Trends and Developments in MAbScreening and CharacterizationScreening and CharacterizationKen Cook

2014MAb Characterization

Pharmaceuticals and Biopharmaceuticals

pharmaceuticalsProduced by chemical synthesis200 - 2,000 daltons1 - 5 reactive groupsRelatively stable

MAb150,000 Da

Precisely defined chemical entities

biopharmaceuticalspGenetically engineeredProduced in living cells2,000 - 2,000,000 daltons10 2000 reactive groups

Biosimilar10 - 2000 reactive groupsModerately to highly labileComplex; a mixture of closely related variants

Aspirin180 Da

Because of their complexity, it is not possible to make identical copies of biologic drugs. These products are therefore referred to as “biosimilar” rather than generic drugs. Developers

Biopharmaceuticals present unique analytical challenges.We have unique analytical capabilities that address these challenges

g g pseek to achieve “similarity” and “comparability”.

We have unique analytical capabilities that address these challenges.

The “Complexity” Challenge in MAb Analytics

Steven Kozlowski, FDA, WCBP2010

Market Trends in Biopharma

• Greater productivity needed in method development

I i d l t i li• Increasing development pipeline for monoclonal antibody (MAb) therapeutics

• Advances in automation in upstream processes such as cell culture and purification process d l tdevelopment

Types of Biopharmaceuticals

Source: PhRMA 2013 Biologics Overview

MAbs are the fastest growing class of drugs

“more than half of biopharmaceuticals in development are antibodies”

by 2016, 6 of the top 10 drugs will be MAbs

“…more than 700 biosimilars/biobetters in the development pipeline…”

Requirements for Biopharma Method Development

• Easy method development• Fast in optimization

• Rapid and simple method• Short runtimesShort runtimes• Easy to set-up and to keep running

• Generic approach

• Instrument speed up options• Instrument speed up options

• Easy method transfer to QA/QCy

Regulatory Requirements

Protein Analytical Chemistry Techniques Used in the Testing of Biological Products

Protein Property Characterization Batch Release/Stability Further Development of Assay

Size / Aggregates Mass spec (intact mass), HPLC SDS-PAGE, SEC Impurity (aggregates, fragments)

Charge CE-IEF, IEC, pH-IEC CE-IEF, IEC, pH-IEC Acylation, deamidation, sialylationvariants

tid i h d h bi i t ti Hydrophobicity peptide mapping, hydrophobic interaction chromatography (HIC) Deamidation, oxidation, (U)HPLC

Concentration Amino acid analysis, HPLC method, ELISA UV A280

LC/MS fl t l b li h id HPAE PAD (IC)Carbohydrate analysis LC/MS, fluorescent labeling, monosaccharidecomposition

HPAE-PAD (IC)(U)HPLC Heterogeneity

2°, 3° Structure Circular dichroism, peptide mapping Disulphide mapping

Peptide Mapping LC/MS N C sequencingPeptide Mapping LC/MS, N- C- sequencing

AAA analysis (U)HPLC-FLD or (U)HPLC-CAD

Binding activity ELISA, Biacore ELISA, Biacore

P t C ll b d C ll b d t Potency Cell-based assays Cell-based potency assay

Identity Western blotting, peptide mapping, (U)HPLC Western blotting, peptide mapping,

Adapted from Camille Dycke et. al., GEN October 15, 2010Adapted from Camille Dycke et. al., GEN October 15, 2010

Topics

• Speeding Up HPLC MAb Characterization AnalysisLC Column Selectivity Developments in Column Chemistry for Mab• LC Column Selectivity – Developments in Column Chemistry for MabAnalysis

• Reducing Method Development Time• High Throughput & Automation Strategies

• Parallel LC Configurations and Multi-Step AutomationI t ti M S i t MAb A l i W kfl• Integrating Mass Spec into MAb Analysis Workflows

• 2D LC – MS Workflows• Current Trends and Developments in Glycan Analysisp y y

• Novel Column Chemistry for HPLC Glycan Analysis• Comparison of LC-based methods

Approaches to Faster LC Separations

• Faster separations can be achieved by…(A) Compressed gradients (e.g. in IEC)

• Can speed up the separation; usually some loss of resolution

(B) Shorter columns• Resolution compromised but often “good enough”

(C) Smaller particle size resinsS d th ti d ith t l f l ti• Speed up the separation, and without loss of resolution

(D) Combinations of the above( )

The Thermo ScientificBio RS System - What is New?

