1 christoph johann wyatt technology europe gmbh analytica conference 2012

1

Christoph Johann

Wyatt Technology Europe GmbH

Analytica Conference 2012

2

A new instrument to improve operation and performance in Flow Field-Flow Fractionation

Introduction

Flow-FFF Theory

Hollow-Fiber Flow-FFF and the Eclipse DUALTEC

Application Examples

Summary


3Analytica Conference 2012

Evolution of Eclipse Instruments

2002

2004

2006 Eclipse 2

.

2009 Eclipse 3+

5 generations of Eclipse instruments since 2002

2011 Eclipse DUALTEC

FFF Focus Day - ILSC 2011 4

molar mass vs. time

BSA - Standard__20070314__203_01gfedcb

time (min)4.0 6.0 8.0 10.0 12.0 14.0 16.0

mol

ar m

ass

(g/m

ol)

51.0x10

miniDAWN 199622 µg BSA

90° LS

Improvement of Signal Qualitymolar mass vs. time

02 BSA 10ug PLC10 350WSC Vc10 Vx30gfedcb BSA - Standard__20070314__203_01.MDFgfedcb

time (min)4.0 6.0 8.0 10.0 12.0 14.0 16.0

mo

lar

mass (

g/m

ol)

51.0x10

HELEOS II 20088.8 µg BSA

90° LS

miniDAWN 199622 µg BSA

.

Improved sensitivity: less sample required

Improved separation: more precise information


Separation takes place in the channel, the different steps of the process will be shown in detail

How Flow-FFF Separation Works


How FFF Separation Works II

FOCUS Step prepares the channel for injection of the sample


Injection of the sample in FOCUS+INJECT mode




Sample injected


Sample injected – by interaction with the cross-flow it is concentrated against the porous bottom wall



Sample injected – the Focusing – Relaxation process takes place



Transport in the channel and separation in ELUTION mode




Elution mode – the sample is transported towards the outlet of the channel



Elution mode – the sample components start to separate



Elution mode – the sample components separate more



Elution mode – the sample components stay close to the membrane throughout the elution process


Eclipse FFF Field-Flow Fractionation (FFF) Principle

Simulation of FFF separation of two particles 3 and 6 nm radius

Separation example.avi

3D-Animation of the separation in an AF4 channel

3D example.avi


Method Optimization

Simulation of FFF separation of two particles 3 and 8 nm in a laminar flow profile

Separation example.avi

Animation of a Flow-FFF Experiment in the Eclipse

Animation of the separation in 3-D


AF4 - Retention Equation

tr retention timew channel thicknessFCR cross-flow rateFOUT flow rate to the detectorDi diffusion coefficient

[1] R. N. Qureshi, Wim Th. Kok, LCGC Europe Jan 2010

ln [1]

Retention depends on the flow rate ratio only, not the length or width of the channel(with given Di and channel height)


AF4 Zone Broadening

st standard deviation of the peak in time units

uCR cross-flow velocity (cross-flow rate divided by channel area

[1]

Zone broadening depends only on instrumental parameters and not only on the ratio of flows, but also on the magnitude of the cross-flow rate


Peak dilution and analysis time

Optimization between improvement of resolution and minimization of TREQ and peak dilution is necessary

TREQ = 839 DILF = TREQ Minimum time required to obtain baseline

separation between two species that differ in Mw by a factor of 2

DILF Dilution factor between the concentration at the membrane c0 and the concentration in the detector c*

R. N. Qureshi, Wim Th. Kok, LCGC Europe Jan 2010 Separation example.avi


How to come up with a method

ln

TREQ = 839 DILF =


General conclusion

For high efficiency the preferred method should have

High cross-flow density

Small channel thickness w

Limiting factor is c0, the concentration at the membrane and detectability

Efficiency has to be sacrificed to avoid those problems

These facts can be illustrated using a simulation software based on FFF theory (called ISIS)


ExampleSample BSA monomer and dimer, 30 µg injected

Fractograms of BSA separation demonstrating that the TREQ is independent on channel length and spacer height, calculation taken from ISIS

SC 350 µm

LC 250 µm


Hollow-Fiber Flow-FFF (HF5)

Wyatt Technology introduces a new Flow-FFF instrument, the Eclipse DUALTEC.

Eclipse DUALTEC allows to apply HF5 and AF4 in one instrument.


Hollow-Fiber Flow-FFF (HF5)

J. Chromatogr. A, 1218 (2011) 4126-4131


Intoduction to HF5

HF5 has been pioneered in 1974 by Lee et al and further developed by Carlshaf and Jönsson in 1988, 1989 and Wim Kok et al.

Advantages:high efficiency (high plate numbers) and high sensitivity because of low peak dilution

low-cost channel, possibly disposable

Problem:Channel construction used to be tedious, no commercial products were available


The HF5 cross-flow is generated by the elution flow, which splits into a longitudinal and a radial direction: no depletion wall, only accumulation wall

Hollow-Fiber Flow-FFF (HF5) Principle

r

zVinrf

Cross-flow

Vout

Hollow Fiber

Channel tube

Cross-flow outlet

Inlet connection

(from injector)

Outlet connection (to

detector) .

