webex proteinas 290610 modificada - agilent.com · fundamento del offgel - separación de...

¿Qué es la Proteómica?

-Proteina + Genoma…………..Proteoma(1994 MarckWilkins)

Proteoma

Dotación completa de t í i l d l Estructuraproteínas, incluyendo las

modificaciones hechas a un conjunto particular de proteínas, producidas por un

ProteómicaEstructura

Funciónorganismo o sistema

- 25.000-30.000 genes……………………………………500.000-1.000.000 proteínas!!

El estudio y comparación sistemáticos del proteoma en diferentes situaciones metabólicas y/o patológicas permite y p g pidentificar aquellas proteínas cuya presencia, ausencia o alteración se correlaciona con determinados estadios fisiológicos.g

Diagnosis+ BIOMARCADORES+

Evolución deenfermedades

Las principales áreas de estudio de la proteómica son:Las principales áreas de estudio de la proteómica son:

1 Id tifi ió d t í t i ió d1-Identificación de proteínas y caracterización de sus modificaciones postraduccionales

2-Proteómica de "expresión diferencial“

3-Estudio de las interacciones proteína-proteína

1-Identificación de proteínas y caracterización de sus modificaciones postraducionales:postraducionales:

Enorme complejidad(gran número).

-Técnicas más comunes:

A El t f i di i lA.Electroforesis monodimensional

SDS-PAGE…1ºDesnaturalizar 2ºCargar 3ºMigrar por peso molecular

B.Electroforesis bidimensional

2D-PAGE…Separación por PI + SDS-PAGE……Proteoma complejo

C.Cromatografía Líquida:

-Por hidrofobicidad; Columnas de Fase reversa.

-Por carga eléctrica: Columnas de intercambio iónico

Por tamaño: Exclusión molecular

-Por tamaño: Exclusión molecular

3.Interacciones proteína-proteína

Interacción proteína-proteína

Señalización celular

OFFGEL Bioanalyzer

¿Estas trabajando con muestras peptidicas o proteicas complejas?

¿Estas interesado en fraccionar grandes cantidades de estas muestras?

¿Estas interesado en obtener mejor sensibilidad en tu LC-MS por técnicas de prefraccionamiento?

¿Estas haciendo digestiones de proteinas in-gel?

¿Estas buscando variaciones en carga de la purificación de¿Estas buscando variaciones en carga de la purificación de proteínas?

Fundamentos del OFFGELProblemática

- Muestras complejas

- Rango dinámico de concentraciones muy amplio:

- Dificulta el fraccionamiento de proteínas de baja abundanciaTécnicas de Separación:Técnicas de Separación:

• Electroforesis convencional en gel 2D:

– Técnica tediosa– Teñido del gel, recorte, extracción manual– Poco reproducible, difícil de automatizar

• Electroforesis OffGEL:

– Técnica de prefraccionamiento nuevo patentado y desarrolladoconjuntamente por Agilent Technologies y Diagnoswiss

– Alta resolución y recuperación de analitos en disolución

Alta resolución y recuperación de analitos en disolución

Fundamento del OFFGEL- Separación de proteínas y péptidos de acuerdo a sus puntos isoeléctricos.

- Utiliza un gradiente de pH inmovilizado sobre un gel (IPG)

Se aplica un potencial las proteínas migran a través del gel hasta que alcanzan un pH = pI- Se aplica un potencial, las proteínas migran a través del gel hasta que alcanzan un pH = pI.

Punto Isoeléctrico: pH al cual la carga neta de la proteína es cero (se calcula en función del número de cadenas laterales básicas y ácidas)

Fundamento del OFFGEL

Instrumentación

Número Muestras: 2 bandejas/ 8 muestras cada • Controlador Local:juna

Fraccionamiento en 12 (baja Rs) ó 24 (Alta Rs)Volumen de cada fracción: 150 µlResolución: 0 1/0 6 pH

Controlador Local: – software preinstalado No requiere PC– métodos preconfigurados

• Datos corriente/voltajeResolución: 0.1/0.6 pHCapacidad de carga: 50 g – 5 mg/muestraTiempo de Fraccionamiento: 8 - 24 hControl de temperatura: 5- 60ºC

– Almacén/ exportacion en excel– Medida para cada muestra individual

• OFFGEL y electroforesis tradicional en gel (IEF)

ge ( )

Metodología de Trabajo

Aplicación:

Técnica OffGELAplicaciones

Respecto a otros productos de Agilent: Bien integrado en flujos de trabajo de proteómica de Agilent El resultado del fraccionamiento OFFGEL puede ser visualizado con el

bi li d 2100 f ibl LC MS á d SDSbioanalizador 2100, y es perfectamente compatible LC-MS o estándar SDS-PAGE

El pI de una fracción es un parámetro de identificación adicional (Spectrum Mill A.03.03 permite el uso del pI para la Autovalidación y como parámetro de bú d MS/MS)búsqueda en MS/MS).