LPG-3400RS/HPG-3x00RS/DGP-3600RS- NEW biocompatible 1034 bar (15,000 psi) pump fluidics

WPS-3000TBRS- NEW biocompatible in-line split-loop (flow-through) 1034 bar (15,000 psi) autosampler

TCC-3000RS/SD- NEW biocompatible 1034 bar 2-pos, 6-port and 10-port, and 6-pos, 7-port valves

Viper Fingertight Fitting Systemp g g g y- NEW biocompatible 1250 bar (18,130 psi) capillaries

Added Bioanalytical Capabilities

• pH and Conductivity Monitoring• Used in protein purification and analysis• Highest accuracy through temperature compensation of

conductivity and pH results• Useful tool for pH gradient analysis in IEC

pH Difficulties With Phosphate Buffers and Blending

10.00

11.00 8 pH2 #17 0 pH

100.0

%C: 0.0 %

9.00 8

7

80.0

10% A

0% A

7.00

8.007

6

543 40% A

20% A

10% A

6.00

60% A

50% A

4.00

5.00

2

1 %B: 0 0 % 0 0

100% A

80% A

0.0 1.3 2.5 3.8 5.0 6.3 7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 21.3 22.5 23.8 25.0 26.3 28.03.00 min

1Flow: 150 µl/min

%B: 0.0 % 0.0

Calibration of Protein A Titre with new Protein A column

Faster Separations Without Loss of Resolution with Smaller Particle Size Resin

• Faster MAb charge variant analysis…by reducing column length, gradient time & particle sizegradient time & particle size

10 0

16.0

A 10 μm, 4x250 mm MAbPac SCX

5.0

10.0

2

0.0 10.0 20.0 30.0 40.0 50.0 58.0mAU

20 0

30.0

6

8

B 3 μm, 4x50 mm MAbPac SCX

10.0

20.0

3 4 5

8

11

910

7

Minutes0.0 5.0 10.0 15.0

-5.0

0.0

312 13 14

101

2 15 16

Minutes

HIC for MAb Analysis

ProPac HIC – Key Application

• Methionine oxidation monitoring

Column: Thermo Scientific™ ProPac ™Column: Thermo Scientific ProPacHIC-10, 4.6 x 100 mm

Eluent: A. 1M (NH4)2 SO4 in 0.1 M NaH2PO4,

80 Main MAb Peak

pH 7.0 B. 0.1 M NaH2PO4, pH 7.0

Flow Rate: 0.75 mL/minMet OxidationInj. Volume: 100 µL (50 µg) Detection: 220 nmSample: MAb

Peak

mA

U

5 10 15 20 25

0

Minutes0

Minutes

Characterization of Aspartic Acid Variants

Valliere-Douglas, et al. (2008) J Chrom. A 1214, 81-89

Speed up of Mab Aggregate Analysis - SEC

55.0

60.0

1 - Gel Filtration dionex 15cm #18 MAb + Caffeine UV_VIS_12 - GEL FILTRATION DIONEX #16 [normalized] Caffiene UV_VIS_1mAU

1 - 2.471

WVL:214 nm

40 0

45.0

50.0 30cm column15cm column Aggregation analysis in under 4

30.0

35.0

40.0

MAb

analysis in under 4 minutes

15 0

20.0

25.0

dimer

5.0

10.0

15.0

Caffeine

-10.0

-5.0

0.0

min

21

Less than 4 minutes!

