UHPLC for Large Bio-Therapeutics

1

The world leader in serving science

Ken Cook

EU Bio-separations Support Expert

June 2015

Various Types of Antibody-Based Therapeutics

Antibody-drug conjugateKadcyla (trastuzumab emtansine)

mAb Fab fragmentCimzia (certolizumab pegol)Lucentis (ranibizumab)

PEGylated mAbCimzia (certolizumab pegol)

2

Fusion proteinEnbrel (etanercept)TNF receptor-Fc fusion

Kadcyla (trastuzumab emtansine)Adcetris (brentuximab)

Bispecific mAbRemovab (catumaxomab)

Properties of Proteins

• Properties:

• Size

• Charge

• Hydrophobicity

+

++

--

-

3

• Hydrophobicity

• Affinity or Recognition +

+

+

-

--

Protein Separation by UHPLC

Ion Exchange Chromatography (IEX)

Size Exclusion Chromatography (SEC)

Size difference?

Charge difference?

Thermo Scientific™

MAbPac™ SEC-1 Column

Thermo Scientific™ ProPac™

WCX-10 ColumnMAbPac SCX-10 Column ProPac SAX-10 Column

4

Hydrophobic Interaction Chromatography (HIC)

Hydrophobicity difference?

Reverse Phase Chromatography (RPC)

MAbPac HIC-10 ColumnMAbPac HIC-20 ColumnMAbPac HIC-Butyl Column

MAbPac RP ColumnThermo Scientific™

ProSwift™ RP Column

• High resolution – small changes in a large molecule

• Speed – many variations to analyse and many potential drug candidates

• Robust analysis

• Global methods to reduce method development time

• Multidimensional possibilities

Requirements for Development of New Solutions

5

• Multidimensional possibilities

• New systems and new chemistries

Biopharma UHPLC Portfolio

Thermo Scientific ™

Dionex ™ UltiMate ™

3000 BioRS SystemEstablished

biocompatible UHPLC. Key benefit: Proven

Vanquish Flex System

Designed for allbiopharma labs. Key benefit: Flexibility

Thermo Scientific ™

Vanquish ™ System

Flagship system. Key benefit: High end

performance

6

Built for Biopharma

20142013 2015

2015 Systems for Bio-Therapuetic Analysis

High Resolution, Cooled Fractionation

7

Distribution of applications

20%

80%

Highly specific Workflows

Standard (biopharma) applications UltiMate 3000

BioRS system

pH and Conductivity

Vanquish Systems

System Biocompatibility by Default

UHPLC is now not limited to small moleculesBiomolecular analysis can benefit from the higher resolution and faster analysis this technique offers.

Thermo Scientific™ Viper™ Fingertight FittingsMP35N 100um i.d. Finger tight and zero dead volume.

Vanquish Parallel Binary PumpIndependent piston drives and variable stroke volume, 1500 bar. – ultra low baseline noise

LightPipe™ Technology in the Vanquish DAD Detector

8

LightPipe™ Technology in the Vanquish DAD Detector Provides you with an unmatched detection experience. Ultra-wide dynamic range, high sensitivity

SamplerZero maintenance ceramic injection valve, loop pre compression, 4 x 96 well plates, charger.

OvenAdiabatic and forced air. 30cm column, active eluent preheating, column switching, ceramic valves.

