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Webinar: Webinar: 'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Characterization using SEC Waters Corporation Waters Corporation January 27, 2012 January 27, 2012 ©2012 Waters Corporation 1

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Page 1: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Webinar:Webinar:'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical

Characterization using SECCharacterization using SEC

Waters CorporationWaters Corporation

January 27, 2012January 27, 2012

©2012 Waters Corporation 1

Page 2: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Waters Commitment

To develop, commercialize and market columns that when used on Waters ACQUITY UPLC® systems, give the speed, sensitivity,

l ti d th d d ibilit th t h t b i l resolution, and method reproducibility that has not been previously achieved for the characterization of biological macromolecules with traditional HPLC.

©2012 Waters Corporation 2

Page 3: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Liquid ChromatographyProtein Separation Modesp

Protein Structure

Primary, Secondary, Tertiary Structure

Net Charge

Carbohydrate Groups

HydrophilicGroups

Hydrophobic Regions

Aromatic Groups

Regions

Disulfide H d

©2012 Waters Corporation 3

Disulfide Linkages

HydrogenBonding

Page 4: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Liquid ChromatographyProtein Separation Modesp

Protein Structure

Primary, Secondary, Tertiary Structure

Net Charge

Carbohydrate Groups

HydrophilicGroups

Hydrophobic Regions

Aromatic Groups

Regions

Disulfide H d

©2012 Waters Corporation 4

Disulfide Linkages

HydrogenBonding

Page 5: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Agendag

Size-Exclusion Chromatography– Theory and practiceTheory and practice

– ACQUITY UPLC Columns for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns

ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns

– Monoclonal Antibody Application

o SEC-MS Applications

– Insulin and Small Protein Applications

– Factors Influencing Component Resolution

– Considerations to extending column life

©2012 Waters Corporation 5

Page 6: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Principles of Size Exclusion Chromatography Principles of Size Exclusion Chromatography of Proteinsof Proteins

Separates proteins by their size in solution (Stokes radius)

Separations are Isocratic

Tends to be used as a “Polishing” isolation step or as an analytical technique to determine presence of protein aggregates

Generally a “lower resolving” technique compared to other methods such as ion-exchange or reversed-phase methods

©2012 Waters Corporation 6

Page 7: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Size Exclusion Chromatographyg p y

No adsorption to surface of particles

Large molecules elute Large molecules elute before small molecules

Large molecules cannot access poresaccess pores

Small molecules access pores within particle

dimer

monomer

©2012 Waters Corporation 7

Page 8: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Common SEC applications:Biotherapeutics Types

Monoclonal Antibodies

Biotherapeutics Types

Antibody Conjugates

Fc Fusion Proteins

Synthetic OligonucleotidesSynthetic Oligonucleotides

Protein Subunit Vaccines

Recombinant Proteins

©2012 Waters Corporation 8

Page 9: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Agendag

Size-Exclusion Chromatography– Theory and practiceTheory and practice

– ACQUITY UPLC Columns for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns

ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns

– Monoclonal Antibody Application

o SEC-MS Applications

– Insulin and Small Protein Applications

– Factors Influencing Component Resolution

– Considerations to extending column life

©2012 Waters Corporation 9

Page 10: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

UPLC Systems for Biopharmaceutical Analysisp y

Wide range of applicationsg pp Complete Solutions

– Instrumentationo UPLC System (s)

• ACQUITY UPLC System• ACQUITY UPLC System• ACQUITY UPLC H-Class System• ACQUITY UPLC H-Class Bio System

o UV, FLR, PDA and MS DetectionsA li ti S ifi Ch i t io Application Specific Chemistries• Developed and designed with applications• QC Tested with application• Optimizes for UPLC

o Software • Data Analysis• Information management

Focused on customer application requirements

©2012 Waters Corporation 10

pp q

Page 11: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Advantages of UPLC Technologyfor SEC Separations

Requires Columns and Instrumentation to Minimize Band Spreading

HPLC

p

Broad BandBroad PeakLess Sensitivity

HPLC

Narrow PeakIncreased Sensitivity

Waters UPLC®

Technology

Less SensitivityLess Resolving Power

Increased SensitivityIncreased Resolving Power

©2012 Waters Corporation 11

Page 12: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Effect of System Dispersion on ACQUITY UPLC BEH200 SEC 1.7 µm separationµ p

UP-SEC0.30

ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm

USP Res= 2.37

AU

0.10

0.20

0.00

ACQUITY UPLC BEH200 SEC 1 7 µm

HP-SEC

USP Res= 1.37

AU

0 10

0.15

0.20

0.25

BEH200 SEC 1.7 µm 4.6 x 300mm

0.00

0.05

0.10

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

©2012 Waters Corporation 12

Large system dispersion decreases resolution Sample: Human polycolonal IgG

Minutes

Page 13: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

ACQUITY UPLC BEH200 and BEH125 SEC1.7 µm Columnsµ

Application Areas

– Molecular weight ranges dependent on pore size:Molecular weight ranges dependent on pore size:

o BEH200: 10,000 to 450,000 Daltons

o BEH125: 1,000 to 80,000 Daltons

– Determination of protein / peptide molecular weight

– Quantitation of protein / peptide aggregates primarily in therapeutic monoclonal antibodies, EPO, and Insulin