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

New Multi-Product MethodpH Gradient IEC

Ion Exchange Protein Elution Mechanisms

IsoelectricPoint (pI)

+Buffer pH typically < pI Cation-Exchange

NH3R +

COO -

Cationic protein binds to

negatively charged cation exchanger

+ ++

++

0Buffer/System pH

Cation ExchangeChromatography

NH3R +

COOH+ ++

3 4 5 6 7 8 9 10

Buffer pH typically > pI Anion-Exchange

Chromatography

2

Anionic proteinbinds to

positively chargedanion exchanger

- -- -

-

–

Chromatography

NH2R

COO -- - -

Protein net charge vs. pH

Mechanism of Salt and pH Elution of Proteins

Improving pH Gradient Cation-exchange Chromatographyof mAbs by Controlling Ionic Strength

Journal of Chromatography A, 1272 (2013) 56– 64

Buffer Development Strategy

• Replace cationic buffer components with zwitterionic buffer species (Good’s Buffers)

• These buffer species contain one quaternary amine group and one sulfonic acid group. They do not bind to the stationary phase in the pH range of 6-10. p g

• They are not repelled by the stationary phase so they can buffer the stationary phase.

MES MOPS TAPS CAPSO6.1 7.2 8.4 9.6

Linear pH Gradient

Programmed gradient vs measured pH

Cytochrome Cy = 1.6923x - 7.2914R² = 0.9929

9.5

10

10.5

ue

Protein pI vs. measured pH at elution

y = 0.1548x + 5.0404R² = 0.9996

9 5

10.5

Programmed gradient vs. measured pH

Trypsinogen

Ribonuclease A

7 5

8

8.5

9

sure

d pH

val

u

8.5

9.5

ed p

H v

alue

L ti 1Lectin - 2

Lectin - 3

Trypsinogen

6

6.5

7

7.5

Mea

s

Measured pH value

Linear (Measured pH Value)

6.5

7.5

Mea

sure

Measured pHLinear (Measured pH)

Lectin - 15.5

7.5 8.5 9.5 10.5

pI value

60.0

55 3

5.50 10 20 30 40

Retention Time [min]

Linear (Measured pH)

30.0

40.0

50.0

nce

[mA

U]

tin-1

-5.

87 -

6.04

-6.2

018

-6.

37

Tryp

sino

gen

-15.

97 -

7.5

leas

eA

-22.

00 -

8.53

toch

rom

e C

-31

.55

-9.9

3

10.0

20.0A

bsor

ban

Lect

Lect

in-2

-6.

97

Lect

in-3

-8.

1

Rib

onuc

l

Cy

0 5 10 15 20 25 30 35 40-5.0

Retention Time [min]

Programmed Gradient and Actual Monitored pH

10.00

10.50 Novartis Method #3 Sample 1 pH

100.0

%C: 0.0 %

%D: 0.0 %

9.00

9.50

1 pH unit i 5 i

8.00

8.50

in 5 minElution points for the same protein 20 minutes apart with the same programed

7.50

sa e p og a edgradient!

6.50

7.00

Origonal Method6.00

Flow: 1.000 ml/min

%B: 0.0 % 0.0

gThermofisher Buffers

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 65.0 70.0 75.0 80.0 85.0 90.05.20 min

Example #2: Herceptin, 5mg/mL,MabPac SCX-10, 10µm 4x250 mm

15.0

30.0

5.0

10.0

0.0

i

%B: 10.0

Salt gradient

0.0 5.0 10.0 15.0 20.0 25.0 30.0 min

15.0

50.0

5 0

10.0

0.0

5.0

%B: 25.0

25.0pH gradient

30 min gradient, MabPac SCX-10, 10 µm, 4x250 mm

0.0 5.0 10.0 15.0 20.0 25.0 30.0 min

4-Protein Standards –Thermo Scientific CX-1 pH Gradient Buffer

1290

10.00

11.002

Lectin-1 - 6.15 - 6.11

609.00

(mA

U)

pH trace

20

40

7.00

8.00

Abs

orba

nce

Ribonuclease A - 22 38 - 8 72

Cytochrome C - 31.88 - 10.15

20

6.001

Lectin-2 - 7.25 - 6.28Lectin-3 - 8.45 - 6.45

Trypsinogen - 16.41 - 7.75

Ribonuclease A - 22.38 - 8.72

2

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0-10 5.00

Retention Time (min)