Consumibles

¿Estas aun realizando geles de proteinas (SDS-PAGE) ?PAGE) ?

¿Necesitas precisión en cuanto al ¿ ptamaño,cuantificación y pureza en tus proteinas?

¿Necesitas comparar resultados?¿Necesitas comparar resultados?

¿Necesitas un método de análisis rápido que i d t di it l i di t t ?proporcione datos digitales inmediatamente?

¿Necesitas sensibilidad equiparable a la tinción de ¿ q pplata?

¿Estas realizando Western-blotting?

¿Estas realizando Western-blotting?

Analisis en gel de Protein & DNA / RNAProtein & DNA / RNA

• Proceso manual

• Dificultad de automatizar• Dificultad de automatizar

• Lento

• No suficiente precisión

• Mala reproducibilidad

• No comparación directa

• No datos digitales

La tecnología lab on-a-Chip integra el análisis típicamente hecho sobreGel y Fotómetros de UV para proporcionar datos digitalescualitativos y cuantitativos en 30-40 minutos.y

Bioanalizador 2100

Mejora la calidad del analisis en gel mediante la tecnología on-Chip electroforética… 1. Cargar muestras 2. Run Analsis 3. Analisis de datos

…reducie el experimento a:

Dispositivo analítico facil de usar que permite la separación y cuantificación del ARN,ADN y

1. Cargar muestras

proteinas basado en un chip de vidrio desechables para electroforesis .

En una sola plataforma se puede medir:

• Tamaño Cuantificación y Pureza de proteinas (5 -230 kDa)Tamaño, Cuantificación y Pureza de proteinas (5 230 kDa)• Tamaño, Cuantificación y Pureza de fragmentos de ADN(25 – 12000 bp)• Comprobar la integridad, separación y cuantificación de los ARNs• Ensayos de fluorescencia con células teñidas(Apoptosis, Transfección, Expresión, )

Agilent 15 Marzo 2010

2100 Bioanalyzer Hardware

Exchangeable cartridge for electrophoresis or flow

t t

16 pin electrodes connected to HV-sources

cytometry assays

Chip holder with heater plate

Selector Chip

Optics for detectionChip

Agilent

Lab-on-a-Chip

Control Activo de Fluidos (Microfluídica)

Volumen de Muestra 1 -4 µl

10 -12 muestras dependiendo de aplicación.p

Separación, tinción, detección de muestras.

Resultados en 5-30 minutos disponibles

No necesidad de un sistema desecho.

N t i ió d

Group/Presentation Title

No contaminación cruzada.

Tree view for navigation between samples and files

Task bar with context sensitive icons for different actions

Tabs for different data displays

Contextmenu bar Single gel lane menu bar g g

for selected E-gram

Customizable gel-like image( h d )

Access to setpoint explorer

(change order)

Customizable result tableCustomizable result table (change order and add additional columns)

Protein LabChip Kits – Specifications (Series II)• Analisis automatizado de 10 muestras de proteinas en menos de 30 minutos

• Dos aplicaciones para diferentes tamaños

– Protein 80: 5 a 80 kDaP t i 230 14 230 kD– Protein 230: 14 a 230 kDa

-Protein250: 10-250kDa

• Resolución del 10% sobre el rango de tamaño.

• Rango linear dinámico grande: (e.g. from 15 - 2000 ng/l CA-II in PBS)

• Sensivilidad equivalente a tinción no-coloidal con Coomassie (R-250)

• Cuantificación relativa y absoluta

• Compatible con variedad de tampones.

