™
Nuvia
HR-S
Strong Cation
Exchange Media
Instruction Manual
Catalog numbers
156-0511
156-0513
156-0515
156-0517
Please read these instructions before you use Nuvia HR-S
cation exchange media. If you have any questions or
comments regarding these instructions, please contact
your Bio-Rad Laboratories representative.
Table of Contents
Section 1: Introduction ................................................. 1
Section 2 : Technical Description ................................ 2
Section 3 : Preparation ................................................. 3
Section 4 : Column Packing ........................................ 4
Section 5 : Column Packing Evaluation ....................... 6
Section 6 : Operation and Maintenance ..................... 7
Section 7 : Regeneration and Sanitization ................. 9
Section 8 : Cleaning in Place (CIP)
and Sanitization ......................................... 9
Section 9 : Storage ....................................................... 9
Section 10: Regulatory Support ................................. 9
Section 11: Ordering Information .............................. 10
Section 1:
Nuvia™ HR-S cation exchange media is a new highresolution chromatography media designed for
intermediate and final polishing purification steps where
separating closely related product impurities (that is,
charged variants) is a challenge. Its exceptional selectivity
and high recovery meet the biomolecule purification needs
at both laboratory and large bioprocess scale.
Nuvia HR-S media joins the family of Nuvia S and Nuvia Q
ion exchange media and may be used for the separation
of proteins, nucleic acids, viruses, plasmids, and other
macromolecules. The unique properties of Nuvia HR-S
media, with its smaller particle size and fast mass transfer,
allow the media to be effective for the resolution of various
biomolecules.
If you have questions about Nuvia media, contact either
your local Bio-Rad process chromatography sales
representative or the Bio-Rad chromatography Technical
Support department for further assistance at
1-510-741-6563.
Introduction
Nuvia HR-S Instruction Manual 1
Section 2
Table 1. Characteristics of Nuvia HR-S media.
Type of ion exchanger Strong cation
Functional group
Total ionic capacity
Dynamic binding capacity*
Shipping counterion
Median particle size 50 ± 10 µm
Recommended linear flow
rate range
Chemical stability
1.0 N NaOH (20°C
0.1 N
NaOH
Compression factor 1.25 (settled bed
pH stability**
Shipping solution 20% ethanol
Regeneration 1–2 M NaCl
Sanitization 0.5–1.0 N NaOH
Storage conditions 20% ethanol or 0.1 N NaOH
* 10% breakthrough capacity determined with 5.0 mg/ml human IgG in 20 mM
NaOAc pH 5.0
** Data derived under accelerated conditions at 60°C.
: Technical Description
Nuvia HR-S
–
–SO
3
100–180 µeq/ml
>70 mg/ml at 300 cm/hr
+
Na
50–200 cm/hr
—
)
(20°C)**
up to 5 weeks
up to 5 years
volume/packed bed volume)
2–14 short term
4–13 long term
2 Nuvia HR-S Instruction Manual
Section
Nuvia HR-S media is supplied fully hydrated in 20%
ethanol + 1 M NaCI as a 50% (v/v) slurry. For column
packing, removal of the shipping buffer is recommended.
Small volumes of Nuvia HR-S media are easily washed in
a Büchner funnel with 4–5 volumes of packing buffer. For
large volumes, cycling through 3–4 settling and decanting
steps using the packing buffer is recommended.
Removal of fines from Nuvia HR-S media is not required
due to the narrow particle-size range. If fines have been
generated during handling, resuspend the slurry and
remove the opaque supernatant before sedimentation is
complete. Repeat several times until supernatant is clear.
: Preparation
3
Nuvia HR-S Instruction Manual 3
Section 4
Nuvia HR-S media can be packed using most
conventional packing methods. A slurry concentration of
20–50% is recommended. The compression factor for
Nuvia HR-S is 1.25. The compression factor is defined as
settled bed height divided by packed bed height.
: Column Packing
Packing Laboratory Scale Columns
This slurry packing method was designed to pack
Nuvia HR-S media in a conventional column with an
internal diameter of 5–15 mm. All buffers should be
degassed. Because a relatively large volume of slurry is
required, it is recommended that a packing reservoir be
used.
1. Prepare degassed 1.0 M NaCl, 20–50 mM buffer salt
(see Table 2), referred to hereunder as the packing
buffer.
2. Nuvia media are shipped as a 50% slurry. Measure the
desired amount of suspended slurry into a graduated
cylinder. Allow the resin bed to settle. Decant the
shipping solution from the settled media.
3. Add sufficient degassed packing buffer to the resin to
make a 20–50% slurry.
4. Mix to suspend the resin. Caution: Do not mix with
a magnetic stir bar as damage may occur. Larger
amounts of slurry may be mixed with a low-shear
marine impeller at low to moderate speed.
5. Add packing buffer to the column to about 2–5 cm
high. Pour in prepared resin slurry.
6. Once a layer of supernatant has developed, insert the
column flow adaptor and flow pack at a linear velocity
of 300–600 cm/hr with packing buffer for at least
10 min. Lower the adaptor to the calculated
compressed bed height.
4 Nuvia HR-S Instruction Manual
Packing Process-Scale Columns
After removing the storage buffer (Section 3), prepare
a 20–50% slurry (v/v) with packing buffer (see Table 2).
