Bio-Rad Bio-Scale Mini UNOsphere SUPrA Affinity Cartridges User Manual

Bio-Scale™ Mini
UNOsphere SUPrA™
Cartridges, 1 and 5 ml

Instruction Manual

Catalog #
732-4200
732-4201
732-4202

For technical support, contact your local Bio-Rad office, or in the
U.S., call 1-800-4BIORAD (1-800-424-6723).

Table of Contents

Section 1...Introduction. .......................................1

Section 2...Connection to Bio-Rad’s Low-

..............Pressure Chromatography

..............Instruments ........................................5

Section 3 ..Connection to Other Liquid

..............Chromatography Systems................10

..............3.1 BioLogic DuoFlow™

Systems.................................11

3.2 HPLC Systems ......................11

3.3 FPLC Systems .......................12

Section 4 ..Getting Started.................................13

4.1 Screening Buffers and
Conditions for UNOsphere

SUPrA....................................14

4.2 Scaling Up the Separation .....16

Section 5 ..Care of the Cartridge........................19

5.1 Cleaning.................................19

5.2 Sanitation...................................

5.3 Autoclaving ............................21

5.4 Storage..................................21

Section 6 ..Technical Assistance ........................22

Section 7 ..Ordering Information.........................23

Section 8 ..References .......................................25

Section 1
Introduction

Bio-Scale™ Mini cartridges have a patent-pending,
double-wall design that provides extra durability and
allows easy, reliable runs with aqueous buffers most
commonly used for protein purification. The
polypropylene Luer fittings and internal sealing
surfaces assure leak-free operation at pressures up
to 45 psi. The cartridges are convenient, disposable,
and supplied ready for use. Cartridges are available
for a variety of chromatographic techniques including
desalting, ion exchange, and affinity chromatography.
See Ordering Information (Section 7) for a listing of
the complete Bio-Scale Mini cartridge product line.

1

UNOsphere SUPrA™ medium is a chromatographic
support based on recombinant protein A. The
media are designed for process-scale purification of
monoclonal antibodies. The protein A ligand is
produced in E. coli without the use of material from
animal origin. The UNOsphere base bead is a
macroporous polymeric bead that is designed for
robust and scaleable applications. See Tables 1 and
2 on pages 4–5 for the technical description of the
product.

UNOsphere SUPrA media are built on the proven
UNOsphere base bead, which insures an easy
scale-up path for process applications. The
outstanding flow pressure performance of
UNOsphere chromatography media allow its use in
process applications without concern for exceeding
the pressure limits of the media or chromatography
system. The flow characteristics of UNOsphere
SUPrA packed in a large column format are shown
in Figure 1.

2

UNOsphere SUPrA affinity chromatography media
come with full regulatory support and are backed by
the support of Bio-Rad’s global application and
development team.

Fig. 1. Flow performance of UNOsphere SUPrA media in
Bio-Rad's EasyPack™ column (20 x 20 cm) packed to 13.1%
axial compression.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 100 200 300 400 500 600 700

3

Pressure (Bar)

Linear Flow (cm/hr)

Table 1. Bio-Scale Mini UNOsphere SUPrA
Cartridge Specifications.

Sizes 1 and 5 ml bed volumes

Dimensions 1 ml: 40 x 5.6 mm, length x inner

diameter

5 ml: 40 x 12.6 mm, lenghth x inner

inner diameter

Maximum pressure tolerance 45 psi

Recommended flow rates 1 ml: 1–2 ml/min (240–480 cm/hr)

5 ml: 5–10 ml/min (140–480 cm/hr)

Maximum flow rate 1 ml: 3 ml/min (730 cm/hr)

5 ml: 15 ml/min (722 cm/hr)

Fittings Female Luer fitting inlet and

male Luer fitting outlet

Column material Polypropylene

Frit material Polyethylene (HDPE)

Shipping condition 20% ethanol

Storage recommendation 20% ethanol

Autoclavability Not autoclavable

4

Table 2. Technical Description of UNOsphere
SUPrA Affinity Chromatography Media.