Month

p p

Staining, Destaining and DetectionTinción, Desteñido y Detección

detection

If no dilution was done the micelles would result in high background and low

detection

X

SDS + dye

sensitivity

proteina

desteñirSDS + dye

detección

proteina

micelas low background good signal to noise ratio SDS conc. b l CMCbelow CMC

Expresión de proteínas

Protein 230 kit

Purificación de proteínas

Protein 230 kit

Se obtienen resultado de tamaño, pureza y concentración

En un solo experimento, en 45 minutos y con 4µl de muestra

Estudio cinético de la eliminación del HIS-tag

Análisis de la capacidad de las columnas

Protein 230 kit

Análisis de anticuerpos

High sensitiveProtein 250 kit

Glicosilación de proteínas

Protein 230 kit

Control de calidad de Anticuerpos

Protein 230 kit

Análisis de alimentos: Variedades de trigo

Protein 230 kit

Análisis de proteínas en leche

Protein 80 kit

Immunoprecipitation/Western Blot:IP/HSP-250IP/HSP 250


Bioanalyzer

Immunoprecipitación: IP/HSP-2501 mg/ml E. coli Lysate+/- Target Protein(s)

His-tagged protein in E. coli background

Anti-His + Protein A magnetic beads

+/ Target Protein(s)

HSP 250 Protein Labeling

ImmunoprecipitationIncubation with specific Antibody0.01% of target cubat o t spec c t body

Incubation with Protein A Beads

Wash with Buffer (3x)

Elution with 50% HSP-250 Sample Buffer

g(-) control

N k f Abp

Direct On-Chip Analysis

No peaks from Abor capture protein!

2100 BioanalyzerHSP-250 AssayC. Wenz & A. Rüfer, Electrophoresis 2009, 30, 4264–4269

IP/HSP250 con GST-tagged PTEN en E. coli

0.1%* Before IP

PTEN control

0.01%

Zoom0.001%

Western Blot con PTEN-GST en E. coli

1 2 3 4 5 6 7

Western Blot

1 2 3 4 5 6 7

IP/HSP250High sensitiveProtein 250 kit

kDa

260

1601108060

kDa

240

150

605040

30

20

95

6345281520 15

LM

1: 1 % PTEN2: 0.1 % PTEN

Advantages of the IP/HSP-250 Method:3: 0.01 % PTEN4: 0.001 % PTEN5: 0.0001 % PTEN6: E. coli only7: PTEN only

- Sensitivity- Specificity- Time-to-result: 3 hours- Cost: less primary & no secondary antibodies

Comparación de métodos para cuantificación

Geles en 2D: Combinación de Offgel y BioanalizadorBioanalizador


E. coli lysate(50 g)

Isoelectric point (pI)

t

OFFGEL Electrophoresis4.3 4.8 5.3 5.8 6.3 6.7 7.2 7.7 8.2 8.7 OFFGEL well pH

kDalecu

lar w

eigh

t

Protein clean up and labeling

p kDa

100

70

50M

ol50

30

15

5

2100 BioanalyzerHiSens Assay

Protein Enrichment by OFFGEL Electrophoresis

OFFGEL load OFFGEL Fraction pH 4.8

bLG

OFFGEL load OFFGEL Fraction pH 4.8

1 % bLG1 % bLGbLG0.1 % bLG

0.01 % bLGno bLG

1 % bLG0.1 % bLG0.01 % bLGno bLG

3 x

Análisis de variedadesde trigo

Protein extractionAlkylation with IAAAcetone precipitatedLabeling at 10 ug/ul total protein (Bradford)de trigo

Wh t T A Wh t T B

Labeling at 10 ug/ul total protein (Bradford)100 ug labeled protein fractionated pH3-10Fractions undiluted analyzed with 250HSP-AssayIsoelectric point (pI)

ight

Wheat Type A Wheat Type B

Mol

ecul

ar w

eM

Mercado Biofarmacéutico

• La industria pharma se mueve de NCE A NBE*

• Aproximadamente 2,500 medicinas de biotech están en la fase de descubrimiento, 900 en pruebas preclínicas y más de 1 600 enpreclínicas y más de 1,600 en ensayos clínicos.

• Medicinas de anticáncer: más de 15001500.

*NCE = new chemical entity

*NBE = new biological entity

49

Mercado Biofarmacéutico

Métodos Cromatográficos; Para proteinas/péptidosseparaciones.

• Intercambio iónico análisis de Isoformas: Separaciones de carga base.carga base.

