Versatile PLRP-S Columns for a Reversed Phase Solution for ... · October 6, 2016 1 Versatile PLRP-S Columns for a Reversed Phase Solution for the Characterization of Monoclonal Antibodies

October 6, 2016

1

Versatile PLRP-S Columns for a Reversed Phase Solution for the Characterization of Monoclonal Antibodies

For Research Use Only. Not for use in diagnostic procedures.

Agenda

October 6, 2016

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• Introduction – mAb’s, Characterization

• Workflow Solutions

• Reverse Phase Challenges

• mAb Intact and Fragment Solutions

• Summary

Critical Quality Attributes and Analytical Methods

Pyro-

Glutamate

Deamidation

/Oxidation

Fragmentation

(Hinge)

Glycosylation

(G0, G1, G2)

Truncation

(Lys 0, 1, 2)

Disulfide

Shuffling

Aggregation

HILIC

IEX

IEX

IEX

RP

SEC

RP

LC/MS Intact mass

Fragment mass

Identification

PTM ID & locations

Glycan ID

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Comprehensive Portfolio of Analytical Instrumentation & Solutions for Biopharma & Biosimilars Discovery > Development > QA/QC

October 6, 2016

Page 4


Sample will be analysed intact or may be reduced and

alkylated (heavy and light chains) or papain digested (Fab

and Fc). The Protein A Biomonolith is ideal for separating mAbs from

fermentation supernatant.

Different pore size of PLRP-S columns provide high efficiency separations over the full range

of protein and peptides

UV only UHPLC workflows for intact proteins may benefit from

the reduced UV intensity of 1260 Infinity DAD, while the

6500 series QTOFs are the first choice for MS characterization.

Workflows for Intact and Fragmented Protein Characterization by RPLC

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LC Columns

Sample Prep –Manual Separation (RP) Detection and Data Analysis

Manual

1260 Infinity DAD

6500 QTOF

Protein A Biomonolith

1290 Infinity II LC

1260 Infinity

Bio-inert LC


Agilent Bio-LC Column Portfolio

Agilent Bio-LC Columns

Affinity

Bio-Monolith Protein A

Protein G

Multiple Affinity Removal System

Reversed Phase

AdvanceBio Peptide Mapping

ZORBAX RRHD 300A 1.8um

Poroshell 300

AdvanceBio

RP mAb

ZORBAX 300SB

ZORBAX Amino Acid Analysis

PLRP-S polymeric reversed phase

AdvanceBio Desalting-RP

Cartridges

HILIC

AdvanceBio Glycan Mapping

ZORBAX RRHD 300-HILIC

Size Exclusion

AdvanceBio SEC

Bio SEC-3

Bio SEC-5

ProSEC 300S

ZORBAX GF-250

ZORBAX GF-450

Ion Exchange

Bio-Monolith

(QA, DEAE, SO3)

Bio mAb

Bio IEX

(SAX, SCX, WAX, WCX)

PL SAX

PL SCX

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AdvanceBio Oligonucleotide

PLRP-S polymeric reversed phase

Reverse Phase Columns: mAb Characterization

Primary Structure Characterization Workflows

7

October 6, 2016

Intact mAb

LC/MS

Heavy / Light Chains

LC/MS, LC-UV

Reduction / alkylation

Peptides

LC/UV

Enzymatic digestion

Fab / Fc Regions

LC/MS, LC/UV

Enzymatic digestion


Challenge Disadvantage Consequence

Long analysis times

Low number of samples run per day

Low throughput

Insufficient Resolution Poor accuracy and precision

for results

Lack of confidence in

analysis results

Short column lifetimes

Must purchase new columns often

Poor use of resources

Method transfer to multiple columns

Different columns for UV and MS

Time consuming

The Challenges

October 6, 2016

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I

M

P

A

C

T

C

O

S

T

!


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October 6, 2016

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LC

Experimental and Instrumentation

• Therapeutic monoclonal antibodies and antibody drug conjugate

- Intact

- Reduction

- FabRICATOR digestion


Accurate-Mass Quadrupole Time-of-Flight MS

PLRP-S, The Versatile Choice

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Characteristic Value

Durable, resilient polymer particles that

deliver reproducible results for long

column lifetimes

Change columns less often, lower cost of

analysis, more confidence in results

Thermal and chemical stability for

separations at extremes of pH and high

temperature

Flexibility for difficult samples, one

column is multi purpose, ultimately lowers

costs

100-4000A pore sizes provide high

efficiency separations over the full range

of protein and peptides

Flexibility of choice for large range of

sample types

Excellent peak shape with various ion

pairing agents including formic acid

Same column and mobile phase for UV

and MS, no redevelopment for different

detectors- impacts cost


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mAb characterization: Intact