• Do a protein concentration assay

• Make up to 8M Urea

• Add DTT or TCEP at 70 C and wait for 30 minutes

• Make up Iodoacetate

• Add Iodoacetate and wait 15 minutes

• Dilute to 1 M Urea

In Solution Trypsin Digestion

9

• Dilute to 1 M Urea

• Make up Trypsin

• Add trypsin to vial and and wait for 4 hours

• Add more Trypsin and digest overnight

• Spin to remove particulates

• Possible SPE to remove detergents and Urea

Thermo Scientific Smart Trypsin Digestion

• Heat stable Trypsin

• 70 ºC heat denaturation of proteins so no need for detergents or urea

• Immobilized Trypsin in throw away PCR tube

• No autolysis

• Less steps and no clean up – much quicker and easier to use

10

• Less steps and no clean up – much quicker and easier to use

• Less modifications

• More reproducible

Undigested Protein RT: 0.00 - 30.02

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bun

dan

ce

8.21575.80

9.08547.83

4.95395.65 7.15

464.70 9.17501.28

3.49441.13

14.92683.01

12.17657.02

10.53490.561.96

538.2613.62

672.57

≤ 90 ºC,≤60 Minutes

Filter LC/MS

Up to 96 Samples, ≤ 60 Minute No Pretreatment Necessary

11

0 2 4 6 8 10 12 14 16 18 20 22Time (min)

0

5

10

15

20

25

3017.23

802.27

19.36813.3115.38

812.42 21.321398.410.63

515.64 20.05823.40

17.78821.23

22.60974.78

• Complete digest of native protein in ≤ 60 minutes• Pretreatment (reduction and alkylation) – optional• Equipment required – thermal cycler• Multiple patents pending

Time for Digestion

Overall time for digestion is faster

Actual hands on preparation time is drastically reduced

Less repetitive manual steps reduces inaccuracy

12

Sample preparation

Protein assay

Digestion

SPE + dry/reconstitute

Spin down

Overlay of Different mAb Digests on the Vanquish System

13

Overlay of 10 Runs of a Trypsin Digest mAb with Retention Time Precision

peak #RT

(min)

RT-RSD (%)

3 3.315 0.0829 5.231 0.06514 6.532 0.01715 6.937 0.02319 10.290 0.02123 12.013 0.01231 14.011 0.01339 15.177 0.01242 15.589 0.010

14

42 15.589 0.01051 17.511 0.00755 17.969 0.01161 18.546 0.01083 20.798 0.01085 21.095 0.01287 22.386 0.00996 24.774 0.012

103 26.155 0.009106 26.155 0.009109 27.529 0.010

Slide 14

JH1 Please format with title-case text.Jim Hegarty; 15.10.2014

Peptide Maps of Herceptin Digest; 6-40min GradientsR

elat

ive

Abu

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Gra

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30%

B /

tota

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tim

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100

0100

0100

0

100

6/16 min6/16 min

8/16 min8/16 min

11/28 min11/28 min

15/28 min15/28 min

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0 05 10 15 20 25 30 35 40 45 50 55 60

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Time (min)

Gra

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30%

B /

tota

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tim

e

0100

0

100

0100

0

15/28 min15/28 min

20/38 min20/38 min

30/48 min30/48 min

40/60 min40/60 min

Can Use Shorter Gradients

Mechanism of Salt and pH Elution of Proteins

16

Comparison of pH Gradient Buffer Systems

In house buffer system 1 In house buffer system 2

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.05.00

6.00

7.00

8.00

9.00

min

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Thermo Scientific CX-1 pH 5.6 and 10.2 Phosphate based

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

Re-equilibration Problems

8.00

8.50

9.00

9.50

10.00

10.50100.0

%C: 0.0 %

5 minutes re-equilibration on a 250mm columnThe ProPac WCX column buffers more against the programmed change in pH.The MAbPac SCX column equilibrates in timeThe ProPac WCX column does not get back down to the initial pH in time and is still dropping on injection

MAbPac SCX column

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0.0 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 16.0 17.0 18.0 19.0 20.0 21.05.00

5.50

6.00

6.50

7.00

7.50

min

21

Flow: 300 µ l/min

%B: 0.0 % 0.0

MAbPac SCX column

ProPac WCX column

Time to equilibrate is different

ProPac WCX is still not at the correct pH on time of injection

Improving Charged Variant Analysis

• Speed of analysis – 60min to 6min or below?

