KrosFlo Research II TFF System

R E S E A R C H I ITFF SYSTEM

KrosFlo Research II TFF System Product Information and Operating Instructions

®

ii KrosFlo® RESEARCH II TFF SYSTEM • PRODUCT INFORMATION AND OPERATING INSTRUCTIONS

Spectrum®’s KrosFlo® Research II Systems and membrane modules meet strict quality control stan-dards and are warranted against defects in material and workmanship for a period of one year fromdate of purchase.

The information contained herein is believed to be accurate and is offered in good faith for the con-venience of the user. PRODUCTS ARE FURNISHED UPON THE CONDITION THAT THE USERASSUMES ALL RISKS AND LIABILITIES AND THAT NEITHER THE SELLER NOR MANUFACTURERSHALL BE LIABLE FOR ANY LOSS OR DAMAGE, DIRECT OR CONSEQUENTIAL, ARISING FROMTHE USE OF THESE PRODUCTS.

Spectrum®, KrosFlo®, MidiKros® and MicroKros® are registered trademarks of SpectrumLaboratories, Inc. C-Flex® is a registered trademark of Consolidated Polymer Technologies, Inc.Excel® is a registered trademark of Microsoft Corporation.

Table of Contents1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. System Configuration and Major Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1 KrosFlo® Research II Pump Drive & Pump Head . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.2 KrosFlo® Research II Pressure Monitor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.3 Flow-path Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.4 Starter Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3. System Options and Parts List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

4. Materials of Construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

5. Basic Concepts of Tangential Flow Filtration (TFF) . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

5.1 Dead-end Filtration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

5.2 Tangential Flow Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

6. Assembling the Flow-path . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

6.1 Constant Volume Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

6.2 Batch Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

7. Integrity Testing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

7.1 Leak Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157.2 Wetting the HF Membrane Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

7.3 Pressure Hold Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

8. Modes of Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

8.1 Batch Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

8.2 Batch Clarification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

8.3 Constant Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

8.4 Diafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

8.5 Dead-end Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

9. Operating the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

9.1 Instructions for Batch Concentration / Clarification . . . . . . . . . . . . . . . . . . . . . . . . . 20

9.2 Instructions for Constant Volume & Diafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

9.3 Instructions for Dead-end Clarification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

10. Pre-Straining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

11. Process Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

12. In-Process Module Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

12.1 Pump Off Cleaning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

12.2 Forward Flushing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

12.3 Reverse Flushing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

13. Module Selection and Scale-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

14. Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

TABLE OF CONTENTS

1KrosFlo® RESEARCH II TFF SYSTEM • PRODUCT INFORMATION AND OPERATING INSTRUCTIONS

INTRODUCTION

2 KrosFlo® RESEARCH II TFF SYSTEM • PRODUCT INFORMATION AND OPERATING INSTRUCTIONS

1. IntroductionThe KrosFlo® Research II Tangential Flow Filtration (TFF) System is the ideal system for research anddevelopment studies for microfiltration and ultrafiltration applications. The system consists of theKrosFlo® Research II Pump and Pump Head, the KrosFlo® Digital Pressure Monitor, and a disposableflow path, all of which have features that ensure efficient and reproducible TFF processes. Theupgraded KrosFlo® Digital Pressure Monitor has both audible low and high pressure alarms for theinlet and permeate and, when combined with the KrosFlo® Research II Pump Drive, has a high pres-sure pump shutoff that helps maintain membrane integrity and achieve high product recovery. ThePressure Monitor also comes standard with KF Comm, a software program that automatically down-loads and graphs the run data including inlet, retentate and transmembrane pressures and flow rates,into an MS Excel® spreadsheet. The disposable flow path eliminates the possibility of cross conta-mination and allows samples to be concentrated down to as low as 2 mls. Other standard featuresinclude: a digital readout of the flow rate on the KrosFlo® Research II Pump, the new easy-to-useKrosFlo® Research II Pump Head, and adjustable holders attached to the pump drive that secure theprocess vessels, Spectrum’s HF modules and the entire flow-path.

The membrane surface areas range from 5 cm2 to 1050 cm2 and are available in the following mem-brane types:

Ultrafiltration:

10 kD Polysulfone 0.5 mm ID50 kD Polysulfone 0.5 mm ID100 kD Polysulfone 0.5 mm ID400 kD Polysulfone 0.5 mm ID500 kD Polysulfone 1.0 mm ID50 nm Polysulfone 0.5 mm ID

Microfiltration:

0.1 µm Mixed Cellulose Ester 0.6 mm ID0.2 µm Mixed Cellulose Ester 0.6 mm ID0.2 µm Mixed Cellulose Ester 1.0 mm ID0.2 µm Polyethersulfone (PES) 0.5 mm ID0.2 µm Polyethersulfone (PES) 1.0 mm ID0.5 µm Polyethersulfone (PES) 0.5 mm ID0.5 µm Polyethersulfone (PES) 1.0 mm ID

The KrosFlo® Research II TFF System offers many conveniences over traditional cross-flow mem-brane systems. The hollow fiber (HF) membrane modules and cross-flow filtration system providefaster and gentler separation that helps avoid membrane fouling and maximizes product recovery.The disposability of the modules eliminates not only the potential for cross-contamination and costsassociated with cleaning and rinsing, but also the difficulties associated with validating re-use mem-branes. Used in conjunction with HF membrane modules, the KrosFlo® Research II TFF Systemoffers the following advantages:

• Faster process times

• Superior filtration dynamics

• Module disposability

• Lower costs

• Direct and easy scale-up for production volumes

SYSTEM CONFIGURATIONS AND MAJOR COMPONENTS


2. System Configurations and Major Components

2.1 KrosFlo® Research II Pump Drive & Pump HeadThe pump drive comes with the drive unit, anattached mounting plate for the KrosFlo® Research IIPump Head and two stainless steel posts (18” and9”) with hardware. The stainless steel posts supportthe module Trilobites (holders) and ReservoirTrilobites (holders) that come with the Starter Kit. Themodule Trilobites can accommodate Spectrum’sMicroKros®, MidiKros®, MiniKros® Sampler/SamplerPlus, and some of the MiniKros® hollow fiber filtrationmodules. The reservoir Trilobites can secure reser-voirs from 8 ml up to 2 L.

• Refer to the provided KrosFlo® Research II Pump Manual for general operating instructions.

• Refer to Section 9 “Operating the System”, for specific instructions on how to use the pump during a filtration process.

2.2 KrosFlo® Research II Pressure Monitor

The pressure monitor comes with a Relay Cable that connects the monitor to the KrosFlo® ResearchII Pump Drive, an RS232 cable for capturing data on a computer, a transformer, three pressure trans-ducers, an operating manual and the KF Comm soft-ware program that logs and graphs the run data inspreadsheet format.

• Refer to the KrosFlo® Research II Pressure Monitor Manual for general operating instructions.

• Refer to Section 9 “Operating the System”, for specific instructions on how to use the pressure monitor during a filtration process.

• Refer to the KF Comm Manual for specific instruc-tions for automatic data collection from the monitor.