4-protein Standards – PO4 Based pH Gradient

1 - 20130626_MABPACSCX4x250_001664_agilentbuffer #3 Lectin+Trypsinogen+RNaseA+CytC UV_VIS_12 20130626 MABPACSCX4x250 001664 agilentbuffer #3 Lectin+Trypsinogen+RNaseA+CytC pH

10 µm, 4 x250 mm60.0

70.0

9.50

10.002 - 20130626_MABPACSCX4x250_001664_agilentbuffer #3 Lectin+Trypsinogen+RNaseA+CytC pHmAU

µ ,

40.0

50.0

8 00

8.50

9.00

pH trace

5 µm 4 x150 mm20.0

30.0

7 00

7.50

8.00pH trace

5 µm, 4 x150 mm

0.0

10.0

6.00

6.50

7.00

12

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0-10.0 5.50min

MAb Charge Variant Separation, 0–100% B

100% B0% B

40.0 10.50

30.09.00

mAU

]

pH trace(a)

20.0

7 00

8.00

bsor

banc

e [m

10.0

6.00

7.00Ab

0 5 10 15 20 25 30 35 40-5.0 5.00

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

MAb Charge Variant Separation, 25–50% B

25% B 50% B

16.0 8.00

10 0

7.75

mAU

]

(c) pH trace

5.0

10.0

7.25

7.50

bsor

banc

e [m

5.0

7.00

Ab

0 5 10 15 20 25 30 35 40-2.0 6.60

Retention Time [min]

Protein Loading with a Salt Gradient2,000 p _ _

mAU WVL:280 nm

1 600

1,800

mAU WVL:280 nm

80.0

%C: 0.0 %

MAbPac SCX 4 x 250mm

1,400

1,600

1,000

1,200

Peak Width

600

800

Resolution

200

400

1 2mg0 3

2

1Flow: 1000 µl/min

%B: 33.3 %

1.2mg

0.3mg

0.1mg

4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00-300 min

Protein Loading with a pH Gradient on the Same Column1,000 3 pH Buffer B test #18 Cap6 UV_VIS_1

900

1,000mAU WVL:280 nm

100.0

%C: 0.0 %

MAbPac SCX 4 x 250mm

700

800

500

600

Peak Width

400

500

Resolution

200

300

%B: 40.0 %

1003

2

1

1 - 12.328 2 - 21.105Flow: 1000 µl/min

25.0

1.2mg

0.3mg0.1mg

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 21.0 22.0 23.0 24.0 25.0-20 min

Effect of Column Length on pH Gradients

The resolution is surprisingly similar even when the column length is changed dramatically. The main difference is the elution time which can be attributed to thewhich can be attributed to the higher capacity of the long column.This is suggesting that the gg gprimary mechanism of separation is the pH gradient itself and the effect on the PI of the proteinof the protein.

Fast Runs-Protein Standards: 20 Min Run vs 10 Min Run

70.0 1 - 2013-10-01_MPSCX-10_5um_sn001050 #2 LTRC, 3:2:3:2, pH calibrated UV_VIS_1mAU WVL:280 nmAz

Flow rate at 1 mL/min, 15min gradient/ 20 min totally cycle time

40.0

20.0

1

-10.0

60.0 2 - 2013-10-01_MPSCX-10_5um_sn001050 #4 LTRC, 3:2:3:2, pH calibrated UV_VIS_1mAU WVL:280 nm

Flow rate at 2 mL/min, 7.5min gradient/ 10 min totally cycle time

12 5

25.0

37.5

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0-10.0

12.5

min

2

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0

Herceptin, 0-100% B140 11 00_ _ _ p , g p

120

140

10.00

11.00mAU

100

9.00

10.00

60

80

8.00

9.00

407.00

20

6.00

-20

0

5.00min

12

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Herceptin, 25-50%B40 0 8 20AU

