SEPAX Cell Processing System Operator s Manual

(1)

SEPAX

Cell Processing System

Operator’s Manual

(2)

(3)

OM – 0114 – 00 – 11 – EN

SEPAX

Cell Processing

System

Operator's Manual

OM-114-EN

(4)

(5)

OM – 0114 – 00 – 11 – EN

1. PREFACE ...1 2. SEPAX S-100 MAIN UNIT...2 3. SINGLE-USE SEPARATION KITS...3 A. USER INSTRUCTIONS CS-470.x... 3A B. USER INSTRUCTIONS CS-480... 3B C. USER INSTRUCTIONS CS-490.x... 3C D. USER INSTRUCTIONS CS-500... 3D E. USER INSTRUCTIONS CS-510... 3E F. USER INSTRUCTIONS CS-530.x... 3F G. USER INSTRUCTIONS CS-600.x...3G H. USER INSTRUCTIONS CS-900.x... 3H I. USER INSTRUCTIONS CS-540.x...3I Z. PREPARATION OF THE CRYOSC CRYOBAG FOR CRYOPRESERVATION.. 3Z 4. THE SEPARATION PROTOCOL ...4 A. USER INSTRUCTIONS PBSC ... 4A B. USER INSTRUCTIONS UCB-HES... 4B C. USER INSTRUCTIONS UCB ... 4C D. USER INSTRUCTIONS UCB-LBC ... 4D E. USER INSTRUCTIONS PBSC POST THAWING WASHING ... 4E F. USER INSTRUCTIONS UCB- POST THAWING WASHING ... 4F G. USER INSTRUCTIONS GENERIC VOLUME REDUCTION...4G H. USER INSTRUCTIONS DENSITY GRADIENT BASED SEPARATION .... 4H Z. USER INSTRUCTIONS PURGE ... 4Z 5. ACCESSORIES ...5 6. MAINTENANCE ...6 7. ERROR MESSAGES ...7

(6)

(7)

Preface OM – 01 – 06 1 - 1

1. PREFACE

1.1 Introduction to the SEPAX system and its features

The SEPAX cell processing system uses a rotating syringe technology that provides both separation through rotation of the syringe chamber (centrifugation) and component transfer through displacement of the syringe piston.

The SEPAX system allows the automated processing of blood component in a functionally-closed and sterile environment.

The separation protocol offers similar cell processing performance to the widely accepted manual separation procedure while ensuring a high level of reproducibility.

The by-products of the blood separation (plasma and red-blood cells) are collected in standard blood bags while the output product (buffy coat or BC) is collected in a cryogenic (or freezing) storage bag.

All bags are connected to the SEPAX single-use kits and form part of the sterile environment. The SEPAX system consists of:

• SEPAX Main Unit The S-100 main (processing) unit provides centrifugal and axial displacement drive to the chamber on the single-use separation kit, as well as drive to the directional valves.

The main unit can be used with the AS-610 Traceability Kit that includes a bar code reader and thermal printer. It can also be used with the SepaxNet SN-100 system that is a powerful traceability tool that transfers procedure data presently stored in the Sepax and Coolmix devices to a centralized standard SQL database.

• Single-use kit Processing kits contain the blood in a sterile environment during the complete operation – valves control the flow of blood components to the correct bag.

1.2 Indication for use

The SEPAX system is a blood cell processing system intended for laboratory use in exclusive combination with a compatible single-use separation kit supplied by Biosafe. The blood to be processed has been previously collected and transported to the laboratory by other means. The Sepax system allows the fast, automated and reproducible separation of blood in a closed and sterile environment. The SEPAX system is not intended for use in transfusion applications at bedside, where blood circulates directly between a patient and the SEPAX unit.

Important note: Sepax Single-use Kits have been sterilized by Ethylene Oxide (EtO). Patients with hypersensitivity to EtO should not receive cord blood processed with these kits. Anaphylactic reactions can occur from hypersensitivity.

1.3 Manufacturer

Biosafe SA is an ISO 9001 / ISO 13485 certified company, working under a number of national and regional directives.

The SEPAX technology is protected by patents EUR 0912250 and US 6123655. Other patents are pending.

(8)

Preface OM – 01 – 06 1 - 2 Biosafe SA • Route du Petit-Eysins 1 • 1262 Eysins • Switzerland

Telephone: +41 22 365 27 27 • Fax: +41 22 365 27 37 www.biosafe.ch • [email protected]

1.4 How to use this manual

This operating manual is organized in functional sections to provide easy access to the information concerning the SEPAX equipment.

Prior to using the SEPAX system this manual should be read in its entirety. The Table of Contents can be used to search for specific information.

1.4.1 Proprietary clause

The content of this manual is the exclusive property of Biosafe SA.

It is strictly forbidden to reproduce and/or disseminate any of the contents without the prior written permission of Biosafe SA.

1.5 Warnings and Precautions

1.5.1 The SEPAX system in general

• To ensure safe and effective use of the SEPAX system operation should only be entrusted to trained personnel.

The SEPAX equipment and single-use kits have not been designed for any modification by the end-user or third party.

Intervention such as modification, revision, maintenance or repair should only be performed by approved Biosafe technicians.

Prior to using any part of the SEPAX system, including the traceability kit or the SepaxNet, the operator should read all of instructions in this manual.

In addition, the operator/user must check that the equipment functions safely and ensure that it is in proper working condition before being used.

The separate manuals for the SepaxNet, the barcode reader, printer and power supply should also be read prior to using the SEPAX system.

A Biosafe representative should be contacted if any doubt exists concerning the use of the SEPAX system.

• Biosafe is not liable for any injury or damage resulting from use of the SEPAX system that does not conform to the indications in this Operator’s Manual

• Biosafe cannot be held responsible for the quality and subsequent effects of products processed on the SEPAX system that have undergone subsequent post-processing.

1.5.2 The single-use separation kit

• The SEPAX system should be used exclusively with SEPAX separation kits, which are supplied sterile and are for single use only.

Each kit should be disposed of in the appropriate manner following blood processing. Biosafe shall not be held responsible for any consequences of single-use kits other than those specified in this document being used with the SEPAX system.

(9)

Preface OM – 01 – 06 1 - 3

1.5.3 Blood manipulations

• If blood spillage or leakage occurs, the product should be discarded (see Chapter 6 for cleaning procedures).

• Gloves and protective clothing should be worn for all blood handling operations.

1.5.4 Electro-Magnetic Compatibility

Recommendations as required by IEC 60601-1-2 §6.8.2.201

Medical Electrical Equipment needs special precautions regarding EMC and need to be installed and put into service according to the EMC information provided in this accompanying document. Portable and mobile RF communications equipment can affect Medical Electrical Equipment. All staff involved has to receive an explanation of the ESD warning symbol and training in ESD precautionary procedure, and that the users hand should be discharged by earth bonding. (Refer to 1.8 Symbol chart and abbreviations).

The Sepax should not be used adjacent to or stacked with other equipment. If adjacent or stacked use is necessary, the Sepax should be observed to verify normal operation in the configuration in which it will be used. Tables below help to determine such conditions.

