The world leader in serving science

Shanhua Lin, Ph.D. R&D Manager Bioseparation, Chromatography Consumables

mAb and ADC Analysis

2

Structure of IgG and Typical Forms of Heterogeneity

3

Protein and mAb Separation by HPLC

Ion Exchange Chromatography (IEX)

Size Exclusion Chromatography (SEC)

Reverse Phase Chromatography (RPC)

Size difference?

Charge difference?

Hydrophobicity difference? Hydrophobic Interaction Chromatography (HIC)

Thermo Scientific™ MAbPac™ SEC-1

MAbPac HIC-10 MAbPac HIC-20 MAbPac HIC-Butyl ProPac HIC-10

YES

YES

YES

YES

MAbPac RP

MAbPac SCX-10 CX-1 pH Gradient Buffer Thermo Scientific™ ProPac ™ WCX-10

4

IEC Analysis for Charge Variant

• ProPac WCX-10 and MAbPac SCX-10 columns

• Separate acidic and basic variants

• CX-1 pH Gradient Buffer Kit provides a platform solution

5

Charge Variant Analysis

10 µm Nonporous Polymeric Beads

Polymeric grafts WCX, SCX, WAX, SAX

Boundary (Cross-linked hydrophilic layer) Core (Highly cross-linked EVB-DVB)

Untreated mAb

Lysine Variants

Acidic Variants Basic Variants

Y Y K K

Y Y K

Y Y

Y Y

Acidic Variants Basic Variants

Carboxypeptidase B digested mAb

ProPac WCX-10 Column

6

Next-generation CEX Column

Lysine Variants

Y Y K K

Y Y K

Y Y

Acidic Variants

Basic Variants

ProPac WCX-10 column, 4×250 mm

MAbPac SCX-10 column, 4×250 mm 60 min. total analysis time

Acidic Variants

Basic Variants

Y Y K K

Y Y K

Y Y

Lysine Variants

Improve resolution or sample throughput through column chemistry

0 10

0

22

MAbPac SCX-10 column, 4×150 mm 15 min. total analysis time

5 15 Minutes

mA

U

MAbPac SCX-10 Column

7

Thermo Scientific CX-1 pH Gradient Buffers

• Dilute buffers 10-fold with DI water • A linear pH gradient (pH 5.6 - 10.2)

is generated by running a linear pump gradient from 100% Buffer A to 100% Buffer B

• It is platform, fast, and high-res!

Buffer A Buffer B

pH 5.6 10.2

Form Liquid Liquid

Concentrate 10X 10X

Shipping condition

Room Temp

Room Temp

Storage condition

4 ~ 8 °C 4 ~ 8 °C

US 8921113 B2: Buffer kit and method of generating a linear pH gradient

8

pH Gradient Buffers – How Do They Work?

+

0

–

5 6 7 8 9 10 11 12

Buffer/System pH

Protein net charge vs. pH

Buffer pH typically > pI

Anion-Exchange Chromatography

Buffer pH typically < pI

Cation-Exchange Chromatography

NH2R

COO -

NH3R +

COOH

Isoelectric Point (pI)

NH3R +

COO -

4

Cationic protein binds to

negatively charged cation exchanger

+ ++

++

+ ++

Anionic proteinbinds to

positively chargedanion exchanger

- -- -

-

- - -

Protein Elution Mechanisms on IEX

pH range covered by CX-1 pH gradient buffers

9

Linear pH Gradient by Zwitterionic Buffer Cocktail

y = 0.1577x + 4.9755R² = 0.9996

5.5

6.5

7.5

8.5

9.5

10.5

0 10 20 30 40

Mea

sure

d pH

val

ue

Retention Time [min]

Measured ValueLinear (Measured Value)

10

Protein Standard – CX-1 pH Gradient Buffer

pH trace

0 5 10 15 20 25 30 35 40 -10

20

40

60

90

5

6

7

8

9

10

11

Lectin-1 - 6.11

Lectin-2 - 6.28

Lectin-3 - 6.45 Trypsinogen - 7.75 Ribonuclease A - 8.72

Cytochrome C - 10.15

MAbPac SCX-10 Column, 4 × 250 mm

0

Peak label: protein name – elution pH

11

Protein Standard – Phosphate-based pH Gradient

pH trace

0 5 10 15 20 25 30 35 40 -10

0

10

20

30.

40

50

60

70

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0 pH

1 2

MAbPac SCX-10 Column, 4 × 250 mm

12

Why pH Gradient Buffers?