• Filtración en Gel: Separaciones por tamaño.• Fase reversa; Separaciones de proteinas intactas y mapeo

ídipeptídico.• IMAC (immobilized metal affinity chrom.)• Cromatografía de afinidad• Cromatografía de afinidad• Cromatografía de Interacción Hidrofobica (HIC)

50

Biologic Manufacturing and QA/QC (HPLC)

Bi R t P Bi l iBio-Reactor producing biologic Purification Step 1 Purification Step 2

Pure Biologic Formulation

QA/QC Analysis by LC/MS• Intact Analysis

– Glycosylation,

QA/QC Analysis by LC/UV

• Protein A: Titer Determination

• Ion Exchange: Analysis of acidic and b i f f bi l i i iti– Post Translational

Modification

• Peptide Mapping

– Enzymatic digestion of protein into peptides for

basic forms of biologic, impurities

• Size Exclusion: Analysis of aggregation and impurities

• Reverse Phase: Analysis of light and heavy chains glycosylation impurities

LC/MS verification of profile changes

p p pverification of composition

heavy chains, glycosylation, impurities

51

5988-8322EN

5989-3336EN

Ion ExchangeBioHPLC ColumnsBioHPLC Columns

54

Ion Exchange ChromatographyHow It Works

• Sample is injected in a mobile phase buffer with a low salt concentration – this binds proteins to the columnp

• Analytes are typically eluted at constant pH with increasing salt gradients (mobile-phase ionic strength) to displace the proteins from the stationary phasephase

• Higher charge proteins bind more strongly and an increased salt gradient is needed to elute themA typical mobile phase will contain NaCl• A typical mobile phase will contain NaCl

• The General Rule for choosing a Bio IEX column– Acidic proteins: SAX or WAX– Basic proteins: SCX or WCX

55

Anion Exchange ChromatographyHow it works

56

• Non-porous PS/DVB particles• Uniform polymeric coating with SCX, WCX, SAX, WAX

layers, designed for protein and peptide separations• Available in 10 µm, 5 µm, 3 µm, 1.7 µm particle sizes• High surface area• High capacity• Equal to or better selectivity for a variety of bio-

molecules compared other columns in the market.• Higher loading than most other analytical IEX vendors• Higher resolution separations of antibodies with smaller

particles• Possibility of faster and higher pressure separations

AntibodiesAntibodiesPeptides in volatile buffersGlycans (glycosylated bio-molecules)Proteins Oligos/PolynucleotidesC ll l t

Aplications

57

Cell lysates2D separations

Agilent Bio IEX

• Non-porous PS/DVB particles• Uniform polymeric coating and WCX layer,

specifically designed for antibody separations • Available in 10 µm, 5 µm, 3 µm, 1.7 µm particleAvailable in 10 µm, 5 µm, 3 µm, 1.7 µm particle

sizes• Equal to or better selectivity for monoclonal

antibodies compared to other columns in the marketmarket

• Higher resolution separations of antibodies with smaller particles

• Possibility of faster and higher pressure separationsseparations

59

Critical QA/QC Methods for Monoclonal AntibodiesSize Exclusion: Aggregation Analysis Ion Exchange: Charge Isoform Analysis

0.025

0.030

0.035 Monomer

AU

0.000

0.005

0.010

0.015

0.020

Dimer

Reversed Phase: Separation of light and heavy chains Peptide Mapping:

P t t l ti l difi ti

-0.005

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

Post-translational modifications

60

Ion Exchange ChromatographyCharge Isoform Analysis of Monoclonal Antibodies

0 50

0.55

0.60

AU

0.30

0.35

0.40

0.45

0.50

0 05

0.10

0.15

0.20

0.25 Acidic Isoforms Basic Isoforms

0.00

0.05

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 26.00 28.00 30.00 32.00 34.00

Column: Agilent Bio MAb, NP5, 4.6mm x 250mmBuffer A: 10 mM Sodium Phosphate, pH 7.50Buffer B: A + 100 mM NaCl, pH 7.50Gradient: 15-95% B in 60 minFlow rate: 0.8 mL/min.Sample: 5 L, 5 mg/mL, mAb

61

2-D HPLC: Cation Exchange and Reversed Phase Chromatography

WasteSCX (SEC)Digest pH < 3

ICAT

PeptidesProtein mixture1) Load peptides on SCX at 0% salt

2) Elute w/ increments of salt (0 1 M 1RP

2) Elute w/ increments of salt (0.1 M - 1 M) using injector program

3) a. Collect fractions and re-inject on RP column (OFF LINE approach)

Mass

RP column (OFF-LINE approach) or

b. Inject directly on RP column (ON-LINE approach) Mass

SpecMS/MS Data2D approach results in more resolved peptides than either

Data Analysissingle dimension Eluted and separated peptides are directly analyzed by MS/MS

62

Size Exclusion BioHPLC ColumnsBioHPLC Columns

63

• 5m Particle, polymeric coated• 100Å, 150Å, 300Å, 500Å, 1000Å,

2000Å pore sizes2000Å pore sizes

• Can be used at low salt concentrations, good for theconcentrations, good for the column, good your HPLC