(10)

Preface OM – 01 – 06 1 - 4 Table 201 – Guidance and manufacturer’s declaration – electromagnetic emission – for all EQUIPMENT AND SYSTEMS (see 6.8.3.201 a) 3))

Guidance and manufacturer’s declaration – electromagnetic emission

The Sepax is intended for use in the electromagnetic environment specified below. The customer or the user of the Sepax should assure that it is used in such an environment.

Emissions test Compliance Electromagnetic environment - guidance RF emissions

CISPR 11

Group 1

The Sepax uses RF energy only for its internal function. Therefore, its RF emissions are very low and are not likely to cause any interference in nearby electronic equipment. RF emissions CISPR 11 Class A Harmonic emissions IEC 61000-3-2 Class A Voltage fluctuations / flicker emissions IEC 61000-3-3 Complies

The Sepax is suitable for use in all establishments other than domestic and those directly connected to the public-voltage power supply network that supplies buildings used for domestic purposes.

Table 202 – Guidance and manufacturer's declaration – electromagnetic immunity – for all EQUIPMENT and SYSTEMS (see 6.8.3.201 a) 6))

Guidance and manufacturer’s declaration – electromagnetic immunity The Sepax is intended for use in the electromagnetic environment specified below. The customer or the user of the Sepax should assure that it is used in such an environment.

Immunity test IEC 60601

test level Compliance level

Electromagnetic environment - guidance Electrostatic discharge (ESD) IEC 61000-4-2 ± 6 kV contact ± 8 kV air ± 6 kV contact ± 8 kV air

Floors should be wood, concrete or ceramic tile. If floors are covered with synthetic material, the relative humidity should be at least 30 %. Electrical fast transient / burst IEC 61000-4-4 ± 2 kV for power supply lines ± 1 kV for input/output lines ± 2 kV for power supply lines

Mains power quality should be that of a typical commercial or hospital environment. Surge IEC 61000-4-5 ± 1 kV differential mode ± 2 kV common mode ± 1 kV differential mode ± 2 kV common mode

Mains power quality should be that of a typical commercial or hospital environment. Voltage dips, short interruptions and voltage variations on power supply input lines < 5 % UT (>95 % dip in UT ) for 0,5 cycle 40 % UT (60 % dip in UT ) for 5 cycles < 5 % UT (>95 % dip in UT ) for 0,5 cycle 40 % UT (60 % dip in UT )

for 5 cycles for 230V only

Mains power quality should be that of a typical commercial or hospital environment. If the user of the Sepax requires continued operation during power mains interruptions, it is recommended that the Sepax be powered from an uninterruptible

(11)

Preface OM – 01 – 06 1 - 5 IEC 61000-4-11 70 % UT (30 % dip in UT ) for 25 cycles < 5 % UT (>95 % dip in UT ) for 5 sec 70 % UT (30 % dip in UT ) for 25 cycles < 5 % UT (>95 % dip in UT ) for 5 sec

power supply or a battery.

Power frequency (50/60 Hz) magnetic field IEC 61000-4-8

3 A/m 3 A/m

Power frequency magnetic fields should be at levels characteristic of a typical location in a typical

commercial or hospital environment. NOTE UT is the a. c. mains voltage prior to application of the test level.

(12)

Preface OM – 01 – 06 1 - 6 Table 204 – Guidance and manufacturer’s declaration – electromagnetic immunity –

for EQUIPMENT and SYSTEM that are not LIFE-SUPPORTING (see 6.8.3.201 b)) Equipment with which the Sepax has been tested in stacked of adjacent configuration and with which stacked or adjacent use is permitted:

Guidance and manufacturer’s declaration – electromagnetic immunity The Sepax is intended for use in the electromagnetic environment specified below. The customer or the user of the Sepax should assure that it is used in such an environment.

Immunity test IEC 60601 test level

Compliance level Electromagnetic environment - guidance

Conducted RF IEC 61000-4-6 Radiated RF IEC 61000-4-3 3 Vrms 150 kHz to 80 MHz 3 V/m 80 MHz to 2,5 GHz 3 V 3 V/m

Portable and mobile RF communications equipment should be used no closer to any part of the Sepax, including cables, than the recommended separation distance calculated from the equation applicable to the frequency of the transmitter.

Recommended separation distance c

P

d

=

1 ,

2

80 MHz to 800 MHz

P

d

=

2 ,

3

800 MHz to 2,5 GHz

where p is the maximum output power rating of the transmitter in watts (W) according to the transmitter manufacturer and d is the

recommended separation distance in metres (m).b

Field strengths from fixed RF transmitters, as determined by an electromagnetic site survey,a should be less than the compliance level in each frequency range.b

Interference may occur in the vicinity of equipment marked with the following symbol:

NOTE 1 At 80 MHz and 800 MHz, the higher frequency range applies.

NOTE 2 These guidelines may not apply in all situations. Electromagnetic is affected by absorption and reflection from structures, objects and people.

(13)

Preface OM – 01 – 06 1 - 7 a

Field strengths from fixed transmitters, such as base stations for radio (cellular/cordless) telephones and land mobile radios, amateur radio, AM and FM radio broadcast and TV broadcast cannot be predicted theoretically with accuracy. To assess the electromagnetic environment due to fixed RF transmitters, an electromagnetic site survey should be considered. If the measured field strength in the location in which the Sepax] is used exceeds the applicable RF compliance level above, the Sepax should be observed to verify normal operation. If abnormal performance is observed, additional measures may be necessary, such as re-orienting or relocating the Sepax.

b_{Over the frequency range 150 kHz to 80 MHz, field strengths should be less than 3 V/m.}

Recommended separation distances between

portable and mobile RF communications equipment and the Sepax

The Sepax is intended for use in an electromagnetic environment in which radiated RF disturbances are controlled. The customer or the user of the Sepax can help prevent electromagnetic interference by

maintaining a minimum distance between portable and mobile RF communications equipment (transmitters) and the Sepax as recommended below, according to the maximum output power of the communications equipment

Separation distance according to frequency of transmitter m Rated maximum output of transmitter W 150 kHz to 80 MHz

P

d

=

1 ,

2

80 MHz to 800 MHz

P

d

=

1 ,

2

800 MHz to 2,5 GHz

P

d

=

2 ,

3

0,01 0,12 0,12 0,23 0,1 0,37 0,37 0,74 1 1,17 1,17 2,33 10 3,69 3,69 7,38 100 11,67 11,67 23,33

For transmitters rated at a maximum output power not listed above the recommended separation distance d in metres (m) can be estimated using the equation applicable to the frequency of the transmitter, where P is the maximum output power rating of the transmitter in watts (W) according to the transmitter manufacturer.

NOTE 1 At 80 MHz and 800 MHz, the separation distance for the higher frequency range applies.

NOTE 2 These guidelines may not apply in all situations. Electromagnetic propagation is affected by absorption and reflection from structures, objects and people.

1.6 Warranty

Biosafe’s products are designed and manufactured to provide reliable performance when properly maintained and used in accordance with the instructions provided in this manual. Each unit is carefully inspected and tested before shipping.

In case of equipment failure or malfunction, Biosafe will replace or repair the concerned equipment according to the agreement in place.

(14)

Preface OM – 01 – 06 1 - 8 Equipment failure or malfunction for reasons other than a manufacturing defect (such as improper handling of the machine or non-compliance with the Operator’s Manual) is not covered under the Biosafe warranty program and such equipment will be replaced or repaired at the charge of the end-user.