2.3 Flow-path KitThree different flow-path kits match the three sizes of filtration modules used in the system:MicroKros® (10 – 50 ml), MidiKros® (20 ml – 200 ml), and MiniKros® Sampler (50 ml – 4+ L). Theappropriate kit that corresponds to the module size to be used should be chosen at the time of pur-chase. The kit comes with enough fittings and tubing to build three flow-paths (depending on theconfiguration). One feed and one process reservoir of different sizes are included with the flow-path.Other sized bottles are available for purchase separately. Filtration modules are sold separately. Forspecific instructions of flow-path construction and options refer to Section 6 “Assembling theSystem”.

2.4 Starter KitThe starter kit includes other items needed to construct and support the flow-path. These includereusable items such as the Reservoir and Module Trilobites, flow restrictors, bulb valve, tie wraps anda tie wrap gun.

SYSTEM OPTIONS AND PARTS LIST


3. System Options and Parts ListThe available system configurations with corresponding catalog numbers are listed below:

KrosFlo® Research II TFF System Selection GuidePart Number Flow-path Type Pump Capacity Tubing F/P CapacitySYR2-X21-01N MicroKros® 2.3 LPM, 110V C-Flex 14 130 ml/min

SYR2-X22-01N MicroKros® 2.3 LPM, 220V C-Flex 14 130 ml/min

SYR2-D21-01N MidiKros® 2.3 LPM, 110V C-Flex 16 480 ml/min


SYR2-S21-01N MiniKros® Sampler Plus 2.3 LPM, 110V C-Flex 17 1.7 L/min


System Parts ListPART DESCRIPTION QTY. PER SYSTEM

KrosFlo® Research II Pump Drive 1Stainless Steel Posts w/ mounting screws

9 inch 1

18 inch 1

Attached Mounting Bracket for Pump Head 1

KrosFlo® Research II Pump Head 1

KrosFlo® Digital Pressure Monitor 1

Disposable Pressure Transducers 3

Transformer 110V/220V 1

Power Cable (either 110V/220V) 1

RS232 Cable 1

Pressure Monitor-Pump Cable 1

KF Comm – Data Collection Software Disc 1

Flow-path Kit (1of 3)

MicroKros®

1/16” Plastic Fittings box w/accessories 1

#14 C-Flex® tubing 18 ft.

MidiKros®



MiniKros® Sampler



MATERIALS OF CONSTRUCTION


PART DESCRIPTION QTY. PER SYSTEM

Starter KitSqueeze Bulb Valve Assembly 1

Tie Wrap Gun 1

Tie Wraps 100

Module Trilobite (holder) 2

Reservoir Trilobite (holder) 2

Flow Control Valve

5/16” to 3/8” OD Tube 2

5/32” to 1/4” OD Tube 2

Bottles

MicroKros® System

15 ml polypropylene bottle with C-Flex® Molded Seal 1

60 ml polypropylene bottle with C-Flex® Molded Seal 1MidiKros® System



MiniKros® Sampler System


2 L polypropylene bottle with C-Flex® Molded Seal 1

4. Materials of Construction PART DESCRIPTION MATERIAL

KrosFlo® Research II TFF System (product contact surfaces)Tubing / Reservoir Closures C-Flex

Reservoirs Polypropylene

Disposable Pressure Transducers Polycarbonate

Plastic Fittings Polypropylene / Polycarbonate

Hollow Fiber Module

Membrane (1of 3) Mixed Cellulose Ester, Polyethersulfone or Polysulfone

Housing Pigmented and Non-pigmented Polysulfone

Potting Polyurethane / Epoxy

End-caps Pigmented Polysulfone

BASIC CONCEPTS OF TANGENTIAL FLOW FILTRATION (TFF)


5. Basic Concepts of Tangential Flow Filtration (TFF)Membranes use the principle of barrier separations to differentiate components based on size.Components larger than the membrane pore are quantitatively held back by the membrane whilesmaller components pass through the membrane structure along with the permeate. Although thereare other methods for driving the separation process such as electric charge (e.g. caustic-chlorinecells) and diffusion (e.g. dialysis and oxygenation devices), Spectrum hollow fiber membrane modulesare designed for applications in which pressure is the driving force.

5.1 Dead-end FiltrationTraditional sieve (or dead-end) filtrations consist of forcing a solution containing suspended solidsdirectly through the membrane structure. Solids retained by the membrane collect on the surface ofthe membrane media, continually reducing the permeation rate and eventually plugging the device.See illustration below.

The driving force for permeation is the pressure difference between the feed and the permeate, calledthe transmembrane pressure (TMP). For dead end filtration this is given by the following equation:

PTMP = Pfeed – Ppermeate

5.2 Tangential Flow FiltrationTangential Flow Filtration (TFF) is an efficient way to separate streams that would become quicklyplugged if processed by dead-ended techniques. When using tangential flow techniques, most ofthe process fluid flows along the membrane surface rather than passing through the membrane struc-ture. Fluid is pumped at a relatively high velocity parallel to the membrane surface. Except for watertreatment applications, only a small percentage of the tangential flow along the membrane surfaceends up as permeate. In most cell and particle separations, for example, only 1% to 5% of the inletflow to the membrane device becomes permeate. The remaining 95% to 99% exits the membranedevice as “retentate”. The retentate is recirculated back to the process reservoir and the module inletsuch that another 1% to 5% can be removed as permeate. This recirculation process continues inrapid succession generating a significant and continuous permeation rate. See the illustration below.

BASIC CONCEPTS OF TANGENTIAL FLOW FILTRATION (TFF)


Various tangential flow membrane geometries include stacked plate and spiral devices which utilizeflat sheet membranes, tubular devices, and shell and tube devices which use hollow fiber mem-branes. In the case of tangential flow separations, the driving force is the transmembrane pressure(TMP), the difference between the average of the module feed and retentate pressures and the per-meate pressure:

PTMP = (Pfeed + Pretentate) / 2 – Ppermeate

Filtrate flow results in a build up of retained components on the membrane inner lumen surface.Generally these components are carried down the length of the hollow fiber and out the end of themodule by the sweeping action of the recirculating fluid. However, under certain conditions a cakelayer accumulates on the surface of the membrane. This boundary layer is composed of solidsand/or solute macromolecules which are retained by the membrane during the course of filtration.This phenomenon, often erroneously referred to as “concentration polarization” can affect moduleperformance by reducing the apparent size of the membrane pore. In other words, the cake layerbecomes the membrane barrier, a “dynamic membrane”.