35.0

40.0

8.00

8.20mAU

25 0

30.07.80

20.0

25.0

7 40

7.60

10 0

15.0

7.20

7.40

5.0

10.0

7.00

-5 0

0.0

6 60

6.80

min

12

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0-5.0 6.60

Different mAb2 Using Fast pH Gradient

138

160 PH gradient_Oct2013 #32 mAb UV_VIS_1mAU

1 - 4.014

WVL:280 nm

100.0

%C: 0.0 %

As fast as CE Analysis!

113

125

As fast as CE Analysis!

75

88

100

50

63

55.0

13

25

382 - 4.268

0

13

Flow: 450 µl/min

%B: 32.0 % 32.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00-20 min

0 to 100% Start for a 10 Minute pH GradientmAU WVL:280 nm

500 9

9 different Mab samples

375 8

125

250 7

6

0 5

4

-250

-125 4

3

500

-375

min

2

1

0.0 0.5 1.0 1.5 2.0 2.5 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 10.0-500 min

Summary

• pH Gradient IEC is perfectly suited as a platform method• Allowing generic methods for multi-product analysis• Even with pI ranges from 5 – 10• Simple and fast method development

• pI value of the unknown MAb can be predicted from the correlation curvepI value of the unknown MAb can be predicted from the correlation curve.

• Easy optimization of the method• Less dependant on sample matrix and sample preparation• High loading capacity for low level analysis as well as

variant fractionationvariant fractionation• Fast high resolution methods using short columns• High capacity methods for fractionation using longer columns• Robust

High-Throughput and Automation Strategies

• Tandem and Parallel LC Configurations• To increase sample throughput of validated methods• To increase sample throughput of validated methods

• Multi-step Automation• To automated multi-step workflow e.g. MAb purification and analysis on a

single LC platform• Reduce hands-on timeReduce hands on time

• Case Studies• Fast MAb Aggregate Analysis• Automated MAb Titer Threshold Method

Parallel LC for Dual Assays Aggregate and Variants

Both AnalysisBoth Analysis with one injection in 10 Minutes!

IEC SEC

Minutes!

IEC SEC

Increases throughput, eliminates the need to duplicate sample plates

System Configuration

A B C

DGPDGP

A B CDual gradient pump‘Two LPG pumps in a single unit’

(upgradeable with solvent selection valves)DGPRightDGPLeft

Fraction collecting autosamplerColumn oven with

UV WPS

g p‘inject – collect – re-inject’column selection valves

up to 6 or 10 columns / positions Injection

collectionUV WPS collection Waste

Prot A

SEC

Prot A

IEC

Typical mAb Workflow

A B C

DGPDGP

A B CSample loading onto protein ADGPRightDGPLeft

UV

Injectioncollection

WasteWPSUV WPS

Prot A

LOAD + WASH

SEC

Prot A

IEC

Typical mAb Workflow

A B C

DGPDGP

A B CElution and fractionationDGPRightDGPLeft

UV

Injectioncollection

WasteWPSUV WPS

Prot A

ELUTE + FRACTIONATE

SEC

Prot A

IEC

Typical mAb Workflow

A B C

DGPDGP

A B CSecond dimension SEC analysis

ion

s on

s DGPRightDGPLeft

80

100

125 mAU

UV 214nmUV 280nm

Agg

lom

erat

prod

ucts

PI

Deg

rada

tiopr

oduc

ts

UV

Injectioncollection

WasteWPS5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0

-10

20

40

60

min

21

AP

UV WPS

Prot A

SEC

Prot A

IEC

Typical mAb Workflow

A B C

DGPDGP

A B CSecond dimension IEC analysisDGPRightDGPLeft

20.0

30.0

mAU

K

K K

UV

Injectioncollection

WasteWPS0.0 20.0 40.0 60.0 80.0

0.0

10.0

min

Acidic Variants

Basic Variants

UV WPS

Prot A

SEC

Prot A

IEC

Key Columns for Biopharma AnalyticsAnalysis Description ColumnsMAb Capture & Titer Analysis