– Determination of size heterogeneity in a sample

©2012 Waters Corporation 13

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BEH™ Technology ParticlesBEH™ Technology ParticlesBridged EthylSiloxane/Silica HybridBridged EthylSiloxane/Silica Hybridg y yg y y

Si

EtO CH2 CH2

Si OOEt

SiOEt

O EtEtO OEtEtO

O

Si

EtO

Si O

O

OEtO

O

Si OOEt

Et

+ Si

EtOEtO

CH2EtO

CH2Si

OEt

OEtOEt

4 Si

EtO

EtO OEtEtO

1

Polyethoxysilane(BPEOS)

EtO OEt nTetraethoxysilane

(TEOS)Bis(triethoxysilyl)ethane

(BTEE)

Bridged EthanesIn Silica Matrix

©2012 Waters Corporation 14

Anal. Chem. 2003, 75, 6781-6788

U.S. Patent No. 6,686,035 B2

Page 15: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

HPLC to UPLC SEC Comparisonp

0.065

0.070

0.065

0.070

HPLC 100% ACQUITY UPLC

0.045

0.050

0.055

0.060

0.045

0.050

0.055

0.060Silica-Diol

SEC 250Å 5µm7.8 x 300 mm

ACQUITY UPLC BEH200 SEC,1.7

µm4.6 x 300mm

AU

0.030

0.035

0.040

AU

0.030

0.035

0.040

0.015

0.020

0.025

0.015

0.020

0.025

2.26 % Aggregate 2.24 %

Aggregate

0.000

0.005

0.010

0.000

0.005

0.010

©2012 Waters Corporation 15

Murine monoclonal antibody - Scaled load

Conditions: 0.4 mL/min; 25mM Sodium Phosphate, pH 6.8, 0.15 M NaCl

Minutes2.00 4.00 6.00 8.00 10.00

Minutes5.00 10.00 15.00 20.00 25.00 30.00

8.00 30.008.00 30.00

Page 16: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Calibration Curves for ACQUITY UPLC SEC Columns

BEH200, SEC, 1.7um

Thyroglobulin (~ 669,000 Da)

IgG (~ 150,000 Da)

Aprotinin (~ 6,500 Da)

BEH125, SEC, 1.7um Uracil (~ 112 Da)

©2012 Waters Corporation 16

Page 17: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Protein Adsorption and SizeProtein Adsorption and Size--Exclusion ChromatographyExclusion Chromatographyg p yg p y

Proteins can interact or adsorb onto the SEC packing material

These interactions create undesired and unpredictable retention of proteins (i.e. proteins not separated by size in solution)p ( p p y )

SEC particles frequently coated with a hydrophilic reagent tominimize non-desired ionic interactions between proteins and minimize non-desired ionic interactions between proteins and packing material

M bil h dditi ( 150 M N Cl) d Mobile phase additives (e.g., 150mM NaCl) may decrease non-desired ionic interactions between proteins and packing material

©2012 Waters Corporation 17

Page 18: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

BEH SEC Particle Overview

The packing material is based on our patented Bridged Ethyl H b id b ti l d ff ti di l b di hi h Hybrid base particle and effective diol bonding, which provide a stable chemistry with minimal secondary interactions.

©2012 Waters Corporation 18

Page 19: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Comparative SEC Column Life

Lysozyme, pKi = 10.7

0 60

0.70

HPLC 100% Silica-Diol

p

Lysozyme,

Suggestive of DIOL Bleed

AU

0.30

0.40

0.50

0.60 HPLC 100% Silica DiolSEC 250Å 4µm4.6 x 300 mm

Injection 19Suggestive of DIOL Bleed

pI = 10.7

0.00

0.10

0.20

Minutes5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

Injection 19Injection 618

©2012 Waters Corporation 19

Page 20: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Comparative SEC Column Life

Lysozyme, pKi = 10.70.60

0.70

HPLC 100% Silica-DiolLysozyme, pI 10 7

Suggestive of DIOL BleedAU

0 20

0.30

0.40

0.50 SEC 250Å 4µm4.6 x 300 mm

Injection 19Suggestive of DIOL Bleed

pI = 10.7

0.22

0.00

0.10

0.20

Minutes5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

jInjection 618

AU

0.10

0.12

0.14

0.16

0.18

0.20

ACQUITY BEH200 SEC, 1.7 µm4.6 x 150 mm

I j ti 19

0.00

0.02

0.04

0.06

0.08

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 7 50 8 00 8 50 9 00 9 50 10 00

Injection 19Injection 618

©2012 Waters Corporation 20

BEH200 shows minimal secondary interactions even after 600 injections

Minutes0.00 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 7.50 8.00 8.50 9.00 9.50 10.00

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Influence of Ionic Strength on Peak Shape and Retention

