5.1.3. Protein Step Elution
7. Thermal Stability and Storage
u
Standard step elution: 100 mM Na acetate, pH 5.0, 4.0 and 3.0.
u
Alternative elution: 100 mM Na acetate, pH 5.6 to 3.0.
Elution using mixed-mode sorbents is accomplished by decreasing pH,
that allows prompting selective desorption of proteins with different relative
hydrophobicity. The optimization strategy is based on identifying a pH value
at which the target protein is desorbed with maximum selectivity.
Typically, the more hydrophilic protein will elute earlier in the step-elution
sequence, while the more hydrophobic protein will elute later.
Optimization of the elution step may require the testing of different buffer
formulations or additives (e.g., 0.5 to 1 M arginine; 10 to 40% (v/v) ethylene
glycol) in order to improve final target protein purity.
5.1.4. Regeneration
Temperature of use
2 to 30 °C
Storage temperature
2 to 8 °C
Storage solution
1 M NaCl with 20% (v/v) ethanol
Caution
Plate must never be frozen
Note
Plate is shipped at ambient temperature
5.2. On MEP HyperCel Mixed-Mode Sorbents
See 5.1.
Part Number
AcroPrep ScreenExpert Plate
MEP, HEA and PPA HyperCel Mixed-Mode Sorbents
96WP3MIXM50
MEP HyperCel Mixed-Mode Sorbent
96WPMEP50
HyperCel STAR AX Anion Exchange Sorbent
96WPSTARAX50
MEP HyperCel PRC Column 5x50, 1 mL
PRC05X050MEPHCEL01
MEP HyperCel PRC Column 8x100, 5 mL
PRC08X100MEPHCEL01
HyperCel STAR AX sorbent is an anion exchanger based on a “salt tolerant” primary
amine ligand. It brings new selectivity options for protein capture or contaminant removal,
in a broad feedstock conductivity range from 2 to 15 mS/cm, avoiding dilution of UF/DF
of the sample.
HEA HyperCel PRC Column 5x50, 1 mL
PRC05X050HEAHCEL01
HEA HyperCel PRC Column 8x100, 5 mL
PRC08X100HEAHCEL01
PPA HyperCel PRC Column 5x50, 1 mL
PRC05X050PPAHCEL01
Guidelines in Table 6 are based on the screening of a feedstock/sample with a conductivity of 10 to 15 mS/cm. “8-run screening” means that half of the screening is carried
out at the conductivity of the undiluted feedstock/sample and the other half (8 conditions
totally), at a conductivity of 5 mS/cm (after dilution/diafiltration of the feedstock/sample).
It is strongly recommended to run several replicates for each process condition.
PPA HyperCel PRC Column 8x100, 5 mL
PRC08X100PPAHCEL01
HyperCel STAR AX PRC Column 5x50, 1 mL
PRCSTARAX1ML
HyperCel STAR AX PRC Column 8x100, 5 mL
PRCSTARAX5ML
AcroPrep Advance 96-Well Filter Plate
8129
Table 6
Guidelines for HyperCel STAR AX Anion Exchange Sorbent
Multi-well Plate Vacuum Manifold
5017
Load
Wash
AcroPrep™ ScreenExpert Plates
For Chromatography Sorbent
Screening and Optimization
Related Products
5.3. On HyperCel STAR AX Anion Exchange Sorbent
Binding
USD 2880
8. Ordering Information
Description
Regeneration of mixed-mode HyperCel sorbents is carried out in 1 M NaOH.
Because AcroPrep ScreenExpert Plates are disposable products, no regeneration
step is necessary, unless screening is focused on optimizing the regeneration step.