Mab capture for analysis workflows; Mab titer determination (concentration) & screening

MAbPac Protein A “The gold standard in antibody analysis”

Charge Variant Analysis

routine charge variant profiling/screening; including lysine truncation, acylation & deamidation; done by CEX & AEX

ProPac WCX-10 MAbPac SCX-10 MAbPac SCX-10RS CX-1 pH Gradient Buffer Kit ProPac SAX-10 Pro-Pac WAX-10 Robust, multi-product , high

resolution pH gradient IEC

superior resolution for most MAb samples tested

Aggregate Analysis

routine screening for Mab aggregates and fragments

MAbPac SEC-1

Glycan Profiling profiling of released glycans Accucore Amide-HILIC

resolution pH gradient IEC

Novel GlycanPac column chemistry separates glycans by y g p g g y

GlycanPac AXH-1 GlycanPac AXR-1

Intact Protein & Subunit Profiling

ADC DAR analysis; glycoform profiling; LC/HC and Fab/Fc analysis; disulfide

ProSwift RP-10R ProSwift RP-2H&4H

size, polarity and charge. Mass Spec compatible.

ProSwift RP-10R monolithicSubunit Profiling LC/HC and Fab/Fc analysis; disulfide mapping

ProSwift RP 2H&4H Accucore 150-C4 MAbPac SEC-1

Sequence & Structural Analysis

primary sequence analysis; peptide mapping; peptide & glycopeptide structural & linkage analysis

Acclaim PepMap PepSwift (PS-DVB) Acclaim RSLC 120, C18

column provides highest resolution and lowest carryover for intact MAb mass analysis.

Analysis g y ,Accucore 150-C18

Trp Oxidation & Deamidation; ADC analysis

targeted analysis of tryptophan oxidation & deamidation

ProPac HIC-10 MAbPac HIC-10

ProPac HIC – novel chemistry for Trp oxidation; orthogonal to IEC and SEC for variant analysis.

Integrating MS into mAb Analysis Workflows

Seamless Integration of Salt-Based SEC, IEC, HIC Methods to MS for Characterization of MAb Products and Impurities

S l A l i

Exact Mass Determination, Bottom-up,and Top-Down Protein CharacterizationAutomated Bio LC-LC/MS

1-D LCProA, SEC, IEC or HIC Data Analysis

Deconvolution of ESI-MS

Sample AnalysisUsing HR/AM Mass Spectrometers

Fraction Collection of MAb Products or Impurities

to zero charge accurate mass

80

90

1002997.31777

2920.44812

2664.49830Products or Impurities[Automated in Autosampler]

z=?

2000 2500 3000 3500m/z

0

10

20

30

40

50

60

70

Rel

ativ

e A

bund

ance

2178.40685

3535.704273619.47065

Automated 2-D LCSPE/Desalting on RP

followed by MS 1311.0 1311.5 1312.0 1312.50

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

z ?

1311.87212R=70792

z=?1311.54737

R=68130z=?

1311.98174R=69867

z=?

1311.43967R=58977

z=? 1312.09147R=67084

z=?

1312.20087R=56981

z=?1311.31799

R=88597z=?

1310.98824R=45346

z=?

1312.42258R=47666

z=?

1312.64646R=43558

z=?