• Resolution – No loss in resolution

• Time to develop the method

• Global applicability

• Robustness

19

• Robustness

Generic Linear pH Gradient with the Vanquish System

MAB 1

MAB 2

MAB 3

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pH 5.6 to 10.2 in 10 minutes

MAB 3

MAB 4

MAB 5

Slide 20

JH3 Please format with title-case text.Jim Hegarty; 15.10.2014

Example 1/5: Cetuximab – Vanquish System Fast Gradients

10 min gradient

5 min gradient

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Number of charge variants and resolution maintained for 2.5 min gradient

3 steps method development1. 10 minutes 0�100% B in 10 minutes

2. 10�35% B in 5 minutes

3. 10�35% B in 2.5 minutes

2.5 min gradient

Example 2/5: Infliximab – Vanquish System Fast Gradients

10 min gradient

5 min gradient

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Number of charge variants and resolution maintained for sub-minute gradient

3 steps method development1. 10 minutes 0�100% B in 10

minutes

2. 20�40% B in 5 minutes

3. 18�27% B in 0.8 minutes

0.8 min gradient

Hydrophobic Interaction Chromatography

Column: MAbPac HIC-10, 4.6 ×100 mmCompetitor A (Ether), 7.5 ×75 mm Competitor B (Butyl), 4.6 ×100 mm

Mobile phase A: 2.0 M ammonium sulfate, 100 mMsodium phosphate, pH 7.0

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

Time (min) %A %B-5.0 60 400.0 60 401.0 60 40

15.0 0 10020.0 0 100

45

Abs

orba

nce

(mA

U)

Competitor A (Ether)

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20.0 0 100

Temperature: 30 ºCFlow rate: 1.0 mL/minInj. volume: 2 µL (4 mg/mL)

Competitor A (Ether): 4 µL Detection: UV (280 nm)Sample: mAb

0 4 8 12 16 20

0

Retention Time (min)

Abs

orba

nce

(

Competitor B (Butyl)

Competitor A (Ether)

MAbPac HIC-10 Column

Global Analysis of Intact mAbs

30

40

50

60

Column: MAbPac HIC-10, 5 µmFormat: 4.6 ×100 mmMobile phase A: 2.0 M ammonium sulfate, 100

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

7.0Gradient:

Time (min) %A %B-5.0 60 400.0 60 401.0 60 40

15.0 0 10020.0 0 100

Abs

orba

nce

(mA

U)

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0 4 8 12 16 20

0

10

20

20.0 0 100

Temperature: 30 ºCFlow rate: 1.0 mL/minInj. volume: 2 µL (4 mg/mL)Detection: UV (280 nm)Sample: mAb1

mAb2mAb3mAb4mAb5

Abs

orba

nce

Retention Time (min)

Aggregate Screening using MAbPac SEC-1 Column

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HIC separation of MAb and Purified Dimer

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Purified Dimer

mAb Sample with Aggregates

Method Speed up of Mab Aggregates with HIC

Column: MAbPac HIC-10, 5 μm

Format: 4.6 ×100 mm

Mobile Phase A: 2 M ammonium sulfate, 100 mM

sodium phosphate, pH 7.0

Mobile Phase B: 100 mM sodium phosphate, pH 7.0

Gradient (a Time (min) %A %B

-5.0 60 40

0.0 60 40

1.0 60 40 15.0 0 100

20.0 0 100

Gradient (b) Time (min) %A %B

-5.0 60 40

0.0 60 40

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0.0 60 40

1.0 60 40

29.0 0 100

34.0 0 100

Flow rate: (a) 1.0 mL/min

(b) 0.5 mL/min

Inj. Volume: 10 μL (4 mg/mL)

Temp.: 30 ºC

Detection: UV (280 nm)

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 / 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 00.0 100 01.0 100 0

15.0 0 100

20

30

40

Abs

orba

nce

(mA

U)

DAR 2

DAR 4

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15.0 0 10020.0 0 100

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

0 4 8 12 16 20

0

10

Abs

orba

nce

(

Retention Time (min)