• Better separation of high molecular weight proteins

• Broader selection than competitors• High stability and long lifetime• Great reproducibility

64

Agilent Bio SEC-5Stability at pH 8.5

Bio SEC-5, 300Å, 4.6x300mmBuffer: 150mM Phosphate, pH 8.5Wavelength: 214 nmFlow Rate: 0 35 mL/min

1. Thyroglobulin2. BSA3. Ribonuclease A4 Uracil

12

3 4

Pre-test, pH7.0

Flow Rate: 0.35 mL/minInjection: 3 uL

4. Uracil

Day 1 AM, pH8.5

Day 1 PM, pH8.5

Day 2 AM, pH8.5

Day 2 PM, pH8.5

Day 3 AM, pH8.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Day 7 AM, pH8.5

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

65

• Unique, 3m particle, polymeric coated• 100Å 150Å 300Å pore sizes• 100Å, 150Å, 300Å pore sizes• Higher resolution than 5µm particle• Higher efficiencies than 5 µm particles• Faster SEC separations• Can be used at low salt concentrations good for theCan be used at low salt concentrations, good for the

column, good your HPLC• Better separation of high molecular weight proteins

66

Agilent Bio SEC-3Improved Efficiency With Smaller Particles

Column Pressure Peak Protein SEC-3, 300Å SEC-5, 300Å

Å

Bio SEC-3(7.8x300mm)

1350 psi

Bio SEC-5(7.8x300mm)

650 psi

Peak Protein ,(7.8x300mm)

,(7.8x300mm)

1 Thyroglobulin 2460 1120

2 BSA Dimer 5100 2720

3 BSA 13090 6590

Column: Bio SEC-3 300Å and Bio SEC-5 300Å

Agilent Bio SEC-3, 300Å,7.8x300mm

3 BSA 13090 6590

4 Ribonuclease A 22000 11160

5 Uracil 38500 27860

Buffer: 150 mM Phosphate buffer, pH 7Flow rate: 1.0 mL/min for 7.8x300 mm Temperature: Ambient (~23° C)Detection: UV 214nm

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Agilent Bio SEC-5, 300Å,7.8x300mm

Injection: 10 µL (3 L for 4.6x300 mm)Sample: 1) Thyroglobulin (1.0 mg/mL), 670 kD; 2) BSA dimer, 132 kD; 3) BSA (1.0 mg/mL), 66 kD; 4) Ribonuclease A (1.0 mg/mL), 13.7 kD, and 5) Uracil(2 5 / L) 120D

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

(2.5 g/mL), 120D.

67

Publication Number 5988-4022EN

Reversed PhaseBioHPLC ColumnsBioHPLC Columns

69

Reverse Phase Mechanism of Bio-Molecules

Peptide in Mobile Phase w/hydrophobic footprint

Peptide adsorbs to Reverse Phase Surface

Peptide desorbs from psurface when organic modifier critical concentration is achieved

70

Reversed Phase Choices for Reversed Phase Separations of Biologics

For Smaller Bio-Molecules Like Peptides• StableBond 300 5µm and 3 5µm 300Å• StableBond 300, 5µm and 3.5µm, 300Å• Totally porous 1.8µm, 80-100Å• Poroshell 120, 2.7µm

For Intact Proteins• Poroshell 300, 5µm• StableBond 300, 5µm and 3.5µm, 300Å• Extend 300, 5µm and 3.5µm, 300Å

71

Poroshell 120 - Peptide Mapping

• Ideal, 120Å pore size for peptides mAU

100

120

DAD1 C, Sig=215,4 Ref=off (D:\DATA\JINGMING\JZ031910\JZ031910 2010-03-19 11-40-48\021-0201.D)

ptid

es

13 6,1

19,2

0

23

2526

27

8 30p p• 80-90% efficiency of sub 2um• At ~40-50% lower pressure

mAU

30

40

50

60

70

*DAD1 C, Sig=215,4 Ref=off (D:\DATA\JINGMING\JZ031910\JZ031910 2010-03-19 11-40-48\021-0101.D)

