After packing or between runs, it can be necessary to remove pyrogens
from the sorbent. The above suggested sodium hydroxide solutions are
effective to remove pyrogens and in most cases to inactivate
microorganisms.
290009
After treatment, the column must be reequilibrated with the normal
sterile pyrogen-free buffer.
Lysine HyperD ®
Thermal Stability and Storage
Temperature of use
2-30°C (36-86°F)
Shipping temperature
Ambient
Storage temperature
2-30°C (36-86°F)
(2-8°C / 36-46°F once opened)
Recommended storage
solution (between runs)
Neutral buffer containing bacteriostatic
agents such as 1 M NaCl and 20%
(v/v) ethanol.
Caution
Must never be frozen
Ordering information
Part Number
5 mL
20059-058
25 mL
20059-036
100 mL
20059-028
1L
20059-010
10 L
20059-044
Affinity Chromatography Sorbent
Product Description
Lysine HyperD sorbent is a high speed high capacity affinity
preparative sorbent for the purification of biological molecules
that bind to lysine, such as plasminogen from human or animal
species plasma. The sorbent provides high binding capacity at
high flow rates.
Lysine HyperD affinity sorbent employs a high-capacity
hydrogel polymerized within the large pores of a rigid bead.
This design combines the desirable characteristics of a soft,
high capacity hydrogel with the high dimensional stability of a
rigid bead. Dynamic binding capacity with Lysine HyperD
sorbent is virtually independent of linear velocity and the rigid
nature of the bead results in very stable beds capable of
handling pressures up to 70 bar (1,000 psi).
Lysine HyperD is available as ready-to-use labpacks
suspended in 1 M sodium chloride with 20 % ethanol as
bacteriostatic.
™
Pack size
BioSepra Lysine HyperD
®
For more information, please contact our technical service.
New York - USA
+1 516.484.5400 phone
+1 516.801.9548 fax
pharmafilter@pall.com
Visit us at www.pall.com
, Pall, BioSepra, HyperD
are trademarks of Pall Corporation.
Portsmouth - UK
+44 (0)23 9230 3303 phone
+44 (0)23 9230 2506 fax
BioPharmUK@europe.pall.com
Filtration. Separation. Solution. is a
service mark of Pall Corporation.
Cergy - France
+33 (0)1 34 20 78 00 phone
+33 (0)1 34 20 78 78 fax
bioseprainfo@pall.com
® indicates a trademark registered
in the U.S.
Triton is a trademark of a subsidiary
of The Dow Chemical Company.
Please contact Pall BioSepra to get
the instructions in your language.
© Pall Corporation 2005-2007
03/2007
4
Properties
Particle size
70 μm (av.)
Ligand
L-lysine
Working pH
3-13
Volume changes due to pH and ionic strength
Non compressible
Pressure resistance
70 bar (1,000 psi)
Main benefits
- Very high productivity as a consequence of a high binding
capacity and the ability to run at high flow rates.
- Rapid packing due to the high density of lysine sorbent which
settles in a few minutes.
- HyperD sorbent is very rigid and allows the use of high flow
rates without pressure increase or shrinking or swelling of the
sorbent.
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- Minimized leakage due to the stable chemical link of the lysine
molecule to the sorbent. Lysine HyperD can be occasionally
washed with 0.1 M sodium hydroxide for cleaning.
Recommended protocol for the purification of
plasminogen from human plasma
z
290009 - 03/2007
™
BioSepra Lysine HyperD
®
Applications
Lysine HyperD is used for the purification of plasminogen from
human or animal species plasma. It can be used to prepare for
example plasmine-free serum for cell culture, by removal of the
plasminogen present in the whole serum. Plasminogen
contains different subclasses that can be discriminated by
gradient elution using e-amino caproic acid.
Lysine HyperD has been developed to improve the productivity
of process-scale molecules such as plasminogen or
plasminogen activators, in allowing a higher binding capacity at
higher flow rates than conventional agarose-based resins.
In Lysine HyperD, the L-lysine moiety is immobilized covalently
by mostly the a-amino groups, via an aminoalkyl linkage. The
general recommendations of use for Lysine HyperD are very
similar to those of conventional lysine-immobilized resins.
Sample
Plasma sample diluted with an equal volume of column equilibration
buffer.
z
Experimental procedure
- Equilibrate the column in a 50 mM phosphate buffer, pH 8.
- Load with above sample.
- Wash with 50 mM phosphate buffer + 0.3 to 0.5 M NaCl to elute
loosely bound contaminants (such as albumin).
- Elute the plasminogen with a gradient of 0.2 to 0.5 M H-amino-caproic
acid in a 50 mM phosphate buffer, pH 8.
- Regenerate with 0.1 M NaOH, 2 column volumes during 30 min.
- Wash with 50 mM phosphate buffer + 0.3 to 0.5 M NaCl, 1-2 column
volumes.
- Equilibrate in starting buffer.
Preparation of the column
- The flasks contain settled sorbent. Gently shake the flask to
obtain an even slurry. If a part of the sorbent remains
sedimented, add fresh buffer to resuspend the sorbent. Do not
use rigid tools to scoop the sedimented sorbent as this may
damage the sorbent.
- Elimination of dissolved gas: carefully degas the suspension
under vacuum while gently swirling (do not use magnetic
stirrer).
- Set up the column and introduce the lower adapter. Connect
a short tubing to the adapter outlet. Fix a syringe to the outlet
tubing and from the syringe, introduce degassed buffer up to 210 mm (0.08-0.04 in.) high. Make sure that air bubbles are
thoroughly eliminated. Clamp the column outlet tubing and
remove the syringe. Pour the degassed suspension (we
recommend a slurry of 70 % of settled sorbent volume) into the
column. Attach the column inlet adapter. Then open the column
outlet and provide flow sufficient to create at least 80 cm (31 in.)
H2O pressure. The column packing is complete when the
sorbent is totally sedimented. For optimized packing, it is
advised to slightly vibrate the tube glass. Do not let the gel bed
dry (add buffer if necessary). Close the column outlet, clamp the
inlet tubing and if desired, connect a pump to the column inlet.
We recommend to operate at 300 cm/h for maximum productivity.
Cleaning and Maintenance
In order to avoid frequent regeneration, it is advisable to introduce into
the column only samples and buffers that are clear and previously
filtered to 0.22 μm. Be sure that changes in pH and ionic strength during
elution do not cause precipitation of sample components.
Nevertheless, after repeated use and if necessary, Lysine HyperD
sorbent may be regenerated in column or in batch. The following
suggestions may be checked first for their degree of efficiency:
Situation
Recommendation
General CleaningIn-Place (CIP)
Wash extensively with 3 M NaCl as a first trial.
Hydrophobic
Contaminants
Wash with 8 M urea, or 6 M guanidine hydrochloride or
an aqueous solution of non ionic detergent (1% Triton*
X100), 1-2 cv.
Unknown
impurities
Wash with 0.1 M sodium hydroxide solution. 1-5 cv of
this solution are generally sufficient to remove alkaline
soluble material. After treatment, neutralize the column
immediately by washing with strong buffer solution.
Flow rate should be between 0.5 and 3 column
volumes/h.
Lysine HyperD sorbent can be packed using peristaltic or
medium pressure pumps. After packing, wash the sorbent
extensively and equilibrate it in the working buffer.
2
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