for the and characterization of protein biopharmaceuticals · strategies for the separation and...

Strategies for the Separation and Characterization of Protein Biopharmaceuticals

Koen SandraWebinar in association with SelectScience and Agilent TechnologiesJanuary 28, 2015

Protein biopharmaceuticals

• Therapeutic macromolecules produced via recombinant DNA technology

• Used in the treatment of life threatening diseases such as cancer, autoimmune diseases, etc.

• Global protein therapeutics market: $100 billion (monoclonal antibodies + other recombinant proteins)

• ±20% of the total pharmaceutical market• Within the current decade, more than 50% of new drug approvals will be biologics

2Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

Protein biopharmaceuticals

• Blockbuster protein biopharmaceuticals:– Insulin: Lantus (Sanofi‐Aventis)

– EPO: Epogen/Aranesp (Amgen)

– Trastuzumab: Herceptin (Roche/Genentech)– Infliximab: Remicade (J&J)

– Adalimumab: Humira (AbbVie)

– Etanercept: Enbrel (Pfizer, Amgen)

– …

• Several of these blockbusters are, or will soon become, open to the market

• This has resulted in an explosion of biosimilar activities


Protein characterization

• Whether being involved in the development of innovator biopharmaceuticals or biosimilars, an in‐depth characterization and analysis of these molecules is required during their development and lifetime

• Analysis is typically more challenging compared to small molecule drugs

• Proteins are large and heterogeneous


• Typical characteristics– Amino acid sequence– Amino acid composition– Structural integrity – Higher order structures– Aggregation– S‐S bridges– N‐ and O‐glycosylation– N‐ and C‐terminal sequence– Charge variants– Deamidation/isomerization– Oxidation– Clipping– …



--NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEW--

C-ter C-ter

N-terN-ter

Hc

Lc

• Typical characteristics– Amino acid sequence– Amino acid composition– Structural integrity – Higher order structures– Aggregation– S‐S bridges– N‐ and O‐glycosylation– N‐ and C‐terminal sequence– Charge variants– Deamidation/isomerization– Oxidation– Clipping– …



--NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK


C-ter C-ter

N-terN-ter

Hc

Lc

• Characteristics determined at different levels



Protein

Peptides Sugars

Amino acid composition Amino acid sequence, modifications, modification sites, disulfide bridges, etc.

N‐glycans

Trypsindigestion

PNGase FAcid hydrolysis

Amino acids

MW, structural integrity, charge variants, aggregation, modifications

A wide range of separation modes

Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar 8

Charge CEX, AEXSize SECHydro(phob/phil)icity RPLC, HIC, HILICAffinity Affinity Chromatography

Reversed‐phase U/HPLC


Proteins

• Challenges encountered in RPLC of proteins



Issue Reason Solution

Peak tailing • Secondary ionicinteractions

• High number of pos charges on proteins

• Stationary phasewith limited access to residual silanols

• Ion‐pairing reagent• Higher temperature

Peak broadening • Low Dm of large molecules

• Limited access topores

• Widepore phases• Higher temperature• Efficient stationary

phase (sub 2 µm, superficially porous)

Adsorption • Hydrophobicity • Less hydrophobicstationary phases

• Stronger solvent• High temperatures

Courtesy of D. Guillarme



LcHc

Fc

Fab

Intact

HcLc

Fab

Fc

10 cm x 2.1 mm x 1.8 µm Zorbax 300 SB-C8Temp: 80°C Flow: 200 µL/min UV: 214 nmSolv A: 0.1% TFASolv B: 0.1% TFA in ACN 30-38.6%B, 2-25 min

RPLC analysis for identity and purity determination of Herceptin

Reversed‐phase U/HPLC – Mass Spectrometry


RPLC-UV-MS of Herceptin Lc and Hc Deconvoluted spectra

Reversed‐phase U/HPLC for comparability assessment


min8 10 12 14 16 18 20

mAU

0

20

40

60

80

100

120

min8 10 12 14 16 18 20

mAU

0

50

100

150

200

250

300

Biosimilar

Originator

Lc Hc

e

f

ab

c

d

g



G0F + C‐terminal Lys

Undergalactosylation observed in biosimilar

3x10

0

0.2

0.4

0.6

0.8

1

2x10

0

2

4

6

Response Units vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9

Widepore Poroshell for comparability assessment


Remicade

Remicade biosimilar

Advance Bio RP-mAb5 cm x 4.6 mm x 3.5 µm 450Å C4Temp: 80°C Flow: 1 mL/min UV: 214 nmSolv A: 0.1% TFA Solv B: 0.1% TFA in 90% ACN30-42.5%B, 0-6 min

