Instructions For Use USD 2937

ScreenExpert RoboColumn®

For High Throughput Chromatography Sorbent Screeningand Optimization

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Table of Contents

1. Introduction............................................................................................................................................ 3

2. Characteristics ..................................................................................................................................... 3

3. Applications .......................................................................................................................................... 4

4. Advice on Handling .............................................................................................................................. 4

4.1 Equipment ....................................................................................................................................... 4

4.2 Sample Preparation ......................................................................................................................... 4

4.3 Experimental Set Up......................................................................................................................... 4

4.4 Te-Chrom Wizard ............................................................................................................................ 4

5. Protocol ............................................................................................................................................... 6

6. Column Scale-Up .................................................................................................................................10

7. Troubleshooting Guide .........................................................................................................................10

8. Ordering Information ............................................................................................................................11

1. Introduction

ScreenExpert RoboColumns are miniaturized chromatography columns pre-packed with Pall chromato -graphy sorbents. They are designed for fully automated and parallel chromatographic separations with robotic liquid handling workstation such as Freedom EVOu workstation (Tecan) or Janus BioTx Pro Plus workstation (Perkin Elmer).

ScreenExpert RoboColumns are packed with anion exchange, mixed-mode or any other Pall chromato -graphy sorbents upon request. The columns are packed by Atoll GmbH and are identical to Atoll MediaScoutuRoboColumns.

Bed containment between two filter frits and professional packing with consideration to individual material compression requirements ensure high efficiency and peak symmetry similar to that of preparative andprocess separation columns.

Up to 96 ScreenExpert RoboColumns can be individually and easily arranged onto a 96-well array plate. As a result of this modular design, ScreenExpert RoboColumns technology is used in a broad spectrum of applications, such as parallel screening and optimization of chromatographic conditions, scale-down experiments, sample preparation.

ScreenExpert RoboColumns are supplied in a package of eight pre-packed units with two removable silicon cover seals for proper storage. A 96-well array plate is available to arrange up to 96 ScreenExpert RoboColumn units.

ScreenExpert RoboColumns are ready-to-use 96-well formatted chromatography columns pre-packed withPall chromatography sorbents. They are available in 200 μL and 600 μL sizes. Characteristics and availablecolumn geometries are detailed in Table 1.

All chromatography sorbents used in ScreenExpert RoboColumns are also available in Pall PRC pre-packedcolumns of 1 mL and 5 mL, for the performance of scale-up studies.

2. Characteristics

Table 1ScreenExpert RoboColumns Characteristics

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ScreenExpert RoboColumnsColumn volume 200 μL 600 μLBed height 10 mm 30 mmColumn inner diameter 5 mmColumn material PolypropyleneChemical stability All commonly used aqueous buffers, pH 1 to 14Avoid Halogenated organic solvents, hexaneStorage solution 20% ethanol /1 M NaClRecommended storage temperature 2 to 8 °CWorking temperature 4 to 30 °CMaximum working pressure Up to 8.0 bar g (116 psi g)Flow rate 16 to 1,000 cm/hr (1)

Laser label Placed on every individual column body, and containing:Product number, sorbent name, column volume in μL

96-Well Array PlateSize 128.3 x 86.0 x 14.0 mmMaterial Polyoxymethylene copolymerNumber of wells 96

(1) The Liquid Transfer menu of the Te-Chromu Wizard enables to set up the flow rate for each individual dispensing and chromatography step (5.1.1.6 Liquid Transfer, Te-Chrom Wizard Software Manual, 396076, en, V1.0). Flow ratecharacteristics rely on the Freedom EVO workstation characteristics, not on the RoboColumns themselves. They mayvary with another automated workstation.

3. Applications

ScreenExpert RoboColumns are suitable for a large variety of applications such as: l Parallel screening of chromatography sorbentsl Parallel screening and optimization of chromatographic conditions in downstream process development

(conductivity, pH)l Scale down experimental workl Determination of dynamic binding capacity l Determination of the optimal residence timel Optimization of the column regeneration procedure

4. Advice on Handling

4.1 Equipment

ScreenExpert RoboColumns are designed for robotic handling. Table 2 on page 5 is a guide for theequipment required for robotic handling of ScreenExpert RoboColumns using the Tecan Freedom EVO workstation.

NOTE: The workstation should hold a liquid handling (LiHa) arm equipped with stainless steel fixed tips.

4.2 Sample Preparation

We recommend loading a clarified sample on ScreenExpert RoboColumns because unclarified samplemay cause clogging of the top and bottom filter frits. Sample filtration through 0.2 μm membrane isrecommended, i.e., using Pall Acrodisc® PF Syringe Filter with hydrophilic Supor® membrane, sterile,0.8/0.2 μm 25 mm (part number 4187).

