Tips and Tricks' for Biopharmaceutical Characterization ... · CH. 2. CH. 2 O. Si EtO OEt Si O O OEt. O. Si O Si OEt O O OEt Et Et. n + Si EtO EtO. CH. 2. EtO. CH. 2. Si OEt OEt

©2012 Waters Corporation 1

'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical Characterization using SECCharacterization using SEC

Waters CorporationWaters Corporation


Waters Commitment

To develop, commercialize and market columns that, when used on Waters ACQUITY UPLC® systems, provide speed, sensitivity, resolution, and reproducibility not previously achieved for characterization of biological macromolecules by traditional HPLC.


Liquid Chromatography Protein Separation Modes

Protein Structure

Primary, Secondary, Tertiary Structure

Carbohydrate Groups

Hydrophobic Regions

Disulfide Linkages

HydrophilicGroups

Aromatic Groups

HydrogenBonding

Net Charge


Liquid Chromatography Protein Separation Modes

Protein Structure

Primary, Secondary, Tertiary Structure

Carbohydrate Groups

Hydrophobic Regions

Disulfide Linkages

HydrophilicGroups

Aromatic Groups

HydrogenBonding

Net Charge


Agenda

Size-Exclusion Chromatography– Theory and practice– ACQUITY UPLC Columns for SEC

o ACQUITY BEH450 SEC, 2.5 µm Columnso ACQUITY BEH200 SEC, 1.7 µm Columnso ACQUITY BEH125 SEC, 1.7um Columns

– Monoclonal Antibody Applicationo SEC-MS Applications

– Insulin and Small Protein Applications– Factors Influencing Component Resolution– Considerations to extending column life


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


Size Exclusion Chromatography

No adsorption to surface of particles

Large molecules elute before small molecules

Large molecules cannot access pores

Small molecules access pores within particle

dimermonomer


Monoclonal Antibodies

Antibody Conjugates

Fc Fusion Proteins

Synthetic Oligonucleotides

Protein Subunit Vaccines

Recombinant Proteins

Common SEC applications: Biotherapeutics Types


Agenda






UPLC Systems for Biopharmaceutical Analysis

Wide range of applications

Complete Solutions– Instrumentation

o UPLC System (s)• ACQUITY UPLC System• ACQUITY UPLC H-Class System• ACQUITY UPLC H-Class Bio System

o UV, FLR, PDA and MS Detectionso Application Specific Chemistries

• Developed and designed with applications• QC Tested with application• Optimized for UPLC

o Software • Data Analysis• Information management

Focused on customer application requirements


Columns and Instruments both Minimize Band Spreading

Broad BandBroad PeakLess SensitivityLess Resolving Power

HPLC

Advantages of UPLC Technology for SEC Separations

Narrow PeakIncreased SensitivityIncreased Resolving Power

Waters UPLC®

Technology


Effect of System Dispersion on ACQUITY UPLC BEH200 SEC 1.7 µm separation

Large system dispersion decreases resolution

Sample: Human polycolonal IgG

ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm

UP-SEC

ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm

HP-SEC

USP Res= 1.37

USP Res= 2.37

AU

0.00

0.10

0.20

0.30

AU

0.00

0.05

0.10

0.15

0.20

0.25

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


ACQUITY UPLC BEH200 and BEH125 SEC 1.7 µm Columns, and BEH450 SEC 2.5 µm Columns

Application Areas– Molecular weight ranges dependent on pore size:

o BEH450: 100,000 to 1,500,000 Daltonso BEH200: 10,000 to 450,000 Daltonso BEH125: 1,000 to 80,000 Daltons

– Determination of protein / peptide molecular weight

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

– Determination of size heterogeneity in a sample


BEHBEH™™ Technology ParticlesTechnology Particles Bridged EthylSiloxane/Silica HybridBridged EthylSiloxane/Silica Hybrid

Bridged EthanesIn Silica Matrix

Polyethoxysilane(BPEOS)

Si

EtO

O

CH2 CH2

Si O

Si

EtO

OEt

Si O

O

OEtO

Si

O

Si

OEt

O

OOEt

Et

Et

n

+ Si

EtOEtO

CH2EtO

CH2Si

OEt

OEtOEt

Tetraethoxysilane(TEOS)