• Traditional salt gradient using ion exchange chromatography

• Needs to be tailored for individual charge variants

• Patented pH buffer formulations

• Fast, robust and reproducible pH gradients

• Ready to use with existing CEX columns and systems

• Simple to optimize and easily automated

• Applicable to the majority of mAbs

0.0 5.0 10.0 15.0 20.0 25.0 30.0

0.0

5.0

10.0

15.0

min

%B: 10.0

30.0

Salt gradient

0.0 5.0 10.0 15.0 20.0 25.0 30.0

0.0

5.0

10.0

15.0

min

%B: 25.0

50.0

25.0

pH gradient

13

Benefit of Linear pH Gradient: Platform Approach

• A platform approach for charge variant analysis, covering the pH range 5.6 to 10.2

• The same pH gradients is applicable to majority of mAb charge variants with pI value between 6-10

• pI value of the unknown mAb can be predicted from the correlation curve

14

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 -20

0

20

40

60

80

100

120

140

160

180

200

5.00

5.50

6.00

6.50

7.00

7.50

8.00

8.50

9.00

9.50

10.00

10.50

pH trace

Abso

rban

ce (

mAU

)

Retention Time (min)

mAb Standards Using Linear pH Gradient

15

Top-selling mAbs Analyzed by pH Gradient Method

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0-10.0

0.0

12.5

25.0

37.5

50.0

70.0

Retention Time (min)

Rituxan

1

2

3

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0

-10.0

0.0

12.5

25.0

37.5

50.0

70.0

Retention Time (min)

Herceptin

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0

-2.0

5.0

10.0

15.0

18.0

Retention Time (min)

Humira

15.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 35.0

-2.0

5.0

10.0

15.0

20.0

Retention Time (min)

Avastin

Platform method for mAb charge variant analysis

16

Benefit of Linear pH Gradient: Simple Optimization

• The method can be simply optimized • By running a shallower pH gradient a higher resolution separation is obtained

(e.g. 50-100%, rather than 0-100%B)

17

0 5 10 15 20 25 30 35 40 -5.0

10.0

20.0

30.0

40.0

5.00

6.00

7.00

8.00

9.00

10.50

Abso

rban

ce [

mAU

]

Retention Time [min]

pH trace (a)

100% B 0% B

*The pH trace at elution was obtained with the Thermo Scientific™ Dionex™ UltiMate™ 3000 pH and Conductivity Monitoring Module (PCM-3000)

mAb Charge Variant Separation, 0–100% B

18

0 5 10 15 20 25 30 35 40 -5.0

0.0

10.0

20.0

25.0

5.00

6.00

7.00

8.50

Abso

rban

ce [

mAU

]

Retention Time [min]

(b)

mAb Charge Variant Separation, 0–50% B

pH trace

0% B 50% B

19

mAb Charge Variant Separation, 25–50% B

0 5 10 15 20 25 30 35 40 -2.0

5.0

10.0

16.0

6.60

7.00

7.25

7.50

7.75

8.00

Abso

rban

ce [

mAU

]

Retention Time [min]

(c) pH trace

25% B 50% B

20

Ultra Fast Method Development

21

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 -100

-75

-50

-25

0

25

60

5.00

6.00

7.00

8.00

9.00

10.50

Run #300

Run #200

Run #100

Run #5

pH trace

Abso

rban

ce [

mAU

]

Retention Time [min]

Repeat Injections of Ribonuclease A: Over 300 Runs

Retention time reproducibility <0.8% RSD

22

CX-1 pH Gradient Buffer Kit (10X): Lot to Lot Consistency

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 -10

20

40

60

80

110

Abso

rban

ce (m

AU)

Retention Time (min)

Lot 1 (130523 / 130528) Lot 2 (130524 / 130529) Lot 3 (130531 / 130530)

23

RP LC/MS Analysis for Intact, mAb Fragments, and ADCs

• MAbPac RP column

• Separate mAb, mAb fragments (LC, HC, Fc, Fab, scFc, and F(ab’)2) and ADC DAR forms

• Often coupled with MS instrument for high resolution accurate mass determination

24

What is it?