Instructions For Use
AcroPrep Advance Filter Plate for Ultrafiltration:
8-Run Screening on HyperCel STAR AX Sorbent
l
350 μL, Omega Membrane 3 K MWCO (10/pkg)
8033
Undiluted Feedstock, 10–15 mS/cm
Diluted Feedstock, 5 mS/cm
l
350 μL, Omega Membrane 10 K MWCO (10/pkg)
8034
Tris-HCl,
pH 7.0
l
350 μL, Omega Membrane 30 K MWCO (10/pkg)
8035
l
350 μL, Omega Membrane 100 K MWCO (10/pkg)
8036
Tris-HCl,
pH 7.0
Tris-HCl,
pH 7.5
Tris-HCl,
pH 8.0
Tris-HCl,
pH 8.5
Tris-HCl,
pH 7.5
Tris-HCl,
pH 8.0
Tris-HCl,
pH 8.5
~0.5 to 2 mg protein per well, adjust at
~0.5 to 2 mg protein per well, adjust at
pH 7.0
pH 7.5
pH 8.0
pH 8.5
pH 7.0
pH 7.5
pH 8.0
pH 8.5
Tris-HCl,
pH 7.0
Tris-HCl,
pH 7.5
Tris-HCl,
pH 8.0
Tris-HCl,
pH 8.5
Tris-HCl,
pH 7.0
Tris-HCl,
pH 7.5
Tris-HCl,
pH 8.0
Tris-HCl,
pH 8.5
10 to 15 mS/cm
5 mS/cm
Elution 1
Binding buffer + 300 mM NaCl
Binding buffer + 150 mM NaCl
Elution 2
Binding buffer + 500 mM NaCl
Binding buffer + 500 mM NaCl
Elution 3
Binding buffer + 1 M NaCl
Binding buffer + 1 M NaCl
For detailed information on the sorbents, refer to Pall datasheets USD 2443, USD 2629 and
USD 2831.
Note: Phosphate buffers with concentrations higher than 20 mM are not recommended
for use with HyperCel STAR AX sorbent because they may compromise the binding of
proteins to the primary amine ligand.
1. Introduction
AcroPrep ScreenExpert Plates are a family of 96-well filter plates pre-filled with Pall
chromatography sorbents. They are designed for early screening of Pall sorbents used in
protein purification.
6. Troubleshooting
In addition to testing different sorbents, many chromatography conditions can also be quickly
tested and optimized in parallel with minimal sample consumption.
1. Clogging
1.1. Make sure that the loading sample and buffers have been filtered through a 0.2 μm filter.
The operation of AcroPrep ScreenExpert Plates involves “batch chromatography” steps, similar
to any column chromatography experiment.
1.2. Clarification of the sample is crucial to avoid clogging of the membrane. Pall recommends Acrodisc
PF Syringe Filter with hydrophilic Supor membrane, sterile, 0.8/0.2 μm 25 mm (part number 4187).
Check that even after clarification, the loading sample is not precipitating once it has been conditioned
into the selected process conditions. Before screening, it is recommended to study sample stability:
adjust the sample at different pH and conductivity and verify that no precipitation occurs for approximately 3 to 4 hours at room temperature.
Rapid screening and optimization of process conditions achieved in AcroPrep ScreenExpert
Plates can be transferred and confirmed or scaled-up using Pall 1 mL or 5 mL PRC prepacked
columns. AcroPrep ScreenExpert Plates can be operated with liquid-handling robotic systems,
or manually using multi-channel pipettes. The recovery of liquid (i.e., samples or buffer deposited
into the wells) can be conducted using vacuum aspiration or centrifugation to draw the liquid
through the membrane at the bottom of each well.
1.3. Dilute the sample if viscosity is too high.
2. Product Description
2. Cross-contamination
2.1. Make sure that the shaking speed and shape do not lead to liquid ejection at the top surface of
the plate.
Table 1
Characteristics of AcroPrep ScreenExpert Plates
2.2. Make sure that the vacuum aspiration is set at -0.3 to -0.5 bar g. Do not exceed -0.7 bar g.
Wells should be all emptied simultaneously, otherwise vacuum system should be optimized.
2.3. If centrifugation is used, make sure that centrifugation speed is not too high. Do not exceed 1000 x g.
2.4. Make sure that the plate does not rub/slip on the collection plate during incubation under agitation.
3. Weeping
3.1. Avoid placing the plate bottom (fluid directors) directly in contact with any surface, especially if the
storage solution has not been removed.
3.2. Make sure to insert a collection plate or an upside-down plate plastic cover (or any other equipment
acting as a spacer) between the plate and the surface.
3.3. Make sure that the plate does not rub/slip on the collection plate during incubation under agitation.
3.4. If the type of fluid is the probable cause of the weeping, blot the bottom of the plate on a soft paper
tissue after fraction collection and between the steps of the chromatography sequence (equilibration,
sample loading, washing and elution steps) to avoid leakage. Alternatively, use a different buffer
formulation (avoid detergents and fluids with low surface tension).