0 06

0.08

p

Conventional 100% Silica-Diol CoatedSEC Column 4.6 x 300 mm 10 mMlysozyme

AU

0.00

0.02

0.04

0.06

Minutes10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00

Minutes

AU

0.04

0.06

0.08

25 mMlysozyme

0.00

0.02

Minutes10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00

0 08

lysozyme

AU

0 00

0.02

0.04

0.06

0.08

100 mMlysozyme

©2012 Waters Corporation 21

0.00

Minutes10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00

Flow rate: 0.5 mL/min; Mobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8

Page 22: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Influence of Ionic Strength on Peak Shape and Retentionp

AU

0 12

0.18

0.24

0 12

0.18

0.24

ACQUITY BEH200 SEC 1.7 µm column, 4 6 150

10 mMlysozyme

A

0.00

0.06

0.12

Minutes5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

0.00

0.06

0.12

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

4.6 x 150mm

AU

0.12

0.18

0.24

0.12

0.18

0.24

25 mM

0.00

0.06

Minutes5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

0.00

0.06

5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

0.240.24

AU

0.06

0.12

0.18

0.06

0.12

0.18

100 mM

©2012 Waters Corporation 22Flow rate: 0.5 mL/min; Mobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8

0.00

Minutes5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

0.005.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00 14.50 15.00

Page 23: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

BEHBEH200 SEC, 1.7um BatchBatch--toto--Batch Batch ReproducibilityReproducibilityp yp y

©2012 Waters Corporation 23

Page 24: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

BEHBEH125 SEC, 1.7um BatchBatch--toto--Batch Batch ReproducibilityReproducibilityp yp y

AU

0.06

0.09

1

3

24

Batch 1 Analyte pl MW

1. Thyroglobulin, 0.1 mg/mL 4.6 669,000

2. Ovalbumin, 0.3 mg/mL 4.5 44,200

A

0.00

0.03

Minutes 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

3. Ribonuclease A, 0.3 mg/mL 9.6 13,700

4. Uracil, 0.05 mg/mL N/A 112

AU

0.03

0.06

0.09

Batch 2

0.00

Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

0.09

AU

0.03

0.06

Batch 3

©2012 Waters Corporation 24

0.00

Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Conditions: 100mM Sodium Phosphate pH 6.8; 0.3 mL/min; 30°C; 4.6x150mm

Page 25: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Column ReproducibilityColumn Reproducibilityp yp yAU

0 00

0.05 Batch 1, Column 1

0.00

AU

0.00

0.05 Batch 1, Column 2

AU

0.00

0.05Batch 1, Column 3

AU

0.00

0.05Batch 2, Column 1

AU

0.00

0.05

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Batch 2, Column 2

©2012 Waters Corporation 25

Minutes

Humanized monoclonal antibody Conditions: 25mM Sodium Phosphate, pH 6.8, 0.15 M Sodium Chloride, 0.4 mL/min Retention times within 0.2min

Page 26: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Agendag

Size-Exclusion Chromatography– Theory and practiceTheory and practice

– ACQUITY UPLC Columns for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns

ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns

– Monoclonal Antibody Application

o SEC-MS Applications

– Insulin and Small Protein Applications

– Factors Influencing Component Resolution

– Considerations to extending column life

©2012 Waters Corporation 26

Page 27: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Monoclonal Antibodyy

©2012 Waters Corporation 27

Adapted from Alain BeckCenter of Immunology

Page 28: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Protein Structure

NNative

UUnfolded

SolubleAggregates Insoluble

AggregatesNative UnfoldedorI

Intermediate

Aggregates

Addressing particulate and aggregation issues of therapeutic protein products, Shi, L, PEGS, May 2011

Soluble aggregates and insoluble particles may affect

©2012 Waters Corporation 28

Soluble aggregates and insoluble particles may affect immunogenicity and efficacy of biotherapeutic

Page 29: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Orthogonal Techniques for Characterization

AUC

Dynamic Light Scattering Counter principle

Static Light Scattering

FFF-MALS

AUCFlow Imaging Microscopy

Light Microscopy

SEC MicroscopeVisual Inspection

nm µm mm cm10 10 10100 100 100

monomersoligomers

subvisible particles Visible particles

Aggregates Particles

©2012 Waters Corporation 29

E. Freud, PDA Visual Inspection Forum, Oct- 2009

Aggregates Particles

Page 30: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Column LifetimeColumn Lifetime

0.015 Dimer = 0.46%USP Res = 2.35

AU

0.000

0.005

0.010 Injection 2

-0.005

0.010

0.015

I j ti 497

mAb

Dimer = 0.49%USP Res = 2.27

AU

0.000

0.005

0 0 0Injection 497

mAb

-0.005

Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50

©2012 Waters Corporation 30

Humanized monoclonal antibody Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm Column: 4.6 x 300 mm

Page 31: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Effect of Flow RateEffect of Flow Rate