The extent of caking is influenced by such fluid variables as the degree of solvation, concentrationand nature of the solids and solutes, fluid temperature and operating variables such as solution veloc-ity along the membrane and transmembrane pressure (TMP). Refer to Section 12. Controlling thisphenomenon is the key to maximizing flux and solute passage and optimizing the process parame-ters. Caking can usually be controlled by ensuring adequate fluid velocity at the liquid-membrane wallinterface. Fluid velocity is controlled by the pumping rate. Recirculation rates depend on the quan-tity of fibers in a module and shear rate considerations. Typically, a shear rate of 12,000 s-1 is usedfor filtration applications and up to 4,000 s-1 is used for perfusion applications. These rates are guidelines only and should be optimized for each process application. Certain applications may work wellat reduced rates while others may require rates that are significantly higher. When protein passagethrough the membrane structure is important, particular attention should be paid to feed (or recircu-lation rate). In general, high feed rates allow more efficient protein passage. Depending on the char-acteristics of the retained components (cells, cell debris, diagnostic particles, etc.), a caking layer canform on the membrane wall that is actually tighter than the membrane pores. In these instances, highrecirculation rate and low transmembrane pressure often help. Variables such as viscosity or shearsensitivity of the solution components may prevent the user from generating enough velocity toreduce membrane caking. Often in these cases, applying a determined amount of back pressure onthe permeate helps to prevent caking. Cell perfusion applications frequently use permeate back pres-sure to prevent caking.


ASSEMBLING THE FLOW-PATH

6. Assembling the Flow-pathThe following are the instructions for assembling the KrosFlo® Research II TFF System flow-paths.Figure 1B is given as a representative layout of the flow-path. For a description of the differencebetween Constant Volume Mode and Batch Mode refer to Section 8.

6.1 Constant Volume ModeRefer to Figures 2, 3 and 4 for assembling either one of the three different size flow-paths (MiniKros®

Sampler, MidiKros® and MicroKros®, respectively) for constant volume mode.

1. Attach one Reservoir Trilobite (holder) to the bottom end of each post on either side ofthe KrosFlo® Research II Pump Drive. The Reservoir Trilobites should be oriented suchthat the front (wide) groove faces forward and its respective thumb screw is directedaway from the pump. Attach both Module Trilobites (holders) to the left post above theReservoir Trilobite. Reservoir and Module Trilobites can be attached to the posts by“hooking” the posts into the groove on the back (narrow) end of the Trilobite and secur-ing the Trilobites in place with the back thumb screw. (Figures 1A and 1B)

2. Attach the HF filtration module by inserting into the front groove on both the ModuleTrilobites. Slide the Trilobites away from each other until the module is held firmly inplace. Secure the Trilobites by tightening the back thumb screws. (Figure 1B)

3. Attach the smaller processing reservoir by inserting into the front groove of the ReservoirTrilobite on the left side of the pump. Secure in place by tightening the front corre-sponding thumb screw. (Figure 1B)

4. Follow the same step for attaching the larger feed reservoir to the Reservoir Trilobite onthe right side off the pump. (Figure 1B)

NOTE: The reservoir Trilobites are designed with a thumb screw to hold the smaller reservoirs byeither the bottle or the cap and to hold the larger reservoirs by the cap only. The smallerreservoirs can be held suspended above the surface of the bench top while the largerreservoirs need to rest on the bench top.

5. Connect the appropriate size HB x HB connector to the tubing coming out of the feedreservoir and further from the pump. Use a 1/4” x 1/4” connector (#1 in Figure 2) for set-ting up the MiniKros® Sampler flow-path or a 1/4” x 1/8” reducer (#7 and #6 in Figures3 and 4, respectively) for setting up the MidiKros® and MicroKros® flow-paths. Connectthe bulb valve assembly to the free end of the fitting.

6. Connect the appropriate size HB x HB connector to the tubing coming out of the feedreservoir and closer to the pump. Use a 1/4” x 1/4” connector (#1 in Figure 2) for theMiniKros® Sampler flow-path, a 1/8” x 1/8” connector (#2 in Figure 3) for the MidiKros®

flow-path or 1/16” x 1/16” connector (#1 in Figure 4) for the MicroKros® flow-path.

7. Connect the stem of the Y-connector (#’s 2, 3 and 2 in Figures 2, 3 and 4, respectively)to the tubing coming out of the processing reservoir and closest to the pump. (The ori-entation of the Y-connector may vary depending on preference.)

8. Cut an appropriate length of tubing (#’s 6, 9 and 9 in Figures 2, 3 and 4, respectively) toconnect from an arm of the Y connector to the free connector on the feed reservoir tub-ing. Prior to connecting, thread a ratchet clamp (#’s 4, 8 and 7 in Figures 2, 3 and 4,respectively) into place near the Y connector. For ease of use, the tubing should bedirected beneath the pump drive as shown in Figure 1B.

Figures 1A & 1B | SYSTEM SCHEMATICS


++

--

--++

PROCESSINGRESERV0IR

FEEDRESERV0IR

HF MODULE

10001000

MODULE TRILOBITE(HOLDER)

RESERVOIR TRILOBITE(HOLDER)

Figure 1A. System Schematic - Top View

Figure 1B. Overall Flow-path Schematic - Front View



NOTE: To minimize hold up volume make sure the Y connector is as close as possible to theprocessing reservoir. To do this it might be preferable to cut the tubing that comes outof the processing reservoir to a minimum length.

9. Assemble the rest of the flow-path including pressure transducers and HF filtration mod-ule for MiniKros® Sampler, MidiKros® and MicroKros® according to Figures 2, 3 and 4,respectively, and the following steps:

MiniKros® Sampler Flow-path (Figure 2, page 12)

i. Cut an appropriate length of tubing (#6) to connect from the free arm of the Y-connec-tor (#2) through the pump head and then to the inlet pressure transducer tee connector(#5, HB x HB x Male Luer). (The orientation of the Y-connector may vary depending onpreference.)

ii. Connect a short piece of tubing from the second HB end of the inlet pressure transducertee connector (#5) to the lower HB inlet port of the MiniKros Sampler HF module (#10).

iii. Connect a short piece of tubing from the upper HB retentate port of the HF module (#10)to a second tee connector (#5) for the retentate pressure transducer.

iv. Attach a HB x HB fitting (#1) to the free tubing coming from the processing reservoir.Cut an appropriate length of tubing to connect from this fitting to the retentate pressuretransducer tee connector (#5). Before connecting tubing thread a flow control valve (#7)and slide into place.

v. Close off the lower permeate side port of the HF module with either a male Luer cap(provided with module) or a short piece of tubing and a HB plug (#3) depending on theconnection type.

vi. Connect a short piece of tubing from the upper HB permeate side port (use Male Luerx HB fitting provided with module if necessary) to a third tee connector (#5) for the per-meate pressure transducer. Connect an appropriate length of tubing from the HB freeend of the tee connector to the desired permeate collection vessel (not provided). Priorto connecting tubing to collection vessel, thread a flow control valve (#7) first followedby a ratchet clamp (#4) and slide into place.

vii. Attach pressure transducers (#8) to the inlet, retentate and permeate pressure trans-ducer tee connectors. Plug the pressure transducer cables into the appropriate labeledjacks on the back of the KrosFlo® Pressure Monitor.

viii. If preferred, suspended tubing can be secured in the tubing notches on the sides of themodule and reservoir Trilobites.

ix. All HB connections may be secured with tie wraps (#9) and a tie wrap gun.