5
USD 2880
Corporate Headquarters
Port Washington, NY, USA
+1 800 717 7255 toll free (USA)
+1 516 484 5400 phone
biopharm@pall.com e-mail
European Headquarters
Fribourg, Switzerland
+41 (0)26 350 53 00 phone
LifeSciences.EU@pall.com e-mail
Asia-Pacific Headquarters
Singapore
+65 6389 6500 phone
sgcustomerservice@pall.com e-mail
Visit us on the Web at
www.pall.com/biopharm
E-mail us at biopharm@pall.com
International Offices
Pall Corporation has offices and plants throughout
the world in locations such as: Argentina, Australia,
Austria, Belgium, Brazil, Canada, China, France,
Germany, India, Indonesia, Ireland, Italy, Japan, Korea,
Malaysia, Mexico, the Netherlands, New Zealand,
Norway, Poland, Puerto Rico, Russia, Singapore,
South Africa, Spain, Sweden, Switzerland, Taiwan,
Thailand, the United Kingdom, the United States,
and Venezuela. Distributors in all major industrial
areas of the world.
To locate the Pall office or distributor nearest you,
visit www.pall.com/contact.
The information provided in this literature was
reviewed for accuracy at the time of publication.
Product data may be subject to change without
notice. For current information consult your local
Pall distributor or contact Pall directly.
© 2013, Pall Corporation. Pall,
, Acrodisc,
AcroPrep, HyperCel, Omega and Supor are
trademarks of Pall Corporation. ® indicates a
trademark registered in the USA.
Filtration.Separation.Solution is a service mark
of Pall Corporation.
1/13, PDF, GN12.8390
USD 2880
Plate size
128 x 86 x 33 mm
Plate material
Polypropylene
Number of wells
96
Well volume
1 mL
Filter media and type
0.45 μm Supor® hydrophilic polyethersulfone membrane (Pall)
Part number for the empty plate
8129
Mode of operation
Vacuum using manifold or centrifuge
Volume of chromatography sorbent per well
50 μL in a total volume of 800 μL
Storage solution per well
750 μL of 1 M NaCl with 20% (v/v) ethanol
Working volume per well
100 to 500 μL
Maximum centrifugation force
700 x g
Maximum vacuum
0.5 bar g (375 mm Hg)
1
Table 2
Types of AcroPrep ScreenExpert Plates and Applications
Separation
Mode
Part Number
Sorbents
Ligand
96WP3MIXM50
MEP, HEA and PPA
HyperCel™ sorbents
x32 wells of each1
MEP: 4-mercaptoethyl-pyridine
HEA: Hexylamine
PPA: Phenylpropylamine
Mixed-mode
MEP HyperCel
sorbent
x96 wells
4-mercaptoethyl-pyridine
Mixed-mode
HyperCel STAR AX
sorbent
x96 wells
Primary amine
Salt tolerant
anion exchange
96WPMEP50
96WPSTARAX50
Applications
Screening of
mixed-mode ligand
selectivity and
optimization
Optimization of
separation conditions
on MEP HyperCel
sorbent
Optimization of
separation by
“salt-tolerant”
anion exchange
It is also recommended to study sample stability before the screening by adjusting
the sample at different pH and conductivity values and verifying that no precipitation
occurs for approximately 3 to 4 hours at room temperature. In all cases, avoid
preparing the sample more than 4 hours before plate processing to avoid any
precipitation due to protein instability.
– Stir the plates for 60 minutes on an orbital shaker at 1100 to 1200 rpm.
The speed and shape of shaking is crucial for all steps, including sample
loading, to be as close as possible from maximum binding. It has been
demonstrated that optimal conditions were reached at 1100 to 1200 rpm
orbital shaking (Pall internal data). Above 1200 rpm, the plate may be ejected
from the shaker. Below 1100 rpm, mixing is not efficient.
4.2. Plate Processing
Incubation time may be shortened to 45 minutes but never below
30 minutes as variability will occur. In any case, do not compare data
obtained at different incubation times.
Plate processing may be automated using a liquid handling robotic system (refer to the
supplier’s instructions for setting up appropriate programs of the robotic system). Vacuum
aspiration or centrifugation can be used to collect liquid through the plate membranes.
Pall recommends vacuum aspiration over centrifugation for fraction collection as it is less
time consuming and is more easily integrated in a robotic system. A vacuum manifold
should ideally be integrated in the robotic workstation. For manual operation, a vacuum
manifold such as Pall’s Multi-well Plate Vacuum Manifold can be used (part number 5017).
Plate Washing
Conduct the washing step twice for 5 minutes using equilibration buffer to ensure
the removal of unbound proteins/biomolecules.
It may be interesting to test successively different washing conditions in addition to
the washing step with equilibration buffer, if major contaminants must be removed.
If centrifugation is used, a centrifuge with a rotor compatible with 1 mL 96-well plate is
needed. Centrifugation steps can be carried out for 1 to 2 minutes at ~500 x g.