AU 0.010

0.020

0.030

0.4 mL/minFlow Rate (mL/min) 0.2 0.35 0.4Average 2.87 2.83 2.79Std Dev 0.04 0.02 0.02% RSD 1.45 0.70 0.57

% Aggregate

0.000

Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

0.030

AU

0.000

0.010

0.020

2.00 2.50 3.00 3.50 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 10.50

0.35 mL/min

AU 0 010

0.020

0.030

Minutes

0 2 mL/min0.000

0.010

Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

0.2 mL/min

©2012 Waters Corporation 31

Triplicate injections overlaid

No observable trend in aggregation with flow rate

Murine monoclonal antibody (63 µg load)

Page 32: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Effect of Particle Size:Effect of Particle Size:Analysis of LMW SpeciesAnalysis of LMW Speciesy py p

0.010BEH200 SEC 1.7µm

LMW peakmAb mAb d

LMW species

AU

0.000

0 010250Å, 4µmHPLC 100% Sili Di l

dimer

AU

0.000

0.010 HPLC 100% Silica-Diol

Å

AU

0.000

0.010 300Å, 5µmHPLC 100% Silica-Diol

AU

0.000

0.010

2 00 4 00 6 00 8 00 10 00 12 00 14 00

290Å, 5µm HPLC 100% Silica-Diol

©2012 Waters Corporation 32

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00

Humanized monoclonal antibody biotherapeutic Conditions: 25 mM Sodium Phosphate, 0.15 M Sodium Chloride Flow rates and Injection volumes scaled for column dimensions

Page 33: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Column StabilityColumn Stabilityyy

Calibration Curve

Protein MWThyroglobulin 669000Ferritin 440000Aldolase 150000BSA 66000

1000000

0 hours

Ovalbumin 44000Carbonic Anhydrase 29000Ribonuclease A 13700Aprotinin 6500Uracil 112

10000Log

Mw

12 hours24 hours36 hours48 hours60 hours

1000.5 1 1.5 2 2.5 3

Elution volume (mL)

©2012 Waters Corporation 33

Protein standard analyzed over 48 hours Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm Elution volume for all proteins within 0.2% RSD

Page 34: Webinar: 'Tips and Tricks' for Biopharmaceutical ...€¦ · Webinar: 'Tips and Tricks' for Biopharmaceutical Characterization using SEC Waters Corporation January 27, ... ©2012

Precautions in SECPrecautions in SEC--MSMS

Protein structure in solution depends on

– pHpH

– Ionic strength

– Buffer and salt

– Additives

Good ionization conditions are different from conditions for biological activitybiological activity

Validation required when buffer is changed

Special uses are valuable

– Fast desalting

– Clips

©2012 Waters Corporation 34

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LC/MS Compatible Mobile Phaseon ACQUITY UPLC BEH200, SEC, 1.7um Q , ,

AU

0.10

0.20PBSPBS

0.00

0.10

0.30

100 M A i F t100 M A i F t

AU

0.10

0.20

100mM Ammonium Formate100mM Ammonium Formate

0.00

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

©2012 Waters Corporation 35

Similar retention time/ peak shape observed with MS compatible mobile phases

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Humanized Monoclonal Antibody:MS Compatible/Native Mobile Phase

0 010

0.015

0.020

0.025

U 1 0 2

1.25e-2

1.5e-2

1.75e-2 26.00100mM Ammonium Formate

AU

-0.005

0.000

0.005

0.010

Time14 00 16 00 18 00 20 00 22 00 24 00 26 00 28 00

AU

0.0

2.5e-3

5.0e-3

7.5e-3

1.0e-2

17.05

20.02

AU 0.010

0.015

0.020

14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

PBS

-0.005

0.000

0.005

Minutes

4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

©2012 Waters Corporation 36

Flow Rates: 100mM Ammonium Formate - 0.15mL/min, PBS- 0.4 mL/min Lower flow rate for MS compatibility

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SEC-MSHumanized Monoclonal Antibodyy

UV @ 2801 2 2

1.4e-2

1.6e-2

1.8e-2

2: Diode Array 280 0.0500Da

Range: 6.757e-125.27

1UV @ 280

AU

4.0e-3

6.0e-3

8.0e-3

1.0e-2

1.2e-2

16.58

2

3

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 17000.0

2.0e-313.22

19.50

1: TOF MS ES+ TIC

7.58e619.493

TIC

%

15.35

16.62

1 2

Scan500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

4

23.7425.06

©2012 Waters Corporation 37

MS: Xevo G2 Q Tof Conditions: 100mM Ammonium Formate, Flow rate: 0.15 mL/min Post UV detection additive: ACN, 0.8% Formic acid

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Extracted SpectrumHerceptin 50%ACN, .4% FA_100mm Amm Form_0.15 mL/min_40CV_AutoQua

1007Oct11_PH_SEC_BEH200_Ext_T_ACN_pt8FA_2_5 907 (15.351) Sm (SG, 10x5.00); Sb (15,2.00 ); Cm (891:932) 1: TOF MS ES+