23565 23570 23575 23580 23585 235900

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

23578.58636

23580.66451

followed by MS m/z m/z

System Configuration

A B C

DGPDGP

A B CDual gradient pump‘Two LPG pumps in a single unit’

(upgradeable with solvent selection valves)DGPRightDGPLeft

Fraction collecting autosamplerColumn oven with

UV WPS

g p‘inject – collect – re-inject’column selection valves

up to 6 or 10 columns / positions Injection

collectionUV WPS collection Waste

RP

SEC

RP

IEC

MAb IEX Fraction Desalting using Monolithic Columns with Consecutive Blanks

120

1 - RP MAB #17 MAb UV_VIS_12 - RP MAB #19 blank UV_VIS_13 - RP MAB #20 blank UV_VIS_1mAU WVL:280 nm

%C: 0.0 %

90

100

110 90.0

%C: 0.0 %

60

70

80

30

40

50

1 - 5.093

10

20

30

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 6.50 7.00-20

-10

0

min

321

Flow : 250 µl/min

%B: 10.0 % 10.0

Accurate MW Determination of Reduced IgG Light Chain

80

85

90

95

100

1303.0917z=18

1465.9155z=16

IgG light chain18+ charge state

45

50

55

60

65

70

75

ve A

bund

ance

1234.6137z=19 1563.5760

z=15

1675.1174z=14

240,000 resolution

10

15

20

25

30

35

40

45

Rel

ativ

1172.8326z=20

1803.8945z=13

1954.1323z=12 2131.7785

z=111117.1264

z=21

240,000 resolution

1200 1400 1600 1800 2000 2200 2400m/z

0

5

10

1302.6 1303.0 1303.4 1303.8m/z

Xtractd l tideconvolution

Measured mass = 23424.4845Target mass = 23428.416g

4 Dalton Mass Deviation 2 S-S?How do we confirm this?How do we confirm this?

Shiaw-Lin Wu, Barry Karger, Barnett Institute, Northeastern University

pH Gradient Separation of Purified IgG on a MAbPac SCX-10 Column

Deconvoluted Results from MS Spectra

mAb Peptide Map – Normal / Stressed Sample

350

200

mAb normal

0

100

21

-100 mAb Stressed

-200

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0-350 min

mAb digest normal and stressed, 5 mg/mL, 30 min gradientAcclaim C18 2.2µm 2.1x250mm SN 1041

Asparagine to IsoAspartic Acid Detection

N+1 shift

Current Trends & New Developments in Glycan Analysis

LC-MS Analysis of Labeled Glycan: HILIC Amide Column

100 19

20

Commercial amide HILIC column (1.7 µm)

ce

15

18

14 &1716 15

e A

bund

anc

14

11a &c

12b, 13 &16

12b

13

Rel

ativ

6,7 & 10

1521 & 22

23

11a &c

1010

1 2 45

9

12a & 13 25

247

266 6 or 7

0 10 20 30 40 50 60Minutes

01 2 9

Conventional HILIC columns do not separate by charge; glycans co eluteConventional HILIC columns do not separate by charge; glycans co-elute

LC-MS Analysis of Labeled Glycan: GlycanPac AXH-1

10014

nce

12 b 20

12a

12b

ve A

bund

an

11a-c

12a-b

1519

Rel

ativ

513

01 2 3

4

5

6

78

910

18 21

2223

24

25 2616

17

P k d i t l “ l t ” ith th h

0 10 20 30 40Minutes0

Mono- Di- Tri - Tetra-Neutral

Peaks grouped into several “clusters” with the same charge

16E6

Charge-based Separation for Easy Quantitative Analysis16E6

34

P k Gl TRelative

Peak Glycan Type %

1 Neutral 0.4

2 Mono-Sialic 8 6

ce C

ount

s

2 Mono Sialic 8.6

3 Di-Sialic 38.4

4 Tri-Sialic 45.4

25

Fluo

resc

enc

5 Tetra-Sialic 7.0

6 Penta-Sialic 0.2

1

5

6

7.00 8.00 9.00 10.00 11.00 12.00MinutesQuantitative Determination of each glycan charge state

Separation of 2AA Labeled N-glycans from IgG by GlycanPac AXH-1 (1.9 µm) Column: pH 5.1 in 25 oC

Column: Thermo Scientific ™ GlycanPac™

AXH-1 (1.9 µm)

Dimension: 2.1x150 mm

1.8E6

3Mobile phase: A: acetonitrile

B: water

C: ammonium Acetate (100 mM, pH =5.1)