1x10

00.5

11.5

22.5

33.5

44.5

1x10

00.5

11.5

22.5

33.5

44.5

Response Units vs. Acquisition Time (min)2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6

Remicade

Remicade biosimilar

3x10

0

0.2

0.4

0.6

0.8

1

2x10

0

2

4

6

Response Units vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9



Differences in hydrophobicity due to a 2-point mutation in the AA sequence of the biosimilar


Remicade

Remicade biosimilar

3x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Response Units vs. Acquisition Time (min)

4 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 5.2 5.3 5.4

Remicade

Remicade biosimilar

Widepore Poroshell – zero carry‐over


1x10

00.20.40.60.8

11.21.41.61.8

22.22.42.62.8

33.23.43.63.8

44.2

Response Units vs. Acquisition Time (min)3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6


Remicade(4 µg o.c.)

Blank

Remicade is a mAb which is prone to carry-over on a substantial number of RPLC columns → No carry-over observed on Widepore Poroshell column



Differences in hydrophobicity due to a 2-point mutation in the AA sequence of the biosimilarcompared


RemicadeRemicade biosimilar Lc

Hc

Ion exchange chromatography


Proteins



WCX analysis (n=5) of Herceptin highlighting charge variants

• Electrostatic interaction between charged side chains and opposite charged ion exchange functionalities• Elution: increase salt concentration or less common pH

pI high(BASIC)

pI low(ACIDIC)

min5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

600

700

800

Replicate 1Replicate 2Replicate 3Replicate 4Replicate 5

Asparagine deamidation

25 cm x 2.1 mm x 5 µm Agilent Bio-mAbTemp: 30°C Flow: 200 µL/min UV: 214 nmMPA: 10 mM phos pH 7.65MPB: 10 mM phos pH 7.65, 100 mM NaCl5-70%B, 0-36 min

+H3N

+Lys

S

A

G

F

Y

P

_

_

_

_

_

+Na

+Na

+Na

min10 15 20 25 30

mAU

0100200300400500600

min10 15 20 25 30

mAU

0100200300400500600

min10 15 20 25 30

mAU

0100200300400500600



WCX analysis of stressed and non-stressed Herceptin

3 days pH 9 stressed originator

Non-stressed originator

1 day pH 9 stressed originator

mAb

mAb with one Lc deamidated

mAb with one Lc deamidated

mAb with both Lc deamidated

mAb with both Lc deamidated

mAb

mAb


min10 15 20 25 30

mAU

0

100

200

300

400

500

600

Ion exchange chromatographyCEX profile of 1 day pH stressed mAb

2

Reduced CEX peak 1: RPLC profile

Reduced CEX peak 2: RPLC profile

LcHc

1

CEX fraction collection, reduction using DTT and transfer to RPLC method

N→D

Size exclusion chromatography


Proteins


Size exclusion chromatographysu

ppor

t

• No interaction with surface• Separation by means of pores having different accessibility for molecules of different size• Elution with solvents that suppress interactions with column packing

SEC analysis of Herceptin highlighting aggregation

mAbmonomer

mAbdimer

Buffer related compound

min0 2 4 6 8 10 12 14 16

mAU

0

10

20

30

40

50

30 cm x 4.6 mm x 3 µm Agilent Bio SEC-3Temp: 24°C Flow: 350 µL/min UV: 214 nmMobile phase: 150 mM phosphate

0.4%

99.6%


SEC for comparability assessment

min0 2 4 6 8 10 12 14 16

mAU

0

200

400

600

800

min0 2 4 6 8 10 12 14 16

mAU

0

200

400

600

800

mAbmonomer

mAbdimer


Originator

Biosimilar


SEC for comparability assessmentmAb

monomer

mAbdimer


min0 2 4 6 8 10 12 14 16

mAU

0

10

20

30

40

50

min0 2 4 6 8 10 12 14 16

mAU

0

10

20

30

40

50

Originator

Biosimilar



Peptides

The power of peptide mapping

• Protein measurement is extremely powerful but does not provide the complete picture nor does it allow to localize modifications

• Which amino acid is glycosylated, oxidized, deamidated, etc.?