23

1

5, 6

7

glyc

opep

tides

10

11,1

213 14 15

,16

17

1819

20

2122

,23

2425 26 27

2829

031 32

35 3637

min0 5 10 15 20

0

20

40

60

80

100

2,3

4

1 5 7 glyc

ope

8 910

11

14,1

516 7

18 21 222

24

229 31 32

33 3435

3736

38

38

p• 2.7um particle size• 2um frit to reduce clogging

min0 10 20 30 40-10

0

10

20 4 8 9

2

30 33 34

2620

3

mAU

0

10

20

30

40

50


373635

34

3231

3029

28

27

252422

23211918

17

1416 159

1012

glyc

opep

tides

3823

45,

67 13 11

2620

• 600 bar pressure limit• The particle has a solid core

min0 20 40 60 80-10

(1.7um) and porous outer layer with a 0.5um diffusion pathpath

72

Monoclonal Antibody Peptide MapPoroshell 120 EC-C18, 3.0 x 150mm. 2.7um

mAU

80

100

120


opep

tides

13

,1 16,1

7

19,2

0

23

2526

27

28 30

35 368

20 min20 min

*DAD1 C, Sig=215,4 Ref=off (D:\DATA\JINGMING\JZ031910\JZ031910 2010-03-19 11-40-48\021-0101.D)min0 5 10 15 20

0

20

40

60

2,3

4

1 5 7 glyc

o

8 910

11

14,

5

18 21 22 24 29 31 3233 34

3733

mAU

20

30

40

50

60

70

23

1

5, 6

7

glyc

opep

tides

10

11,1

213 14 15

,16

17

1819

20

2122

,23

2425 26 27

2829

031 32

435 36

3738

50 50 minminOptimal run Optimal run

min0 10 20 30 40-10

0

10

20 4 8 9 30 33 34

2620mAU50


25s 2620

Optimal run Optimal run time most time most

peaks peaks resolvedresolved

10

20

30

40

50

373635

34

3231

3029

28

27

22422

23211918

17

1416 159

1012

glyc

opep

tides

3823

45,

67 13 11

100 min100 min

min0 20 40 60 80-10

0

73

Bio-Monolith HPLC ColumnsColumns

74

Agilent Bio-Monolith HPLC Columns• Analytical separations of macro bio-molecules: viral

ti l DNA tib di (I G d I M) d th

particles, pDNA, antibodies (IgG and IgM) and other large proteins

• Polymer-based, monolith disk columns for rapid on-off chromatographic separations of large molecules

• Monolith disc is 5.2 mm in diameter and 4.95 mm in l th dilength disc.– continuous channels, eliminating diffusion mass

transfer– convective mass transfer, a few orders of

magnitude faster than diffusive transport

• The large channels (1200-1500nm) enable excellent separation power and flow characteristics, especially for the separations of large bio-molecules

• Flow-rate Independent separation, no diffusion, no d id lpores and no void volume

75

Bio-Monolith Phases and Target ApplicationsA il Bi M li h HPLC C l A li iAgilent Bio-Monolith HPLC Column Applications

Column Description Key Applications Bio-Monolith QA

The quaternary amine bonded phase (Strong Anion Exchange) is fully charged over a working pH range of 2-13, binding bio-molecules with negative charges The Bio-Monlith QA column is designed for

HSA Purity Adenovirus process monitoring

and quality control Monitoring DNA imp ritcharges. The Bio-Monlith QA column is designed for

a wide range of applications, particularly process monitoring and quality control of viruses, DNA and large proteins.

Monitoring DNA impurity removal

Monitoring endotoxin removal IgM purification monitoring and

quality control

Bio-Monolith DEAE

The diethylaminoethyl bonded phase (Weak Anion Exchange) offers increased selectivity of bio-molecules with negative charge over a working pH range of 3-9. Applications include process monitoring and quality control of bacteriophage and plasmid DNA purifications.

Process monitoring and quality control of bacteriophage purification

Process monitoring and quality control of plasmid DNA purification p p

p

Bio-Monolith SO3

The sulfonyl bonded phase (Strong Cation Exchange) is fully charged over a working pH range of 2-13, binding bio-molecules with positive charges. Applications include fast and high resolution

l ti l ti f l bi l l h

Fast and high resolution analytical separations of large molecules such as proteins, antibodies

analytical separations of large bio-molecules such as proteins and antibodies.

Hemoglobin A1c fast analytics

Bio-Monolith Protein A

The Protein A affinity column is designed for the analytical separation of all IgG (human and mouse), except for IgG class3. Enables rapid separation and quantitation of IgG

Quantitative Determination of Human IgG (fermentation titer calculation)

quantitation of IgG.