Composition Highly crosslinked polyacrylamide polymer

Particle size range 53–61 µm

Ligand Recombinant protein A

Coupling chemistry Epoxy

Dynamic binding capacity* 150 cm/hr 30 ± 3 mg/ml

300 cm/hr 25 ± 2 mg/ml
450 cm/hr 20 ± 2 mg/ml

Chemical stability** 10 mM hydrochloric acid

6 M guanidine hydrochloride

0.1 M arginine (pH 2.8)

0.1 M citrate (pH 2.8)

0.1 M glycine (pH 2.8)

Working pH range 3–11

Cleaning-in-place (CIP) 6 M guanidine hydrochloride

10 mM hydrochloric acid

0.1 M sodium hydroxide
1 M acetic acid/20% ethanol

Recommended mobile phase 100–600 cm/hr
velocity range

Temperature stability 4–40°C

Delivery conditions 50% slurry in 20% ethanol

Storage conditions 4–8°C

* Minimum 20 mg/ml at 300 cm/hr; 10% breakthrough capacity determined

with 1.0 mg/ml polyclonal human IgG in 1.1 x 10 cm column.

** No significant change in chromatographic performance after 24-hour storage

at room temperature.

5

Section 2
Connecting to Bio-Rad's
Low-Pressure
Chromatography
Instruments

The Bio-Scale™ Mini cartridges are ideal for use with
Bio-Rad's BioLogic™ LP system, Econo gradient
pump, and Model EP-1 Econo pump, and all low­pressure chromatography instruments. Bio-Scale
Mini cartridges can be conveniently connected
directly to the system using the Luer lock fittings on
the cartridge.

1. Install 1.6 mm ID tubing in the pumphead.
Adjust platen pressure screw (on pumphead).
Using a screwdriver or coin, turn the screw
counterclockwise as far as it will go, then turn
clockwise three full turns. Assemble with fittings
and lock rings as shown in Figure 1.

6

(Use orange lock rings and medium size barb
fittings with 1.6 mm tubing.)

Fig. 1. Biologic LP setup.

7

Platen pressure

screw

See detail

2 3

1

9

456

7 8

0

C .

Alarm

Lock ring

Tubing

Luer fitting

2. To maximize gradient accuracy and apply

samples efficiently, install 1.6 mm ID tubing from
the pump to the MV-6 sample inject valve (if
available). If using the MV-6 sample inject valve,
turn the knob counterclockwise as far as it will
go so it will now correspond to the printed
diagram on the valve. (See Figure 2.)

Fig. 2. Connecting to a MV-6 valve.

8

SAMPLE LOOP

INJECT
PORT

MV-6

BIOLOGIC LP

SYSTEM OR ECONO

GRADIENT PUMP

TO

INJECT

VALVE

FILL

TO

COLUMN

WASTE

BIOLOGIC LP

SYSTEM OR ECONO

GRADIENT PUMP

SAMPLE LOOP

INJECT
PORT

FILL

MV-6

INJECT

TO

VALVE

"INJECT" POSITION"FILL" POSITION

TO

COLUMN

WASTE

9

Fig. 3. Column and fittings.

Inlet fitting

Outlet fitting

3. Connect the inlet of the cartridge to the male

Luer fitting on the MV-6 sample inject valve. (See
Figure 2.) If not using the MV-6 sample inject
valve, connect a barb to male Luer fitting on the

1.6 mm ID tubing, then connect to the top of the
female Luer on the Bio-Scale mini cartridge. For
optimum performance, a cartridge should be
mounted vertically with the arrow on the
cartridge pointing downward.

4. Connect the cartridge outlet to the 1.6 mm ID

tubing leading to the BioLogic LP optics module
or Econo UV monitor. It is recommended to use
the shortest length (approximately 10 cm) of

1.6 mm ID tubing. Connect a barb to female
Luer fitting to the 1.6 mm ID tubing, then
connect to the bottom of the male Luer on the
Bio-Scale Mini cartridge.

10

Section 3
Connecting to Other Liquid
Chromatography Systems

The Bio-Scale™ Mini cartridges can be connected
to any liquid chromatography system, provided that
the maximum pressure limit (3 bar, 45 psi, or
300 KPa) of the cartridges is not exceeded. It is
recommended that the system pressure limit be set
according to the cartridge pressure limit. Pressures
in excess of 3.4 bar are usually caused by
restrictions in tubing or detector cells downstream
from the cartridge. Bio-Rad offers two fittings kits for
easy connection of a Bio-Scale Mini cartridge to a
BioLogic DuoFlow™, HPLC- or FPLC-type system.