• This can be assessed at peptide level following proteolytic digestion with e.g. trypsin

• Peptide measurement is also more powerful towards identity/sequence confirmation


Tryptic peptides Herceptin

Ligh

t Cha

in

B(1

-214

)H

eavy

Cha

in

A(1

-449

)


Hc

Lc


--NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

• 62 identity peptides• Modifications• Incomplete and aspecific cleavages• ...

> 100 peptides

Reversed‐phase U/HPLC: Peptide map


Detailed reversed-phase HPLC peptide map for Herceptin

identity and purity assessment

25 cm x 2.1 mm x 2.7 µm AdvanceBio C18Temp: 60°C Flow: 300 µL/min UV: 214 nmSolv A: 0.05% TFASolv B: 0.05% TFA in ACN 1-45%B, 2-35 min5 µL (2.4 µg)F3C C

O

O-

F3C CO

O-

+H3N

+Lys

S

A

G

F

Y

P

silic

a

C18

• Solvophobic interaction between non‐polar side chains and non‐polar surface• Electrostatic interaction between charged side chains and adsorbed TFA• Elution: increase concentration of organic solvent


T27

T32

T18

T46

T2

T4T12T20T36T39T48T49

T13

T38T8

T14

T10

T58

T44

T51

T40

T34T23

T62

T11

T19

T24

T30

T56T59 T2

2-23

T22

T5T7

T37-38

T26

T53

T33T25

T57

T29

T47

T21

T45

+ gl

ycan

s

T55

T35

T41

T15T1

T3

T50

T31

T16

T21 pyroGlu

T26

deam

T3 d

eam

T43’

T43

T42

T61

T3’T54T45

T6

Large undigested material*

*

**

T41

ox

* Sample preparation related peaks


LC‐MS based peptide mapping


Compounds extracted out of dataset

• Matching of peptides on sequence (MassHunter BioConfirm)

– Experimental workflow

– In‐silico workflow



protein peptides Experimental MW

Theoretical MWpeptides

Digestion

In-silicodigestion (user defined)

Peptide ID

protein

Mass spectrometry

In-silicomodifications (user defined)



Compounds matched onto protein sequence

Compounds extracted out of dataset



• Sequence coverage: 98.8% (655 out of 663 amino acids covered)

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Hc

(A-c

hain

)Lc

(B-c

hain

)


T45unglycosylated

Originator

T45 glycosylated 99,02 %

T45 unglycosylated 0,98 %

T38

T51T24

T47

T55

T16T6

T37-38

T27

T8

T19

T59

T2

T45 + glycans



T45 + G0

T45 + G0F

T45 + G1Fa

T45 + G2F

T45 + G1Fb

EEQYNSTYR

• T45 glycopeptide separation

C‐ter C‐ter

N‐terN‐ter

Hc

Lc

C‐ter C‐ter



T56

T33

T21

T43

T42

T61

T15

T34T23

T25

T3

T3 d

eam

T3’

T26T31

T26

deam

T26

deam

T41T4

1 ox

T18

T13

T14

T62

T22T29

T1T50

T54

T30

T62

+ K

C‐ter C‐ter

N‐terN‐ter

Hc

Lc

C‐ter C‐terM→Mox

N→D

+ K

N→D


Comparability assessment


Originator

Biosimilar



Originator

Biosimilar

T45 + G0

T45 + G0F

T45 + G1Fa

T45 + G2FT45 + G1Fb

T45 + G0F

T45 + G0T45 + G1F




Originator

Biosimilar

ASQDVNTAVAWYQQKPGK Originator Biosimilar

T3 native 91,75 % 86,92 %

T3 deamidation 8,25 % 13,08 %

ASQDVDTAVAWYQQKPGK

ASQDVNTAVAWYQQKPGK

ASQDVDTAVAWYQQKPGK

• Identification of modification sites

The power of mass spectrometry


Native

Deamidation



Originator

Biosimilar

T62

T62

T62+K--NHYTQKSLSLSPGK

• RPLC x RPLC peptide map of Herceptin

Comprehensive 2D‐LC (LC x LC)


Analytical and Bioanalytical Chemistry, issue 1, 2015

• RPLC x RPLC for comparability assessment


Strategies for the Separation and Characterization of Protein Biopharmaceuticals - Webinar

Originator 1 Originator 2 Clone (biosimilar)

