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1 Christoph Johann Wyatt Technology Europe GmbH Analytica Conference 2012.

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Presentation on theme: "1 Christoph Johann Wyatt Technology Europe GmbH Analytica Conference 2012."— Presentation transcript:

1 1 Christoph Johann Wyatt Technology Europe GmbH Analytica Conference 2012

2 2 Introduction Flow-FFF Theory Hollow-Fiber Flow-FFF and the Eclipse DUALTEC Application Examples Summary Analytica Conference 2012

3 3 Evolution of Eclipse Instruments Eclipse Eclipse 3+ 5 generations of Eclipse instruments since Eclipse DUALTEC

4 FFF Focus Day - ILSC miniDAWN µg BSA 90° LS Improvement of Signal Quality HELEOS II µg BSA 90° LS miniDAWN µg BSA. Improved sensitivity: less sample required Improved separation: more precise information

5 Analytica Conference Separation takes place in the channel, the different steps of the process will be shown in detail How Flow-FFF Separation Works

6 Analytica Conference How FFF Separation Works II FOCUS Step prepares the channel for injection of the sample

7 Analytica Conference Injection of the sample in FOCUS+INJECT mode How Flow-FFF Separation Works

8 Analytica Conference How Flow-FFF Separation Works Sample injected

9 Analytica Conference Sample injected – by interaction with the cross-flow it is concentrated against the porous bottom wall How Flow-FFF Separation Works

10 Analytica Conference Sample injected – the Focusing – Relaxation process takes place How Flow-FFF Separation Works

11 Analytica Conference Transport in the channel and separation in ELUTION mode How Flow-FFF Separation Works

12 Analytica Conference How Flow-FFF Separation Works Elution mode – the sample is transported towards the outlet of the channel

13 Analytica Conference How Flow-FFF Separation Works Elution mode – the sample components start to separate

14 Analytica Conference How Flow-FFF Separation Works Elution mode – the sample components separate more

15 Analytica Conference How Flow-FFF Separation Works Elution mode – the sample components stay close to the membrane throughout the elution process

16 Analytica Conference 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

17 Method Optimization FFF Focus Day - ILSC Simulation of FFF separation of two particles 3 and 8 nm in a laminar flow profile SeparationSeparation example.avi Animation of a Flow-FFF Experiment in the EclipseExperiment Animation of the separation in 3-D

18 FFF Focus Day - ILSC AF4 - Retention Equation t r retention time wchannel thickness F CR cross-flow rate F OUT flow rate to the detector D i diffusion coefficient [1] R. N. Qureshi, Wim Th. Kok, LCGC Europe Jan 2010 [1] Retention depends on the flow rate ratio only, not the length or width of the channel (with given D i and channel height)

19 AF4 Zone Broadening t standard deviation of the peak in time units u CR cross-flow velocity (cross-flow rate divided by channel area FFF Focus Day - ILSC [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

20 Peak dilution and analysis time Optimization between improvement of resolution and minimization of TREQ and peak dilution is necessary FFF Focus Day - ILSC TREQMinimum time required to obtain baseline separation between two species that differ in Mw by a factor of 2 DILFDilution factor between the concentration at the membrane c 0 and the concentration in the detector c* R. N. Qureshi, Wim Th. Kok, LCGC Europe Jan 2010 SeparationSeparation example.avi

21 How to come up with a method FFF Focus Day - ILSC

22 General conclusion For high efficiency the preferred method should have High cross-flow density Small channel thickness w FFF Focus Day - ILSC Limiting factor is c 0, 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)

23 Example F CR (ml/min) u CR (10 5 m/s) F OUT (ml/min) TREQ (min) DILFPeak Width (min) SC 350 µm LC 250 µm FFF Focus Day - ILSC Sample BSA monomer and dimer, 30 µg injected

24 Example FFF Focus Day - ILSC Sample 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

25 FFF Focus Day - ILSC 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.

26 FFF Focus Day - ILSC Hollow-Fiber Flow-FFF (HF5) J. Chromatogr. A, 1218 (2011)

27 27 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 FFF Focus Day - ILSC 2011

28 28 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 z V in rfrf Cross-flow V ou t Hollow Fiber Channel tube Cross-flow outlet Inlet connection (from injector) Outlet connection (to detector). FFF Focus Day - ILSC 2011

29 A single fiber is sealed inside a PET housing, no glue is used. The sealing mechanism works for fibers of different diameters. (German patent ) HF5 Channel Design Analytica Conference

30 30 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. FFF Focus Day - ILSC 2011

31 31 HF5 –AF4 Retention Equation Retention R in Flow-FFF is a function of the mean layer thickness AF4 R = 6 HF5 R = 4 In HF5 the same retention is achieved with 33% lower concentration at the membrane. This leads to reduced overloading and aggregation effects. c0c0 c = ½ c 0.….