11

3.1 BioLogic DuoFlow Systems

The Bio-Scale Mini cartridge to BioLogic system
fittings kit (catalog #732-0113) includes ¼–28
female to male Luer and ¼–28 female to female
Luer to connect one Bio-scale Mini cartridge to the
BioLogic DuoFlow system.

3.2 HPLC Systems

The Luer to 10–32 adaptor fittings kit (catalog
#732-0112), provides fittings necessary to connect
the cartridge to nut and ferrule type fittings found
on most HPLC systems. Alternatively, the cartridge
can be connected to HPLC systems via a low dead
volume

1/16 in union with a new piece of stainless

steel tubing attached to the union. Simply slip a
short length of the 0.8 mm ID tubing over

1/16 in OD

stainless steel tubing to a distance of 1 cm.

12

3.3 FPLC Systems

The Luer to M6 adaptor fittings kit (catalog
#732-0111), provides fittings necessary to connect
the cartridge to the M6 fittings found on FPLC or
related systems.

13

Section 4
Getting Started

4.1 Screening Buffers and Conditions for

UNOsphere SUPrA™

Buffers

A1: 0.02 M sodium phosphate, 0.02 M sodium citrate,
pH 7.5.

A2: 0.02 M boric acid, 0.02 M sodium phosphate,

0.02 M sodium citrate, 1.0 M sodium sulfate, pH 9.0.

B: 0.02 M sodium citrate, 0.1 M sodium chloride,
pH 2.5.

Conditions

Equilibrate column: with 10 CV buffer A1 or A2 (see
note below).

Inject: MAb sample either as is or diluted 1:10 into
buffer A1 or A2 (see below).

14

Wash: buffer A1 or A2 until effluent absorbance
returns to baseline.

Elute: in a 10 CV linear gradient to 100% B, or
Desired % buffer B.

Strip: with 5 CV buffer B.

Use buffer A1 for binding human and guinea pig
IgG. Use buffer A2 for all others. The recommended
column equilibration interval is excessive under
most conditions, but should be used as a default
until specific equilibration requirements are established
for your particular system. In addition, be aware of
solubility limitations of antibodies that require high
salt concentrations (A2 buffer) for binding.
Characterize product solubility thoroughly under
loading conditions. If the antibody fails to remain
fully soluble for the longest possible duration from
equilibration to completion of sample load when
adjusted to load conditions, then load by using
online dilution technique.

15

Initial selectivity screening should be conducted with
a linear gradient. Knowledge of a monoclonal's
subclass may suggest a particular range of conditions,
but variation from one monoclonal to another is
sufficient to risk incomplete or no elution.

The choice of citrate for the low pH buffer is
predicated on the broad pH range achievable with
phosphate/citrate systems. If a higher pH range is
required, add boric acid to the binding buffer.
(Note: if CHT™ ceramic hydroxyapatite is used as
the subsequent polishing column, once scouting is
finished replace citrate with glycine or other
nonchelating salt for further process development.
Citrate buffers are incompatible with CHT.)

4.2 Scaling Up the Separation

For quick scale up, two or three cartridges of the
same type can be connected in series.
Backpressure will increase with cartridges in series,
so care should be taken to maintain pressures
≤45 psi.

16

Bio-Scale™ Mini cartridges are available in 1 and 5 ml
cartridge format. The UNOsphere SUPrA media are
also available in larger amounts from 25 ml bottles
to bulk quantities.

Section 5
Care of the Cartridge

5.1 Cleaning

During operation it is recommended that the column
bed is periodically cleaned to remove bound
substances that can adversely impact the separation
performance of the column. The accumulated
substances fall into two general categories: a)
difficult to remove precipitated or denatured substances,
and b) substances that are hydrophobically bound
to the column bed. To ensure that all bound substances
are released and washed out of the column, the
following cleaning-in-place (CIP) protocols are
recommended.