A single fiber is sealed inside a PET housing, no glue is used. The sealing mechanism works for fibers of different diameters. (German patent 10 2010 043 877.4)

HF5 Channel Design


10 2010 043 877.4


Hollow Fiber Membranes

Hollow Fibers are a high-tech product, they are very reproducible and robust

Hollow Fiber membranes have ideal properties for FFF application in terms of flux, stability and lack of interaction with the sample, e.g. antibodies.


HF5 –AF4 Retention Equation

Retention R in Flow-FFF is a function of the mean layer thickness l

AF4 R = 6 lHF5 R = 4 l

In HF5 the same retention is achievedwith 33% lower concentration at themembrane.

This leads to reduced overloading and aggregation effects.

c0

c = ½ c0

l

.….


HF5 – AF4 Sensitivity and Efficiency

In Flow-FFF efficiency (maximum Plate Numbers N) depends mainly on cross flow velocity and retention time.

The HF5 fiber material is polysulfone, which has a very high flux (maximum cross flow rate per unit area). Therefore high cross flow rates are possible.

Peak dilution is proportional to detector flow rate, which can be smaller in HF5 because of low channel volume.

High plate number + low dilution = high sensitivity

.....


HF5 Limitations

HF5 requires low sample load.HF5 is not as flexible in terms of channel height and membrane material.

HF5 and AF4 complement each other, therefore we developed the DUALTEC principle.


HF5 Flow Schematic

The position of the focus point is determined by measuring the focus flow rate entering the channel

Technical specification of the flow schematic has been filed for patent in Germany No. 10 2010 041 222.8.

D etector

F lowContro ller

F low M eter

O utlet Port

In le t Port

M ain Pum p

W aste1

2

3 4

5

6

FocusNeedle Valve

PressureSensor

PressureSensor

C rossflowAutosam pler

Only one pump is needed to generate the inject flow and cross-flow


Focusing Visualization

Visual inspection is not necessary any more with the DUALTEC innovation.

.

FFF Focus Day - ILSC 2011

DUALTEC Autofocus

382011 Copyright Wyatt Technology Europe GmbH – All Rights reserved

The Eclipse DUALTEC

Integration with Agilent 1260 and ChemStation ® as well as with Dionex Ultimate andChromeleon®


Benefits of the Eclipse DUALTEC

Sensitivity

Productivity

Flexibility



Sensitivity

Productivity

Flexibility


Example – Carbonic Anhydrase

Separation of carbonic anhydrase, (1 mg/ml) with hollow fiber cartridge, injection amounts increase from 1 µg to 5 µg, UV signal at 280 nm

concentration vs. time

2 2010-11-08 CAH single vc 035 vx 85 ff 85 1 ul lHF10 50 mM NH4HCO3]gfedcb 4 2010-11-08 CAH single vc 035 vx 85 ff 85 2 ul lHF10 50 mM NH4HCO3]gfedcb6 2010-11-08 CAH single vc 035 vx 85 ff 85 3 ul lHF10 50 mM NH4HCO3]gfedcb 8 2010-11-08 CAH single vc 035 vx 85 ff 85 4 ul lHF10 50 mM NH4HCO3]gfedcb10 2010-11-08 CAH single vc 035 vx 85 ff 85 5 ul lHF10 50 mM NH4HCO3]gfedcb

time (min)8.0 10.0 12.0 14.0 16.0

con

cen

trat

ion

(g

/mL

)

0.0

-65.0x10

-51.0x10

-51.5x10

-52.0x10


Sensitivity comparison between HF5 and AF4

Carbonic anhydrase, peak height as a function of injection amount compared to calculated peak heights on AF4 channels SC and LC with 350 µm channel height

UV peak heights at 280 nm as a function of injection amount

y = 4,165x - 0,516

R2 = 0,9999

y = 1,9701x + 5E-15

y = 1,1493x + 2E-15

0

5

10

15

20

25

0 1 2 3 4 5 6

Injection amount [µg]

Pe

ak

he

igh

t [m

AU

]

HF ID 0.8 mm

HF calculated

AF4 SC calculated

AF4 LC calculated


Sensitivity with respect to light scattering signals

Peak height of the 90° LS signal for BSA monomer separated on HF5 and AF4 channel SC with 490 µm spacer – experimental data

Light scattering signal at 90 ° as a function of injection amount

y = 0,6102x + 0,2298

R2 = 0,9956

y = 0,0994x + 0,0182

R2 = 0,9998

0

0,5

1

1,5

2

2,5

3

3,5

0 5 10 15 20 25

Injection amount [µg]

Lig

ht

scat

teri

ng

sig

nal

at

90°

[mV

]

HF ID 0.8 mm

flat channel 490 µm


Separation of protein mix on HF5– peak identification

1

2

34

5

6

7

min0 5 10 15 20 25 30 35

mAU

0

5

10

15

20

25

30

VW D 1 A , W avelength=215 nm (PR O TEIN M IX\17112010\D EF_LC 2010-11-17 18-50-10\17112010_PM _013.D )