• Molecular weight

• Glycosylation profile

• Impurity/homogeneity


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High Efficiency Intact mAb: LC/UV PLRP-S • Narrow peak shapes with both TFA and FA


1 x10

0

0.5

1

1.5

2

2.5

3

3.5

1 x10

0

0.5

1

1.5

2

2.5

Response Units vs. Acquisition Time (min)

1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6

Width = 0.24

Width = 0.18

TFA

FA

PLRP-S 1000Å, 2.1 x 50 mm, 5µm

UV (280nm)

High Efficiency Intact mAb: LC/UV & LC/MS PLRP-S

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• Single buffer system with FA as ion pairing agent

• For UV and MS detection

1x10

0

1

2

3

4

0

1

2

3

4

min1 2 3 4 5 6 7 8 9 10

8x10

0

1

2

3

4

5

6

0

1

2

3

4

5

6

1 2 3 4 5 6 7 8 9 10 min

mAU

PLRP-S, 1000Å,

2.1 × 50 mm, 5 μm

PLRP-S, 1000Å,

2.1 × 50 mm, 8 μm

TICUV (280nm)

G0F/G0F

G0F/G1F

G0F/G2F

G1F/G2F

5x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

3136.7026

3880.6849

Counts vs. Mass-to-Charge (m/z)

2400 2600 2800 3000 3200 3400 3600 3800 4000

7x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

…

147243.34147405.43

147081.32147565.42

Counts vs. Deconvoluted Mass (amu)

147000 147100 147200 147300 147400 147500 147600 147700

Deconvoluted spectraMass spectrum

Width = 0.24

Width = 0.24

FWHM: 0.1

FWHM: 0.105

counts


Agilent Application Note: 5991-7163EN

October 6, 2016

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High Efficiency Intact ADC: LC/UV & LC/MS PLRP-S

• Single buffer system with FA as ion pairing agent

• For UV and MS detection

8x10

0

1

2

3

4

5

6

8x10

0

1

2

3

4

5

6

Counts vs. Acquisition Time (min)

1 2 3 4 5 6 7 8 9 10

1x10

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

Response Units vs. Acquisition Time (min)

1 2 3 4 5 6 7 8 9 10

PLRP-S, 1000Å,

2.1 × 50 mm, 5 µm

PLRP-S, 1000Å,

2.1 × 50 mm, 8 µm

TICUV (280nm)

Width = 0.58

Width = 056

FWHM: 0.25

FWHM: 0.27

4x 10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

Counts vs . Mass-to-Charge (m/z )2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400

4x 10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

…

152223.44151108.99

153182.99

149350.36

154135.99

155099.12

148431.15

Counts vs . Deconvoluted Mass (amu)148000 149000 150000 151000 152000 153000 154000 155000 156000

DAR 0

DAR 1

DAR 2

DAR 3 DAR 4

DAR 5

DAR 6

DAR 7

DAR 8

DeconvolutedspectraMass spectrum



October 6, 2016

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mAb characterization: fragments

F(ab’)2

Fc

2 * Fab

ScFc

DTT reduction Papain digestion

FabRICATOR

LC HCIntact

+Cys

• Fragment generation - Chemical and enzymatic reactions

• Molecules more amenable to chromatographic and mass spectrometric measurement

• Modifications can readily be traced back to specific domains.


October 6, 2016

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High Resolution mAb Fragments: LC/UV & LC/MS PLRP-S

• Separation of light and heavy chain fragments

TIC

UV (280nm)

LC HC

LC HC

1x10

0

0.2

0.4

0.6

0.8

1

1.2

1x10

0

0.2

0.4

0.6

0.8

1

2 3 4 5 6 7 8 9

5x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

…

50675.00

50512.32

50838.42

50409.52


50400 50500 50600 50700 50800 50900

G0F/G0F

G0F/G1F

G0F/G2F

9x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

…

23035.94

23057.48 23090.96

23111.91


22950 23000 23050 23100 23150 23200 23250

Heavy Chain (HC)

Light Chain (LC)

Deconvoluted spectra

min

mAU

counts

Rs = 4.2

Reduced mAb


Agilent Application Note: 5991-7163EN PLRP-S 1000Å, 2.1 x 50 mm, 5µm

October 6, 2016

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High Resolution ADC Fragments: LC/UV & LC/MS PLRP-S

• Separation of different drug conjugated species

Reduced ADC (Cys conjugated)

UV (280nm)

TIC Agilent DAR Calculator

Drug to Antibody Ratio (DAR) = 4.0



High Resolution of Reduced ADC: LC/MS PLRP-S

October 6, 2016

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October 6, 2016

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Agilent Application Note: 5991-7192EN *JOURNAL OF PHARMACEUTICAL SCIENCES 104:1362–1372, 2015