4.3 Experimental Set Up

When using the high throughput process development approach for ScreenExpert RoboColumns, it is suggested to use Design of Experiments (DoE) criteria for the experimental set up. This enables to screen many different chromatographic parameters such as resin type, pH, conductivity/ionicstrength, etc., efficiently and simultaneously.

For experimental set-up and the configuration of the 96-well array plate, the Te-Chrom Wizard interfaceis recommended.

4.4 Te-Chrom Wizard

The Te-Chrom Wizard is a dialog-based graphic user interface offered by Tecan and based on theFreedom EVOwareu software.

The Te-Chrom Wizard was developed for the use of ScreenExpert RoboColumns units and providesthe possibility to configure hardware, plate layout, and the chromatographic process as well as to setall process relevant chromatographic parameters, e.g., flow rate, volume without direct script writing.

An overview of some of the Te-Chrom Wizard functions is shown in Figure 1.

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Equipment

96-Well Array Plate

Te-Chrom Module (A)

Te-Chrom Shuttle

Te-Chrom Wizard(Courtesy of Tecan)

Plate Reader

Reagent and Sample Reservoirs

Collection Plate

Details

Arrange up to 96 individualScreenExpert RoboColumns onto the 96-well array plate according to application requirements.

Use the Te-Chrom module (A) to integrate the 96-well arrayplate onto the robotic worktable. The Te-Chrom module is heightadjustable and contains a wasteoption (B).

Arrange up to 96 individualScreenExpert RoboColumnsonto the 96-well array plate according to application requirements.

Use the Te-Chrom Wizard toconfigure the chromatographicprocess and to set all processrelevant parameters, e.g., volume, flow rate, etc.

Use an internal or external plate reader for data evaluation

A broad spectrum of troughs,tubes and plates can be positioned onto the roboticworktable by using the appro priate Tecan carrier.

Use any type of multi-well platewhich matches the ANSI/SDSstandard (UV- or non-UV-readable).

Tips and Tricks

l Do not mix short (200 μL) and long (600 μL) ScreenExpert RoboColumns units.

l Make sure that ScreenExpert RoboColumns units are well clickedinto the 96-well array plate before using them.

l Use the Te-Chrom module in combination with a plate stacker Te-Stacku for the optimized handling and storage of collectionplates.

l Use the waste container to drain off, e.g., conditioning, regen eration solvents.

l Make sure that the 96-well array plate is in an appropriate distance to the collection plate before starting the experiment.

Use the robotic manipulator (RoMa) arm to transport the collectionplate from a pre-defined transfer position to any destination on therobotic worktable, e.g., plate reader, hotels, auto sampler, etc., forfurther analysis, e.g., UV measurement, ELISA, HPLC, MS, etc.

l Assign labels, e.g., sorbent name and column types by using theplate layout configurator.

l Use the neutralization option to adjust the pH of the fractions afterthe acidic elution of mAbs.

NOTE: Confirm suitability for every specific application withTecan

Determine the precise volume of fractions for subsequent data plotting.

Use an 8-, 48- or 96-deep well reservoir for buffer solution /preparation.

l Use a half area 96-well microplate to collect fractions in a volumerange from 25 to 175 μL.

l Use a standard 96-well microplate to collect fractions in a volumerange from 75 to 340 μL.

l Use 96-deep well reservoirs to collect high volume fraction up to 2 mL.

Table 2Recommended Equipment for Automated Handling of ScreenExpert RoboColumns

A

B

Figure 1Te-Chrom Wizard Functions

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5. Protocol

This protocol is designed as a general guideline for working with ScreenExpert RoboColumns. Adjustmentmay be required depending on type of application, sample, chromatography sorbent type and ScreenExpertRoboColumn dimension.

1. Remove cover seals

• Gently peel off the bottom seal and the row holder from the rowof eight ScreenExpert RoboColumns.

• Assemble the ScreenExpert RoboColumn units onto the 96-wellarray plate. Up to 12 rows of 8 units can be assembled. Do notmix different formats (200 and 600 μL) on the same array.

• Gently peel off the top seal of each row of 8 RoboColumn units.

2. Remove excess storage solution

• Remove remaining storage solution in the top part of thecolumns by blotting the RoboColumn arrays upside down on a soft paper tissue.

Hardware configuration

Plate layout

Process step editor

Liquid transfer

Plate handling

Reporting

3. Te-Chrom – Adjusting the operating height

• Press and hold the fastener (C) to set the Te-Chrom to the required height of the Te-Chrom module scale (D).

NOTE: Make sure that the ScreenExpert RoboColumn array is as close as possible to the collection plate before starting the experiment.