Bis(triethoxysilyl)ethane(BTEE)

4 Si

EtO

EtO OEtEtO

1

Anal. Chem. 2003, 75, 6781-6788

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


HPLC to UPLC SEC Comparison

Murine monoclonal antibody - Scaled load Conditions: 0.4 mL/min; 25mM Sodium Phosphate, pH 6.8, 0.15 M NaCl

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

Minutes2.00 4.00 6.00 8.00 10.00

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

Minutes5.00 10.00 15.00 20.00 25.00 30.00

2.26 % Aggregate 2.24 %

Aggregate

HPLC 100% Silica-Diol

SEC 250Å 5µm7.8 x 300 mm

ACQUITY UPLC BEH200 SEC,1.7

µm4.6 x 300mm

8.00 30.008.00 30.00


Calibration Curves of ACQUITY UPLC BEH450, BEH200, and BEH125 SEC Columns


Protein Adsorption and SizeProtein Adsorption and Size-- Exclusion ChromatographyExclusion Chromatography

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)

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

Mobile phase additives (e.g., 150mM NaCl) may decrease non- desired ionic interactions between proteins and packing material


BEH SEC Particle Overview

The packing material is based on our patented Bridged Ethyl Hybrid base particle and effective diol bonding, which provide a stable chemistry with minimal secondary interactions.


Lysozyme, pKi = 10.7

Suggestive of DIOL Bleed

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

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

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

Injection 19Injection 618

Comparative SEC Column Life


Lysozyme, pI = 10.7


BEH200 shows minimal secondary interactions even after 600 injections

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

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

ACQUITY BEH200 SEC, 1.7 µm4.6 x 150 mm


Lysozyme, pKi = 10.7

Suggestive of DIOL BleedAU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

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

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


Comparative SEC Column Life


Lysozyme, pI = 10.7


Column StabilityColumn Stability

Calibration Curve

100

10000

1000000

0.5 1 1.5 2 2.5 3

Elution volume (mL)

Log

Mw

0 hours12 hours24 hours36 hours48 hours60 hours

Protein MWThyroglobulin 669000Ferritin 440000Aldolase 150000BSA 66000Ovalbumin 44000Carbonic Anhydrase 29000Ribonuclease A 13700Aprotinin 6500Uracil 112

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


AU

0.00

0.02

0.04

0.06

0.08

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

AU

0.00

0.02

0.04

0.06

0.08

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

AU

0.00

0.02

0.04

0.06

0.08

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

Influence of Ionic Strength on Peak Shape and Retention

Conventional 100% Silica-Diol Coated SEC Column 4.6 x 300 mm

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

10 mM

25 mM

100 mM

lysozyme

lysozyme

lysozyme


Influence of Ionic Strength on Peak Shape and Retention

AU

0.00

0.06

0.12

0.18

0.24

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

0.18

0.24

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

AU

0.00

0.06

0.12

0.18

0.24

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

0.18

0.24

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

ACQUITY BEH200 SEC 1.7 µm column, 4.6 x 150mm

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

10 mM

25 mM

AU

0.00

0.06

0.12

0.18

0.24

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

0.18

0.24

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

100 mM

lysozyme


BEHBEH125 SEC, 1.7um BatchBatch--toto--Batch Batch ReproducibilityReproducibility

AU

0.00

0.03

0.06

0.09

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

1

3

24

AU

0.00

0.03

0.06

0.09

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

AU

0.00

0.03

0.06

0.09

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

Batch 1

Batch 2

Batch 3

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

Analyte pl MW

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

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

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

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

BEH125 SEC Protein StandardPart No. 186006519


BEHBEH200 SEC, 1.7um BatchBatch--toto--Batch Batch ReproducibilityReproducibility



AU

0.00

0.05

0.10

AU

0.00

0.05

0.10

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

21

3 4 5

6

ACQUITY BEH200 SEC(300mm)

ACQUITY BEH200 SEC and BEH450 SEC(150mm + 150mm)

ACQUITY BEH450 SEC(300mm)

Compounds: 1. Thyroglobulin Dimer (1,340 KDa), 2. Thyroglobulin (667 KDa), 3. IgG (150 KDa), 4. BSA (66 KDa), 5. Myoglobin (17 KDa), 6. Uracil (112 Da)