Follow the column manufacturer’s recommendations with
one major exception: do not recirculate the Nuvia slurry
through the packing pump.
Use a low-shear hydrofoil impeller for automated mixing
or a plastic paddle for manual mixing. The best overall
performance of Nuvia HR-S will be obtained with a
compression factor of 1.25, defined as settled bed height
divided by the packed bed height.
Bio-Rad recommends using process columns with axial
compression capabilities for best results. For stall-packing
type columns, additional compression may be required to
achieve a compression factor of 1.25.
After the desired compression is achieved, flow condition
the column with fresh packing or equilibration buffer for
3 column volumes (CV) in upflow followed by 3 CV in
downflow at the process flow rate. After flow conditioning,
evaluate column efficiency using your standard operating
procedures or the procedure described in Section 5.
Detailed packing procedures for process scale
columns can be obtained by contacting your Bio-Rad
representative.
Nuvia HR-S Instruction Manual 5
Section 5
: Column Packing
Evaluation
When column packing is complete, equilibrate the
column until baseline conductivity is stable. To test the
effectiveness of column packing, inject a sample of a low
molecular weight, unretained compound (for example,
acetone or 1 M NaCl). If acetone is used as the test
marker (use a UV absorbance monitor set at 280 nm), the
equilibration buffer must have a salt concentration
<100 mM. If 1 M NaCl is the test marker (use a
conductivity monitor), then the equilibration buffer salt
concentration should be 100–200 mM. The sample
volume should be 1–3% of the total column volume.
Column testing should be operated using the same linear
velocity used to load and/or elute the sample.
To obtain comparable height equivalent to a theoretical
plate (HETP) values among columns, the same conditions
must be applied.
N = Number of theoretical plates
N = 5.54(Ve/W½h)
L = Bed height, cm
HETP = L/N
Ve = Peak elution volume or time
W½h = Peak width at peak half height in volume or time
Ve and W½h should always be in the same units
Peaks should be symmetrical and the asymmetry factor as close
as possible to 1.
Peak asymmetry factor calculation:
As = b/a
a = Front section of peak width at 10% of peak height
bisected by line denoting V
b = Latter section of peak width at 10% of peak height
bisected by line denoting V
As equal to 0.8–2.5 should be easily achieved under normal
operating conditions.
6 Nuvia HR-S Instruction Manual
2
e
e
Section 6
: Operation and
Maintenance
A linear flow rate of 100 cm/hr with a 20 cm bed is a
recommended starting point for Nuvia HR-S. Purification
may be optimized by changing the pH, flow rate, or ionic
strength of the elution buffer, modifying the gradient profile,
or experimenting with different buffer salts.
Note: Due to the smaller particle size of Nuvia HR-S
media, it is recommended to run the process at flow rates
no greater than 200 cm/hr to minimize backpressure and
headspace formation.
Figure 1 shows the effect of flow rate on backpressure.
Fig. 1. Nuvia HR-S media pressure/flow performance for a 20 cm
diameter x 20 cm bed height column packed to a compression
factor of 1.25.
Nuvia HR-S Instruction Manual 7
All
buffers
commonly used for ion
chromatography can be used with
(see Table
functional
2).
Buffering ions
group on the ion
that have
exchanger
results.
exchange
Nuvia
media
the same
charge
will produce the best
as the
Table 2.
chr
Buffer Buffering Range
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
Common
omatography
buffers for
.
ion exchange
8 Nuvia HR-S Instruction Manual
Section 7
: Regeneration and
Sanitization
After each run, the packed bed should be washed with 2–6
bed volumes of 1–2 M NaCl or until absorbance returns to
baseline to remove reversibly bound material. The column can
then be sanitized in 1.0 N NaOH at 50–100 cm/hr; a minimum
contact time of 120 min is recommended.
Section 8
: Cleaning in Place
(CIP) and Sanitization
If a column no longer yields reproducible results, the
media may require thorough CIP and sanitization after
regeneration 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, 6 M guanidine
hydrochloride, and 1 to 5% SDS.
Section 9
For long-term storage, Nuvia HR-S media should be
equilibrated with 0.01–0.1 N NaOH or 20% ethanol
(see Table 1).
Section 10
Regulatory support files are available for Nuvia HR-S
media. If you need assistance validating the use of Nuvia
media in a production process, contact your local Bio-Rad
representative.
: Storage
: Regulatory Support
Nuvia HR-S Instruction Manual 9
Section 11
: Ordering
Information
Catalog # Description
156-0511 Nuvia HR-S Media, 25 ml
156-0513 Nuvia HR-S Media, 100 ml
156-0515 Nuvia HR-S Media, 500 ml
156-0517 Nuvia HR-S Media, 10 L
732-4707 Foresight™ Nuvia™ HR-S Plates, 20 µl
732-4831 Foresight Nuvia HR-S RoboColumn Units,
200 µl
732-4832 Foresight Nuvia HR-S RoboColumn Units,
600 µl
732-4723 Foresight Nuvia HR-S Column, 1 ml
732-4743 Foresight Nuvia HR-S Column, 5 ml
Larger volumes
are
available
Triton is a
10 Nuvia HR-S Instruction Manual
and
upon request.
trademark
special packaging
of
Union Carbide
Corporation.
for
industrial applications
Life Science
Group
Sig 121210033316 Rev A US/EG
Bio-Rad
Laboratories, Inc.
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