DAR 0

DAR 6

DAR 8

6 min gradient, MAbPac RP Column with ADC Mimic

10.0

15.0

20.0

25.0 2 - MABPACRP_2PT1X50_BETA01 #35 HER-MMAE UV_VIS_1mAU WVL:280 nm

55.0

0.1% TFA + 70% IPA + 20% MeCN: 0.0 %

Peak 1: 0 MMAE, 2.04%Peak 2: 1 MMAE, 3.89%Peak 3: 2 MMAE, 3.94%Peak 4: 2 MMAE (isomer), 8.89%Peak 5: 3 MMAE, 25.11%Peak 6: 4 MMAE, 55.73%

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0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00-5.0

0.0

5.0

10.0

min

2

1

Flow: 600 µ l/min

0.1% TFA + 9.9% H2O + 90% MeCN: 35.0 % 35.0

HER

HER-MMAE

Peak 6: 4 MMAE, 55.73%

400

600

800

Cetuximab Separation on a MABPac RP Reverse Phase Column

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0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 12.0-100

200

400

min

Accurate Monoisotopic Mass Measurement Using Ultra High Resolution

LC-MS of Antibody Light Chain and Heavy Chain

+23

1005.5 1006.0 1006.5 1007.0 1007.5m/z

0

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926.0595z=25

890.4806z=26 964.5199

z=24

857.5000z=27

1006.4988z=23

1102.3079z=211052.2487

z=22

798.5352z=29

1157.3224z=20771.8840

z=30 1218.1823 45

50

55

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G0F G1F+45

+45

+45 G1F

+45

1135.0 1135.2 1135.4 1135.6 1135.8 1136.0 1136.2 1136.4 1136.6 1136.8 1137.0m/z

0

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Q Exactive Plus MS, 280K, protein modeThermo Scientific Q Exactive ™ Plus MS, 140K

0.7 ppm

800 900 1000 1100 1200 1300 1400m/z

0

10

20

30 z=30 1218.1823z=19

1285.9696z=18 1361.4380

z=17

m/z

G0F G1F

G2F

2.3 ppm 1.8 ppm

20.2 ppm

1126 1128 1130 1132 1134 1136 1138 1140 1142 1144 1146m/z

0

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35

40Rel

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G2F+45

System Configuration for Multiple Chemistries

UV

A B C

DGPRightDGPLeft

A B C

WPS

Dual gradient pump‘Two LPG pumps in a single unit’

(upgradeable with solvent selection valves)

Fraction collecting autosampler‘inject – collect – re-inject’

Column oven with column selection valvesup to 6 or 10 columns / positions Injection

collection

32

UV WPS collection Waste

SEC

IEC

Prot A

Wide Range of Bio Analytical UHPLC Characterization Methods

2 minutes 60 minute digest 0.00 0.13 0.25 0.38 0.50 0.63 0.75 0.88 1.00 1.13 1.25 1.37 1.50 1.62 1.75 1.87 2.00 2.12 2.25 2.37 2.50 2.62 2.75 2.87 3.00

-200

0

125

250

375

500

625

750

875

1,000

1,125

1,250

1,375

1,600 PHILOGEN #4 IgG UV_VIS_1mAU

min

WVL:280 nm

Flow: 1000 µ l/min

%B: 0.0 %

100.0

0.0

%C: 0.0 %

mAb Capture Peptide Mapping Glycan Analysis

33

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00-5.0

0.0

5.0

10.0

15.0

20.0

25.0 2 - MABPACRP_2PT1X50_BETA01 #35 HER-MMAE UV_VIS_1mAU

min

2

1

WVL:280 nm

Flow: 600 µ l/min

0.1% TFA + 9.9% H2O + 90% MeCN: 35.0 %

55.0

35.0

0.1% TFA + 70% IPA + 20% MeCN: 0.0 %

2 minutes 6 minutes4 minutes

ADC AnalysisCharged Variant AnalysisAggregate Analysis

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