4. Te-Chrom – Loading the array plate containing ScreenExpertRoboColumns

• Move the locking slider (E) to the left mechanical stop.

• Load the ScreenExpert RoboColumn array (F).

• Move the locking slider (E) to the right mechanical stop to lock the RoboColumn array.

NOTE: Make sure that all RoboColumns are well fixed into the 96-well array plate before starting the experiment.

5. Te-Chrom Wizard – Experimental set up

A. Hardware configuration

• Select the appropriate configuration of the Te-Chrom.

• Check if your Te-Chrom is equipped with a waste tray.

• Define a transfer position for the collection plate.

B. Plate layout

Click load to import an existing plate layout. To create a new layout, follow the actions below:

• Select the ScreenExpert RoboColumn labware.

• Define the required RoboColumn matrix.

• Select the RoboColumn dimension.

• Set a desired label, e.g., resin name.

• To import or export the plate layout, click the Save button.

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E

D

C C

F

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C. Process configuration

• Define the chromatographic workflow by setting a desired number of process steps.

• Use the Continuous Fraction Collection option to collect fractions over multiple plates, without leaving gaps betweenplates pipetted in different process steps and to optimize the consumption of collection plates.

D. Configure your process steps

• Edit a process step name, e.g., Equilibration.

• Check the Process Full Plate option accordingly:

– When checked: The full ScreenExpert RoboColumn array will be processed.

– When not checked: The RoboColumn array is processed column per column.

• Set parameters for tip washing.

• Set individual chromatographic parameters:

– Select the source of liquid.

– Set the dispensing volume.

– Set the required liquid class for aspiration.

– Set the required linear flow rate (cm/hr) or volumetric flow rate (μL/sec).

NOTE: Calculate the required residence time (τ) by dividing the RoboColumn volume (CV) by the volumetric flow rate (uv). τ = CV/uv.

CAUTION! Be sure to use the correct units for uv and CV: uv in mL/min and CV in mL, or uv in μL/min and CV in μL.

• Check the waste option accordingly:

– When checked: the waste tray will be engaged by the RoMa(Robotic Manipulator) / LiHa (Liquid Handling).

– When not checked: the flow-through will be collected in a desired collection plate.

• Check the Neutralization option to perform a pH adjustmentafter, e.g., acidic elution of mAbs.

• Check the Data Collection option to evaluate the collectionplate in a required reader device by, e.g., UV measurement.

Equilibration

Load

Post Load Wash

Elution 1

Elution 2

Regeneration

Storage

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2

0

1009080706050403020100

0 0.2 0.4 0.6 0.8 1.0 1.2V (mL)

Elut

ion

Buf

fer (

%)

ODλ

= 28

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1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8

• Select the number of fractions according to the given dispensing volume for the chromatography step if the wasteoption is not checked.

• Repeat these steps to configure all chromatographic processsteps.

E. Plate handling

• Add the required amount of source plates.

• Add the required amount of destination plates.

F. Reporting

• Set output folder.

• Select output format.

After the experimental set-up, the Te-Chrom wizard will generate a chromatographic method in Freedom EVOware software automatically.

6. Run chromatographic method

NOTE: Make sure that all required solvents and consumables, e.g., collection plates, are in the designated position on the robotic worktable, before running the chromatographic method.

7. Data evaluation

• Determine the precise volume of the collected fractions by using an appropriate plate reader.

• Determine the protein concentration by using UV measurement.

NOTE: Transfer the collection plate to any location on the roboticworktable or to secondary devices, e.g., auto sampler for furtheranalysis, e.g., HPLC, MS, ELISA, CGE/SDS-PAGE after pathlength detection by using the RoMa.

8. Data plotting

• Plot the UV data against the fraction volume to create apseudo-chromatogram.

NOTE: Make sure that blank values of the used solvents are subtracted from the UV data before plotting.

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9. Regeneration and storage

• For appropriate regeneration of ScreenExpert RoboColumns,please refer to Pall recommendations for the corres pondingchromatography sorbent.

• For appropriate storage of ScreenExpert RoboColumns, pleaserefer to Table 1.

6. Column Scale-Up

All chromatography runs developed on ScreenExpert RoboColumns can be directly transferred onto PRCpre-packed columns (1 or 5 mL). Make sure to maintain residence time constant during scale-up.

7. Troubleshooting Guide

Fault

ScreenExpert RoboColumns are clogged

ScreenExpert RoboColumns are leaking

ScreenExpert RoboColumns are lifted up from the 96-wellarray plate during the pull out of the robotic needles

Low protein recovery in the elution fraction(s)

Possible Cause

l The sample is too viscous.

l There is too much cell debris in the sample.

l The column has been regenerated and used too many cycles.

l Columns are clogged.

l The o-ring sealing is damaged.

l The insertion depth of the robotic needle is incorrect.

l ScreenExpert RoboColumns are not well fixed into the 96-well array plate.

l The residence time for sample loading is too low.

l The choice of the sorbent and/or processconditions is not appropriate.

