; Injection volume: 10 mL

5

min 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

175

200

225

250

275

300

325

350

375

400

Herceptin

Cysteine ADC 1 cleavable linker

Cysteine ADC 2 non-cleavable linker

Gradient: 2-22 min, 30-48%B

Herceptin/ADC1/ADC2 Separation- small size 2.1 x 50 mm

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Column: Proteomix® RP-1000 (5 µm, 1000 Å, 2.1 x 50 mm) Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN; Flow rate: 0.6 mL/min; Detector: UV 210 nm; Column temperature: 80 ˚C; Column pressure: 70 bar; Sample: Herceptin, ADC 1 and ADC 2 diluted in water; Injection volume: 0.5 mL for Herceptin, 1 mL for ADC 1 and ADC 2

min 1 2 3 4 5 6

mAU

100

150

200

250

300

Herceptin

Herceptin-ADC1

Herceptin-ADC2

Unconjugated mAb

0-5 min, 34-51% B

6

Minimum sample carry over

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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.

Column: Proteomix® RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN; Flow rate: 1.0 mL/min; Detector: UV 210 nm; Column temperature: 80 °C; Sample: Cysteine ADC 1 1 mg/mL diluted in water; Injection volume: 8 mL

7

min 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

180

200

220

240

260

280

300

320 2-22 min, 30-48%B

Blank

ADC

Minimum sample carry over in the blank injection after sample run.

Herceptin and its lysine ADC separation

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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.

Column: Proteomix® RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN; Flow rate: 1.0 mL/min; Detector: UV 210 nm; Column temperature: 80 °C; Sample: Herceptin and lysine ADC 1 mg/mL diluted in 0.1% TFA; Injection volume: 10 mL

8

min 0 2.5 5 7.5 10 12.5 15 17.5 20

mAU

20

40

60

80

100

Lysine-ADC

Herceptin

2-22 min, 25-55% B Each in full scale

Unconjugated mAb

MAb/ADC fragments: Reduced mAb Herceptin and Cysteine ADC 1

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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.

Column: Proteomix® RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN; Flow rate: 1.0 mL/min; Detector: UV 210 nm; Column temperature: 80 °C; Sample: Herceptin and ADC1 2 mg/mL reduced with 20 mM DTT, incubated at 65 °C for 20 minute; Injection volume: Reduced herceptin 2 mL , Reduced herceptin ADC 1 5 mL

9

min 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

180

200

220

240

260

280

300

320

Gradient: 2-22 min, 30-48% B

Reduced Herceptin

L0

H0

Reduced ADC 1

Possible subunits for reduced ADC: L0,L1, H0, H1, H2, H3

Mab fragment: Fab/Fc separation with 40 ˚C and 80 ˚C

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Column: Proteomix® RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN; Flow rate: 1.0 mL/min; Detector: UV 210 nm; Column temperature: 40, 80 ˚C; Sample: mAb (digested by papain), 1 mg/mL diluted in 0.1% TFA; Injection volume: 20 mL

10

min 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

200

250

300

350

400

450

40 ˚C

80 ˚C

2-22 min, 30-40% B

Temperature effect: mAb separation with 25°C /40°C / 80°C

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Column: Proteomix® RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN Flow rate: 1.0 mL/min; Detector: UV 210 nm; Sample: mAb 1mg/mL; Injection volume: 20 mL

11

min 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

250

500

750

1000

1250

1500

1750

2000

2250

40 °C

80 °C

25 °C

min 6 8 10 12 14 16

mAU

0

20

40

60

80

100

120

140

160

2-22 min, 20-70%

Temperature effect: Herceptin Cysteine ADC 2 Separation

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Better Surface Chemistry for Better Separation © Sepax Technologies, Inc.

Column: Proteomix® RP-1000 (5 µm, 1000 Å, 4.6 x 100 mm); Mobile phase: A: 0.1% TFA in water; B: 0.1% TFA in 100% ACN; Flow rate: 1.0 mL/min; Detector: UV 210 nm; Column temperature: 25, 80 ˚C; Sample: Herceptin Cysteine ADC 2 1 mg/mL diluted in 0.1% TFA; Injection volume: 20 mL

12

min 0 2.5 5 7.5 10 12.5 15 17.5 20 22.5

mAU

100

200

300

400

500

600

700

80 ˚C

25 ˚C

2-22 min, 20-70%