Bio-Scale™ Nuvia™ S
Cartridges
1 and 5 ml
Instruction Manual
Catalog #732-4420
732-4421
732-4422
732-4423
Bio-Rad Technical Support
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Luer-Lok is trademark of Becton, Dickinson and Company.
Contents
Section 1 Introduction........................... 1
Section 2 Connecting to Bio-Rad’s
Section 3 Connecting to Other Liquid
Section 4 Preparing a Cartridge for Use.........
Low-Pressure Chromatography
Instruments...........................5
Chromatography Systems ...........10
3.1 BioLogic DuoFlow™ Systems..........11
3.2 HPLC Systems .......................11
3.3 FPLC Systems .......................12
1 3
4.1 Sample Preparation...................14
4.2 General Purification Protocol..........15
4.3 Purification Scale-Up .................17
Section 5 Cleaning-in-Place (CIP) and
Section 6 Ordering Information................. 2 0
Section 7 Related Reading.................... .23
Sanitation............................18
5.1 Autoclaving........................... 19
5.2 Storage.............................. 1 9
Section 1 Introduction
Bio-Scale™ Mini cartridges have a patented*
double-wall design that provides extra
durability and allows easy, reliable runs with
the aqueous buffers most commonly used for
protein purification. The polypropylene luer
fittings and internal sealing surfaces ensure
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 Section 6, Ordering
Information, for the complete Bio-Scale Mini
cartridge product line.
*
U.S. patent 7,208,087.
1
The design of the Bio-Scale Mini cartridges
offers:
n
Convenience — the cartridge is ready for
equilibration in the buffer of choice
n
Versatility — luer fittings allow connection
to any chromatography system or directly
to a Luer-Lok syringe
Bio-Scale Mini Nuvia
™
S cartridges are
packed with Nuvia S ion exchange support.
This ultra-high capacity ion exchange
support is built on the UNOsphere
™
platform
and is designed for purification of proteins,
nucleic acids, viruses, plasmids, and other
macromolecules. Nuvia S support may be
used at any stage of the purification process.
2
Table 1. Bio -Sc ale Mi ni Nuv ia S cartr idge s peci ficat ion s.
Sizes 1 and 5 ml bed volumes
Dimensions 1 ml: 40 x 5.6 mm inner diameter (ID)
5 ml: 40 x 12.6 mm ID
Max pressure 45 psi
tolerance
Recommended 1 ml: 1–2 ml/min (240–280 cm/hr)
flow rates 5 ml: 5–10 ml/min (140– 480 cm/hr)
Max flow rate 1 ml: 4 ml/min (970 cm/hr)
5 ml: 20 ml/min (963 cm/hr)
Fittings Female luer fitting inlet
Male luer fitting outlet
Column material Polypropylene
Frit material Polyethylene (HDPE)
Shipping conditions 20% ethanol + 0.1 M NaCl
Storage 20% ethanol or 0.1 N NaOH
Autoclavability Not autoclavable
3
Table 2. Nuvi a S medi a spec ifica tio ns.
Type of exchanger Strong cation
Functional group –SO
–
3
Total ionic capacity 90–150 µeq/ml
Dynamic binding capacity* ≥110 mg/ml (300 cm/hr)
Shipping counterion Na
+
Median particle size 85 ± 15 µm
Recommended linear flow rate** 50 –600 cm/hr
Chemical stability
1.0 N NaOH (20°C) Up to 1 week
0.1 N NaOH (20°C)
***
pH stability
2–14 short-term
***
Up to 5 yr
4–13 long-term
Shipping 20% ethanol + 0.1 M NaCl
Regeneration 1–2 M NaCl
Sanitation 0.5 –1.0 N NaOH
Storage 20% ethanol or 0.1 N NaOH
*
10% breakth rough capacity determined with 4.5 mg/ml human IgG in
40 mM NaOAc, 30 m M NaCl, pH 5.0.