Flow: 0.4 mL/min

3

8

Flow: 0.4 mL/min

Temp: 30 oC

Injection: 5 pmoles

Detection: fluorescence detector

Sample: 2AB Labeled N glycan from IgG

13

ence

Cou

nts

Sample: 2AB Labeled N-glycan from IgGTime (min) % A % B C%

Flow Rate(mL/min)

-10 81 18 1 0.4

9

17

Fluo

resc

e

0 81 18 1 0.4

25 74 18 8 0.4

35 62 18 20 0.4

0

1 2 4

5

67

10

11

1214 15

16

10.0 20.0 30.0

0

Minutes

Separation of N-glycan by Thermo Scientific™ Acclaim® Glycan AXR Column 2.1x150mm, 1.9 um

700,000counts

Time (min)

% A %B %D Flow (mL/min)

0 0 5 95 0.4

20 4 18 78 0 4

Eluent: A: Acetonitrile B: Ammonium formate (0.1M, pH = 4.4) D; water

0.4 mL/min

20 4 18 78 0.4

24 0.7 30 69.3 0.4

44 6 30 64 0.4

60 15 30 55 0.4

44 numbers of peaksPeaks width is better than

3 l3um column

-100 000

-50,000

0

min

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 52.0 54.0 56.0 58.0 60.0-100,000

Comparison of the Three New Glycan Columns

Glycan HILIC WAX 1.9um

Glycan WAX – RP 1.9um

GlycanPac AXH-1, AXR-1

• High resolution columns for separation and structural characterization of biologically relevant glycansof biologically relevant glycans

• UHPLC column suitable for high-throughput analysis

• UHPLC-FLD for fluorescently labeled N-glycans

• LC-MS and LC-MS/MS for structural characterization of both labeled and native N- and O-glycans from proteins by MS detection

Trend Toward HR/AM MS for Intact IgG Mass Measurement e.g. Glycoform Analysis

80859095

100 2745.7720

2851.3544

75

80

85

90

95

100 2745.7720

2797.56972695.8919

R: 17.5K

4045505560657075

elat

ive

Abu

ndan

ce

2907.25952556.4844

2965.37642471.3405

3025 86322391 628825

30

35

40

45

50

55

60

65

70

Rel

ativ

e A

bund

ance

510152025303540R

e 3025.86322391.6288

2680 2700 2720 2740 2760 2780 2800 2820m/z

0

5

10

15

20

25

1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

-7 ppmG0F+G1F

-0 7 ppm 8 5G0F+G0F G0F+G2F (or 2G1F)

0.7 ppm -8.5 ppm

-0.9 ppm

Protein Deconvolution 1.0

G1F+G2FG2F+G2FG0+G0F

G0+G02xMan5

5.0 ppm0.9 ppm

Q ExactiveO bit MS

G1F+G2F+SAIn-depth characterization, comparability studies e.g. process change; originator vs. biosimilars comparability

Orbitrap MS

Summary• Unique construction of ProPac ion-exchange phases enables high resolution separations

of protein isoforms and other closely-related protein variants• Protein A column for rapid capture and Titre of IgG• The ProPac HIC has improved hydrolytic stability compared to other silica-based HIC

columns with better resolution than polymer-based HIC columns• The ProPac SEC column enables high performance protein separations by size for

aggregate anal sis in less than 4 min tesaggregate analysis in less than 4 minutes• Dual analysis can be carried out with short runs using different chemistries• ProSwift monolithic RP columns useful for fast high resolution separations of large

proteins with ultra low carryoverproteins with ultra low carryover.• GlycanPac columns for unique separation and resolution of Glycans• Bio-Compatible inert Viper connections for ultra low dispersion

The U3000 BioRS system allow biocompatible Mab UHPLC analysis and automated 2• The U3000 BioRS system allow biocompatible Mab UHPLC analysis and automated 2 dimensional capture and analysis steps. Doing the work of multiple instruments in one.

Thank You—Q&A

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