Biosafe shall under no circumstances be liable for consequential or economical damage that may be an indirect or direct consequence of a defective part.

1.7 Customer support

All SEPAX equipments supplied with a copy of the Operator’s Manual.

In addition, competent technical staff will provide end-user training prior to use and will always be available for specific questions or clarifications

For assistance in technical or application issues, please contact your local representative or call Biosafe Customer Service in Switzerland + 41 22 365 2727

1.8 Symbol chart and abbreviations

Operating instructions must be observed by user

Class I _{Equipment energized from an external electrical power source} Type B equipment providing a degree of protection against electrical shocks particularly regarding allowable leakage current

0123

CE marking of the SEPAX instrument and kits

~

Alternating current

O Power off

I

Power on

STERILE EO Sterilization with ethylene oxide Expiry date

Not reusable, for single use only.

Date of manufacture

REF Product number

LOT

Batch designation Refer to chapter 6.2

Pins of connectors identified with the ESD warning symbol should not be touched. Connections should not be made to these connectors unless the users hand is discharged by earth

(15)

2. SEPAX S-100 MAIN UNIT

2.1 General description

The SEPAX S-100 main unit that provides centrifugal and axial displacement drive to the chamber on the single-use separation kit, as well as drive to the directional valves.

The main components of the SEPAX S-100 are:

• Centrifuge motor and cabinet including separation chamber ‘pit’ (the actual separation chamber is part of the single-use separation kit)

• Pneumatic pump system providing positive and negative pressure to displace the separation chamber piston - the unit is equipped with a line pressure monitor

• Charge-Coupled Device (CCD) to allow precise measurement of the volume in the separation chamber

• Rotary pins drives to position the stopcocks on the single-use separation kit • Optical line sensor to monitor the different components passing through the tubes.

• Computer system (CPU) for controlling the automated process, including memory card and RS-232 communication port

• User interface with liquid crystal display (LCD), membrane keypad and speaker

(16)

2.2 Operating and storage requirements

Please read the following instructions carefully before installing the SEPAX equipment

2.2.1 Environmental specifications

The SEPAX system must only be operated under the following environmental conditions:

Mode Operation Storage & Transport Temperature +7°C to +27°C 0°C to 50°C

Relative

humidity 30% to 75%, non-condensing 20% to 75%, non-condensing Maximum

Altitude 2000m 2000m

The SEPAX S-100 should:

. only be operated on a flat, stable, horizontal and clean surface . be used in an open environment to allow sufficient ventilation . cleaned regularly (see Chapter 6 for cleaning instructions) . kept upright during transport.

. be connected to an earthed power supply directly (no adapters or extension leads)

The SEPAX S-100 should not be exposed to:

- direct sunlight or strong light sources - liquids or corrosive substances - physical shocks or vibrations. - heavy weights

- Other equipment that contain magnets or that generate magnetic or electromagnetic fields (such as mobile/cellular phones).

(17)

2.3 SEPAX S-100 Main Processing Unit – Components

8a. By-product bag hooks

10. Input product bag support

11. Bubble chamber support

12. Data card slot 2. Optical line sensor

3a. Separation chamber pit covers 4. Air filter 3b Separation chamber pit 9. Handles 8b. Collection bag hook 7. Line pressure sensor 1. Keyboard 6. Rotary pins 16. IBM PS2 port 15. Serial ports 13. Power switch 14. Cable socket The machine OM – 02 – 08 2 - 3

(18)

1. Keyboard

Keyboard, with 6 keys that allow the operator to select options, such as traceability parameters (see 2.3.1 Key Board).

2. Optical line sensor

The equipment uses the different light absorbance of the separated components to manage the automated separation procedure.

3. Separation chamber pit & covers

The separation chamber pit is where the single-use kit separation chamber is installed. The separation chamber pit covers are closed around the separation chamber rotary seal to ensuring stability during the procedure – the SEPAX S-100 informs the operator if the pit covers are not closed.

The SEPAX S-100 provides both electrical centrifugal drive and pneumatic axial drive to the separation chamber for separation of the input product and transfer of each component to the appropriate bag/container.

A Charge-Coupled Device detector (CCD), situated in the pit and positioned alongside the separation chamber, monitors the position of the chamber piston allowing real-time volume measurements.

4. Air filter

The air filter prevents dust from entering the machine.

5. LCD display

The display provides continuous information concerning progress of the separation procedure and any procedure anomalies.

6. Rotary pins

Two rotary pins position the stopcocks on the single-use separation kit, to direct flow of the separated components to the appropriate bag.

7. Line pressure sensor

The line pressure sensor monitors pressure in the single-use separation kit tubing, to avoid over-pressure.

8. Bag hooks

Hooks are provided on which the collection and by-product bags are hung during the separation procedure.

9. Handle

Handles on each side of the SEPAX-100 allow easy transport - the SEPAX S-100 only weighs 13kg (29lbs).

10. Input product bag support

Support to hang the input (or source) bag.

11. Bubble chamber support

Support to hang the bubble chamber.

12. Data card slot

(19)

The machine OM – 02 – 08 2 - 5 START START START START START START STARTSTART

ENTER

No

PCMCIA card slot onto which is down-loaded procedure and input product data.

13. Power switch

On/off button.

14. Cable socket

Socket for the power supply cable.

15. Serial ports

2 x serial ports, one for connection to thermal printer or the connection boxes of the SepaxNet SN-100 system, the second for use by service technician.

16. IBM PS2 port

Serial interface connector for connection to barcode reader.

2.3.1 Keyboard Functions

The keyboard has six keys with the following functions:

KEY

ACTION

- Print barcode hardcopy of manually-entered traceability references

STOP

- Emergency STOP. Stops the procedure

- Moves the display cursor up

- Moves the display cursor down

ENTER

- Validates selection

(20)

The machine OM – 02 – 08 2 - 6 2.4 Technical specifications of the SEPAX

2.4.1 Dimensions (approximate): Width: 29 cm (11.4”) Length: 36 cm (14.2”) Height: 37 cm (14.6”) 2.4.2 Weight: 13kg (29lbs) 2.4.3 Power:

Use only the original certified cable for the power supply.

The SEPAX equipment should always be connected to an Uninterruptible Power Supply (UPS)

Input range: 100 to 240 VAC Input frequency: 50 - 60 Hz Consumption: 200 VA Leakage current: < 500 μA

(The SEPAX S-100 automatically adjusts for the local supply voltage)

Fuse: T2AH

2.4.4 Centrifuge:

Max speed: 8000 rpm

Speed range: 1700 - 8000 rpm Over speeds protection: 8800 rpm

(21)

The separation kit OM – 03 – 07 3 - 1

3. THE SINGLE-USE SEPARATION KIT

3.1 General description

• The separation kits are composed of a proprietary separation chamber, tubing and collection/by-product bags.

• The separation chamber is a syringe pump that enables both the centrifugation of the processed sample and the transfer of the sample from the initial bag to the various collection and by-product bags in a closed and sterile environment – the separation chamber is the same for all separation kits

• The kits are individually packed and sterilized, and have a shelf life of 2 years.