**
Elution flow rates <150 cm/hr minimize viscosity-induced backpressure.
***
Data derived under accelerated conditions at 60ºC.
4
Section 2 Connecting to Bio-Rad’s Low-Pressure Chromatography Instruments
Bio-Scale™ Mini cartridges are ideal for use
with Bio-Rad’s BioLogic
gradient pump, Model EP-1 Econo pump, and
all low-pressure chromatography instruments.
They can be connected using the convenient
Luer-Lok fittings on the cartridge.
1. Install 1.6 mm ID tubing in the pumphead.
Adjust the platen pressure screw (on the
pumphead). Using a screwdriver or coin,
turn the screw counterclockwise as far as
it will go, then turn it clockwise three full
turns.
™
LP system, Econo™
5
2. Assemble with fittings and lock rings as
shown in Figure 1 (use orange lock rings
and medium size barb fittings with 1.6 mm
tubing).
Platen pressure
screw
Lock ring
Tubing
Luer fitting
See detail
Fig. 1. BioLogic LP sys tem setup.
6
3. 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 corresponds to the
diagram on the valve (Figure 2).
To BioLogic
LP system or
Econo grad ient
pump
To BioLogic
LP system or
Econo grad ient
pump
Fig. 2. Connecting to an MV-6 valve.
7
4. Connect the inlet of the cartridge to the
male luer fitting on the MV-6 sample inject
valve (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, mount the cartridge
vertically, with the arrow on the cartridge
pointing down.
5. Connect the outlet to the 1.6 mm ID tubing
leading to the BioLogic LP optics module
or Econo UV monitor. Use the shortest
length (approximately 10 cm) of 1.6 mm ID
tubing. Connect a barb to female luer to
the 1.6 mm ID tubing, then connect to the
bottom of the male luer on the Bio-Scale
Mini cartridge.
8
Inlet fitting
Fig. 3. Column and fittings.
Outlet fitting
9
Section 3 Connecting to Other Liquid Chromatography Systems
Bio-Scale™ Mini cartridges can be connected
to any liquid chromatography system
provided that the pressure limit (3 bar, 45 psi,
or 300 kPa) of the cartridges is not exceeded.
Set the system pressure limit 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 fittings kits for connecting
Bio-Scale Mini cartridges to BioLogic
DuoFlow
™
, HPLC-, or FPLC-type systems.
10
3.1 BioLogic DuoFlow Systems
The luer to BioLogic system fittings kit
(catalog #732-0113) includes ¼–28 female to
male and ¼–28 female to female luer fittings
to connect one Bio-Scale Mini cartridge to
the BioLogic DuoFlow system (Figure 4).
Fig. 4. Components of the luer to BioLogic
system fittings kit (catalog #732-0113).
3.2 HPLC Systems
The luer to 10–32 adaptor fittings kit (catalog
#732-0112) provides fittings for connecting
the cartridge to the nut- and ferrule-type
fittings found on many HPLC systems.
Alternatively, connect the cartridge to HPLC
systems via a low dead volume,
11
1
/16 in union
with a new piece of stainless steel tubing
attached to the union. Slip a short length of
0.8 mm ID tubing over
1
/16 in OD stainless
steel tubing to a distance of 1 cm.
3.3 FPLC Systems
The luer to M6 adaptor fittings kit (catalog
#732-0111) provides fittings to connect a
cartridge to the M6 fittings found on FPLC or
related systems.
Alternatively, make a connection using
Upchurch Scientific adapters: two ¼–28
to metric adaptors (P-621), one ¼–28 to
male luer (P-619), and one ¼–28 to female
luer (P-628). Connect the luers to the ¼–28
metric adaptors. Attach the adaptor with
the male luer to the column inlet line of the
FPLC system and the adaptor with the female
luer to the FPLC column outlet. To prevent
tubing or cartridge failure, do not exceed the
maximum recommended flow rate.