1

2

34

5

6

7

1. Carbonic anhydrase (1mer)2. BSA (1mer) + Carbonic anhydrase (2mer)3. Carbonic anhydrase (3mer)4. BSA (2mer)5. Apoferritin (1mer)6. Thyroglobulin (1mer) + Apoferritin (2mer)7. Apoferritin (3mer) + Thyroglobulin (2mer)

Total protein amount injected was 0.5 µg, longitudinal flow rate 0.2 ml/min, cross-flow rate 0.85 ml/min, UV signal 215 nm


Separation with narrow-bore fiber (250 µm radius)chromatograms

21 2011-08-18 Wte ab vc 022 vx 045 ff 022 015µg 50 mM PBS 150 mM NaCl pH 7,2

volume (mL)0.0 0.5 1.0 1.5

Rela

tive S

cale

0.0

0.5

1.0 UV

Peak half width 100 µlRetention time 3 minutesInjection amount 150 ng

Sample is a monoclonal antibody for therapeutic use


Monomer-Dimer Separation

chromatograms

21 2011-08-18 Wte ab vc 022 vx 045 ff 022 015µg 50 mM PBS 150 mM NaCl pH 7,2

volume (mL)0.4 0.6 0.8 1.0 1.2 1.4

Rela

tive S

cale

0.000

0.005

0.010

0.015

0.020UV

Sample is a monoclonal antibody for therapeutic use - dimer amount < 1% or < 2 ng



Sensitivity

Productivity

Flexibility


Eclipse DUALTEC - Productivity

Disposable channel cartridge for HF5 allows to install a new channel with minimal downtime. No operator skill is needed (in contrast to replacing a membrane).

AutoFocus places the focusing zone at the user defined place without manual intervention or visualization experiments.

Short run time is possible.

Low consumption of sample and solvent, which is important in method development

HF5 is ideal for temperature controlled operation.



Sensitivity

Productivity

Flexibility


Eclipse DUALTEC - Flexibility

Automatic switching between two separation modes is a standard feature.

Scale-up of separation in terms of sample load can be done without additional experiments by transferring the method from HF5 to AF4 using ISIS (Intelligent Separation Improvement System).

Possible combinations:

HF5 – AF4: method development vs. higher sample load

HF5 – HF5: doubles lifetime of the channel before manual replacement)

AF4 – AF4: two channel geometries can be compared

FFF – SEC: comparison to column separation


Applications


Analysis of MAb Aggregation

Characterization of MAb aggregation is of vital importance.

Separation of aggregates and their quantification is difficult and results may depend on the method applied (SEC, AF4).

HF5 results presented here on an MAb indicate that HF5 is well suited for aggregate quantification.


MAb IgG1 separated on HF5

Vial #2 (2 µg)

77,3% Recovery

18,9 % Recovery

3,95 % Recovery


HF5 AF4

0 5 10 15

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

UV

abs

orba

nce

@ 2

15 n

m(m

AU

)

Time (min)

2μg 5 μg 10 μg

Vial # 2

0 5 10 15

0

20

40

60

80

100

120

140

160

180

UV

abs

orba

nce

@ 2

15 n

m (m

Au

)

Time (min)

1μg 2 μg 5 μg 7 μg

Vial # 2

(21/01/2011) (28/01/2011)

Comparison of HF5 and AF4


Analysis of oligomers in IgG1

Although prepared from the same stock, two samples of the four aliquotes have got much higher amount of dimer and trimer.

Vial # 2&3

Vial # 1&4

zoom

FFF Focus Day - ILSC 2011

Separation of a mix of 3 latex samples, with 35, 50 and 100 nm radius with the HF5 channel, particle size determined with Multi-Angle Light Scattering (MALS)

Particle Characterizationgeometric radius vs. time

1 2011-03-08 latex 25 50 100 nm vc 065 vx 035__035 18 min 006 ff 035 4 min gr foc 5 min2 ul lhf2 02% SDSgfedcb

time (min)0.0 5.0 10.0 15.0 20.0 25.0

ge

om

etr

ic r

ad

ius

(n

m)

30.0

40.0

50.0

60.0

70.0

80.090.0

100.0

UV

A new instrument for Hollow-Fiber- (HF5) and asymmetrical Flow-FFF (AF4) is introducedHF5 is characterized by

High sensitivity coming from high plate numbers and low peak dilutionImproved productivity with a disposable channel, short analysis time and low eluent consumption

The Eclipse DUALTEC has the flexibility to switch between two separation modes, allowing specifically to combine FFF and SEC separation experiments for the same sampleThe new development makes Flow-FFF accessible for every chromatography user

Summary


1 christoph johann wyatt technology europe gmbh analytica conference 2012

Documents

flowfff experiment

sample slide

channel slide

crossflow rate slide

new flowfff instrument

isis slide

separate slide

method fff focus day