76

Bio-Monolith Protein A Column: Rapid IgG Quantification in Cell Culture Production and Purification Process Monitoring

190

240

]

1mL/min2 mL/min

The Calibration Curvey = 728.55xR2 = 0.9975

1400

1600

90

140

Rel

ativ

e A

bsor

banc

e [m

AU

]

400

600

800

1000

1200

Peak

Are

a

Th I G l ti k d t t

-10

40

0 0.5 1 1.5 2 2.5

Time [min]

0

200

0 0.5 1 1.5 2 2.5

C [mg/mL]

• Figure 3 illustrates results from a 2-fold dilution series of human IgG concentrate.

• The initial IgG concentration was 2 mg/mL. IgG peak areas were integrated and plotted.

• The linear range of the assay easily covers the production ranges necessary to accommodate

• The IgG elution peak was measured at two flow rates, 1 ml/min and 2 ml/min that correspond to 10 column volumes/min and 20 column volumes/min, respectively.

• The results about the presence and theproduction ranges necessary to accommodate developmental and manufacturing cell cultures.

The results about the presence and the concentration of IgG in the sample can be reliably obtained within minutes.

77

Multiple AffinityMultiple Affinity Removal System

http://www.chem.agilent.com/en-us/products/consumables/columns/lcandlc-

78

ms/multipleaffinityremoval-human14/pages/default.aspx

Protein Biomarker Discovery and Validation

Cancer Cells

Blood Vessel WallBlood Vessel Wall

High Abundant Protein (Albumin)

Leached Protein (Potential Biomarker)

Agilent Restricted

Protein Biomarker Application

Sample Prep Simplify/DepleteExtraction of unique proteins from samples and simplify sample for analysis

Extract protein from Blood serum/plasma samples Extract protein from tissue or blood with

FFPE Protein Extraction Solution or PPS Silent

Surfactant

Blood serum/plasma samples (or CSF, urine) deplete high-

abundant proteins with Multiple Affinity Removal

System

Fractionate Separate/LC Prep IdentifyReduction of complexity and enrichment

3100 OFFGEL

Fractionate, concentrate, and desalt proteins with mRP-C18 3100 OFFGEL

Agilent 1200 Series Liquid

Chromatography

Agilent 6000 Series Mass

Spectrometry w/ HPLC Chi

Quality Control

with mRP-C18

Digest with Proteomics Chromatography

2100 Bioanalyzer HPLC-ChipGrade Trypsin

Agilent Restricted

Agilent Multiple Affinity Removal SystemFY2004 FY2006FY2005 FY2007

“Original” Top-6

FY2004 FY2006FY2005 FY2007

Human Serum

Spin Tube format

“High Capacity” Top-6 Human Serum

Level-II Human-14“High Capacity”

Top-7 Human PlasmaSpin Tube format

Mouse-3Spin Tube format

Original MARS

• Simple two buffer system• 10 minute protocolOriginal MARS

Increasing Capacity and Depth While Maintaining Performance

• 10 minute protocol• Reproducible• Low collection volumes• Only need centrifuge

Selectivity (specific Ab-Ag recognition, no Pr-Pr complex) Reproducibility (run-to-run and lot-to-lot of product) Reliability and increased productivity (quick and easy) Recovery of sample (minimal loss)

• Robust

y p ( )

Agilent Restricted

Human-14 Column - Aids in analysis of plasma/serum proteome

LL L

L LL L

H

H

LLLH

HH

H HHH LL

LL

plasma/serum proteome

L L

IgGTransferrin

Fibrinogen

H H HL

Human-7 Human-14IgG Fibrinogen

1-Antitrypsin

IgA

1-Acid Glycoprotein

Haptoglobin Complement C3Transthyretin

Apolipoprotein AII

6% Remaining for Analysis

15% Apolipoprotein AI

Albumin2-Macroglobulin

IgM

94% f HAP d l t dO l 85% f HAP d l t d 94% of HAP depletedOnly 85% of HAP depleted

Agilent Restricted

Multiple Affinity Removal System

LLL

Low Abundant Proteins

Total Serum/Plasma Protein

LL LL

LLLL LLLL

L

HL

LH H

H

H

HH

LL

HHLL

LLHH HH

HH

HH

HHHH

HHHHH

H

HHHHHHHHHH

HH

LL LL

LLLL LLLL

HHHLH

H HHH H

LLHH

HH HHHHHH HH

HH HH HH

HHHH HHHH HHHH

HH HH HH

HHHH

HHHH HHHH HHHH

HHHHHHHH

HHH

High Abundant Proteins

Agilent Restricted

OFFGEL Incremento de la sensibilidad en MSejemplo de trabajo

OFFGEL

ejemplo de trabajo

Incremento de 4x en la detección de proteínas trasel fraccionamiento con elel fraccionamiento con el OFFGEL