2

1

3 45

6

2

1

3 4 5

6

Combining Pore Sizes for Added Method Development Flexibility



Column ReproducibilityColumn ReproducibilityAU

0.00

0.05

AU

0.00

0.05

AU

0.00

0.05

AU

0.00

0.05

AU

0.00

0.05

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

Batch 1, Column 1

Batch 1, Column 2

Batch 1, Column 3

Batch 2, Column 1

Batch 2, Column 2

Humanized monoclonal antibody

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

Retention times within 0.2min


Agenda






Monoclonal Antibody

Adapted from Alain BeckCenter of Immunology


Protein Structure

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

NNative

UUnfolded

orI

Intermediate

SolubleAggregates Insoluble

Aggregates

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


Orthogonal Techniques for Characterization

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

nm µm mm cm10 10 10100 100 100

monomersoligomers

subvisible particles Visible particles

Aggregates Particles

SEC

Static Light Scattering

FFF-MALS

AUC

Dynamic Light Scattering

MicroscopeVisual Inspection

Flow Imaging Microscopy

Counter principle

Light Microscopy


AU

-0.005

0.000

0.005

0.010

0.015

AU

-0.005

0.000

0.005

0.010

0.015

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

Column LifetimeColumn Lifetime

Humanized monoclonal antibody


Column: 4.6 x 300 mm

Injection 2

Injection 497

Dimer = 0.46%USP Res = 2.35

mAb

mAb

Dimer = 0.49%USP Res = 2.27


AU

0.000

0.010

0.020

0.030

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

AU

0.000

0.010

0.020

0.030

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

AU

0.000

0.010

0.020

0.030

Minutes2.00 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

Effect of Flow RateEffect of Flow Rate

Triplicate injections overlaidNo observable trend in aggregation with flow rate Murine monoclonal antibody (63 µg load)

0.4 mL/min

0.35 mL/min

0.2 mL/min

Flow 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


Effect of Particle Size:Effect of Particle Size: Analysis of LMW SpeciesAnalysis of LMW Species

AU

0.000

0.010

AU

0.000

0.010

AU

0.000

0.010

AU

0.000

0.010

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00

250Å, 4µmHPLC 100% Silica-Diol

BEH200 SEC 1.7µm

300Å, 5µmHPLC 100% Silica-Diol

290Å, 5µm HPLC 100% Silica-Diol

LMW peakmAb mAb dimer

LMW species

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


Agenda






Precautions in SECPrecautions in SEC--MSMS

Protein structure in solution depends on– pH– Ionic strength– Buffer and salt– Additives

Good ionization conditions are different from conditions for biological activity

Validation required when buffer is changed

Special uses are valuable– Fast desalting– Clips


AU

0.00

0.10

0.20

AU

0.00

0.10

0.20

0.30

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

LC/MS Compatible Mobile Phase on ACQUITY UPLC BEH200, SEC, 1.7um

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

100mM Ammonium Formate

PBS


PBS


AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

AU

-0.005

0.000

0.005

0.010

0.015

0.020

Minutes4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

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

1.25e-2

1.5e-2

1.75e-2 26.00

17.05

20.02

Humanized Monoclonal Antibody: MS Compatible/Native Mobile Phase

Flow Rates: 100mM Ammonium Formate - 0.15mL/min, PBS- 0.4 mL/min

Lower flow rate for MS compatibility


PBS


SEC-MS Humanized Monoclonal Antibody

MS: Xevo G2 Q Tof

Conditions: 100mM Ammonium Formate, Flow rate: 0.15 mL/min

Post UV detection additive: ACN, 0.8% Formic acid

UV @ 280

TIC

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

%

4

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

AU

0.0

2.0e-3

4.0e-3

6.0e-3

8.0e-3

1.0e-2

1.2e-2

1.4e-2

1.6e-2

1.8e-2

2: Diode Array 280 0.0500Da

Range: 6.757e-1

13.22

25.27

16.58

19.50

1: TOF MS ES+ TIC

7.58e619.49

15.35

16.62

23.7425.06

1 2

3

1

2

3


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

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

%

0

100

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

%

0

100

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

%

0

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

2353.5356

3530.13483706.4951

3801.68433901.6064

3905.8140

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+ 1.97e3

2652.85842648.5669

2460.41241251.3574

2801.4185

2968.89673154.9690

3370.08893616.4006

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

1985.9363

2056.27692166.3523 2382.8904

2647.5525

Extracted Spectrum

Deconvoluted molecular weight determined using MaxEnt1

Intact IgG MW 148,221Peak 1

ClipMW 100,764Peak 2

Low MW SpeciesPeak 3


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• No Sample Concentration required