Action

l Increase dilution of the cell paste before lysis,or dilute after the lysis.

l Centrifuge and/or filter the sample if unclarifiedsample has been used.

l Reduce the number of cycles.

l See above.

l Check the taught X- and Y coordinates forScreenExpert RoboColumn labware for precision.

l Check the insertion depth (16 mm from the top of the column) of the robotic needle.

l Make sure that all ScreenExpert RoboColumnsare well fixed into the 96-well array plate beforestarting the experiment.

l Decrease the flow rate for sample loading.

l Check and adapt if needed the sorbent and/orprocess conditions.

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8. Ordering Information

Description Part Number

ScreenExpert RoboColumn MEP HyperCel 200 μL, row of 8 SR2MEP

ScreenExpert RoboColumn HEA HyperCel 200 μL, row of 8 SR2HEA

ScreenExpert RoboColumn PPA HyperCel 200 μL, row of 8 SR2PPA

ScreenExpert RoboColumn HyperCel STAR AX 200 μL, row of 8 SR2STARAX

ScreenExpert RoboColumn MEP HyperCel 600 μL, row of 8 SR6MEP

ScreenExpert RoboColumn HEA HyperCel 600 μL, row of 8 SR6HEA

ScreenExpert RoboColumn PPA HyperCel 600 μL, row of 8 SR6PPA

ScreenExpert RoboColumn HyperCel STAR AX 600 μL, row of 8 SR6STARAX96-well RoboColumn array plate SR96WAP

Complementary Products

AcroPrep™ ScreenExpert Plates

MEP, HEA and PPA HyperCel Mixed-Mode Sorbents 96WP3MIXM50

MEP HyperCel Mixed-Mode Sorbent 96WPMEP50HyperCel STAR AX Anion Exchange Sorbent 96WPSTARAX50

Multi-Well Plates

AcroPrep Advance 96-Well Filter Plate 8129

AcroPrep Advance Filter Plate for Ultrafiltration

350 μL, Omega™ 3K MWCO (10/pkg) 8033

350 μL, Omega 10K MWCO (10/pkg) 8034

350 μL, Omega 30K MWCO (10/pkg) 8035350 μL, Omega 100K MWCO (10/pkg) 8036

PRC Pre-packed Columns

PRC Column 5x50 MEP HyperCel, 1 mL PRC05X050MEPHCEL01

PRC Column 8x100 MEP HyperCel, 5 mL PRC08X100MEPHCEL01

PRC Column 5x50 HEA HyperCel, 1 mL PRC05X050HEAHCEL01

PRC Column 8x100 HEA HyperCel, 5 mL PRC08X100HEAHCEL01

PRC Column 5x50 PPA HyperCel, 1 mL PRC05X050PPAHCEL01

PRC Column 8x100 PPA HyperCel, 5 mL PRC08X100PPAHCEL01

PRC Column 5x50 HyperCel STAR AX, 1 mL PRCSTARAX1MLPRC Column 8x100 HyperCel STAR AX, 5 mL PRCSTARAX5ML

Syringe Filter

Acrodisc PF Syringe Filter with hydrophilic Supor membrane, sterile, 0.8/0.2 μm 25 mm 4187

Visit us on the Web at www.pall.com/biopharmE-mail us at biopharm@pall.com

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The information provided in this literature was reviewed for accuracy at the time ofpublication. Product data may be subject to change without notice. For current information consult your local Pall distributor or contact Pall directly.

Corporate HeadquartersPort Washington, NY, USA+1 800 717 7255 toll free (USA)+1 516 484 5400 phonebiopharm@pall.com e-mail

European HeadquartersFribourg, Switzerland+41 (0)26 350 53 00 phoneLifeSciences.EU@pall.com e-mail

Asia-Pacific HeadquartersSingapore+65 6389 6500 phonesgcustomerservice@pall.com e-mail

© 2014, Pall Corporation. Pall, , Acrodisc, AcroPrep, HyperCel, Omega andSupor are trademarks of Pall Corporation. Filtration.Separation.Solution. is aservice mark of Pall Corporation. ® indicates a trademark registered in the USAand TM indicates a common law trademark. u MediaScout, RoboColumn andRoboColumns are trademarks of Atoll GmbH. Freedom EVO, Freedom EVOware,Te-Chrom and Te-Stack are trademarks of Tecan Group.

7/14, GN14.9172 USD 2937