MidiKros® Flow-path (Figure 3, page 13)

i. Cut an appropriate length of tubing (#9) to connect from the free arm of the Y-connec-tor (#3) through the pump head and then to the inlet pressure transducer tee connector(#1, HB x HB x HB). (The orientation of the Y-connector may vary depending on pref-erence.)

ii. Connect a short piece of tubing from the second HB end of the inlet pressure transducertee connector (#1) to the lower HB inlet port of the MidiKros® HF module (#13).

iii. Connect a short piece of tubing from the upper HB retentate port of the HF module (#13)to a second tee connector (#1) for the retentate pressure transducer.



iv. Attach a HB x HB fitting (#2) to the free tubing coming from the processing reservoir.Cut an appropriate length of tubing to connect from this fitting to the retentate pressuretransducer tee connector (#1). Before connecting tubing thread a flow control valve(#10) and slide into place.

v. Close off the lower permeate side port of the HF module with a male Luer plug (#6).

vi. Use a male Luer x HB fitting (#5) to connect a short piece of tubing from the upper per-meate side port to a third tee connector (#1) for the permeate pressure transducer.Connect an appropriate length of tubing from the HB free end of the tee connector tothe desired permeate collection vessel (not provided). Prior to connecting tubing to col-lection vessel, thread a flow control valve (#10) first followed by a ratchet clamp (#8) andslide into place.

vii. Connect a small piece of tubing to the stems of all 3 tee connectors (#1) with a HB xfemale Luer connectors (#4) attached to the free end of each tubing piece. Attach apressure transducer (#11) to each of the three HF x female Luer connector (#4). Plugthe three pressure transducer cables into the appropriate labeled jacks on the back ofthe KrosFlo® Pressure Monitor.

viii. If preferred, suspended tubing can be secured in the tubing notches on the sides of themodule and reservoir Trilobites.

ix. All HB connections may be secured with tie wraps (#12) and a tie wrap gun.

MicroKros® Flow-path (Figure 4, page 14)

i. Remove stop-cocks from all 3 pressure transducers (#10). Attach a pressure trans-ducer directly to both the lower inlet and upper retentate Luer ports of the MicroKros®

HF module (#11). Attach HB x female Luer connectors (#3) to both free male ends ofthe attached pressure transducers.

ii. Cut an appropriate length of tubing (#9) to connect from the free arm of the Y-connec-tor (#2), through the pump head and then to the HB x female Luer connector (#3) onthe bottom end of the inlet pressure transducer. (The orientation of the Y-connector mayvary depending on preference.)

iii. Attach a HB x HB fitting (#1) to the free tubing coming from the processing reservoir.Cut an appropriate length of tubing to connect from this fitting to the retentate pressuretransducer HB x female Luer connector (#3). Before connecting tubing thread a flowcontrol valve (#8) and slide into place.

iv. Close off the lower permeate side port of the HF module with a male Luer plug (#5).

v. Attach the third pressure transducer (#10) (w/o stop-cock) to the upper permeate sideport. Attach a HB x male Luer connector (#4) to the free end of the pressure transduc-er. Connect an appropriate length of tubing from the HB free end of the connector tothe desired permeate collection vessel (not provided). Prior to connecting tubing to col-lection vessel, thread a flow control valve (#8) first followed by a ratchet clamp (#7) andslide into place.

vi. Plug the pressure transducer cables into the appropriate labeled jacks on the back ofthe KrosFlo® Pressure Monitor.

vii. If preferred, suspended tubing can be secured in the tubing notches on the sides of themodule and reservoir Trilobites.


Figure 2 | MINIKROS® SAMPLER FLOW-PATH

500ml

2000ml

Y CONNECTOR WITH HOSE BARBS

STRAIGHT THROUGH CONN.WITH HOSE BARBS

TEE CONNECTOR WITHLUER THREAD

RATCHET CLAMP

SQUEEZE BULB

RESERVOIRS WITHCLOSURES

PLUG WITH HOSE BARB

TUBING

ITEM DESCRIPTION QTY

PRESSURE TRANSDUCERWITH STOPCOCK

TIE WRAP

FLOW CONTROL VALVE

MODULE

78

9

5 4

3

106

1

2

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PERMEATE

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Figure 2. MiniKros® Sampler Flow-path


Figure 3 | MIDIKROS® FLOW-PATH



TEE CONNECTOR

RATCHET CLAMP

SQUEEZE BULB


TUBING



TIE WRAP

FLOW CONTROL VALVE

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PERMEATE

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Figure 3. MidiKros® Flow-path


Figure 4 | MICROKROS® FLOW-PATH



RATCHET CLAMP

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TUBING



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MODULE

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Figure 4. MicroKros® Flow-path

INTEGRITY TESTING


6.2 Batch Mode (Figure 5, page 16)

For batch refer to Figure 5, “MiniKros® Sampler Flow-path (Batch Mode)” as a representative layoutand follow steps 6.1.6 and 6.1.7, keeping in mind that since there is no feed reservoir, the Y-con-nector will not be necessary. The tubing will instead connect directly from a HB connector on theprocessing reservoir tubing, through the pump head and to the inlet pressure transducer fitting.The retentate line will instead connect from the outlet end of the HF module, through a pressuretransducer fitting and then to the other processing reservoir tubing. The bulb valve assembly, usedfor priming and integrity testing the system, will need to be attached (only when in use) to the endof the syringe tip filter that is connected to the stop-cock on the processing reservoir.

7. Integrity TestingAll Spectrum HF membrane modules are integrity tested in our class 10,000 cleanroom prior to pack-aging and shipping. Membrane integrity tests generally measure the capillary pressure of wettedpores. It is strongly recommended to integrity test all HF membrane modules again prior to use. Drymodules must be fully primed to ensure complete membrane wetting, otherwise a false integrity fail-ure will be indicated due to non-wetted or incompletely wetted pores. For wetting and integrity test-ing procedures please refer to the “Hollow Fiber Module—Preparation and Instruction Guide”(Document 400-12058-000) provided with each hollow fiber module. For specific instructions on howto use the KrosFlo® Research II System to wet out the module and to perform an integrity test seebelow.

7.1 Leak TestThe system seals should first be pressure tested with air prior to filling the systems with fluids.

1. Close the ratchet clamp on the permeate line. Open the shut-off clamp on the feed line.Open the bulb valve vent.

2. Turn on the pressure monitor and ensure that it is tared to zero.

3. Close the vent on the bulb valve and generate a pressure of approximately 5 psi.

4. If there is no significant pressure drop (<0.1 psi/min) then the system has no leaks andis ready for use.

5. If the pressure decay is greater than 0.1 psi/min, then check to make sure all the sealsand fittings are secure. Adjust connections as necessary to eliminate pressure decay.

6. After assuring system is free of air leaks, relieve the pressure by opening the vent on thebulb valve.

7.2 Wetting the HF Membrane Module

1. Fill the feed reservoir with the appropriate wetting agent: 100% IPA or 70% EtOH for drypolysulfone membranes, or DI water for the other membrane types (prewetted polysul-fone, mixed cellulose ester or polyethersulfone).