HCMass Spectrum LC

Mass Spectrum

LC

HC

HC-HC

LC

HC-HC

HC

UV280

TIC

Conditions: System, ACQUITY UPLCTM with TUV optical detector and Synapt G2 QTof MSFlow Rate: 0.2 ml/min 0.1%TFA and 0.1%FA in 30% ACN


Agenda






- -

-

AU

0.00

0.50

1.00

1.50

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

AU

0.00

0.20

0.40

0.60

0.80

1.00

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

ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm

BioSuite125 UHR SEC 4.6 x 300mm

BSA

(66,

000)

Ova

lbum

in(4

4,00

0)Ca

rb. An

hyd .

(29

,000

)

Myo

glob

in(1

6,90

0)

Ubi

quiti

n(8

,565

)RA

SG (

898)

G-G

-G

(189

)G

-G (

132)

Ura

cil (

112)

A2

14

AU

0.00

0.50

1.00

1.50

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

AU

0.00

0.50

1.00

1.50

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

AU

0.00

0.20

0.40

0.60

0.80

1.00

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

AU

0.00

0.20

0.40

0.60

0.80

1.00

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

ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm

BioSuite125 UHR SEC 4.6 x 300mm

BSA

(66,

000)

Ova

lbum

in(4

4,00

0)Ca

rb. An

hyd .

(29

,000

)

Myo

glob

in(1

6,90

0)

Ubi

quiti

n(8

,565

)RA

SG (

898)

G-G

-G

(189

)G

-G (

132)

Ura

cil (

112)

A2

14

Resolution of Proteins and Peptides (Aqueous)

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

BEH125 column provides increased resolution throughout the lower end of the peptide mass range (132 29,000).


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

G-GG-G

-GRASG

Aprotin

inUbiq

uitin

Cytoch

rome C

Myoglo

binCarb

. Anh

yd.

Ovalbu

min

BSABSA D

imer

Insuli

n Mon

omer

Insuli

n Dim

er

Peptide/Protein

%R

SD/R

eten

tion

Tim

e R

ange

% RSD Retention Time Range

ACQUITY UPLC BEH125 SEC 1.7µm Column Reproducibility

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.


-

-

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00

BioSuite 125 4µm UHR 4.6 x 300 mm column

ACQUITY UPLC BEH125 SEC 1.7µm 4.6 x 300 mm column

USP Rs= 1.94

USP Rs= 3.29

Resolution of Small Protein

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


.

AU

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

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

AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

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

AU

0.00

0.10

0.20

0.30

0.40

0.50

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 = 3KFlow Rate = 0.5 mL/min

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

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 = 15KFlow Rate = 0.4 mL/min

ACQUITY UPLC BEH125 1.7µm(4.6 x 300 mm)

HMWP 10µm(7.8 x 300 mm)

HPLC/UPLC Column Comparison

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

(Waters HMWP) tested to perform in the European Pharmacopoeial method.

Increase in HMW resolution observed in shorter run-times


AU

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

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

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

AU

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

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

Injection 853

Injection 26

BEHBEH125 SEC, 1.7um Column Life Insulin Analysis

ACQUITY UPLC BEH125, SEC 1.7µm4.6 x 300 mm


0

1

2

3

4

5

6

0 100 200 300 400 500 600 700 800 900

Injection Number

Ret

entio

n Ti

me

(min

)

0.0

1.0

2.0

3.0

4.0

5.0

USP

Res

olut

ion

Retention Time USP Resolution

Column Stability for Insulin Analysis

Over 800 injections the retention time of the insulin monomer peak and the resolution between insulin monomer and dimer peaks are maintained.