Only separation kits provided by Biosafe for specific protocols (applications) should be used with the Sepax system

3.2 Possible kits for the different protocols

Protocols Kits

UCB UCB-HES UCB-LBC PBSC PBSC-CW UCB-CW Vol. Red. Ficoll

CS-470.x1 X X CS-4803 X X X X CS-490.x X X X X X CS-5003 X2 X2 X2 X2 X2 CS-5103 X2 X2 X2 X2 X2 CS-530.x X X X CS-600.x X X CS-900.x X CS-540.x4 X X X CS-430.x X X CS-570.x4 X X

1 _{Works only with SEPAX machines equipped with a stopcock blocker.} 2

Up to a total volume of 70 ml (CS-500) or 20 ml (CS-510).

3_{Discountinued} 4

Functionnaly closed

Only the separation kits CS-530.x and CS-540.x and the Protocol UCB-HES are available on the US market.

3.3 Storage requirements

The separation kit must be stored in a clean and dry place, free from biological contaminants and chemical vapours – for storage conditions see Section 2.2.1.

(22)

The separation kit OM – 03 – 07 3 - 2

3.4 Single-use Kit components

Each kit includes a separation chamber and associated harness. The harness includes:

• Bubble chamber with a micro aggregate filter

• Set of tubing lines with roller and slide clamps

• Stopcock manifold

• Collection/by-product bags

• Line pressure monitor with a micro-filter

• Spike for connecting the sample bag to the kit (Biosafe advises use of a sterile connection device

3.4.1 Cord blood collection bag

The SEPAX system is compatible with standard input bags.

20cm of tubing should be left on the input bag at the time of collection to facilitate connection of the bag to the single-use separation kit.

If a spike is to be used, the input bag must have a standard blood bag membrane port (septum) with a diameter of 4-6 mm.

3.5 Instructions for Use (IFU)

Each box of 6 single-use separation kits includes Instructions for Use corresponding to that particular kit.

Instructions for Use should be read carefully prior to opening the kit blister pack.

Verify expiry date and check the general condition of the kit / blister before use DO NOT use kits that shown sign of damage or mishandling

(23)

CS-470.x OM – 3A – 04 3A - 1

SEPAX cell separation kit CS-470.1

Configuration

A: Separation chamber 200ml

B: Line pressure monitor luer with microbial filter

C: Stopcock

D: Bubble chamber

E: Spike to source bag

F: Buffy coat collection vial with injection site and

microbial filter. Warning: this is not a cryovial!

E

F

D

C

B

A

(24)

(25)

CS-480 OM – 3B – 02 3B - 1

SEPAX cell separation kit CS-480 (Discontinued)

Configuration

A: Separation chamber 200ml

B: Line pressure monitor luer with microbial filter C: Stopcock manifold

D: Bubble chamber E: Spike to source bag F: 500ml PVC collection bag G: Spike to buffy-coat collection bag

C

G

B

A

F

D

E

(26)

(27)

CS-490.x OM – 3C – 05 3C - 1 SEPAX cell separation kit CS-490.1

D: Bubble chamber E: Spike to source bag F: 500ml PVC collection bag G: Spike to output bag H: Luer lock to output bag I: Injection site

The tube between the injection site (I) and the spike (G) is double-coated type and may be difficult to seal with certain types of sealers. That portion of tube is not compatible with sterile connection device (SCD).

A

B

C

D

E

F

G

H

I

(28)

CS-490.x OM – 3C – 05 3C - 2 CS-490.1 General Notes:

The CS-490.1 is a separation kit conceived for GVR and PBSC protocols in cellular products processing.

- The CS-490.1 kit can be connected to the cellular product input bag with a sterile connection device: to use a sterile connection device, the input bag must have at least 20cm of available tubing to be inserted in the sterile connection device with the tubing between the spike port and the bubble chamber in the input bag line (“E” in figure n.1). This operation must be done under LAMINAR FLOW.

- If 20cm (8”) of tubing is not available, the connection of the input bag will require use of the spike

connector pre-installed on the CS-490.1 kit: this operation must be done under LAMINAR FLOW.

STEP 1: Required material

Prior to starting the procedure, ensure that the following material is within easy reach:

• Sepax S-100 main processing unit with AS-610 traceability kit

• Cellular product input bag (not supplied by Biosafe)

• Output Bag. (not supplied by Biosafe) A freezing bag or a transfer bag can be used. Be sure that the bag has enough capacity to contain your final product

• CS-490.1 single-use kit

Figure 1

• Sterile Connection Device if necessary (not supplied by Biosafe)

• Tubing sealer (not supplied by Biosafe)

• Tubing stripper (not supplied by Biosafe)

• All laboratory material related with the cell count operations STEP 2: Sampling of Cellular product Input bag

Prior connection of the initial bag to the CS-490.1 kit, follow your validated procedure to perform initial cell count.

(29)

CS-490.x OM – 3C – 05 3C - 3 STEP 3: CS-490.1 kit sterility verification

Before opening the kit blister pack under laminar flow, ensure that the sterility indicator on the Tyvek® cover is brown, indicating that the kit is sterile.

If the sterility indicator is blue, the kit should not be used and the operator should inform Biosafe of the lot number in question.

STEP 4: CS-490.1 kit integrity verification

Under laminar flow spread the kit out in order to identify the tubing lines and components as in figure 1.

Inspect visually the kit: if ruptures or kinks are detected or kit components are missing (clamps, caps, etc.), the kit should not be used and the operator should inform Biosafe of the lot number in question.

STEP 5: Closing of the roller clamp

Close ONLY the red roller clamp placed on the input line. Leave the other clamps open.

Figure 2 STEP 6: Output bag connection

There are three ways to connect the output bag. The output bag will contain the final product, TNC concentrate also called “buffy coat”, at the end of the procedure.

• If you have a sterile connection device, you can use it to connect the output bag to the kit, the output bag needs a 10 cm tubing so to use with the SCD (see Instructions for Use supplied with the sterile connection device). The sterile connection should be made between the stopcock and the luer lock. This operation must be done under LAMINAR FLOW.

• If a sterile connection device is not available, you can use a spike. First connect the spike to output bag to the kit by using the luer lock (figure 3). The CS -490.1 kit can then be connected to the output bag using spike connection in the output bag line. This operation must be done under LAMINAR FLOW.

• It’s possible to connect a bag directly on the luer-lock of the output line. In that case there is no sampling port available. This operation must be done under LAMINAR FLOW.

Remark: with a SCD or luer lock connection, you will loose the sampling port on the output line. Be sure that a sampling port on your output bag is available.

(30)

CS-490.x OM – 3C – 05 3C - 4 Figure 3

STEP 7: Input Bag connection

There are two ways to connect the input bag.

• If you have a sterile connection device, you can use it to connect the input bag to the kit (see Instructions for Use supplied with the sterile connection device). The sterile connection should be made between the spike pre-installed spike connector and the bubble chamber in the input bag line, as shown in figure 4. This operation must be done under LAMINAR FLOW.

• If a sterile connection device is not available, the CS-490.1 kit can be connected to the input bag using the pre-installed spike connector in the input bag line (“E” in figure n.1). This operation must be done under LAMINAR FLOW conditions.

Figure 4

Once the input and the output bags have been connected, you kit should be similar to the one on the figure 5. In that example, the output bag has been connected via spike.

