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Bio-Scale™Mini CHT
™
Ceramic Hydroxyapatite
Cartridges, 5 ml
Instruction Manual
Catalog # 732-4322
732-4324
732-4332
732-4334
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5.2 Wash Alternatives ..................20
5.3 Autoclaving ............................20
5.4 Storage ..................................20
Section 6 ..Technical Support.............................21
Section 7 ..Ordering Information.........................22
Section 8 ..References .......................................24
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The Bio-Scale Mini CHT cartridges are packed with
Type I or Type II ceramic hydroxyapatite supports.
These supports are based on hydroxyapatite, a
form of calcium phosphate used in chromatographic
separations of biomolecules. CHT ceramic
hydroxyapatite is a spherical, macroporous form of
hydroxyapatite. Unlike most other chromotagraphic
absorbents, CHT is both the ligand and the support
matrix. CHT Type I has a higher protein binding
capacity and greater capacity for acidic proteins.
CHT Type II has a lower protein binding capacity
but provides better resolution of nucleic acids and
certain other proteins. The Type II material also has a
very low affinity for albumin and is especially suitable
for the purification of many species types and classes
of immunoglobulins. Applications of hydroxyapatite
chromatography include the purification of different
subclasses of monoclonal and polyclonal antibodies,
antibodies that differ in light chain composition,
antibody fragments, isozymes, supercoiled DNA from
linear duplexes, and single-stranded from double
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Table 2. Product Description
TTyyppee II TTyyppee IIII
FFuunnccttiioonnaall ggrroouuppss
Ca
2+
, PO4, OH Ca2+, PO4, OH
OObbsseerrvveedd ddyynnaammiicc bbiinnddiinngg ccaappaacciittyy
llyyssoozzyymmee ((LLyyss))
≥ 25 mg Lys/g CHT ≥ 12.5 mg Lys/g CHT
NNoommiinnaall ppoorree ddiiaammeetteerr
600–800 Å 800–1,000 Å
MMaaxxiimmuumm bbaacckkpprreessssuurree
100 bar (1,500 psi) 100 bar (1,500 psi)
N
Noommiinnaall mmeeaann ppaarrttiiccllee ssiizzee
20 ± 2, 40 ± 4, and 80 ± 8 µm
BBuullkk ddeennssiittyy
0.63 g/ml 0.63 g/ml
OObbsseerrvveedd ddyynnaammiicc bbiinnddiinngg ccaappaacciittyy
IIggGG
25–60 mg IgG/ml CHT* 15–25 mg IgG/ml CHT*
TTyyppiiccaall lliinneeaarr fflloow
w rraattee rraannggee
50–1,000 cm/hr
ppHH ssttaabbiilliittyy
6.5–14
BBaassee ssttaabbiilliittyy
at least 21 months in 1 N NaOH
RReeggeenneerraattiioonn
500 mM sodium phosphate, pH 7
1,000 mM trisodium phosphate, pH 11–12
AAuuttooccllaavvaabbiilliittyy ((bbuullkk))
121°C, 20 min in phosphate buffer, pH 7
SSaanniittiizzaattiioonn
1–2 N NaOH
RRe
eccoommmmeennddeedd ccoolluummnn ssttoorraaggee
0.1 N NaOH
SShheellff lliiffee ((ddrryy,, uunnuusseedd mmaatteerriiaall))
85 months stored dry, sealed, and at room
temperature
* 40 µm particles, 300 cm/hr, 5 mM sodium phosphate, pH 6.5
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(Use orange lock rings and medium size barb
fittings with 1.6 mm tubing.)
Fig. 1. Biologic LP setup.
6
Platen pressure
screw
See detail
23
6
1
9
45
.
78
0
C
rm
la
A
Lock-ring
Tubing
Luer fittimg
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3. Hold the cartridge vertically with outlet in the
downward direction. 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, 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
to the 1.6 mm ID tubing, and then connect to
the bottom of the male luer on the Bio-Scale
Mini cartridge.
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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 fitting kits for
easy connection of a Bio-Scale Mini cartridge to a
BioLogic DuoFlow, HPLC- or FPLC-type system.
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3.3 FPLC Systems
The luer to M6 adaptor fittings kit (catalog number
732-0111) provides fittings necessary to connect
the cartridge to the M6 fittings found on FPLC or
related systems.
Alternatively, connection can be made by using the
following Upchurch Scientific
®
Quick Connect Luer
Adaptors: two Upchurch P-621 adaptor, 1/4–28, to
metric adaptors, one Upchurch P-619 adaptor,
1/4–28, to male luer, and one Upchurch P-628
adaptor, 1/4–28, to female luer. Assemble the luers
to the 1/4–28 metric adaptors. Attach the adaptor
with the male luer to the column inlet line of the
FPLC system and the one with the female luer to
the FPLC column outlet. To prevent tubing or
cartridge failure, do not exceed the maximum
recommended flow rate.
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5. Equilibrate the cartridge with running buffer and
operatate the cartridge according to the CHT
instruction manual or according to your protocol.