AU

-0.026

-0.024

-0.022

AU

0.000

0.002

0.004

0.006

AU

-0.044

-0.042

-0.040

Minutes4.00 5.00 6.00

Minutes6.00 7.00 8.00 9.00

ACQUITY UPLC BEH125 SEC 1.7 µm

Control Control

Sample 1 Sample 1

Sample 2 Sample 2

Fragment

Effect of Pore Size: Insulin

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

ACQUITY UPLC BEH200SEC 1. 7µm


AU

-0.026

-0.024

-0.022

AU

0.000

0.002

0.004

0.006

AU

-0.044

-0.042

-0.040

Minutes4.00 5.00 6.00

ACQUITY UPLC BEH125, 1.7µm4.6 x 300mm

Minutes6.00 7.00 8.00 9.00 10.00

BioSuite125 UHR, 4µm4.6 x 300mm

Minutes14.00 16.00 18.00 20.00

Insulin HMWP, 10µm7.8 x 300mm

Control

Sample 1

Sample 2

Control

Sample 1

Sample 2

Control

Sample 1

Sample 2

Rs= 2.21

Rs= 1.93

Rs= 1.92

Rs= 2.08

Rs= 1.95

Rs= 1.88

Rs= 3.37

Rs= 2.63

Rs= 2.63

HMW

Fragment

Effect of Particle Size: Insulin

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


Agenda






Factors Influencing ResolutionFactors Influencing Resolution

Resolution increases with lower injection volumes

Resolution increases with lower 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 difference


AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50

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

Humanized IgG (20 mg/mL), 4.6 x 150 mm column


5 µL, 100µg

10 µL, 200µg

15 µL, 300µg

21.0%

21.2%

Aggregates

21.8%

Injection Volume, Total Load

15 µL, 300µg

Aggregates

21.8%

Injection Volume, Total Load


AU

-0.020

-0.010

0.000

0.010

0.020

0.030

0.040

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

AU

-0.020

-0.010

0.000

0.010

0.020

0.030

0.040

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

Effect of Column Length:Monoclonal Antibody

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

300 mm

150 mm98.88%

98.76%USP Res= 2.811.22%

USP Res=2.071.12%

mAbaggregates

mAbaggregates


AU

0.00

0.10

0.20

0.30

AU

0.00

0.05

0.10

0.15

0.20

0.25

AU

0.00

0.05

0.10

0.15

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

0.2 mL/minRs= 2.4~1500 psi

0.4 mL/minRs= 1.8~3000 psi

0.8 mL/minRs= 1.3~6000 psi

IgG dimer

Effect of Flow Rate on RsEffect of Flow Rate on Rs

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


AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

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

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

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

3.7815

3.0235

2.6550

USPInjection Volume

3.7815

3.0235

2.6550

USP Res

Injection Volume

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.80

1.90

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

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.80

1.90

Minutes3.50 4.0

04.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.80

1.90

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

3.231.25

3.150.625

3.262.5

3.2610

USP Res

Concentration

(mg/ mL)

3.231.25

3.150.625

3.262.5

3.2610

USP Res

Concentration

(mg/ mL)

Effect of Sample Load : Myoglobin

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)

Increased injection volumes can result in a significant loss of resolution in UPLC-SEC analyses.

Effect of Volume Load Effect of Concentration


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

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


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

Different cations of chloride salt additive

Buffer: 10mM sodium phosphate, pH 6.8 and 200mM of additive

Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil


Agenda






UP-SEC Recommendations

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

o Eliminate system corrosiono Best sample recovery

• 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


Excipients

Added to increase protein stability, minimize protein-protein interactions

Inhibit adsorption of proteins to vials

Can affect protein aggregation

Can affect biotherapeutic efficacy and immunogenicity

Common excipients– Carbohydrates (Sucrose, Treahlose)– Surfactants (Triton X-100, Polysorbate (Tween) 80, Polysorbate

(Tween) 20, Brij 35, Puronic F-68)– Human serum albumin


0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0 100 200 300 400 500 600 700 800 900 1000

Injection Number

USP

Pla

te C

ount

Column Lifetime

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.