4.34e33448.14043025.98782907.2991

2797.63792601.3782

2471.37262353.8545

3530.13483706.4951

3801.68433901.6064

3905.8140

p

Intact IgG MW 148 221

m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800

%

0

2353.5356 4118.3599

7Oct11 PH SEC BEH200 Ext T ACN pt8FA 2 5 982 (16.619) Sm (SG, 10x5.00); Cm (969:996) 1: TOF MS ES+

MW 148,221Peak 1

%

100_ _ _ _ _ _ _p _ _ ( ) ( , ); ( )

1.97e3

2652.85842648.5669

2460.41241251.3574

2801.4185

2968.89673154.9690

3370.08893616.4006

ClipMW 100,764Peak 2

100

m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800

0

7Oct11_PH_SEC_BEH200_Ext_T_ACN_pt8FA_2_5 1152 (19.493) Sm (SG, 10x5.00); Cm (1116:1184) 1: TOF MS ES+ 1.22e41537.7053

1489.6832

1478 2386

1643.7091

1702.38311765 3279

Peak 2

%

1478.2386 1765.3279

1985.9363

2056.27692166.3523 2382.8904

2647.5525

Low MW SpeciesPeak 3

©2012 Waters Corporation 38

m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800

0

Deconvoluted molecular weight determined using MaxEnt1

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SECSEC--UVUV--MSMS: : A generic methodology for A generic methodology for screening intact and reduced antibodiesscreening intact and reduced antibodies

Desalting LC/HC Resolution Detect Clips

HC Detect Clips• No Sample Concentration required

LCUV

280

HC HC

HC-HC

TIC

Mass Spectrum LC

Mass Spectrum

LCHC HCHC-HC

©2012 Waters Corporation 39

Conditions: System, ACQUITY UPLCTM with TUV optical detector and Synapt G2 QTof MS

Flow Rate: 0.2 ml/min 0.1%TFA and 0.1%FA in 30% ACN

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Agendag

Size-Exclusion Chromatography– Theory and practiceTheory and practice

– ACQUITY UPLC Columns for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns

ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns

– Monoclonal Antibody Application

o SEC-MS Applications

– Insulin and Small Protein Applications

– Factors Influencing Component Resolution

– Considerations to extending column life

©2012 Waters Corporation 40

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Resolution of Proteins and Peptides (Aqueous)

1.50ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm1.501.50ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm

AU

0 00

0.50

1.00

AU

0 00

0.50

1.00

AU

0 00

0.50

1.00

0.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

0.80

1.00BioSuite125 UHR SEC 4.6 x 300mm

A2

14

0.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

0.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

0.80

1.00

0.80

1.00BioSuite125 UHR SEC 4.6 x 300mm

A2

14

AU

0.00

0.20

0.40

0.60

AU

0.00

0.20

0.40

0.60

AU

0.00

0.20

0.40

0.60

- -

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min

©2012 Waters Corporation 41

-

/ BEH125 column provides increased resolution throughout the lower end of the

peptide mass range (132 29,000).

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ACQUITY UPLC BEH125 SEC 1.7µm Column Reproducibility

1

% RSD Retention Time Range

p y

0.6

0.7

0.8

0.9

n Ti

me

Ran

ge

0.2

0.3

0.4

0.5

%R

SD/R

eten

tion

0

0.1

Peptide/Protein

©2012 Waters Corporation 42

Table 1. Retention time reproducibility for 5 ACQUITY UPLC BEH125 SEC 1.7 µm columns (4.6 mm x 30cm) using aqueous and organic (insulin separation method only) mobile phases.

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Resolution of Small Protein

BioSuite 125 4µm UHR 4.6 x 300 mm column

ACQUITY UPLC BEH125 SEC 1.7µm 4.6 x 300 mm column

0.60

0.70

0.80

0.60

0.70

0.80

0.60

0.70

0.80

0.60

0.70

0.80

4.6 x 300 mm column 4.6 x 300 mm column

USP Rs= 1.94

AU

0.20

0.30

0.40

0.50

AU

0.20

0.30

0.40

0.50

AU

0.20

0.30

0.40

0.50

AU

0.20

0.30

0.40

0.50

USP Rs= 1.94

USP Rs= 3.29

0.00

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

0.00

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

0.00

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

0.00

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

-

- Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min, sample 2 mg/mL

©2012 Waters Corporation 43

sample 2 mg/mL USP monomer/aggregate resolution was 1.7 times greater on the BEH125 1.7µm SEC

column as compared to 4 µm pore diol-coated silica column.

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HPLC/UPLC Column Comparison:Insulin

0.007

0.008

0.009

0.010

AU

0.05

0.10

0.15

0.20

0.25

0.30

ACQUITY UPLC BEH125 1.7µm(4.6 x 300 mm)

AU

0.002

0.003

0.004

0.005

0.006 0.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Rs = 3.7USP Plate Count = 15K

0.000

0.001

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

0.018

0.020

0.30

0.40

0.50

USP Plate Count 15KFlow Rate = 0.4 mL/min

HMWP 10µm

AU

0004

0.006

0.008

0.010

0.012

0.014

0.016 AU

0.00

0.10

0.20

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Rs = 2.1USP Plate Count = 3K

HMWP 10µm(7.8 x 300 mm)

.