Figure 5

SCD

Connection

Spike

(31)

CS-490.x OM – 3C – 05 3C - 5 STEP 8: CS-490.1 kit installation on the Sepax

For instructions on installation of the CS-490.1 kit on the Sepax S-100 main unit and launching of the automated separation procedure, refer to the Operator’s Manual (Section 4.6) provided with the Sepax S-100.

The GVR protocol will perform automatically the priming of the lines. If you are using the GVR protocol, go directly to step 10.

If you are using the PBSC protocol, the priming of the lines should be performed manually, in that case go to step 9.

STEP 9: Manual Priming of the line (PBSC protocol only)

The tubing line between the source bag and the white stopcock has to be primed manually with blood. The display shows “Prime Bubble Chamber” (Figure 6)

Figure 6

Squeeze the bubble chamber, without opening the roller clamp, and release it 2-3 times until the bubble chamber is half-filled with blood (Figure 7) then press “Enter”.

Figure 7

Lower the input bag position by adjusting the holder position. This adjustment will decrease the flowing speed of the blood during the priming (Figure 8).

(32)

CS-490.x OM – 3C – 05 3C - 6 Press the key (Figure 9): the blood flows towards the stopcock; press a second time the up-key when the blood is between the blue and the white stopcocks: the blood stops flowing (Figure 10).

Figure 9

Figure 10 •

• If the priming succeeded, select START PBSC in the main menu, Figure 11.

• If the blood did not reach the blue stopcock, you can redo the priming by selecting REDO PRIMING in the Menu, Figure 11.

• If the blood went too far, figure 12, select REDO PRIMING, and use the gravity force to draw back the blood to the initial bag (figure13. After that by selecting REDO PRIMING again, prime again the tubes by bringing the blood between the blue and white stopcock.

(33)

CS-490.x OM – 3C – 05 3C - 7 Figure 13

AUTOMATED PROCEDURE

Warning: Avoid touching the machine and the kit: Moving bag, tubes, stopcock and covers may cause errors.

STEP 10: Mixing of the input bag – GVR protocol only

That step is only true for the GVR protocol. If you are running the PBSC protocol, go directly to step 11.

After each sedimentation cycle the procedure will pause and will begin to beep. The operator should then mix the input bag and press on the ENTER key so to resume the procedure (Figure 14 and 15).

Figure 14 Figure 15

(34)

CS-490.x OM – 3C – 05 3C - 8 STEP 11: Post Procedure – Remove Bags and Filter

At the end of the automated procedure, the message REMOVE BAGS AND AIR FILTER - ENTER appears on the display. Please do so, as shown on the figures 16 and 17 and press “ENTER”:

Figure 16 Figure 17

STEP 12: Post Procedure – Line Stripping

The message STRIP BC LINE - ENTER appears on the display. Strip the BC line as shown in figure 18. Then Press Enter.

The STRIP RBC LINE - ENTER message will appear on the Sepax display. Strip the RBC line as shown in figure 19. Then Press Enter.

The STRIP PLASMA LINE - ENTER message will appear on the Sepax display. Strip the Plasma line as shown in figure 20. Then Press Enter

(35)

CS-490.x OM – 3C – 05 3C - 9 Figure 20

STEP 13: Post Procedure – Close Clamps and remove kit

The message CLOSE ALL CLAMPS- ENTER appears on the display. Close all the four clamps, one for each line, then press “ENTER”.

The message REMOVE KIT- ENTER appears on the display Dismount the kit (figure 21) and press enter. The protocol will exit and the display shows the Main Menu. You can then start a new procedure.

Figure 21

(36)

CS-490.x OM – 3C – 05 3C - 10 STEP 14: Buffy-coat sampling

Prior sample taking, the final product (buffy-coat) must be mixed in order to obtain a homogeneous and representative sample.

If you used the spike to connect your output bag to the kit, it’s possible to take the sample, under laminar flow, by using the dedicated syringe port placed on the “Y” connector for needle

insertion, as shown in figure 22, and then follow your validated procedure.

If you used A SCD or the luer lock to connect your output bag to the kit, you will need to use a port of the bag so to take your sample.

(37)

CS-500 OM – 3D – 03 3D - 1

Configuration

D: Bubble chamber E: Spike to source bag F: 500ml PVC collection bag G: Buffy-coat collection bag

A

C

B

F

E

D

F

G

(38)

(39)

CS-510 OM – 3E – 02 3E - 1

Configuration

D: Bubble chamber E: Spike to source bag F: 500ml PVC collection bag G: Buffy-coat collection bag

A

C

B

F

E

D

F

G

(40)

(41)

CS-530. x OM – 3F – 08 3F - 1

SEPAX cell separation kit CS-530.0 / CS-530.3

Configuration

Fig. 1 A: Separation chamber 200ml

D: Bubble chamber

E: Spike to input product bag

F: Output bag with 25 ml nominal volume G: letter not used for this kit

H: 500ml PVC by-product (plasma) bag I: Luer port for AK-10 accessory J: DMSO extension line

K: AK-10, DMSO Vial Spike

B

A

C

D

E

F

H

J

I

K

(42)

CS-530. x OM – 3F – 08 3F - 2 CS-530.0 General Notes:

The CS-530.0 is a separation kit conceived for UCB -HES and UCB protocols in Umbilical Cord Blood processing.

- The CS -530.0 kit can be connected to the UCB input bag with a sterile connection device: to use a sterile connection device, the UCB input bag must have at least 20cm of available tubing to be inserted in the sterile connection device with the tubing between the spike port and the bubble chamber in the UCB input bag line (“E” in figure n.1). This operation must be done under LAMINAR FLOW.

- If 20cm (8”) of tubing is not available, the connection of the UCB input bag will require use of the spike port pre-installed on the CS-530.0 kit: this operation must be done under LAMINAR FLOW.

• UCB input bag (not supplied by Biosafe)

• If UCB-HES protocol is used, Hydroxy-Ethyl-Starch HES solution (HES 450-0.7-6%) corresponding to 20% of the UCB input volume (UCB + CPD anti-coagulant)

• Sterile connection device (not supplied by Biosafe)

• Sterile tubing sealer (not supplied by Biosafe)

• All laboratory material related with the cell count operations STEP 2: Sampling of UCB input bag

Prior connection of the initial UCB bag to the CS-530.0 kit, follow your validated procedure to perform initial cell count.

STEP 3: CS-530.0 kit sterility verification

Before opening the kit blister pack, ensure that the sterility indicator on the Tyvek® cover is brown, indicating that the kit is sterile.

Spread the kit out in order to identify the tubing lines and components as shown above.

NOTE :

(43)

CS-530. x OM – 3F – 08 3F - 3 STEP 5: CS-530.0 kit preparation

Close ONLY the red roller clamp placed in the input UCB line and the two blue clamps on the DSMO extension line (“J” in figure n.1).

Leave the other 2 white clamps open.

STEP 6: If UCB-HES protocol is used: Hydroxy-Ethyl-Starch (HES) preparation If you are not using the UCB-HES protocol, go directly to step 8.