Column and guard Column without guard


AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50

Effect of Column Guard on Lifetime : Monoclonal Antibody

mAb formulation with excipients (Tween 80)

Improved mAb peak tailing with use of guard column

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00



No Guards Guards Replaced every 200 injections


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

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– 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 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


Injection 10

Injection 627

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

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

Bacterial growth

Injection 10

Injection 627

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


AU

0.000

0.005

0.010

0.015

AU

0.000

0.010

0.020

0.030

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

Interaction with Flow Cell under Native Conditions

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

Standard Teflon AF 10mm flow cell

Titanium 5 mm flow cell


Auto•Blend Plus™ Technology AQUITY H-Class Bio

Program methods directly in units of pH and Molarity

Calculation of required proportions from physical constants– pH is calculated using Henderson-Hasselbalch equation with

pKa provided– Typically use pKa corrected for salt concentrationOR:– Empirical calibration table covering operating range of buffer

and salts selected

Proportions calculated at each pump stroke for best fidelity

Independent gradients for pH and salt concentration

Can produce a near true linear pH gradients as required


Instrument Control Method Auto•Blend™ to Auto•Blend Plus™

Click to Convertto Auto•Blend PlusTM


Typical Gradient Table Auto•Blend™ Plus


Auto•Blend Plus™ PH and Salt Gradients


Calibration Options Auto•Blend™ Plus

Select to Use Empirical rather than Henderson- Hasselbalch


Auto•Blend™ Plus Calibration Options - Delivered pH

6.35

6.45

6.55

6.65

6.75

6.85

6.95

7.05

7.15

1 2 3 4 5 6 7 8 9

MeasuredpH

Fraction Number

Programmed

pK

Corrected pK

Empirical


Auto●Blend Plus™ Method Development

AU

0.00

0.05

0.10

AU

0.00

0.06

0.12

AU

0.00

0.05

0.10

AU

0.00

0.05

0.10

AU

0.00

0.05

0.10

Minutes0.00 5.00 10.00 15.00

pH 6.1

pH 7.6

pH 7.1

pH 7.0

pH 6.9

A mixture of proteins was separated using cation exchange chromatography- alpha- Chymotrypsinogen A (peak A), Ribonuclease A (peak B), and Cytochrome C (peak C) –

A

A

A+B

A

A

B

B

B

B

C

C

C

C

C


SEC of Humanized Monoclonal AbSEC of Humanized Monoclonal Ab Effect of AutoBlend Plus pH AdjustmentEffect of AutoBlend Plus pH Adjustment

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

AU

0.00

0.10

0.20

0.30

0.40

0.50

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

A: 17.26%B: 2.74%C: 20.00%D: 60.00%

A: 2.74%B: 17.26%C: 20.00%D: 60.00%

pH6.0

pH7.6

125mM NaH2 PO4

125mM Na2H2 PO4

1000mM NaCLH2 O


0.000

0.005

0.010

0.015

0.000

0.005

0.010

0.015

0.000

0.005

0.010

0.015

0.000

0.005

0.010

0.015

Minutes4.00 5.00 6.00 7.00 8.00

Minutes4.00 5.00 6.00 7.00 8.00

Minutes4.00 5.00 6.00 7.00 8.00

UV

Ab

sorb

ance

@ 2

80

nm

150 mM 250 mM 350 mMpH

6.0

6.5

7.0

7.5

[NaCl]

HMW

Monomer

LMW 1 LMW2

Developing a Robust SEC Method using AutoBlend Plus


Reference Material

Care and Use– 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 A– “Controlling contamination in LC/MS and HPLC/MS Systems,”

715001307– “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 Size-Exclusion and Ion-Exchange Chromatography Systems”, Waters Technical Brief, 720004182en


Summary: Waters ACQUITY UPLC SEC System Solution

New SEC column chemistries in 125Å, 200Å and 450Å pore sizes based on BEH particles– 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


Acknowledgements

Paula Hong

Kenneth Fountain

Ed Bouvier

Sue Serpa

Bill Warren

Documents

Tips and Tricks' for Biopharmaceutical Characterization ... · CH. 2. CH. 2 O. Si EtO OEt Si O O OEt. O. Si O Si OEt O O OEt Et Et. n + Si EtO EtO. CH. 2. EtO. CH. 2. Si OEt OEt