32 FFF Focus Day - ILSC 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.....

33 FFF Focus Day - ILSC HF5 – AF4 Channel Comparison Yellow: cross flow rate at constant flow density Green: maximum feasible cross flow density HF5 AF4HF5

34 FFF Focus Day - ILSC 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.

35 FFF Focus Day - ILSC 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 Only one pump is needed to generate the inject flow and cross-flow

36 FFF Focus Day - ILSC Focusing Visualization Visual inspection is not necessary any more with the DUALTEC innovation..

37 DUALTEC Autofocus FFF Focus Day - ILSC 2011

38 2011 Copyright Wyatt Technology Europe GmbH – All Rights reserved 38 The Eclipse DUALTEC Integration with Agilent 1260 and ChemStation ® as well as with Dionex Ultimate and Chromeleon®

39 FFF Focus Day - ILSC Benefits of the Eclipse DUALTEC Sensitivity Productivity Flexibility

40 FFF Focus Day - ILSC Benefits of the Eclipse DUALTEC Sensitivity Productivity Flexibility

41 FFF Focus Day - ILSC 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

42 FFF Focus Day - ILSC Carbonic Anhydrase Separation Results *Recovery of the SEC separation on a Superdex column was 70%, indicating the protein purity of the standard is 70%. Recovery was calculated using the UV detector response at 280 nm wavelength with an extinction coefficient of 1600 ml/(g cm).

43 FFF Focus Day - ILSC 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

44 FFF Focus Day - ILSC 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

45 FFF Focus Day - ILSC Separation of protein mix on HF5– peak identification 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

46 FFF Focus Day - ILSC Separation with narrow-bore fiber (250 µm radius) Peak half width100 µl Retention time 3 minutes Injection amount 150 ng Sample is a monoclonal antibody for therapeutic use

47 Monomer-Dimer Separation FFF Focus Day - ILSC Sample is a monoclonal antibody for therapeutic use - dimer amount < 1% or < 2 ng

48 FFF Focus Day - ILSC Benefits of the Eclipse DUALTEC Sensitivity Productivity Flexibility

49 FFF Focus Day - ILSC 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.

50 FFF Focus Day - ILSC Benefits of the Eclipse DUALTEC Sensitivity Productivity Flexibility

51 FFF Focus Day - ILSC 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

52 2 inlet ports to the channel Tip-Injection AF4 Hollow-Fiber Flow-FFF (HF5) Software controllable selection between two separation modes Eclipse Flow-FFF Overview FFF Focus Day - ILSC inlet ports to the channel Central down stream injection (CDI) AF4 Semi-Preperative Channel Frit-Inlet AF4 (FI-AF4) Metal free system

53 Applications FFF Focus Day - ILSC

54 FFF Focus Day - ILSC 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.

55 FFF Focus Day - ILSC Aggregation behavior of MAb IgG1 Sample is a monoclonal antibody for therapeutic use currently on the market. A stock sample with 38 mg/ml was taken thawed from -80°C, diluted 1:100 in mobile phase (PBS 10 mM, NaCl 25 mM) and again frozen at - 20 °C and aliquots analyzed after different storage time. Vial 4°C analyzed the same day Vial -20°C for 4 days Vial -20°C for 8 days Vial -20°C for 11 days

56 FFF Focus Day - ILSC MAb IgG1 separated on HF5 Vial #2 (2 µg) 77,3% Recovery 18,9 % Recovery 3,95 % Recovery

57 FFF Focus Day - ILSC HF5 AF4 2μg 5 μg 10 μg Vial # 2 1μg 2 μg 5 μg 7 μg Vial # 2 (21/01/2011) (28/01/2011) Comparison of HF5 and AF4

58 FFF Focus Day - ILSC 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

59 FFF Focus Day - ILSC Measurements performed on the same sample (Vial # 2), 2μg After three hours After three days The amounts of 2 mer and 3 mer progressively descrease with elapsing time while the 1 mer increases. Reversible aggregates ? (2 mer & 3 mer ) UV 215 nm First day Comparison of IgG1 oligomer concentration with time

60 Eclipse Frit-Inlet AF4 Frit-Inlet AF4 is an alternative way to achieve separation with AF4 without a focusing step A frit is placed at the inlet in the upper plate The sample is injected through the tip of the channel A flow stream equivalent to the cross-flow is introduced through the top-frit Relaxation is achieved on the fly without stopped-flow focusing FFF Focus Day - ILSC

61 FI-AF4 – Relaxation Principle Relaxation is achieved while the sample is transported throught the first section of the AF4 channel with the frit inlet flow coming from the top FFF Focus Day - ILSC

62 Resolution of FI-AF4 Separation efficiency is a little less compared to focusing AF4, shown is a separation of BSA monomer and dimer FFF Focus Day - ILSC

63 Possible problem of sample aggregation in AF4 Conventional AF4 can lead to sample aggregation. Example of increasing molar mass with higher sample load of hyaluranic acid (data taken from poster presented at the ILSC 2011) FFF Focus Day - ILSC

64 Benefit of FI-AF4 FI-AF4 minimizes the problem of sample aggregation. Hyaluranic acid sample does not increase in molar mass with higher sample load (data taken from poster presented at the ILSC 2011) FFF Focus Day - ILSC

65 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 Characterization FFF Focus Day - ILSC 2011

66 A new instrument for Hollow-Fiber- (HF5) and asymmetrical Flow-FFF (AF4) is introduced HF5 is characterized by High sensitivity coming from high plate numbers and low peak dilution Improved 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 sample The new development makes Flow-FFF accessible for every chromatography user Summary Analytica Conference 2012


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