In accordance with your internal validation procedure, prepare a Hydro-Ethyl-Starch (HES) solution (HES 450-0.7-6%) corresponding to 20% of the UCB input volume (UCB + CPD anti-coagulant). For example, if the volume of the UCB input bag is 100ml including CPD, you should prepare 20ml of HES solution.

Fill a syringe with the prepared HES, wait for the HES to reach room temperature. STEP 7: If UCB-HES protocol is used: HES injection in the UCB input bag

Under LAMINAR FLOW conditions, the operator should insert the syringe’s needle in the dedicated port of the UCB input bag, and then manually agitate the UCB input bag with one hand while injecting the HES with the other hand: this ensures a homogenous HES dispersion in the bag.

Inject the HES into the UCB input bag at a rate of approximately 2 seconds per ml.

STEP 8: UCB input bag connection

Connect the UCB input bag to the CS-530.0 kit with a sterile connection device (see Instructions for Use supplied with the sterile connection device).

The sterile connection should be made between the spike pre -installed spike connection and the bubble chamber in the UCB input bag line (“E” in figure n.1).

If a sterile connection device is not available, the CS-530.0 kit can be connected to the UCB input bag using the pre-installed spike connection in the UCB input bag line (“E” in figure n.1) under LAMINAR FLOW conditions.

STEP 9: CS-530.0 kit installation and UCB or UCB-HES automated procedure

For instructions on installation of the CS-530.0 kit on the Sepax S-100 main unit and launching of the automated separation procedure, refer to the Operator’s Manual (Section 4.6) provided with the Sepax S-100.

Once the procedure is complete, the Sepax S-100 guides the operator through a manual stripping of the buffy-coat (BC) bag line.

(44)

CS-530. x OM – 3F – 08 3F - 4 STEP 10: Post -procedure actions

At the end of the automated procedure, with the kit still installed on the S-100, the “STRIP BC LINE – ENTER” message will appear on the S-100 display and the stopcock valves will be in the position shown in Fig. 2 and 3.

Before pressing “ENTER”, manually squeeze the air from the BC bag until there is no air in the bag, as shown in Fig. 3.

While holding the BC bag in this position with one hand, press the “ENTER” button on the S-100 with the other hand.

The BC bag can then be released as the stopcock on the S-100 has been closed preventing return of the air.

Fig. 2 Fig. 3

Continue following the instructions on the S-100 display panel until instructed to “DISMOUNT KIT”.

STEP 11: Removal of the CS-530.0 kit from the Sepax S-100

Close the WHITE clamp above the Y connector and as CLOSE to the Y-connector as possible. Seal 3 times above the white clamp. Sealing 3 times and cutting out the line in the middle seal ensures safety in case of imperfect seal.

After sealing, detach the BC bag line with its components as shown in Fig. 4.

(45)

CS-530. x OM – 3F – 08 3F - 5 STEP 12: Buffy-coat sampling

Prior sample taking, the buffy-coat must be mixed in order to obtain a homogeneous and representative sample: press manually 30 times the small (5ml) section of the buffy-coat bag. To take the sample, under laminar flow use the dedicated syringe port placed on the “Y” connector for needle insertion and then follow your validated procedure.

STEP 13: DMSO preparation

Preparation of the DMSO solution for cryopreservation must be performed following your validated procedure.

The quantity of DMSO solution to be injected in the BC cryobag is 5ml or your validated volume. Note: If you inject DMSO by syringe pump, you will need to add an extra volume of 1.5 ml. (ex: If you need to add 5 ml, you put 5 + 1.5 ml = 6.0 ml in the syringe). This volume corresponds to the dead volume that remains in the tube.

STEP 14A: DMSO injection into the BC bag by automated Syringe pump

Connect one end of the DMSO extension line to the luer-lock syringe containing the DMSO cryosolution and the other end to the luer-lock on the kit. Check that the blue clamp on the DMSO extension line is closed.

Install cryobag on the Coolmix device, install the syringe in the syringe pump device and select the flow rate following the User Instructions of the syringe pump device. Inject DMSO with the syringe pump.

If you are using the Coolmix automated cooling and mixing device, refer to the Coolmix Operator Manual, for display settings and automated procedure.

The Coolmix is not available on US market

STEP 14B: DMSO injection into the BC bag by Gravity feed

Insert the DMSO Vial Spike (AK-10) in the DMSO vial and place the spike/vial in the DMSO Vial Adapter. Please check that you are using the correct DMSO vial adapter. If you use the CS-530.0 and the 7ml DMSO vial you need the DMSO vial adapter with the green spot. If you use the 8ml DMSO vial, use the DMSO vial adapter with the red spot. See fig 5.

with 7ml DMSO vial… with 8ml DMSO vial… If adding DMSO by syringe pump, go to step 14A.

(46)

CS-530. x OM – 3F – 08 3F - 6 …use DMSO Vial Adapter 530 for 7ml vial …use DMSO Vial Adapter 530 for 8ml vial

Fig 5.

Connect one end of the DMSO extension line to the DMSO Vial Spike (AK-10) and the other end to the luer-lock on the kit. Install the kit and DMSO vial assembly on the Coolmix - see fig. 6. If you are using the Coolmix automated cooling and mixing device, refer to the Coolmix Operator Manual, for display settings and automated procedure.

Fig. 6

The Coolmix is not available on US market

After the kit and DMSO vial assembly installation on the Coolmix, open:

• the air vent on the DMSO Vial Spike

• the DMSO extension line BLUE clamp

• the BLUE clamp on the kit where the DMSO extension line is connected

Strip gently and carefully the DMSO extension line until the DMSO flow reaches the Coolmix cover.

Press Start Mix and Start Timer on Coolmix - the DMSO will be mixed with the BC as it flows into the BC bag.

After the end of the DMSO injection, strip once again the DMSO extension line and close the DMSO extension line clamps (WHITE and BLUE).

(47)

CS-530. x OM – 3F – 08 3F - 7

SEPAX cell separation kit CS-530.1

Configuration

B: Line pressure monitor luer with 0.2µm filter C: Stopcock manifold

D: Bubble chamber

E: Spike to input product bag

F: Biosafe CryoSC cryobag (storage capacity 10-30ml, DMSO included) G: letter not used for this kit

H: 500ml PVC by-product (plasma) bag I: Luer port

J: DMSO extension line

B

A

C

E

F

H

J

I

D

(48)

CS-530. x OM – 3F – 08 3F - 8 CS-530.1 General Notes:

The CS-530.1 i s a single-use separation kit conceived for UCB-HES and UCB protocols in Umbilical Cord Blood processing.

- The CS-530.1 kit can be connected to the UCB input bag with a sterile connection device (SCD): to use a sterile connection device, the UCB input bag must have at least 20cm of available tubing. This tubing needs to be placed in the SCD device with the tubing of the CS-530.1 kit located between the spike port and the bubble chamber in the UCB input bag line (“E” in figure n.1). This operation must be done under LAMINAR FLOW.

- If 20cm (8”) of tubing is not available, the connection of the UCB input bag will require use of the spike port pre-installed on the CS-530.1 kit: this operation must be done under LAMINAR FLOW.