12
Section 4 Preparing a Cartridge for Use
Bio-Scale™ Mini Nuvia™ cartridges contain
20% ethanol + 0.1 M NaCl (v/v) as the
shipping solution. The fully hydrated support
is ready to use after equilibrating the cartridge
in the buffer of choice. To perform a buffer
exchange, connect the cartridge to a liquid
chromatography system or peristaltic pump
and condition it as follows:
1. Set the pump flow rate to 1–2 ml/min
(240–280 cm/hr) for the 1 ml cartridge or
5–10 ml/min (140–480 cm/hr) for the 5 ml
cartridge.
2. Wash the cartridge with degassed low-salt
buffer for 2 min.
13
3. Wash with degassed high-salt buffer for
5 min.
4. Equilibrate with low-salt buffer for 5 min.
5. Reduce the flow rate to the rate that will be
used in the purification protocol.
4.1 Sample Preparation
Correct pH and ionic strength are necessary
for consistent and reproducible results.
Sample can be exchanged into the starting
buffer or diluted to the starting buffer
concentration. This can be achieved by
diluting the sample to the ionic strength of the
starting buffer, dialyzing against the starting
buffer, or exchange into the starting buffer.
Buffer exchange can be accomplished using
a number of products (Table 3). The choice of
product will depend on sample volume. Filter
all samples through a 0.45 µm filter prior to
cartridge application.
14
Table 3. P rodu cts fo r buf fer exc hang e.
Sample
Volume Product MW Cutoff Cata log #
50–100 µl Bio-Spin® 6 6 kD 732-6002
columns
Bio-Spin 30 30 kD 732-6006
columns
0.1–3 ml Bio-Scale 6 kD 732-5304
Mini P6 cartridges
Up to 3 ml Econo-Pac® 10DG 6 kD 732-2010
cartridges
Unlimited Bio-Gel® P-6DG gel 6 kD 150-0738
4.2 General Purification Protocol
Ion exchange chromatography is usually
performed using increasing salt gradients
or pH gradients to elute the sample
components. For best results and increased
cartridge life, samples and buffers should be
degassed and filtered through a 0.45 µm filter.
Common buffers are listed in Table 4.
15
Table 4. C ommon ion exch ange buffers .
Catio n Buf fer Ra nge
Acetic acid 4.8–5.2
Citric acid 4.2–5.2
HEPES 6.8–8.2
Lactic acid 3.6 –4.3
MES 5.5–6.7
MOPS 6.5–7.9
Phosphate 6.7–7.6
PIPES 6.1–7.5
TES 6.8–8.2
Tricine 7.8–8.9
An appropriate starting point for purifying
samples is a linear gradient of 0–0.4 M NaCl
spanning 1–20 column volumes at 120 cm/hr,
0.5 ml/min for the 1 ml cartridge and
2.5 ml/min for the 5 ml cartridge. Separation
can be optimized by changing the gradient
profile.
16
At the end of each run, the cartridge can
be regenerated with 1.0 M NaCl followed by
starting buffer. Return to the desired flow rate
and proceed with the next separation.
4.3 Purification Scale-Up
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. Bio-Scale Mini cartridges
are available in 1 and 5 ml cartridge formats.
Nuvia S support is also available in larger
amounts, from 25 ml bottles to bulk quantities
for scaling-up methods developed using
the cartridges. Nuvia S media are fully
supported with regulatory support files. In
addition, Bio-Rad carries an extensive line of
empty chromatography columns for use in
laboratory and process scale applications.
17
Section 5 Cleaning-in-Place (CIP) and Sanitation
If a column no longer yields reproducible
results, the media may require thorough CIP
and sanitation to remove strongly bound
contaminants. Acceptable CIP agents include
25% acetic acid, 8 M urea, 1% Triton X-100,
6 M potassium thiocyanate, 70% ethanol,
30% isopropyl alcohol, 1 N NaOH, and 6 M
guanidine hydrochloride.