NOTE: Rinsing the module can also be done offline to avoid contaminating the flow-path whenalcohol is used. Use a proper sized beaker or similar container as the feed and return.

2. Open the shut-off clamps for the feed line. Close the shut-off clamp for filtrate line.Close the vent on the bulb valve.


Figure 5 | MINIKROS® SAMPLER FLOW-PATH (BATCH MODE)

NOT USED IN THISSCHEMATIC


TEE CONNECTOR WITHLUER THREAD

RATCHET CLAMP

SQUEEZE BULB


PLUG WITH HOSE BARB

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7

Figure 5. MiniKros® Sampler Flow-path (Batch Mode)


3. Open the stop-cock on the processing reservoir. Use the squeeze bulb to generatepressure in the system and drive the wetting agent into the processing reservoir. Afterthe process reservoir fills most of the way up, close the stop-cock.

4. Relieve the pressure on the feed reservoir by opening the vent on the squeeze bulb valve.

5. Turn the pump on forward rotation (counter-clockwise) and increase the velocity until aflow pressure is generated of approximately 5 psi. At this point, air pockets will be forcedout of the flow-path and will collect in the processing reservoir, thus lowering the fluidlevel.

6. Open the filtrate shut-off clamp and collect 2 ml of fluid per cm2 of membrane surfacearea (refer to “Hollow Fiber Module—Preparation and Instruction Guide”). If wetting withalcohol, make sure to rinse out the alcohol with 2 ml/cm2 of DI water after the system isdrained.

7. If using the KF Comm data collection software open the ‘Module Characteristics’ work-sheet and begin collecting data to get baseline Normal Water Permeability (NWP) data.The permeate flow (Qpermeate) and Temperature need to be manually entered. The ‘NWP’worksheet shows the graph.

8. Before shutting off the pump, always close the shut-off clamps for both the feed and thefiltrate lines.

NOTE: Whenever the system is filled with fluid and the pump is turned off, the shut-off clampsfor both the feed and filtrate lines should be closed first. Because the retentate volumeis small, pressure without recirculation can plug the membrane. Although there is nopressure due to pumping, hydrostatic pressure can convert the HF module into dead-end mode.

9. Drain fluid from the system.

i. Close the feed and filtrate shut-off clamps.

ii. Lift the cap above the processing reservoir and turn on the pump to drive the remain-ing fluid in the flow-path tubing into the reservoir.

iii. Shut-off the pump once all the fluid has emptied into the process reservoir. Empty theprocess reservoir and reconnect.

iv. Drain fluid from HF module extracapillary space by opening both sideports on the module.

7.3 Pressure Hold Test

1. Wet the membranes in accordance with Section 7.2. Pre-wetted UF membranes can beintegrity tested right out of the package without having to perform the wetting proceduredescribed in 7.2.

2. Close the filtrate and open the feed shut-off clamps.

3. Use the inflation bulb (with vent closed) to generate pressure in the feed reservoir until 5psi (0.3 bars) registers on the inlet pressure transducer. Do not exceed 30 psig (2 bars).Verify that the pressure does not decay more than 0.5 psi/min. If it does, then there is aleak in the system set-up. Tighten all seals, clamps and connections and try again.

INTEGRITY TESTING


MODES OF OPERATION

4. Once the system integrity is assured, open the filtrate shut-off clamp. (Note: If using thedata collection software KF Comm begin collecting data in the Integrity Test worksheet.)If the HF membrane module is integral the pressure should only shift a little but thenremain steady. A pressure degradation of less than 0.5 psi/min may result from gas dif-fusion through the wetted pores. This is acceptable.

5. If the pressure drops rapidly then there may be one or more broken fibers. If the pres-sure drops slowly but greater than 0.5 psi/min, then there is either one broken fiber, apin-hole leak or an incompletely wetted membrane. Try wetting the module againaccording to Section 7.2 and then perform another Pressure Hold Test. If the pressuredrops faster than 0.5 psi/min again then use a different membrane module.

6. As an alternative to using the pressure monitor, submerging the end of the filtrate line inwater can also detect a breach of membrane integrity. While the system is under pres-sure, open the filtrate shut-off clamp. Rapid bubbling indicates a loss of integrity or anincompletely wetted membrane. Try wetting the membrane and performing the pressurehold test again.

7. If rapid bubbling occurs again, then replace the membrane module. Very slow and slightbubbling is the result of gas diffusion through the wetted pores and indicates an integralmembrane.

8. Modes of OperationThe basic operating modes for the KrosFlo® Research II System are:

• Batch Concentration of cells, virus, precipitates, proteins or diagnostic particles

• Batch Clarification of cells, cell debris, virus, precipitates or proteins

• Topped-off Batch for reduced volume operations

• Diafiltration (washing) of cells, cell debris, virus, precipitates, proteins or diagnostic particles

• Dead-end Filtration to recover extra product following clarification

8.1 Batch Concentration (product in the retentate)Generally, the term “concentration” is applied to applications where the material retained by the mem-brane is (or contains) the desired product. As the process fluid is recirculated through the membraneand back to the feed reservoir, the feed reservoir becomes the process reservoir in which the volumediminishes due to the removal of permeate and the product is concentrated. This mode of operationis used, for example, with fermentation recoveries in which the desired product is the cell itself or asan initial processing step in which the product is intracellular.

8.2 Batch Clarification (product in the permeate)The term clarification is generally used for applications in which the desired product is in the perme-ate, such as soluble proteins. This mode of operation is used, for example, to harvest animal cell fer-mentations in which desired product is secreted by the cells or microbial fermentation in which theproduct has been released into solution by cell lysis.

When operated in concentration or clarification modes, the membrane quantitatively removes solidslarger than the pores of the membrane and allows the passage of soluble materials that are smaller


than the membrane pores. Set-up is the same for both batch concentration and batch clarificationprocesses. The retentate is returned to the feed reservoir, acting as a process reservoir, and moreclarified product permeates through the membrane. The degree of concentration, called the con-centration factor (CF) or volume reduction factor (VRF) is given by the following equation where Vi isthe initial volume and Vf is the final volume: CF = VRF = Vi / Vf.

8.3 Constant VolumeA disadvantage of cross-flow batch operations is the relatively high flow rate required for efficient tan-gential flow, 20 to 100 times the permeate rate. As a result, it is difficult to achieve high concentra-tion factors without foaming or vortexing in the process reservoir. A way of avoiding this problem isto operate the membrane in constant volume mode, where a smaller intermediate reservoir is utilizedas the process reservoir to maintain a constant “working” volume. While the pump creates a posi-tive pressure that drives the filtration, it also creates an equal negative pressure (vacuum) that pullsthe feed in at the same rate. So there is an overall volume movement from the feed reservoir throughthe process reservoir and out the filtrate line. The module simply inhibits the passing of any materiallarger than the membrane pore size and concentrates it in the working volume in the process reser-voir.