• Separation of different drug conjugated species


Reduced ADC (Cys and Lys conjugated)


(PLRP-S column)

October 6, 2016

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High Resolution mAb Fragments: LC/UV & LC/MS PLRP-S

• Separation of scFc and F(ab’)2 fragments

TIC

UV (280nm)


FabRICATOR digested mAb

F(ab’)2

ScFc

F(ab’)2

ScFc

1x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1 2 3 4 5 6 7 8 9 10

Rs = 7.5

6x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

…

25201.2025363.46

25525.30

24998.09

24836.22 25654.96


24700 24900 25100 25300 25500 25700 25900

7x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

…

96721.64

Counts vs. Deconvoluted Mass (amu)96550 96650 96750 96850 96950

F(ab’)2

ScFc

min

mAU

counts



October 6, 2016

20

• Agilent AdvanceBio Desalting-RP cartridges are designed and manufactured

by Agilent for on-line removal of salt ions prior to MS detection in (U)HPLC

analysis of biomolecules.

• The cartridges contain a 10 μm, 1000Å reversed-phase polymeric particle that

will effectively remove the salt and improve the quality of the MS data generated.

• These cartridge style columns can be used on their own on any LC system to

desalt collected fractions

• Also on Agilent 1290 Infinity II 2D-LC systems as the second dimension to desalt

after an affinity/ion exchange/size exclusion first dimension separation.

AdvanceBio Desalting-RP Cartridges

Advantages

Use MS detection with traditionally non-MS friendly methods like IEX, SEC, and Affinity

Save time by allowing you to desalt your samples on-line

Save instrument maintenance and reduce down time by avoiding salt going to your MS

Improve the quality of your data to improve confidence in your data


October 6, 2016

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AdvanceBio Desalting-RP Columns Make a Difference

0

1

2

3

4

5

6

Mass-to-charge ratio (m/z)

200 600 1000 1400 1800 2200 2600 3000 3400 3800 4200 4600 5000 5400 5800

Counts

x104MS profile with AdvanceBio Desalting-RP cartridgeshowing high-quality detection

MS profile without AdvanceBio Desalting-RP cartridgeshowing signal suppression

4x10

0

1

2

3

4

5

6

7

8

4x10

0

1

2

3

4

5

6

7

8 1 2 2 3

4x10

0

1

2

3

4

5

6

7

8 12 23

Counts vs. Acquisition Time (min)2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930

1 2 2 3

4x10

0

1

2

3

4

5

6

7

8

9

4x10

0

1

2

3

4

5

6

7

8

4x10

0

1

2

3

4

5

6

7

8

Counts vs. Mass-to-Charge (m/z)2400 2600 2800 3000 3200 3400 3600 3800

WasteMS

Waste

WasteMS

Waste

WasteMS

Waste

# injection 1

# injection 2

# injection 3

Reproducibility High quality MS with AdvanceBio Desalting-RP cartridges

• Use of desalt column provides improvement in signal intensity (6X greater MS signal)

• Injection to injection repeatability shows consistent performance of AdvanceBio Desalting-

RP cartridges



October 6, 2016

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Batch-to-Batch reproducibility

Lot 0006268734 Lot 0006299268

Deconvoluted mass spectrum

• Exceptional batch-to-batch molecular weight consistency

Lot 0006268734 Lot 0006299268

Deconvoluted mass spectrum

AdvanceBio Desalting-RP Columns


Cytochrome C mAb

October 6, 2016

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300 mM NaCl

0

0.5

1.0

1.5

2.0

2.5

50597.5

50759.7

50921.550451.2

50597.5

50759.7

50922.550451.8

Counts

x105

0

0.4

0.8

1.2

50597.5

50759.3

50921.150451.9

Deconvoluted mass (amu)

50100 50200 50300 50400 50500 50600 50700 50800 50900 51000 51100 51200 51300 51400

150 mM NaCl

Counts

x105

0

0.4

0.8

1.2

No NaCl

Counts

x105

G0FG1F

G2F

• The relative quantification between the

samples is similar regardless of the

presence or absence of NaCl

Effect of the salt concentration

Relative quantification

Glycoform 0 mM NaCl 150 mM NaCl 300 mM NaCl

% G0F 49.6 50.9 50.0

% G1F 41.5 41.2 41.5

% G2F 8.9 7.9 8.5

AdvanceBio Desalting-RP cartridges


Heavy chain deconvoluted mass spectrum


October 6, 2016

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Examples of 2D-LC/MS workflows Biocolumns


October 6, 2016

Agilent Technologies

25

On-line desalting 2D LC/MS of innovator mAb 1D – IEX, 2D – Desalting-RP

2D MS profiles

1D CEX profile (UV 280 nm) (Agilent Bio MAb NP5, 4.6 × 250 mm, PEEK)