• Sepax S-100 main processing unit

• UCB input bag (not supplied by Biosafe)

• If UCB-HES protocol is used, Hydroxy-Ethyl-Starch HES solution (HES 450-0.7-6%) corresponding to 20% of the UCB input volume (UCB + CPD anti-coagulant)

• Sterile connection device SCD (not supplied by Biosafe)

• Sterile tubing sealer, also compatible with EVA tubing (not supplied by Biosafe)

• All laboratory material related with the cell count operations STEP 2: Sampling of UCB input bag

Prior connection of the initial UCB bag to the CS-530.1 kit, follow your validated procedure to perform initial cell count.

STEP 3: CS-530.1 kit sterility verification

Before opening the kit blister pack, ensure that the sterility indicator on the Tyvek® cover is brown, indicating that the kit is sterile.

Spread the kit out in order to identify the tubing lines and components as shown above.

(49)

CS-530. x OM – 3F – 08 3F - 9 STEP 5: CS-530.1 kit preparation

Close ONLY the red roller clamp placed in the input UCB line and the two blue clamps on the DSMO extension line (“J” in figure n.1).

Leave the other 2 white clamps open.

STEP 6: If UCB-HES protocol is used: Hydroxy-Ethyl-Starch (HES) preparation If you are not using the UCB-HES protocol, go directly to step 8.

In accordance with your internal validation procedure, prepare a Hydroxy-Ethyl-Starch (HES) solution (HES 450-0.7-6%) corresponding to 20% of the UCB input volume (UCB + CPD anti-coagulant). For example, if the volume of the UCB input bag is 100ml including CPD, you should prepare 20ml of HES solution.

Fill a syringe with the prepared HES solution; wait for the HES solution to reach room temperature.

STEP 7: If UCB-HES protocol is used: HES injection in the UCB input bag

Under LAMINAR FLOW conditions, the operator should insert the syringe’s needle in the dedicated port of the UCB input bag, and then manually agitate the UCB input bag with one hand while injecting the HES with the other hand: this ensures a homogenous HES dispersion in the bag.

Inject the HES into the UCB input bag at a rate of approximately 2 seconds per ml.

STEP 8: UCB input bag connection

Connect the UCB input bag to the CS -530.1 kit with a sterile connection device (see Instructions for Use supplied with the sterile connection device) under LAMINAR FLOW conditions.

The sterile connection should be made between the spike and the bubble chamber in the UCB input bag line (“E” in figure n.1).

If a sterile connection device is not available, the CS-530.1 kit can be connected to the UCB input bag using the pre-installed spike connection in the UCB input bag line (“E” in figure n.1) under LAMINAR FLOW conditions.

STEP 9: CS-530.1 kit installation and UCB-HES automated procedure

For instructions on installation of the CS-530.1 kit on the Sepax S-100 main unit and launching of the automated separation procedure, refer to the Operator’s Manual (Section 4.6) provided with the Sepax S-100 device.

Once the procedure is complete, the Sepax S-100 guides the operator through a manual stripping of the buffy-coat (BC) bag line.

(50)

CS-530. x OM – 3F – 08 3F - 10 STEP 10: Post -procedure actions

At the end of the automated procedure, with the kit still installed on the S-100, the “STRIP BC LINE – ENTER” message will appear on the S-100 display and the stopcock valves will be in the position shown in Fig. 2 and 3.

Before pressing “ENTER”, manually squeeze the air from the BC bag until there is no air in the bag, as shown in Fig. 3.

While holding the BC bag in this position with one hand, press the “ENTER” button on the S-100 with the other hand.

The BC bag can then be released as the stopcock on the S-100 has been closed preventing return of the air.

Fig. 2 Fig. 3

Continue following the instructions on the S-100 display panel until instructed to “DISMOUNT KIT”.

STEP 11: Removal of the CS-530.1 kit from the Sepax S-100

Close the WHITE clamp above the Y connector and as CLOSE to the Y-connector as possible. Seal 3 times above the white clamp. Sealing 3 times and cutting out the line in the middle seal ensures safety in case of imperfect seal.

After sealing, detach the BC bag line with its components as shown in Fig. 4.

(51)

CS-530. x OM – 3F – 08 3F - 11 STEP 12: Buffy-coat sampling

Prior sample taking, the buffy-coat must be mixed in order to obtain a homogeneous and representative sample: press manually 30 times the small (5ml) section of the buffy-coat bag. To take the sample, under laminar flow use the dedicated syringe port placed on the “Y” connector for needle insertion and then follow your validated procedure.

STEP 13: DMSO preparation

Composition and preparation of the DMSO solution for cryopreservation must be performed following your validated procedure.

The quantity of DMSO solution to be injected in the BC CryoSC cryobag is indicated in the table below or follow your validated volume. To inject DMSO by syringe pump, you will need to prepare a syringe filled with with an extra 1.5 ml to the volume wanted in order to compensate the dead volume that remains in the tubing (DMSO extension line) after DMSO injection. (ex: If you need to add 5 ml, you put 5.0 + 1.5 ml = 6.0 ml in the syringe).

Output volume [mL] DMSO solution* [mL] Total volume [mL]

16 min

4

20

5

25

24 max

6

30

* 55% w/v DMSO and 5% w/v Dextran 40

Note: Minimum output volume for UCB -HES and UCB protocols is 20ml and 10 ml, respectively. In order to use the Coolmix for an efficient mixing, the minimum volume before DMSO addition should be 16 ml (see table above).

Note: Addition of DMSO by gravity feed is not possible

STEP 14: DMSO injection into the BC bag by automated Syringe pump

Connect one end of the DMSO extension line to the luer-lock syringe containing the DMSO cryosolution and the other end to the luer-lock on the kit. Check that the blue clamp on the DMSO extension line is closed.

Install CryoSC cryobag on the Coolmix device. Prime manually the DMSO extension line tubing by pushing the syringe until the DMSO solution reaches the cover of the Coolmix. Then install the syringe in the syringe pump device and select the flow rate following the User Instructions of the syringe pump device. Start injection of the DMSO solution with the syringe pump device.

If you are using the Coolmix automated cooling and mixing device, refer to the Coolmix Operator Manual, for display settings and automated procedure.

(52)

(53)

CS-600.x OM – 3G – 04 3G - 1 SEPAX cell separation kit CS-600.1

Configuration

D: Double input line with female luer lock connectors and clamps E: Washing solution input with spike and clamp.

F: 1000ml PVC waste collection bag

G: Double output line with spike, injection site and clamps.

H: 2 x extensions for input bag connection with double spike, male luer lock connector and clamps. For use with cryobag requiring a double connection during the thawing process.

I: 2 x extensions for input bag connection with a single spike, male luer lock connector and clamp. For use with cryobag with a single connection during the thawing process.

CS-600.1 General Notes:

The CS-600. 1 is a separation kit conceived for PBSC Washing and UCB Washing protocols to perform routine wash of thawed PBSC or cord blood products.

Two different working procedures are described. One requires use of a Sterile Connection Device (SCD) to perform sterile connections outside the laminar flow. The other describes when no SCD is available and connections must be performed under controlled environment (laminar flow). It is strongly recommended to use the SCD to minimize the time between thawing and the beginning of the washing procedure.

Note for PBSC Washing: the input bag volume capacity should be at least the double of the initial volume of the product to be washed.

The UCB Washing and the PBSC Washing are using the same kit, the CS-600.1. But the protocols are different. Thus we describe the handling of the kit for each protocol in 2 different parts. Please refer to the protocol that you are using.