In the example of harvesting from a fermenter or stirred-tank bioreactor, attempts are often made touse the bioreactor vessel as the process reservoir with poor results. The fermenter exit and entranceports are frequently too small for adequate recirculation flow. The return line must be modified so thatit is submerged at high concentrations (low volumes). And foaming or vortexing prevents achievingthe desired concentration factor and results in a relatively large amount of unprocessed broth. Morerapid and higher concentration can be achieved by using a secondary process reservoir and operat-ing in a constant volume mode.

8.4 Diafiltration Materials that pass through the membrane can be washed away from materials that are retained bythe membrane (cells, particles, etc.). The technique is used to recover additional product in clarifica-tion applications, and to achieve better product purity in concentration applications. For best effi-ciency, the wash buffer should be free of the solute that is being recovered or removed. Diafiltrationmay be accomplished either by adding buffer at the same rate as the permeation rate (constant vol-ume diafiltration) or by reducing the volume in the feed reservoir and adding more buffer to regain theoriginal volume (discontinuous diafiltration). The amount of diafiltration performed can be expressedby the volume of wash buffer added divided by the batch volume, i.e. the number of “wash volumes”.During a constant volume diafiltration in which soluble components pass freely through the mem-brane, each wash volume of permeate removed reduces the solute concentration by a factor of e(2.718..). For example, a four time wash volume diafiltration will reduce the concentration of soluteby a factor of e4, i.e. 50-fold or over 98%. Using this technique, the concentration of solute can bemonitored in the permeate until the desired level of purification of product recovery is achieved.Components that are partially retained by the membrane cannot be diafiltered or washed with thesame efficiency. The membrane rejection (or retention) is defined as:

Rejection = 1 – Concentration in the permeateConcentration in the retentate

As the membrane rejection of an undesired component increases, diafiltration becomes less effectiveas a technique for removing it.

MODES OF OPERATION


OPERATING THE SYSTEM

Figure 6. The Effect of Solute Rejection on Diafiltration Efficiency

The system set-up for diafiltration is the same as for topped-off batch mode except that wash bufferenters the process reservoir instead of more product.

8.5 Dead-end FiltrationWhile the normal mode of operation involves tangential flow, Spectrum cross-flow membrane mod-ules can also be run in a dead-end mode. In the dead-end mode, the retentate line is capped orclosed with a valve so that all of the solution being processed is forced through the membrane wall.Though the efficiencies of tangential flow separation are lost, in certain circumstances such as at theend of a clarification run where maximum permeate recovery is desired, switching to dead-end modecan be advantageous.

Conventional tangential flow membrane devices that have high membrane costs must be cleanedand re-used to be economically feasible. Therefore, dead-end techniques must be avoided on re-usemembranes because they eliminate the ability to clean the membrane and increase the chances ofsample cross-contamination. Spectrum disposable membrane modules, however, lend themselvesto this mode of product recovery. Since the modules are priced to be disposable, membrane clean-ing is not necessary prior to use.

9. Operating the SystemThere are two basic system set-ups and operating instructions for the different modes. “Batch” oper-ating instructions are used when the KrosFlo® Research II TFF System is used for batch clarificationand batch concentration. “Constant Volume” operating instructions are used when the system isused for topped-off batch and diafiltration modes. Finally, for clarification applications the system canbe converted to “Dead-end” Filtration to achieve maximum product recovery. This mode can also beused as a post-process integrity test for the membrane module.

9.1 Instructions for Batch Concentration / Clarification

1. Assemble the system according to the instructions in Section 6.2.

2. Integrity Test: Test the integrity of the system and the membrane module according tothe instructions in Section 7.

3. Introduce the sample to be filtered into the reservoir and seal the lid.



4. Priming: Close the permeate shut-off clamp and make sure the second permeate port(drain) is closed. Turn the pump on in the forward direction and slowly increase the ratejust high enough to prime the system but low enough to avoid foaming and vortexing ofthe solution. Continue until a clear, bubble free stream is coming out through the reten-tate line back to the feed reservoir. It is important to eliminate all air from the recircula-tion loop to avoid product shear.

5. Setting Operating Parameters: Adjust the pump rate to the desired velocity after the airis eliminated. An easy and quick guide to use is given in the header of the KF Commworksheet. Type the model number of the filter or select the filter from the pull downmenu. The recommended feed flow rates at specific shear rates are given on the rightside of the header. For fouling (eg. protein) applications or cell applications, shear ratesof 12,000 s-1 or a maximum shear rate of 4,000 s-1 are recommended respectively.

6. If using the KF Comm data acquisition software, open the ‘Trial Data’ worksheet andbegin collecting data. The permeate flow rate (Qpermeate) and total volume of permeate(Vpermeate) will need to be manually entered at their respective times to get graphs of theFlux Rate decay and the Pressures vs. Volumetric Transfer (VT). OR, equivalently, themass of the permeate (Mpermeate) can be manually entered at the respective times usingan appropriate scale (not provided). A separate graph using the Mpermeate can be madeoffline to also measure the flux decline.

7. Check for system leaks and tighten fittings and connections if necessary.

8. Open the permeate shut-off clamp. Filtrate should start coming out the permeate line.

9. Adjust the pressure if necessary by using the flow restrictor on the retentate line to induceback pressure.

10. Concentration / Clarification: Allow the system to run in batch mode while monitoring thefiltrate (flux) rate until the desired concentration factor has been achieved or the desiredvolume has been filtered through the membrane module. In batch mode, the flux ratemay diminish as the retentate material concentrates in the process reservoir.

11. If the retentate starts to vortex or foam, stop processing the batch. For clarification appli-cations, the system can run dry. When the batch processing is complete, close the per-meate shut-off clamp and turn off the pump.

12. Product Recovery: To recover retentate, lift the lid above the feed reservoir and turn thepump on at a low rate using air to drive the retentate in the recirculation loop into thefeed reservoir.

13. To recover filtrate in the extracapillary space of the membrane module, open the perme-ate shut-off clamp as well as the drain shut-off clamp on the module lower side-port.Drain filtrate into the filtrate reservoir used during the filtration process.

14. Post-process Integrity Test: Since Spectrum hollow fiber modules are disposable, assur-ance of membrane integrity can be achieved by performing a post-process integrity test.

NOTE:: A post-process integrity test renders the module non-reusable.



9.2 Instructions for Constant Volume & Diafiltration

1. Assemble the system according to the instructions in Section 6.1.

2. Integrity Test: Test the integrity of the system and the membrane module according tothe instructions in Section 7.

3. Fill the processing reservoir with the process fluid to be washed.

i. Open the shut-off clamp on the feed line and close the shut-off clamp on the permeateline.

ii. Open the stop-cock on the processing reservoir. Use the squeeze bulb to generatepressure and drive the sample into the processing reservoir. Allow the processing reser-voir to fill to the desired level then close the stop-cock.

4. Establish a pump velocity (corresponding to suggested recirculation rates based on adesired shear).

5. The membrane inlet pressure and velocity of process fluid should be adjusted for eachspecific application. Transmembrane pressure can be increased by increasing the recir-culation rate, pressurizing the feed tank, or pulling a vacuum on the filtrate line.