• 2D-LC/MS Characterization of innovator mAb Charge Variants Using Ion Exchange and

Reversed-Phase Chromatography (Agilent AdvanceBio Desalting-RP cartridges)

• Analysis of charge variants of innovator mAb using heart-

cutting 2D-LC with MS detection

(Agilent AdvanceBio Desalting-RP cartridges)


First dimension:UV signal (280 nm)

Time (min)6 8 10 12 14 16 18 20

Absorbance

(mAU)

0

50

100

150

200

250

300

0

0.5

1.0

1.5

2.0

2.5

3.0

3.51 2 2 3

Acquisition time (min)

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Waste MS Waste

Counts

x108

Second dimension:MS (TIC)

Second dimension:m/z spectrum

Counts

x104

0

1

2

3

4

5

6

Mass-to-charge ratio (m/z)

2200 2600 3000 3400 3800 4200 0

1

2

3

4

5

147079.24

147241.30

147404.41

147566.22

146936.93148026.91

Deconvoluted mass (amu)

146800 147200 147600 148000

Counts

x104Second dimension:Deconvoluted spectrum


October 6, 2016

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1D CEX profile (UV 280 nm)

(Agilent Bio MAb NP5, 4.6 × 250 mm, PEEK)

2D MS profiles


Multiple heart-cutting 2D-LC/MS analysis (IEX → Desalting-RP) 1D – IEX, 2D – Desalting-RP

• Effective online desalting approach using Agilent AdvanceBio Desalting-RP

cartridges for identification of the mAb charge variants

• 128 Da shift between peaks 1, 2, and 3 -

corresponds to C-terminal lysine truncation


min 6 8 10 12 14 16 18 20

mAU

0

5

10

15

20

25

30

35

Peak 1

Peak 2

Peak 3

Acidic variants Basic variants

Main peak

4 x10

0

1

2

3

4

5 147244.85

147082.69

147407.70

4 x10

0

0.5

1

1.5

2

2.5

147371.22

147532.66

147695.74

4 x10

0

0.4

0.8

1.2

1.6

2

2.4 147337.84

147500.82

147662.57

147826.06

Counts vs. Deconvoluted Mass ( amu ) 146800 147200 147600 148000 148400

147208.32

Peak 1

Peak 2

Peak 3

147569.98

G0F/G0F

G0F/G1F

G0F/G2F or (G1F) 2

G1F/G2F

G0F/G0F G0F/G1F

G0F/G2F or (G1F) 2

G1F/G2F

G0F/G0F

G0F/G1F

G0F/G2F or (G1F) 2

G1F/G2F


October 6, 2016

Agilent Confidential

27

On-line desalting 2D LC/MS of innovator mAb 1D – Bio-Monolith Protein A, 2D – Desalting-RP

2D MS profiles

1D Protein A profile (UV 280 nm) (Agilent Bio-Monolith column Protein A)

2D-LC/MS Characterization of innovator mAb Using Protein A and Reversed-Phase

Chromatography (Agilent AdvanceBio Desalting-RP cartridges)


• Analysis of innovator mAb captured on Agilent Bio-Monolith

column Protein A using heart-cutting 2D-LC with MS detection


min1 2 3 4 5 6 7

mAU

0

20

40

60

80

4x10

0

1

2

3147080.03

147404.02147241.87

147565.34

147729.55


146800 147200 147600 148000 148400 148800

8 x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2 1 1 2 2 3 3

Counts vs. Acquisition Time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Waste MS

Waste

4 x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Counts vs. Mass-to-Charge (m/z)

2200 2400 2600 2800 3000 3200 3400 3600 3800 4000

October 6, 2016

Agilent Confidential

28

Summary


The versatility of PLRP-S column in the characterization of mAb has been

demonstrated.

Reversed-phase methods using the PLRP-S column have been shown to be

excellent for both mAb intact and fragment levels analysis.

PLRP-S is suitable for both UV and MS detection, excellent peak shape with FA.

Agilent AdvanceBio Desalting-RP cartridges effectively remove salts for better

MS sensitivity.

Agilent AdvanceBio Desalting-RP cartridges provide reproducible MS results.

Agilent AdvanceBio Desalting-RP cartridges allow automated 2D LC in Multiple

Heart-Cutting 2D-LC/MS methods for characterization of mAbs.

October 6, 2016

29

Questions?Contact us at: www.agilent.com/chem/advancebio-query


https://www.agilent.com/en/promotions/agilent-advancebio-columns

Documents

Versatile PLRP-S Columns for a Reversed Phase Solution for ... · October 6, 2016 1 Versatile PLRP-S Columns for a Reversed Phase Solution for the Characterization of Monoclonal Antibodies