C

D

E

A

B

F

G

H

I

(54)

CS-600.x OM – 3G – 04 3G - 2 SEPAX cell separation kit CS-600.1 with PBSC Washing

Washing several bag - recommendations

The PBSC Washing protocol allows the washing of 1 or 2 bags per washing cycle. It’s possible to have 2 washing cycles per procedure. It means that with the same kit, it’s possible to wash from 1 to 4 bags.

If you wash several bags, be sure to wash bags containing product from the same patient. If you perform a 2 washing cycle procedure, it’s easier to have a sterile connection device to connect the bags to the kit. If you don’t have a SCD, you will have to dismount the kit from the machine and connect the bag(s) for the second cycle under sterile conditions.

• One or several input bags with product from the same patient (not supplied by Biosafe)

• One or two output bags (not supplied by Biosafe)

• 2 CS-600.1 single-use kit, 1 for backup

• washing solution (not supplied by Biosafe), please check the chapter 4E to estimate the volume needed

• Sterile connection device recommended (not supplied by Biosafe)

• Tubing sealer (not supplied by Biosafe)

• Orbital Shaker (not supplied by Biosafe)

• All laboratory material related with the cell count operations

• Material related to the thawing of the product STEP 2: Sepax parameter setting

In order to reduce the time between the thawing of the input product and the beginning of the washing procedure, we recommend setting the parameters of the protocol ready.

Please check the chapter 4E of the operator manual for more details about parameter selection. Select the PBSC washing protocol on the main menu and check the parameters.

NOTE:

• The dilution ratio should be set to 1.0. Do not change the dilution ration parameter without validation.

Fig. 2

• It is recommended to set the output volume to at least 100ml.

• The input volume represents the volume processed by cycle. If 2 bags are processed in parallel (during the same cycle), enter the sum of their volume.

(55)

CS-600.x OM – 3G – 04 3G - 3 STEP 3: CS-600.1 kit sterility verification

Before opening the kit blister pack under laminar flow, ensure that the sterility indicator on the Tyvek® cover is brown, indicating that the kit is sterile.

Under laminar flow spread the kit out in order to identify the tubing lines and components as in figure 1.

STEP 5: CS-600.1 kit preparation under laminar flow

Under laminar flow, close ALL the clamp s of the single-use kit, the clamps on the extension lines included.

That protocol can wash from 1 to 4 bags of PBSC product in two sequential cycles. Always wash bags containing the product from the same patient.

Usually when performing 2 washing cycles, 1 or 2 output bags will be used. If you do only one cycle, you will use one output bag.

Connect the output bag(s) to the CS-600.1 kit (spike of line “G” in Fig. 1). You can connect up to two output bags as there are two spikes available. If you connect only one output bag, seal and detach the unused spike.

Connect the washing solution bag to the washing solution spike (spike of line “E” in Fig. 1, see Fig. 3). Use 2.5% albumin in isotonic solution. Please refer to the Operator’s Manual chapter 4E provided with the Sepax S-100 to see the quantity of washing solution needed.

(56)

CS-600.x OM – 3G – 04 3G - 4 DO NOT THAW the unit before it is necessary.

You can connect 1 or 2 input bags that you will process in one washing cycle. The CS-600.1 kit is provided with 2 different input bag lines (H and I on fig. 1). Choose the one corresponding to your input bag.

Take the mono-spike line (I) if you wash one bag per cycle, shown on fig. 4. Take the double-spike line (H) if you wash two bags per cycle, shown on fig. 5.

Fig. 4 Fig. 5

STEP 6A: Connection of the Input Bag if SCD is available

Do that step only if a sterile connection device is available.

After having selected the input bag line that you will use, go directly to step 7. DO NOT THAW THE PRODUCT at this stage.

Leave the extension lines (H and I) under the laminar flow. You will use them later. STEP 6B: Connection of the Input Bag if SCD is not available

Do that step only if a sterile connection device is not available.

Prior connection of the initial bag to the CS-600.1 kit, follow your validated procedure to thaw and then perform initial cell count of the PBSC product.

Connect the chosen input bag(s) line to the input bag with the spike and then connect the line to the kit with the luer-lock connection. This operation must be done under LAMINAR FLOW. If you plan to do only one washing cycle, seal and detach the unused tube (Fig. 6).

Fig. 6

Seal here the unused tube

(57)

CS-600.x OM – 3G – 04 3G - 5 STEP 7: CS-600.1 kit installation on the Sepax

For instructions on installation of the CS-600.1 kit on the Sepax S-100 main unit, parameter settings and launching of the automated separation procedure, refer to the Operator’s Manual (Section 4.6) provided with the Sepax S-100.

If your input bag(s) is already connected to the kit (SCD not available) put it/them on an orbital shaker (fig. 7). Do not switch on the shaker at this stage.

Fig. 7

STEP 8: Automatic ki t test and priming Start kit test by pressing “Enter” (Fig. 8).

Fig. 8

When asked for, open washing solution line clamp, the waste line clamp and the output bag line clamp (Fig. 9). Press Enter. If you plan to do 2 washing cycles, open only one output bag clamp. The second output bag will be used for the second washing cycle.

Connection without SCD: input bag is placed on a orbital shaker

Washing solution

(58)

CS-600.x OM – 3G – 04 3G - 6 Fig. 9

Select either “Standard wash” or “High wash” and then press “Enter”. For details, please check chapter 4E.

The machine then primes the kit and the washing solution for primary dilution is prepared in the separation chamber. Once the priming is finished, the Sepax displays (Fig. 10):

Fig. 10

STEP 9: input bag connection WITH SCD device

Prior connection of the initial bag to the CS-600.1 kit, follow your validated procedure to thaw and then perform initial cell count of the PBSC product. This operation must be done under LAMINAR FLOW.

If you plan to perform two sequential washing cycles, only thaw the bag(s) that will be processed during the first cycle (1 or 2 bags).

Connect the input bag(s) to the chosen input bag line by using the spike. This operation must be done under LAMINAR FLOW (fig. 11).

Fig. 11

If your input bag is already connected to the kit with the spike, press “Enter* and go to step 10. If connecting the input bag with an SCD device, go to step 9.

SEPAX Cell Processing System Operator s Manual

SEPAX

Cell Processing System

Operator’s Manual

SEPAX

Cell Processing

System

Operator's Manual

TABLE OF CONTENTS

1. PREFACE

P

d

=

1

,

2

P

d

=

2

,

3

P

d

=

1

,

2

P

d

=

1

,

2

P

d

=

2

,

3

0123

~

I

LOT

2. SEPAX S-100 MAIN UNIT

MENU

MENU

MENU

MENU

MENU

MENU

MENU

ENTER

ENTER

ENTER

ENTER

ENTER

ENTER

ENTER

KEY

ACTION

STOP

ENTER

3. THE SINGLE-USE SEPARATION KIT

SEPAX cell separation kit CS-470.1

A: Separation chamber 200ml

B: Line pressure monitor luer with microbial filter

C: Stopcock

D: Bubble chamber

E: Spike to source bag

F: Buffy coat collection vial with injection site and

microbial filter. Warning: this is not a cryovial!

E

F

D

C

B

A

C

G