6. Membrane inlet pressure should not exceed 20 psi, the maximum transducer pressure.The KrosFlo® Digital Pressure Monitor has a programmable high pressure warning alarmand a programmable high pressure pump shut-off that can be used to ensure that pres-sure does not exceed a user-defined limit.

7. Re-adjust the processing reservoir volume to a desired level by introducing more samplefrom the feed reservoir as described in step 3.

NOTE: The level of fluid in the processing reservoir plus the volume in the tubing in the pro-cessing loop will determine the final volume of the sample after diafiltration. Below arethe procedures to adjust the fluid level of the processing reservoir:

LOWERING PROCESSING RESERVOIR FLUID LEVEL

• During the filtration run close the feed shut-off clamp.

• Make sure the filtrate clamp is open.

• Open the stop-cock on the processing reservoir.

• When desired reservoir volume is reached, close the stop-cock on the processing reservoir and open the feed shut-off clamp. To avoid vortexing and bubbling keep the fluid level in the processing reservoir above the dip tubes that extend toward the bottom of the reservoir.

RAISING PROCESSING RESERVOIR FLUID LEVEL

• During the filtration run make sure the feed shut-off clamp is open and the filtrate line is closed.

• Open the stop-cock on the processing reservoir.

• When desired reservoir volume is reached, close the stop-cock on the processing reservoir and open the filtrate line.

Whenever the processing reservoir volume fills or drains with the stop-cock closed then there is a leak in the system.

PRE-STRAINING


8. Open the shut-off clamps for both the feed and filtrate lines.

9. Use the KF Comm data acquisition software to maintain a record of the operating con-ditions.

10. After the desired number of washings has been achieved the system can be drainedaccording to the instructions in Section 7.2.9.

9.3 Instructions for Dead-end Clarification

1. With the pump turned off, open the pump head. Close the retentate and permeate shut-off clamps.

2. Apply up to 20 psi pressure to the head space of the feed reservoir (the bulb valve canprovide up to 5 to 10 psi). Open the permeate shut-off clamp.

3. Collect permeate until there is no fluid remaining or until the membrane module plugs.

4. Drain the remaining permeate from the module extracapillary space by opening the drainshut-off clamp on the lower side-port of the module.

5. To release the pressure in the system, open the feed reservoir stop-cock.

10. Pre-StrainingSome process streams may require a strainer to prevent the lumen of the hollow fibers from becom-ing occluded by solution components larger than the diameter of the hollow fibers. If the fiber lumensbecome blocked, the pressure between the pump and the module will increase and the permeaterate will decline. In extreme cases, the resulting pressure build-up between the pump and the inletto the module can result in membrane module failure. A strainer should be used on the fluid prior toentering the process reservoir.

For applications where large particles or agglomerates are a concern, a test should be performedprior to processing. Install a 50 mesh screen in place of the membrane module. Circulate theprocess fluid at a high rate. If the feed pressure begins to increase while the pumping rate is con-stant, the screen is being blocked. If this is the case, the process fluid must be strained prior to enter-ing the process reservoir.

Strain the process fluid using a 50 mesh stainless steel screen. If the pressure remains constant afterpre-straining the feed, the pre-strainer worked. If there is difficulty in pre-straining the process fluid,a larger mesh strainer can be used in tandem with modules having 1 mm fiber diameters. There are,however, some fluids that plug screens quickly but do not adversely affect the membrane.


PROCESS OPTIMIZATION

11. Process OptimizationAs a pressure-driven process, the two most important variables for TFF are recirculation velocity andtransmembrane pressure (TMP). In terms of flux and passage efficiency, these two variables are atodds with one another. Higher recirculation rates, which generate the convective forces parallel tothe membrane surface, act to cleanse the membrane. Conversely TMP, which generates the drivingforce for permeation, acts to form a cake layer. Optimum operation of the module depends on estab-lishing an equilibrium between the two parameters. Optimization requires that the pressure dropthrough the module be monitored. This pressure drop (∆P) is the generated by the fluid resistance ofthe membrane channels and is defined by the equation:

∆P = Pin - Pout

Pressure drop is generated by the recirculation rate through the module and is related to the charac-teristics of the fluid, such as viscosity and density, as well as the characteristics of the module, suchas membrane diameter and length. The Transmembrane pressure (TMP) is the average pressure dif-ferential generated across the membranes and is defined by the equation:

TMP = (Pin - Pout) / 2 – Ppermeate

TMP is generated by the recirculation rate though the module plus any downstream restrictions. Thefollowing method produces a graph of Steady State Flux vs. TMP that generally resembles Figure 7:

Figure 7. Typical Flux and Solute Passage Curve

The first section of the curve (1) is the pressure dependent region and indicates that a cake layer hasnot yet been formed. In the pressure dependent range solute passage is generally unrestricted; rais-ing the TMP increases the flux. The top section (2) of the curve is the pressure independent rangeand indicates that a cake layer has been formed. In the pressure independent range solute passageis controlled by the cake layer. Raising the TMP does not increase the flux. The mid-section of thecurve (3) shows the onset of cake layer formation. The optimum TMP is the highest flux where solutepassage is close to 100%. The following is a step-wise approach to optimizing the operating condi-tions:

1. With the permeate line clamped off and no downstream restriction, begin the run with thehighest possible recirculation rate. This may be dictated by one or more considerations;such as, inlet pressure to the module, pump capacity, solution shear sensitivity, etc.


2. Open the permeate line. Allow the permeate rate to reach steady state, which normallytakes 5 to 10 minutes. Then measure the flux and take a permeate sample to determinesolute passage. Collect the data (recirculation rate, transmembrane pressure, and flux)using KF Comm on a computer. Collect the solute passage data separately.

3 Increase the TMP of the module by adding back pressure. This can be done using aretentate back pressure valve or by applying fluid or head pressure to the process ves-sel. Generally this increase should be in 0.5 bar increments. If the module was alreadybeing run at the maximum inlet pressure of 2 bars (30 psig), then the initial recirculationrate will have to be decreased.

4. Establish a new equilibrium, measure the flux, take another sample of the filtrate andrecord the recirculation rate and transmembrane pressure.

5. Repeat Steps 11.3 and 11.4 until either flux does not increase or until the passage ofdesired solutes begins to decline. Optimum is just prior to the point of flux decline anddecline of solute passage.

In certain instances even the highest recirculation rates and lowest TMP’s may generate caking whichwill restrict passage of solutes. In these cases, restricting or metering the permeate will reduce trans-membrane pressure (TMP) by increasing the permeate pressure. (Refer to the TMP equation.) Whenpermeate pressure is other than atmospheric, the pressure transducer must be connected to the per-meate line. In Step 11.2 above, partially open the permeate line then proceed to Step 11.3.

TMP is increased in this case by opening the permeate line or speeding the metering pump incre-mentally and checking the permeate for solute passage. Continue to increase TMP in this manneruntil solute passage declines. Optimum is just prior to the point of flux decline.

12. In-Process Module CleaningEven under optimum conditions, a slow decrease in the permeate rate may occur. There are threetechniques for restoring permeate flux. These include: pump off; forward flushing; and reverse flush-ing. All three, however, stop or reverse permeation (which must be considered in evaluating the ben-efit of these techniques).