0.000

0.002

0.004

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

USP Plate Count = 3KFlow Rate = 0.5 mL/min

Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Injection volume:

©2012 Waters Corporation 44

(Waters HMWP) tested to perform in the European Pharmacopoeial method. Increase in HMW resolution observed in shorter run-times

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BEHBEH125 SEC, 1.7um Column LifeInsulin Analysis

0.004

0.005

Injection 26

y

ACQUITY UPLC BEH125, SEC 1.7µm4 6 x 300 mm

AU

0.000

0.001

0.002

0.003

j4.6 x 300 mm

-0.002

-0.001

Minutes2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00

0.005

AU

0.001

0.002

0.003

0.004

Injection 853

-0.002

-0.001

0.000

2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00

©2012 Waters Corporation 45

Comparable absolute retention time change observed for both columns Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Sample: Human

Insulin ( 4mg/mL), Injection volume: 5 µL

Minutes50 3 00 3 50 00 50 5 00 5 50 6 00 6 50 00 50 8 00 8 50 9 00

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Column Stability for Insulin Analysis

Retention Time USP Resolution

y y

3

4

5

6

ime

n)

3.0

4.0

5.0

utio

n

0

1

2

3

Ret

entio

n T

(mi n

0.0

1.0

2.0

USP

Res

olu

0 100 200 300 400 500 600 700 800 900

Injection Number

Over 800 injections the retention time of the insulin monomer peak

©2012 Waters Corporation 46

and the resolution between insulin monomer and dimer peaks are maintained.

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Effect of Pore Size:Insulin

ACQUITY UPLC BEH125 SEC 1.7 µm

ACQUITY UPLC BEH200SEC 1. 7µm

AU

-0.026

-0.024

-0.022 Control Control

AU

0 000

0.002

0.004

0.006Sample 1 Sample 1Fragment

0.000

AU

-0.042

-0.040 Sample 2 Sample 2

-0.044

Minutes4.00 5.00 6.00

Minutes6.00 7.00 8.00 9.00

Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v) Wavelength : 276 nm

©2012 Waters Corporation 47

(v/v/v), Wavelength : 276 nm

The HMW and insulin fragments are better resolved on the 125Å pore diameter column as compared to the 200Å pore diameter

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Effect of Particle Size:Insulin

-0.022

ACQUITY UPLC BEH125, 1.7µm4.6 x 300mm

BioSuite125 UHR, 4µm4.6 x 300mm

Insulin HMWP, 10µm7.8 x 300mm

Control Control Control

AU

-0.026

-0.024 Rs= 2.21 Rs= 2.08Rs= 3.37HMW

AU

0 000

0.002

0.004

0.006Sample 1 Sample 1 Sample 1

Rs= 1.93Rs= 1.95

Rs= 2.63

Fragment

0.000

AU

-0.042

-0.040 Sample 2 Sample 2Sample 2

Rs= 1.92Rs= 1.88

Rs= 2.63

-0.044

Minutes4.00 5.00 6.00

Minutes6.00 7.00 8.00 9.00 10.00

Minutes14.00 16.00 18.00 20.00

©2012 Waters Corporation 48

Improved resolution of HMW and Fragment peaks observed with BEH125 1.7 um column Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v),

Wavelength : 276 nm, Column dimensions: 4.6 x 300mm BEH125 and BioSuite125, 7.8 x 300mm Insulin HMWP, Flow rate: 0.4 mL/min (HMWP: 0.5 mL/min), Injection volumes: scaled

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Agendag

Size-Exclusion Chromatography– Theory and practiceTheory and practice

– ACQUITY UPLC Columns for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns

ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns

– Monoclonal Antibody Application

o SEC-MS Applications

– Insulin and Small Protein Applications

– Factors Influencing Component Resolution

– Considerations to extending column life

©2012 Waters Corporation 49

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Factors Influencing ResolutionFactors Influencing Resolutiongg

Resolution increases with lower injection volumes

Resolution decreases with increasing flow rate Resolution decreases with increasing flow rate– Ideal flow rate is lower than typically running, however will sacrifice

speed

Resolution increases with column length

Baseline resolution typically achieved at 50%-100% molecular weight differenceg

©2012 Waters Corporation 50

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Loading Capacity:Loading Capacity:Undiluted Monoclonal Antibody Undiluted Monoclonal Antibody BiopharmaceuticalBiopharmaceutical

2 40

2.60

pp

Injection Volume, Total LoadInjection Volume, Total Load

1.80

2.00

2.20

2.40

15 µL, 300µg15 µL, 300µg

AU1.20

1.40

1.6010 µL, 200µg

Aggregates

21.8%

Aggregates

21.8%

0 0

0.60

0.80

1.00

5 µL, 100µg

21.0%

21.2%

0.00

0.20

0.40

1 50 2 00 2 50 3 00 3 50 4 00 4 50

©2012 Waters Corporation 51

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50

Humanized IgG (20 mg/mL), 4.6 x 150 mm column Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm

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Effect of Column Length:Monoclonal Antibody

0.040

y

150 mm98.88%USP Res=2 07

AU

0.000

0.010

0.020

0.030 USP Res=2.071.12%

-0.020

-0.010

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

300

mAbaggregates

0.010

0.020

0.030

0.040 300 mm

98.76%USP Res= 2.811.22%

AU

-0.020

-0.010

0.000

mAbaggregates

©2012 Waters Corporation 52

Minutes3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00

Murine monoclonal antibody (load: 6.4 µg - 150mm; 12.7 µg -300mm) Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8;214 nm

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Effect of Flow Rate on RsEffect of Flow Rate on Rs

0.30 0.2 mL/minRs= 2.4 IgG dimer

AU

0.00

0.10

0.20 ~1500 psigdimer

AU

0.10

0.15

0.20

0.25

0.4 mL/minRs= 1.8

~3000 psi

0.00

0.05

0.15 0.8 mL/min

AU

0.05

0.10Rs= 1.3

~6000 psi

©2012 Waters Corporation 53

0.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Resolution increases with lower flow rates Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8;280 nm Column: BEH200 SEC 1.7 µm, 4.6 x 150mm

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Effect of Sample Load :Myoglobin

1 40

1.50

1 40

1.50

USPInjection USPInjection 1 70

1.80

1.90

1 70

1.80

1.90

1 70

1.80

1.90

USP Concentration USP Concentration

y g

Effect of Volume LoadEffect of Concentration

0.90

1.00

1.10

1.20

1.30

1.40

0.90

1.00

1.10

1.20

1.30

1.40

3.0235

2.6550

USPInjection Volume

3.0235

2.6550

USP Res

Injection Volume

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.10

1.20

1.30

1.40

1.50

1.60

1.70

3.262.5

3.2610

Res(mg/ mL)

3.262.5

3.2610

Res(mg/ mL)

AU

0.40

0.50

0.60

0.70

0.80

AU

0.40

0.50

0.60

0.70

0.80 3.7815 3.7815

AU

0.40

0.50

0.60

0.70

0.80

0.90

1.00

AU

0.40

0.50

0.60

0.70

0.80

0.90

1.00

AU

0.40

0.50

0.60

0.70

0.80

0.90

1.00 3.231.25

3.150.625

3.231.25

3.150.625

0.00

0.10

0.20

0.30

Minutes

3.00 3.50 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

0.00

0.10

0.20

0.30

Minutes

3.00 3.50 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

0.00

0.10

0.20

0.30

Minutes3.50 4.0

04.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00

0.00

0.10

0.20

0.30

Minutes3.50 4.0

04.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50

0.00

0.10

0.20

0.30

Minutes3.50 4.0

04.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00

Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min, sample 2 mg/mL, Column: ACQUITY UPLC BEH125 1.7µm SEC , 4.6 x 300 mm column

Myoglobin: 5 mg/mL (volume load, and 20 uL injection volume (concentration)

©2012 Waters Corporation 54

Myoglobin: 5 mg/mL (volume load, and 20 uL injection volume (concentration) Increased injection volumes can result in a significant loss of resolution in UPLC-SEC

analyses.

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Effect of Salt Anion on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shapep

Different anions of sodium salt additive

©2012 Waters Corporation 55

Different anions of sodium salt additive Buffer: 10mM sodium phosphate, pH 6.8 and 200mM of additive ( unless otherwise noted) Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil

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Effect of Salt Cation on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shapep

Different cations of chloride salt additive

©2012 Waters Corporation 56

Buffer: 10mM sodium phosphate, pH 6.8 and 200mM of additive Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil

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Agendag

Size-Exclusion Chromatography– Theory and practiceTheory and practice

– ACQUITY UPLC Columns for SEC

o ACQUITY BEH200 SEC, 1.7 µm Columns

ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns

– Monoclonal Antibody Application

o SEC-MS Applications

– Insulin and Small Protein Applications

– Factors Influencing Component Resolution

– Considerations to extending column life

©2012 Waters Corporation 57

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UP-SEC Recommendations

H Class Bio system– Biocompatible system for the analysis of biological moleculesBiocompatible system for the analysis of biological molecules

o Eliminate system corrosion

o Best sample recovery

Limit sample adsorption• Limit sample adsorption

• Limit damage to molecules, especially oxidation

o Eliminate adduct formation in MS detection

– Based on ACQUITY UPLC H-Class System (Quaternary)

– True UPLC performance

– Compatible for all modes of chromatography

– Incorporates Auto●Blend Plus™ Technology

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Excipientsp

Added to increase protein stability, minimize protein-protein interactionsinteractions

Inhibit adsorption of proteins to vials

Can affect protein aggregation

Can affect biotherapeutic efficacy and immunogenicity

Common excipients– Carbohydrates (Sucrose, Treahlose)y ( , )

– Surfactants (Triton X-100, Polysorbate (Tween) 80, Polysorbate (Tween) 20, Brij 35, Puronic F-68)

– Human serum albuminHuman serum albumin

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Column Lifetime

9000

6000

7000

8000

9000

nt

3000

4000

5000

6000

USP

Pla

te C

oun

0

1000

2000

3000

Column and guard Column without guard

00 100 200 300 400 500 600 700 800 900 1000

Injection Number

Figure 1: Effect of using a 30 mm guard column on column efficiency for

©2012 Waters Corporation 60

Figure 1: Effect of using a 30 mm guard column on column efficiency for a monoclonal antibody. The arrows indicate where the guard column was changed.