4646

• RPLC x RPLC for comparability assessment



Originator 2

47

T62+K

T62T62

T62+K

MS Originator MS Biosimilar

Hydrophilic interaction chromatography


Glycans

N‐glycan analysis workflow


2-AB

2-AB

Glycan profile

LC-FLD (MS)2-AB labelingPNGase F

Clean-up

Clean-up2-Aminobenzamide (2-AB)

HILIC: 2‐AB labeled N‐glycans


• Hydrophilic partitioning between aqueous layer and mobile phase• Elution: increase water concentration

silic

a

Glycan

Glycan

AdvanceBio Glycan Mapping column15 cm x 2.1 mm x 2.7 or 1.8 µm Temp: 55°C Flow: 400 µL/min Fluorescence detectionSolv A: 100 mM NH4-formate pH 4.5Solv B: ACN 80-60%B, 0-38 min

HILIC: 2‐AB labeled N‐glycans


min8 10 12 14 16 18 20 22 24

LU

-0.5

0

0.5

1

1.5

2

2.5

3

min8 10 12 14 16 18 20 22 24

LU

-0.5

0

0.5

1

1.5

2

2.5

3

1.8 µm fully porous particles

2.7 µm superficiallyporous particles

G1Fa

G1Fb

G2F

G0F

G0

G0-

Glc

NA

c

G0F

-Glc

NA

c

G1-

Glc

NA

cMan5

G1aG1F-GlcNAc

G1b

G0

G0-

Glc

NA

c

G0F

-Glc

NA

c

G1-

Glc

NA

c

G0F

Man5G1a

G1F-GlcNAc

G1Fa

G1Fb

G2F

The power of mass spectrometry


AB

AB

AB

ABAB

ABAB

AB

AB

AB

AB

AB

AB

AB

AB

• MS/MS spectrum of 2AB‐labeled G0F

min10 15 20 25 30

LU

0

2.5

5

7.5

10

12.5

15

17.5

min10 15 20 25 30

LU

02468

10121416



Originator

Biosimilar


G0

G0F

G1Fa

G1FbG2F


min10 15 20 25 30

LU

0

2

4

6

8

min10 15 20 25 30

LU

02468

10

min10 15 20 25 30

LU

02.5

57.510

12.515

min10 15 20 25 30

LU

02468

1012

min10 15 20 25 30

LU

02468

1012

54

Biosimilar

Biosimilar ‐ Cell culture optimization• Bringing glycosylation within originator specifications by adding

uridine, galactose and manganese to the CHO growth medium

G0

G0F

G1F G2F

Biosimilar: 4x

Biosimilar: 24x

Biosimilar: 8x

Biosimilar: 16x


Biosimilar ‐ Cell culture optimization

1.8 µm fully porous HILIC particles

GlycanRelative intensity

Biosimilar Biosimilar4x

Biosimilar8x

Biosimilar16x

Biosimilar24x

Specificationsoriginators

% G0‐GlcNAC 0.55 0.49 0.47 0.53 0.61 0.77 +/‐ 0.09

% G0F‐GlcNAc 5.35 2.72 2.15 1.78 1.67 1.88 +/‐ 0.87

% G0 1.57 2.67 1.98 2.35 2.02 4.35 +/‐ 0.29

% G1‐GlcNAc 0.39 0.32 0.35 0.45 0.51 1.07 +/‐ 0.34

% G0F 70.80 48.91 46.39 44.19 43.72 44.39 +/‐6.47

% Man5 8.22 5.79 4.95 6.21 7.14 1.89 +/‐ 0.35

% G1a + % G1F‐GlcNAc 0.55 1.35 1.34 1.51 1.44 2.61 +/‐ 0.30

% G1b 0.17 0.32 0.29 0.37 0.38 0.69 +/‐ 0.20

% G1Fa 7.76 22.66 25.58 25.83 25.91 27.30 +/‐ 3.48

% G1Fb 3.62 8.11 8.70 8.75 8.62 9.13 +/‐ 0.89

% G1F 11.38 30.77 34.28 34.57 34.53 36.44 +/‐ 4.38

% G2F 1.01 6.66 7.80 8.03 7.98 5.92 +/‐ 2.47

• Bringing glycosylation within originator specifications by adding uridine, galactose and manganese to the CHO growth medium

Acknowledgement


• Isabel Vandenheede, Emmie Dumont and Pat Sandra (RIC, Kortrijk, Belgium)