4.1 Sample Preparation
Proper 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's concentration. This can be
achieved by diluting the sample to the ionic strength
of the starting buffer, dialyzing against the starting
buffer, or exchanging it into the starting buffer.
Buffer exchange can be accomplished using the
Bio-Scale Mini P6 cartridge, Bio-Spin
®
6 or
Bio-Spin 30 columns, Econo-Pac 10DG desalting
columns, or Bio-Gel
®
P-6DG gel filtration gel. The
choice of product will depend on sample volume.
All samples should be filtered through a 0.45 µm
filter prior to cartridge application.
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calcium. Common buffers for hydroxyapatite
chromatography are listed in Table 4.
An appropriate starting point for purifying samples is
a linear gradient from 5–500 mM sodium phosphate
pH 6.8, spanning 10 to 20 column volumes at 2.0 ml/min
for the 5 ml cartridge. For separations of monoclonal
antibodies from aggregates, an alternative purification
protocol is eluting with a linear gradient from 0-2 M
sodium chloride in 5-20 mM sodium phosphate,
pH 6.5. The separation can be optimized by changing
the gradient profile. At the end of each run the
cartridge can be regenerated with 500 mM potassium
or sodium phosphate buffer, neutral pH; or 400 mM
trisodium phosphate, pH 11–12; followed by starting
buffer. Return to the desired flow rate and proceed
with the next separation.
4.3 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. CHT ceramic
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CCHHTT SSuuiittaabbiilliittyy
ppHH BBuuffffeerr 1100 ccyycclleess 5500 ccyycclleess
7.0 MES + 5 mM PO
4
+ n/a
7.0 Acetate + 5 mM PO
4
+ n/a
7.0 Imidazole + 5 mM PO
4
++
7.0 Glycine + 5 mM PO
4
+ n/a
7.0 Arginine + 5 mM PO
4
+ n/a
7.0 HEPES + 5 mM PO
4
+ n/a
7.0 Tris + 5 mM PO
4
+ n/a
7.5 Phosphate (5 mM) + n/a
7.5 MES + 5 mM PO
4
+ n/a
7.5 Imidazole + 5 mM PO
4
++
7.5 Acetate + 5 mM PO
4
+ n/a
7.5 HEPES + 2 mM PO
4
+ n/a
7.5 HEPES + 5 mM PO
4
+ n/a
7.5 Tris + 2 mM PO
4
+ n/a
7.5 Tris + 5 mM PO
4
++
8.5 Tris + 5 mM PO
4
+ n/a
* All experiments performed in small scale columns. Each cycle used approximately 35 column
volumes of buffer to simulate an equilibration and long gradient, as well as five column volumes
of 1 N NaOH to simulate regeneration.
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5.2 Wash Alternatives
Perform wash alternatives with any of the following
alternative buffers in place of step 1. Continue with
step 2 listed in Section 5.1.
• 1–2 M KCI or NaCI
• 6 M urea
• 8 M guanidine-HCI
All the wash alternative buffers should contain 5 mM
phosphate at neutral pH.
5.3 Autoclaving
Bio-Scale Mini cartridges are not autoclavable.
5.4 Storage
After the cartridges are washed with deionized
water, Bio-Scale Mini CHT cartridges should be
purged and stored with 0.1 N NaOH. Higher
concentrations of NaOH may be used if desired.
Used CHT, after being regenerated and sanitized,
can be stored in up to 1.0 N NaOH at room
temperature.
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Section 7
Ordering Information
Prepacked Bio-Scale Mini Cartridges*
Description 5 x 1 ml 1 x 5 ml 5 x 5 ml
CHT Ceramic Hydroxyapatite, Type I, 40 µm 732-4322 732-4324
CHT Ceramic Hydroxyapatite, Type II, 40 µm 732-4332 732-4334
UNOsphere
™
Q Support 732-4100 731-4102 731-4104
UNOsphere S Support 732-4110 731-4112 731-4114
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. Please contact your local Bio-Rad
representative.
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Section 8
References
P. Gagnon et al., A Ceramic Hydroxyapatite-Based
Purification Platform, BioProcess International 4 (2),
50–60 (2006)
T. Ogawa et al., Effect of pH on Gradient Elution of
Proteins on Two Types of Macro-Prep Ceramic
Hydroxapatite, Prep Tech ’95, Industrial Separation
Science Conference, East Rutherford, NJ (1995)
S.R. Shepard et al., Discoloration of ceramic
hydroxyapatite used for protein chromatography, J
Chromatography A 891, 93–98 (2000)
E. Dolinski et al., Purification of a Fusion Protein by
Ceramic Hydroxyapatite Chromatography, Second
International Conferences on Hydroxyapatite and
Related Products, San Francisco, Ca (2001)
M. Gorbunoff et al., The Interaction of Proteins with
Hydroxyapatite I: Role of Protein Charge and
Structure, Analytical Biochemistry136, 425–432 (1984)
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