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Effect of Column Guard on Lifetime :Monoclonal Antibody

0 24

y

0 28

No Guards Guards Replaced every 200 injections

0.18

0.20

0.22

0.24

0 20

0.22

0.24

0.26

0.28

Injection 2Injection 902

Injection 6Injection 488

AU0.12

0.14

0.16

AU

0 12

0.14

0.16

0.18

0.20

0.04

0.06

0.08

0.10

0 04

0.06

0.08

0.10

0.12

0.00

0.02

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50

0.00

0.02

0.04

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

©2012 Waters Corporation 61

mAb formulation with excipients (Tween 80)

Improved mAb peak tailing with use of guard column

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BEH200 SEC, 1.7um Care and Use:(Ways to extend column life)( y )

Preparation of SEC Mobile Phase and Needle Wash– Pre filter through <0 .2 um filter (i.e, Don’t inject particulates)

Use high purity water– Use high purity water– Replace mobile phases weekly and do not “top off”

Ramp up and down flow to column over 1min to minimize “bed shock”

Attention to SEC Eluent Inlet Filters– Use titanium, NOT stainless steel– Inlet filters can be major source of bacterial contaminationInlet filters can be major source of bacterial contamination

o Consider occasional sinker replacement or 70% alcohol “pull through” to prevent problems

Column Storage Considerations- Overnight: Continuously flush with the mobile phase at 10% of the maximum recommended flow rate- Extended: Store in the HPLC grade water with 10% methanol

©2012 Waters Corporation 62

Extended: Store in the HPLC grade water with 10% methanol

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Bacterial growth

0 12

0.14

g

Injection 6270.10

0.12

Injection 627

AU

0.06

0.08

Injection 100.02

0.04

Injection 10

0.00

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

©2012 Waters Corporation 63

System pressure increases slightly over lifetime (~50 psi) Analysis of frit indicated bacterial growth

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Interaction with Flow Cell under Native Conditions

0.015

Tit i 5 fl ll

AU

0.005

0.010 Titanium 5 mm flow cell

0.000

0.030 Standard Teflon AF 10mm flow cell

AU

0.010

0.02010mm flow cell

0.000

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00

SEC-PDA chromatogram of bovine serum album (BSA) (5 mg/mL in H20) shows the effect of flow cell

©2012 Waters Corporation 64

SEC PDA chromatogram of bovine serum album (BSA) (5 mg/mL in H20) shows the effect of flow cell material on peak shape. BSA monomer exhibits extensive peak tailing.

Conditions: 25mM sodium phosphate, 150mM sodium chloride, pH 6.8, 0.4 mL/min, Injection volume: 4 µL, Wavelength: 280 nm; Column: ACQUITY UPLC BEH200 SEC 1.7 µm column, 4.6 x 300mm

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Reference Material

Care and Use– Size Exclusion and Ion-Exchange Chromatography of Proteins using Size Exclusion and Ion Exchange Chromatography of Proteins using

the ACQUITY UPLC™ System,” 715002147, REV. A

– “Size Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC H-Class System, ” 715002909, Rev Athe ACQUITY UPLC H Class System, 715002909, Rev A

– “Controlling contamination in LC/MS and HPLC/MS Systems,” 715001307

– “Improving the Lifetime of UPLC Size-Exclusion Chromatography Improving the Lifetime of UPLC Size Exclusion Chromatography Columns Using Short Guard Columns,” Waters Technical Brief, 720004034en

– “Guidelines for Routine Use and Maintenance of Ultra-Performance Guidelines for Routine Use and Maintenance of Ultra Performance Size-Exclusion and Ion-Exchange Chromatography Systems”,Waters Technical Brief, 720004182en

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Summary:Waters ACQUITY UPLC SEC System Solution New SEC column chemistries in 125 and 200Å pore size based on

BEH particles

Reduced secondary interaction– Reduced secondary interaction

– Improved physical and chemical column lifetime

– Improved column-to-column reproducibility

– Improved resolution

– Improved throughput

UPLC-SEC provides improved resolution, sensitivity, and higher throughput as compared to tradition HPLC– Improved resolution of monoclonal antibody aggregates and clipped forms

Complete system solution includes column chemistry and system– UPLC columns specifically designed for bioseparations

– ACQUITY H-Class Bio System designed for the bioapplications

– Auto●Blend Plus™ Technology provides convenience and efficiency

©2012 Waters Corporation 66