• The colleagues from the biopharmaceutical industry who trigger and inspire us in developing and applying chromatographic and mass spectrometric methodologies and strategies to tackle their challenging requests

• Maureen Joseph, Gina Goggings, Linda Lloyd, Phu Duong (Agilent Technologies, Wilmington, Delaware)

www.richrom.com

[email protected]

Strategies for the Separation and Characterization of Protein Biopharmaceuticals

Improved LC Column Choices for Bioseparations

January 28, 2015

February 12, 2015

Confidentiality Label

1

Strategies of Column Selection for Separation and Characterization of Protein Biopharmaceuticals

February 12, 2015


2

Issue Challenge Reason SolutionPeak tailing

• Lowerresolution

• Less accuracy

• Secondary ionicinteractions

• High number of pos charges on proteins

• Stationary phase with limitedaccess to residual silanols

• Ion-pairing reagent• Higher temperature

Peak broadening

• Lower resolution

• Reduced sensitivity

• Low Dm of large molecules

• Limited access topores

• Widepore phases• Higher temperature• Efficient stationary phase

(sub 2 µm, superficially porous)

Adsorption • Poor recovery• Less sensitivity

• Hydrophobicity • Less hydrophobic stationaryphases

• Stronger solvent• High temperatures

Column parameters are important to solve problems of efficiency/resolution and recovery for improved LC and LC/MS characterization of proteins.

Reversed-phase LC Issues and Solutions

Reversed Phase Column Choices Improve Efficiency/Resolution and RecoveryAdvanceBio RP-mABThe optimum high speed, large molecule resolution for use with both HPLC and UHPLC systems• Superficially porous, 3.5um particle

The most popular phases for proteins, plus a unique selectivity.• C4, C8, Diphenyl

ZORBAX RRHD 300Å, 1.8umFast, high resolution UHPLC analysis of proteins, including intact mAbs, and protein fragments• Totally porous 1.8um silica particles with

300Å pores• 1200 bar for UHPLC use• Suitable for intact, fragments and digests

The most popular phases for proteins plus a unique selectivity.• SB-C3, SB-C8, SB-C18, Diphenyl• StableBond bonding for long lifetime with

TFA ion-pairing reagent

February 12, 2015


3

3.5 um3.0 um

0.25 um

450Å pores

Superficially porous particle

Fast Intact mAb Analysis and Comparison of PhasesShort 3 minute separation, with each phase unique.

Method Parameters Column dimensions: 2.1 x 100 mmMobile phase A: 0.1% TFA in water/IPA (98/2)Mobile phase B: IPA/acetonitrile/MPA (70/20/10)Flow rate: 1.0 mL/min

Gradient: 10-58% B in 4 min, 1 min wash at 95% B, 1 min re-equilibration at 10% BSample: 5 μL injection of Humanized Recombinant Herceptin Variant IgG1 Intact from Creative Biolabs (1 mg/mL) Temperature: 80 °CDetection: UV @ 254nm

February 12, 2015

4

min1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

mAU

0

20

40

60

80

100

120

140

DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D)

min1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

mAU

-4

-2

0

2

4

6

8

10

12

14

DAD1 H, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-21 08-02-26\1443508-52-0006.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-20 09-06-44\1435601-25-0037.D) DAD1 E, Sig=254,8 Ref=off (AEM_PS450_...\AEM_PS450_IGG-INTACT_MD_4 2014-08-19 15-36-09\DIP143501-3-047.D)

AdvanceBio RP-mAb C4AdvanceBio RP-mAb SB-C8AdvanceBio RP-mAb Diphenyl

Agilent Technologies

2 2.5

Reversed Phase Peptide Mapping – Resolution Maximized with SPP AdvanceBio Peptide MappingUHPLC column efficiency and resolution for your complete peptide map • HPLC and UHPLC• 2.7um superficially porous particle• LC and LC/MS• Minimal peak tailing• Optimal C18 bonding

February 12, 2015


5

8x10

0

0.75

1.75

2.75

3.75

Acquisition Time (min)1 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930313233343536373839

40 min. Analysis0.2mL/min140 Bar

8x10

0

0.6

1.6

2.6

3.6

4.6

Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.56 6.5 7 7.5 8 8.5 9 9.510 10.511 11.512 12.513

14 min. Analysis0.6mL/min433 Bar

TIC2.1 x 100mm, 2.7um

2.7um1.7um

0.50 um

120Å pores

Choices for Other Modes of Chromatography - SEC

SEC – for aggregation analysis• Columns are porous silica with typical

lengths of 250 or 300 mm• Typical flow rate is 1.0 mL/min on a

7.8 mm ID column or 0.35 mL/min on a 4.6 mm ID column

• To increase resolution (through increased pore volume) run columns in series (costs time!)