12.1 Pump Off CleaningTurning the pump off will allow a loosening of the cake layer on the membrane. Re-starting the pumpwill flush loosened material from the membrane lumen surface. Although allowing one minute beforere-starting the pump is usually sufficient, the optimal time will vary with the solution being processedand should be investigated for each individual case.

12.2 Forward FlushingClosing the permeate shut-off clamp while recirculating will clean the membrane in the downstreamhalf of the module. Closing the permeate line causes the permeate pressure to rise and exceed theretentate pressure in the down stream half of the module. In this region the permeate will flow fromthe outside of the fiber back inside. This loosens and carries away caked material. Normally forwardflushing for one minute is sufficient to clean the downstream half of the membrane. Open the per-meate line again and continue processing.

The principle of forward flushing can be explained as follows: when the permeate line is closed, thenet filtration rate in the module is zero. But permeation still occurs internally. The inlet half of the mem-brane module (the high pressure end) generates permeate that back flushes the downstream half ofthe membrane module (the low pressure end). This phenomenon is called Sterling Flow.

IN-PROCESS MODULE CLEANING

MODULE SELECTION AND SCALE-UP


Reversing the pump with the permeate line closed will back flush the other half of the module and isan effective cleaning technique.

12.3 Reverse FlushingTo clean both halves of the module simultaneously, operate the pump in the reverse direction suchthat the recirculation flow enters the module from the retentate end of the module and exits the inletend. Open the permeate line and allow the permeate to be pulled back into the module through thepermeate port for 1 minute. Then switch the pump to the forward direction again and continue pro-cessing.

Reversing the pump induces a negative pressure along the entire length of the membrane fibers andcauses permeate to be pulled back through the walls of the membrane and into the retentate. Thisflow reversal will flush caked material back into the bulk process fluid. Using caution, the membranecan also be back flushed by pumping permeate directly into the extracapillary space of the module.This must be done with caution at 0.7 bars differential (10 psid) maximum or the membrane hollowfibers can be permanently damaged.

Note that reverse flushing will cause the bulk process fluid volume to increase in the processing reser-voir. Precautions must be taken to accommodate for this volume increase.

13. Module Selection and Scale-upThe KrosFlo® Research II TFF System can successfully be used to determine the membrane surfacearea needed for scale-up. The performance of the membrane module depends on many processingvariables; such as, the fluid type being processed, the conditions of the fluid (temperature, % solids,viscosity, etc.), fluid volume, the recirculation rate, the extent of caking, etc. For scale-up purposesthe amount of surface area and module size needed to process a batch depends on the following:

• The steady state flux

• The batch size to be processed

• The desired process duration

• The time required for equipment turn-around (cleaning, rinsing, etc.).

Steady state flux can usually be determined by a trial run with the fluid to be processed. The batchsize and the desired time to process the batch are determined by the user. Spectrum KrosFlo® mod-ules are especially useful for small volume scale-up studies.

It is generally prudent to include a safety factor in designing the process. Oversizing the membranesurface area by 10% to 30% is generally sufficient to account for problems associated with fluid vari-ability.

In order to determine the amount of membrane surface area required for scale-up, the steady stateflux must be determined. Accuracy of the scale-up procedure is dependent on several variablesbeing constant from the test runs to the final process. These variables include solution composition,recirculation rate, processing temperature and operating pressures. Probably the most importantconsideration is that the solution being tested is representative of the scale up fluid to be processed.Steady state flux occurs when the permeate rate remains essentially constant over a period of 15 to30 minutes.

MODULE SELECTION AND SCALE-UP


By knowing the steady state flux, the batch size to be processed and the desired processing dura-tion, a simple calculation can be made to determine the necessary membrane surface area:

Required surface area = Filtrate volume desired Required time x Steady state flux

Units are:

Required surface area = square meters (1 m2 = 10,000 cm2)

Filtrate volume desired = liters

Required time = hours

Steady state flux = liters per square meter hour (L/m2hr)

Example 1:

Clarify animals cells from 1.5 liters of bioreactor broth

The steady state flux is 90 L/m2hr.

The desired processing time is 45 minutes or 0.75 hours.

Required Surface Area = 1.5 liters = 0.022 m2 or 220 cm2

0.75 hours x 90 L/m2hr

Referring to Spectrum’s product catalog, part no. M52M-260-01N, a MiniKros® Sampler Plus mod-ule with 460 cm2 is sufficient for this application and will actually process 1.5 liters of permeate in 0.36hours (22 minutes).


ORDERING INFORMATION

14. Ordering Information

KrosFlo® Research II TFF System Components & Accessories

KrosFlo® Research II TFF SystemsPart Number Flow-path Type Pump Capacity Tubing F/P CapacitySYR2-X21-01N MicroKros® 2.3 LPM, 110V C-Flex 14 130 ml/min

SYR2-X22-01N MicroKros® 2.3 LPM, 220V C-Flex 14 130 ml/min





System Components & AccessoriesPart Number Description

ACPM-201-01N KrosFlo® Pressure Monitor, 110V; w/ alarm, auto shut-off, KF Comm, 3 transducers & RS232

ACPM-202-01N KrosFlo® Pressure Monitor, 220V; w/ alarm, auto shut-off, KF Comm, 3 transducers & RS232

ACPM-499-03N Disposable Pressure Transducer for KrosFlo® Pressure Monitor (3/pkg)

ACR2-021-01N KrosFlo® Research II Pump, 2.3 LPM, 110V (pump head not included)

ACR2-022-01N KrosFlo® Research II Pump, 2.3 LPM, 220V (pump head not included)

ACR2-H3S-01N KrosFlo® Research II Pump Head, 3 SS Rollers

ACR2-XFP-01N MicroKros® Flow-path Fittings Kit (Size 14 tubing & fittings)

ACR2-DFP-01N MidiKros® Flow-path Fittings Kit (Size 16 tubing & fittings)

ACR2-SFP-01N MiniKros® Sampler Plus Flow-path Fittings Kit (Size 17 tubing & fittings)

ACTO-015-01N Process Reservoir 15 ml Polypropylene




ACTO-2PP-01N Process Reservoir 2 L Polypropylene

ACTO-4PP-01N Process Reservoir 4 L Polypropylene

ACTO-12P-01N Process Reservoir 12 L Polypropylene

NOTE: Contact Spectrum for assistance selecting the appropriate HF Module, Flow-path, Reservoir Bottles and Disposables for your application.

400-11545-000 Rev. 00 • 052608-152210

SpectrumLabs.com Americaphone (310) 885-4600 (world-wide) e-mail [email protected] www.spectrumlabs.com

SpectrumLabs.com Europe phone +31 (0) 76 5719 419 e-mail [email protected] www.spectrumlabs.eu

SpectrumLabs.com Chinaphone (+86) 21 68810228 e-mail [email protected] www.spectrumlabs.cn

SpectrumLabs.com Japanphone +81-77-552-7820e-mail [email protected] www.spectrumlabs.jp

KrosFlo Research II TFF System

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