1. Sanitize the support in the column with
2–4 bed volumes of 1.0 N NaOH at
50–100 cm/hr while maintaining a contact
time of at least 40 min.
18
2. To reequilibrate the column, wash it with
2–4 bed volumes of 0.5–2 M NaCl solution
(containing 50–100 mM buffer).
3. If lipid removal is required, wash the
column with a 20–70% ethanol solution at
50 cm/hr.
5.1 Autoclaving
Bio-Scale™ Mini cartridges are not
autoclavable.
5.2 Storage
After washing the cartridges with
deionized water, Bio-Scale Mini ion
exchange cartridges should be purged,
stored in 20% ethanol solution, and
capped for extended storage.
19
Section 6 Ordering Information
Bio-Scale™ Mini Cartridges
For a list of available cartridges*, visit
www.bio-rad.com/cartridges/.
Descr iptio n 1 x 1 ml 5 x 1 ml 1 x 5 ml 5 x 5 ml
Nuvia™ S 732-4420 732-4 421 732-4422 732-4423
UNOsphere™ Q ---- 732-4100 731-4102 731-4104
UNOsph ere S ---- 732-4110 731-4112 731-4114
UNOsph ere Rapid S ---- 732-4400 732-44 01 732-4402
UNOsph ere SUPrA™ 732-4200 732-4201 732-4202 ---Macro-Prep® High Q ---- 732-4120 732-4122 732-4124
Macro-P rep High S ---- 732-4130 732-4132 732-4134
Macro-P rep DEAE ---- 732-4140 732-4142 732-4144
CHT™ Type I ---- ---- 732-4322 732-4324
CHT Type II ---- ---- 732-43 32 732-4334
CFT™ Type II ---- ---- 732-4405 732-4406
Bio-Gel® P-6 ---- ---- 732-4502 732-450 4
Affi-Prep® protein A ---- 732-4600 732-4602 ----
Profinity™ IMAC ---- 732-4610 732-4612 732-4614
DEAE Af fi-Gel® Blue ---- ---- 732-46 32 732-4634
Affi-Gel Blue ---- ---- 732-4642 732-4644
* Media are available in volumes for process scale chromatography;
inquire with your local Bio-Rad representative
20
Fittings Kits
Catalog # Description
732-0111 Luer to M6 Adaptor Fittings
Kit, includes luer to M6 fitting to
connect 1 cartridge to an FPLC
system
732-0112 Luer to 10–32 Adaptor
Fittings Kit, includes luer to
polypropylene/PTFE 10–32
fittings to connect 1 cartridge
to an HPLC system
732- 0113 Luer to BioLogic™ System
Fittings Kit, includes ¼–28
female to male luer and ¼–28
female to female luer to connect
1 cartridge to the BioLogic
DuoFlow
™
system
21
731-8226 3.2 mm Barb to Male Luer,
pkg of 25
731-8225 1.6 mm Barb to Male Luer,
pkg of 25
731-8224 0.8 mm Barb to Male Luer,
pkg of 25
731-8223 3.2 mm Barb to Female Luer,
fits inlet and outlet of EconoColumn chromatography
columns and low-pressure
tubing, pkg of 25
731-8222 1.6 mm Barb to Female Luer,
pkg of 25
731-8221 0.8 mm Barb to Female Luer,
pkg of 25
22
Section 7
Related Reading
Harris ELV and Angal S (1989). Protein
Purification Methods: A Practical Approach
(Oxford: IRL Press).
Scopes RK (1987). Protein Purification:
Principles and Practice (Second Edition)
(New York: Springer-Verlag).
Snyder LR and Kirkland JJ (1979).
Introduction to Modern Liquid
Chromatography (Second Edition)
(New York: Wiley).
Gagnon P (1997). Avoiding InstrumentAssociated Aberrations in Purification ScaleUp and Scale-Down, BioPharm 10, 42–45.
23
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