• To increase resolution, use smaller particle sizes (3um)

• To reduce secondary interactions and improve recovery maximize inertness

BioSEC-3, 7.8 x 300mm, 3um• 3 m particle• Proprietary hydrophilic coating to

prevent secondary interaction• 100Å, 150Å, 300Å pore sizes• High efficiency and resolution• Faster SEC separations• Can be run with low salt buffers

February 12, 2015


6

Improved SEC Efficiency With Smaller Particles

Column: Bio SEC-3 300Å and Bio SEC-5 300ÅBuffer: 150 mM Phosphate buffer, pH 7Flow rate: 1.0 mL/min for 7.8 x 300 mm Temperature: Ambient (~23 °C)Detection: UV 214nmInjection: 10 µL (3 L for 4.6 x 300 mm)Sample: 1) Thyroglobulin (1.0 mg/mL), 670 kD; 2) BSA dimer, 132 kD; 3) BSA (1.0 mg/mL), 66 kD; 4) Ribonuclease A (1.0 mg/mL), 13.7 kD, and 5) Uracil (2.5 g/mL), 120 D.

Min0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Agilent Bio SEC-3, 300Å,7.8 x 300 mm93 bar

Agilent Bio SEC-5, 300Å,7.8 x 300 mm45 bar

Peak Protein Efficiency Gain

SEC-3, 300Å (7.8x300mm)

SEC-5, 300Å (7.8x300mm)

1 Thyroglobulin 2.2X 2460 1120

2 BSA Dimer 1.9X 5100 2720

3 BSA 2.0X 13090 6590

4 Ribonuclease A 2.0X 22000 11160

5 Uracil 1.4X 38500 27860

Choices for Other Modes of Chromatography - IEX

IEX – for charge variants• Typically non-porous 10um

polymeric particles, 250mm long• Separation with high salt or pH

changes• Non-specific interactions with

surfaces can reduce resolution• Metal ions eluting from instrument

can lower resolution and cause column poisoning

• To increase resolution choose smaller particle sizes (5 and 3um)

• To maximize recovery choose inert systems

BioMAb 3 or 5um 4.6 mm• Non-porous PS/DVB particles• Uniform polymeric hydrophilic coating

and WCX layer, specifically designed for antibody separations with minimal non-specific interactions

• Fully Bio-inert choices for maximum recovery - 10 µm, 5 µm with PEEK

• Also available in 3 µm and 1.7 µm particle sizes for highest resolution

February 12, 2015


8

Improving Ion Exchange Chromatography (IEX)

Better peak shape and higher efficiency were achieved with a smaller particle size on the Agilent Bio MAb 5 vs. Bio MAb 10 column.

9Characterization of mAb

2/12/2015

min0 5 10 15 20 25 30 35

mAU

0

100

200

300

400

500

1.4

31

1.5

59

2.7

25

24.8

28

26.5

60

33.9

02

05-30% B in 30min

Incomplete separation due to protein/ system interactions. Massive fronting and tailing makes quantitation impossible.

Resolve charge variants with BioInert LC and column.

Choices for Other Modes of Chromatography – HILICReleased glycan analysis by HPLC-FLD

1260 Infinity Bio-inert HPLC/FLD

AdvanceBioGlycan Mapping,

2.7 um


2.7 um+


1.8 um


1.8 um+

1290 Infinity UHPLC

(superficially porous)

10

(totally porous)

Why Amide HILIC?Selectivity is stable over column lifetimeHigh peak capacityRT increases with size, depending on monosaccharide type and position

Columns can Reduce Challenges in Chromatography for the Characterization of Monoclonal Antibodies

Titer determination and purification Affinity Chromatography

Protein identification and impurity profilingReversed-phase chromatography (RP)

Glycan analysisHydrophilic interaction chromatography (HILIC)

Charge variant analysisIon exchange chromatography (IEX)

Aggregation analysisSize exclusion chromatography (SEC)

February 12, 2015

11

Agilent Technologies

for the and characterization of protein biopharmaceuticals · strategies for the separation and...

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