Serum Proteomics Workflow – Front to Back

Blood Sample from Patient

Serum/Plasma sent to lab

Remove High Abundant Protein

Fractionation of Proteins via

mRP

Fractionation of Proteins and/or

Trypsin enzyme di ti f1 D (Reverse Phase) Proteins and/or

Peptides via OGE

digestion of proteins into

peptides

1-D (Reverse Phase) or 2-D (Ion Exchange

+ Reverse Phase) Chromatography

Mass Spectrometer + Software ID peptides and verify presence of protein (quantitation)

Agilent Restricted

Overlay of chromatograms from run 1, 50, 100, 150 & 200 on a Human 14 column

No1rm.

2500

Bound Fraction4.6 mm ID x 100 mm column

2000 Flow-throughFraction

Column performs reproducibly for 200+ runs

1000

1500

Plasma Injection

Elution

500

1000 Elution1 ml/min

Re-equilibration0.125 ml/min

min0 5 10 15 20 25 30 35

0

min0 5 0 5 0 5 30 35

Great reproducibility after 200 uses – nearly identical performance

Agilent Restricted

5989-7839EN

Publication Number 5988-9911ENPublication Number 5988 9911EN

mRP (Macroporous Reverse Phase) Column

High Recovery Protein Fractionation

mRP Column

Agilent Restricted

mRP-C18 Protein Fractionation ColumnR Ph l f t i ti dReverse Phase column for protein separation and fractionation. The silica based particles and recommended LC methods have been optimized for:

• Highest recoveries of protein samples (95% - 99% of loaded sample)• Highest resolution separationsg p• Reproducibility• High sample loading capacity (3X higher than most standard RP columns))

Key Applications:• Positioned to be used after MARS protein depletion for further fractionationfractionation • Will simultaneously concentrate & de-salt (mass spec ready)• Used for variety of sample types and purposes (whole cell lysates and for recovery of membrane protein fractionation)lysates and for recovery of membrane protein fractionation)

Agilent Restricted

Comparison of mRP with ZORBAX SB300-C8

mRP-C18mAU

30

A li iA1

hemopexin

SB300-C8Norm.

40

15

20

25Acid-glycoprotein ApolipoproteinA1

complement component C420

30

sorb

ance

(280

nm

)

banc

e (2

80 n

m)

0

5

10

i0 10 20 30 40 0

0

10

Ab

Abs

orb

Sample: 270ug flow-through (6M urea/5.0% AcOH) of immunodepleted human serum from Multiple Affinity Removal System columnColumns: Panel A – Zorbax SB300-C18 (300 A, 5.0um), 4.6 mm x 50 mm i.d., SS; Panel B – mRP-C18 (macroporous, 5um), 4.6 mm 50 i d PEEK 0 75 L/ i DAD 280

Sample: 270ug flow-through (6M urea/5.0% AcOH) of immunodepleted human serum from Multiple Affinity Removal System columnColumns: Panel A – Zorbax SB300-C18 (300 A, 5.0um), 4.6 mm x 50 mm i.d., SS; Panel B – mRP-C18 (macroporous, 5um), 4.6 mm

min0 10 20 30 40 50

Retention Timemin0 10 20 30 40 50

Retention Time

x 50 mm i.d., PEEK, 0.75mL/min., DAD 280nmMobile Phase & Conditions: A-0.1% TFA/water, B-0.08%TFA/ACN, Temp 80° C, gradient:5-30%B in 5min., 30-55%B in 33min., 55-100%B in 4min.1D SDS PAGE: Collected 36 fractions (1.0 min. time slices) from immunodepleted human serum RP separation

( ) ( p )x 50 mm i.d., PEEK, 0.75mL/min., DAD 280nmMobile Phase & Conditions: A-0.1% TFA/water, B-0.08%TFA/ACN, Temp 80° C, gradient:5-30%B in 5min., 30-55%B in 33min., 55-100%B in 4min.1D SDS PAGE: Collected 36 fractions (1.0 min. time slices) from immunodepleted human serum RP separation

Significant improvement in resolution – more defined fractions

Agilent Restricted

Publication Number 5989-0228EN

Agilent BioSeparations Selection Guide

5990-3534EN

Currently beingCurrently being updated with new BioHPLC

l !columns!

February 2010Agilent Confidential

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