MAbPac RP columns

Column Chemistry

Phenyl

Substrate Spherical, polymer, wide-pore (1,500 Å)

Particle size

4 µm

Pore size 1,500 Å Formats 100 mm: 3.0 + 2.1mm ID

50 mm: 3.0 + 2.1mm ID 10mm Guard Cartridges: 3.0 + 2.1mm ID

25

SiteClick™ Enzyme-based N-glycan Labeling of Antibody

Azide-Activated Antibody

Antibody •Polyclonal •Monoclonal •Recombinant

Site-Specifically Labeled Antibody

Gal-T(Y289L) UDP-GalNAz β-galactosidase

sDIBO-Val-Cit-pAB-MMAE

N3

N3

N3

N3

O

O

HN

S OOO

O

NH

O

O

O

O

OHN

O

HN

NH

NH2O

NH

O NHN

NO O

NHN

O

OO

O

O

O

O

(CH3CH2)3NH

NN

N

N3 N3

N3

26

Site-selective Antibody-drug Conjugates (ADCs) DAR Forms

DAR 1

DAR 2

DAR 3

DAR 4

27

LC/UV Analysis of ADC

Column: MAbPac RP, 4 µm Format: 2.1 × 50 mm Mobile 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 0.0 65 35 0.5 65 35 4.5 45 55 5.0 45 55 5.5 65 35 6.0 65 35 Flow rate: 0.6 mL/min Temperature: 80 ºC Inj. volume: 2 µL Detection: UV (280 nm) Sample: a. Trastuzumab b. Trastuzumab-MMAE

-2.0

5.0

12.0

0.50 1.00 2.00 3.00 4.00 4.50 -5.0

10.0

25.0

(a)

(b)

Retention Time (min)

28

LC/MS Analysis of ADC

2950 3000 3050 3100 3150 3200 3250

m/z

0

50

100 0

50

100 0

50

100 0

50

100

Rel

ativ

e Ab

unda

nce

0

50

100 0

50

100 3242.41 3172.00 3104.48 3039.85 2977.80

3175.55 3107.78 2981.16 3043.27

3209.69 3141.27 2953.08 3075.87 3098.20

3013.12 3033.70 3235.89 3165.45

3127.68 3194.17

3002.59 3064.18 3105.03 3170.88 3239.77 3041.58 2977.71

3118.75

3197.87 3131.09

3182.06

3006.21 3069.68 3232.09 3156.83 3021.45

3105.18 2983.64 3169.86 3237.45 3043.16

3027.89

3108.56

2968.60 3090.80 3179.52 3154.48 3221.20 2992.43 3062.25 3122.04 3195.93

3237.30 3169.93 3043.17 2983.59 3105.26

3147.21 3214.04 3021.41 3082.89

3240.81 3173.29

2962.08 2986.74

3139.68

3046.60 3163.88

3115.21 3190.26

3232.52

3067.48

3100.44 2977.99

3146.61 3023.28 3212.12 3083.76 2965.15

3196.41 3131.01

3150.02 3087.05

3008.33 3068.62

3215.53 3026.44

3248.18 2973.83 3042.22 3115.73 3166.15

3224.81

3124.25 3187.95 3063.00 3004.06

3238.48 3172.49 3109.06 2989.49 3191.33

3047.96

3127.33

3222.69 3033.15 3093.96 2975.07 3156.91 3009.77 3069.21

(a) Peak 1, DAR 0

(b) Peak 2, DAR 1

(e) Peak 5, DAR 3

(d) Peak 4, DAR 2

(c) Peak 3, DAR 2

(f) Peak 6, DAR 4

29

Deconvolution of mAb and Intermediate

(a) Trastuzumab

(b) Trastuzumab-azide

4-N3

1-N3

2-N3

3-N3

G0/G0F G2F/G2F

G1F/G1F (G0F/G2F)

G0F/G1F

G0F/G0F G1F/G2F

30

Deconvolution of Multiple DAR Forms

1 x DIBO-Val-Cit-pAB-MMAE

(a) DAR 1

(b) DAR 2

1 x DIBO-Val-Cit-pAB-MMAE

(c) DAR 3

(d) DAR 4 1 x DIBO-Val-Cit-pAB-MMAE

31

SiteClick™ Enzyme-based N-glycan Labeling of Antibody

Azide-Activated mAb Unconjugated mAb ADC, DAR = 2.0

GlycINATOR UDP-GalNAz GalT(Y289L) DIBO-Toxin

N3 N3 Toxin Toxin

Poster number: P-202-TH Presenting: Thursday, January 26 from 7:30am – 8:30am. Title: High Resolution LC/MS Separation and Characterization of Chemoenzymatic Site-specific Engineered Antibody-drug Conjugates (ADCs)

32

• Chris Pohl

• Xiaodong Liu

• Julia Baek

• Yoginder Singh

• Brian Agnew

• Terry Zhang

• Jonathan Josephs

Thank you!