17

CIP Protocols

The following protocols are suggested to remove
precipitated or denatured substances from the bed.

Wash the bed with 2–5 column volumes in reverse
flow with one of the following solutions:

• 6 M guanidine hydrochloride

• 10 mM hydrochloric acid

• 0.1 M sodium hydroxide

• 1 M acetic acid/20% ethanol

Followed by a reverse flow wash with at least
5 column volumes of binding buffer, neutral pH (7–8).

To remove any hydrophobically bound substances
from the bed, wash the column with 2–5 column
volumes in reverse flow of a nonionic
surfactant/detergent, followed by a reverse-flow wash
with at least 5 column volumes of neutral pH binding
buffer.

18

Suggested contact time per cycle is 15 min at room
temperature.

5.2 Sanitization

To reduce the potential for microbial contamination
of the cartridge, the column can be periodically
washed with a solution consisting of 0.1 M sodium
hydroxide. Allow to stand for 1 hour, then wash with
buffer until a neutral pH is reached.

5.3 Storage

To store UNOsphere SUPrA™ for long periods,
equilibrate the media with a 20% ethanol/water
solution and store at 4ºC.

5.4 Autoclaving

Bio-Scale™ Mini cartridges are not autoclavable.

19

Section 6
Technical Assistance

For additional information and technical assistance,
contact your local Bio-Rad representative as listed
on the back cover of our catalog, or in the U.S., call
technical support at 1-800-4BIORAD.

20

Section 7
Ordering Information

Bio-Scale™ Mini Cartridges*

Description 5 x 1 ml 1 x 5 ml 5 x 5 ml 1 x 1 ml

UNOsphere™Q Support 732-4100 731-4102 731-4104
UNOsphere S Support 732-4110 731-4112 731-4114
UNOsphere Rapid S 732-4400 732-4401 732-4402
UNOsphere SUPrA 732-4201 732-4202 — 732-4200

Macro-Prep®High Q Support 732-4120 732-4122 732-4124
Macro-Prep High S Support 732-4130 732-4132 732-4134
Macro-Prep DEAE Support 732-4140 732-4142 732-4144

Bio-Gel®P-6 Support — 732-4502 732-4504

Affi-Prep®Protein A Support 732-4600 732-4602 —

Profinity™IMAC Support 732-4610 732-4612 732-4614

Affi-Gel®DEAE Blue Support — 732-4632 732-4634
Affi-Gel Blue Support — 732-4642 732-4644

* For the most up-to-date list of cartridge offerings, please visit

us online at www.bio-rad.com/cartridges/.

• Larger package sizes of media are available for process-scale

chromatography. Inquire with your local Bio-Rad representative.

21

Fittings Kits

22

Catalog # Description

732-0111 Luer to M6 Adaptor Fittings Kit, includes Luer to

732-0112 Luer to 10–32 Adaptor Fittings Kit, includes Luer

732-0113 Luer to BioLogic™ System Fittings Kit, includes

M6 fitting to connect to an FPLC system

to polypropylene/PTFE 10–32 fittings to connect
1 cartridge to an HPLC system

¼–28 female to male Luer and ¼–28 female to
female Luer to connect one cartridge to the
BioLogic DuoFlow™ system

Section 8
References

1. Harris ELV and Angal S, Protein Purification

Methods: A Practical Approach, IRL Press,
Oxford (1989)

2. Scopes RK, Protein Purification: Principles and

Practice (Second Edition), Springer-Verlag, New
York (1987)

3. Snyder LR and Kirkland JJ, Introduction to

Modern Liquid Chromatography (Second
Edition), Wiley, New York (1979)

4. Gagnon P, Avoiding Instrument-associated

Aberrations in Purification Scale-up and
Scale-down, BioPharm 10, 42–45 (1997)

5. Gagnon P, Purification Tools for Monoclonal

Antibodies Validated Bio Systems (1996)

23

FPLC is a trademark of GE Healthcare Group Companies.
Luer-Lok is trademark of Becton, Dickinson and Co. Triton
is a trademark of Union Carbide Corp. Upchurch is a trademark
of Upchurch Scientific.

24

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Laboratories, Inc.

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