webex proteinas 290610 modificada - agilent.com · Fundamento del OFFGEL - Separación de...
Transcript of webex proteinas 290610 modificada - agilent.com · Fundamento del OFFGEL - Separación de...
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¿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!!
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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
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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
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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
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-Por tamaño: Exclusión molecular
3.Interacciones proteína-proteína
Interacción proteína-proteína
Señalización celular
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OFFGEL Bioanalyzer
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¿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?
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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
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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)
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Fundamento del OFFGEL
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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)
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ge ( )
Metodología de Trabajo
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Aplicación:
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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
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¿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?
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¿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 2010Page 21
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
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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
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Expresión de proteínas
Protein 230 kit
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Purificación de proteínas
Protein 230 kit
Se obtienen resultado de tamaño, pureza y concentración
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En un solo experimento, en 45 minutos y con 4µl de muestra
Estudio cinético de la eliminación del HIS-tag
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Análisis de la capacidad de las columnas
Protein 230 kit
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Análisis de anticuerpos
High sensitiveProtein 250 kit
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Glicosilación de proteínas
Protein 230 kit
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Control de calidad de Anticuerpos
Protein 230 kit
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Análisis de alimentos: Variedades de trigo
Protein 230 kit
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Análisis de proteínas en leche
Protein 80 kit
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Immunoprecipitation/Western Blot:IP/HSP-250IP/HSP 250
High sensitiveProtein 250 kit
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%
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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
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Geles en 2D: Combinación de Offgel y BioanalizadorBioanalizador
High sensitiveProtein 250 kit
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
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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
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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
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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)
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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
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5988-8322EN
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5989-3336EN
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Ion ExchangeBioHPLC ColumnsBioHPLC Columns
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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
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Anion Exchange ChromatographyHow it works
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• 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
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Cell lysates2D separations
Agilent Bio IEX
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• 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
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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
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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
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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
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Size Exclusion BioHPLC ColumnsBioHPLC Columns
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• 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
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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
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• 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
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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.
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Publication Number 5988-4022EN
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Reversed PhaseBioHPLC ColumnsBioHPLC Columns
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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
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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
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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
DAD1 C, Sig=215,4 Ref=off (D:\DATA\JINGMING\JZ031910\JZ031910 2010-03-19 11-40-48\021-0301.D)
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
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Monoclonal Antibody Peptide MapPoroshell 120 EC-C18, 3.0 x 150mm. 2.7um
mAU
80
100
120
DAD1 C, Sig=215,4 Ref=off (D:\DATA\JINGMING\JZ031910\JZ031910 2010-03-19 11-40-48\021-0201.D)
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
DAD1 C, Sig=215,4 Ref=off (D:\DATA\JINGMING\JZ031910\JZ031910 2010-03-19 11-40-48\021-0301.D)
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
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Bio-Monolith HPLC ColumnsColumns
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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
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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 RestrictedPage 79
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 RestrictedPage 80
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 RestrictedPage 81
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 RestrictedPage 82
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 RestrictedPage 83
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 RestrictedPage 85
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 RestrictedPage 86
5989-7839EN
Page 87
Publication Number 5988-9911ENPublication Number 5988 9911EN
Page 88
mRP (Macroporous Reverse Phase) Column
High Recovery Protein Fractionation
mRP Column
Agilent RestrictedPage 89
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 RestrictedPage 90
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 RestrictedPage 91
Publication Number 5989-0228EN
Page 92
Agilent BioSeparations Selection Guide
5990-3534EN
Currently beingCurrently being updated with new BioHPLC
l !columns!
February 2010Agilent Confidential
Page 94