Plate Elution
As many successive elution steps as desired for investigation may be carried out
on a single well (elution 1 to elution n), e.g.,
4.2.1. Plate Preparation
1
See Table 3
Table 3
Sorbent Distribution on Mixed-Mode Screening Plate, Part Number 96WP3MIXM50
Well Number
1 to 4
5 to 8
9 to 12
A to H
MEP HyperCel Sorbent
HEA HyperCel Sorbent
PPA HyperCel Sorbent
The plates are designed in accordance with the standards set forth by the Society of
Biomolecular Screening for multi-well plates. They have a rigid construction which facilitates
ease of use in automated systems. Plate well geometry design prevents cross-talk or cross
contamination of samples. The advanced fluid director on the bottom of the plate also minimizes
cross contamination of the filtrate during processing.
Before opening the plate (removing the top and bottom foils), make sure to
resuspend the sorbent in the storage solution by inverting the plate bottom side
up and tapping the plate several times. Finally revert the plate top side up.
– If sample load is 100 μL, use 100 μL elution buffer
The top and bottom foils of the AcroPrep ScreenExpert Plate must be entirely
removed and processing of the entire plate is recommended. Once the foils have
been removed, it is strongly recommended to remove the 750 μL of storage
solution from each well to avoid weeping. The storage solution can be removed
using vacuum aspiration at -0.3 to -0.5 bar g (225 to 375 mm Hg) for 30 to 40
seconds or using centrifugation for 1 to 2 minutes at ~500 x g.
Elution steps corresponding to one process condition should always be run at
least twice: first elution to recover proteins/biomolecules eluted at the condition
tested, and second elution to avoid carry-over from an elution condition to
another. To shorten elution process, it is suggested to carry out the first step for
10 minutes and the second, for 5 minutes only.
If only part of the plate must be processed (e.g., columns 1 to 6), it is possible to
remove only the foils from columns 1 to 6 using a cutter. In that case, it is necessary
to verify that the sealing of the foils of unprocessed wells is maintained. Partial
peeling of the foil may compromise the sealing.
The maximum working volume of supernatant recommended is 500 μL per
well. The amount of storage solution per well is above the recommended
working volume only to ensure appropriate storage of the sorbent in each
well, and should be removed as soon as the plate is open. We recommend
avoiding direct contact between the plate bottom outlets and any surface
until the storage solution is removed to prevent weeping from the well.
3. Equipment
u
AcroPrep ScreenExpert Plate
u
Flat bottom 96-well collection plate (350 μL or 1 mL deep-well depending on the size of the
fractions to collect). Plate can be non-readable or UV-readable. UV readable collection plate
enables direct absorbance reading at 280 nm for mass balance evaluation.
If plate is processed using a liquid-handling robotic system:
u
4.2.2. Running the Experiment
Liquid handling robot including:
– 96-well plate vacuum manifold
– Stirring plate or an orbital shaker adaptable to microplates
Plates are processed as described in Table 4. Each step should be carried out at
the temperature intended for the final process (i.e., room temperature in most
cases) under orbital shaking at 1100 to 1200 rpm.
If plate is processed manually:
u
Multi-channel pipettes and tips
u
Stirring plate or orbital shaker adaptable to microplates
Table 4
Recommended Sequence Applied on AcroPrep ScreenExpert Plates
A collection plate or upside-down plate plastic cover (or any other equipment acting
as a spacer) should be fixed to the surface of the shaker to prevent direct contact
between the fluid directors and any surface. Fix it firmly to avoid vibrations during
shaking. Similarly, the plate receiver part of the shaker should be designed in a
manner that the plate does not vibrate or rub on the collection plate/spacer.
u
Step
1
Equilibration
400
5
x3
2
Sample loading
200
60
x1
3
Washing
200
5
x2
Washing (optional) 200
5
x2
A vacuum manifold (Pall Multi-Well Plate Vacuum Manifold, part number 5017) or a
centrifuge
4
Volume (μL)
Duration (min)
Number of Cycles
Elution 1
200
10
x1
u
pH meter
Elution 1’
200
5
x1
u
Conductimeter
Elution 2
200
10
x1
u
Refrigerator (2 to 8 °C) or ice
Elution 2’
200
5
x1
u
High throughput (HT) analytical tools: HT screening of sorbents generates a high number of
fractions to analyze. To avoid bottlenecking development process, analytics should be based
as much as possible on HT analytical tools.
Elution 3
200
10
x1
Elution 3’
200
5
x1
Elution n
200
10
x1
Elution n’
200
5
x1
Other equipment :
4. Protocol
For standard process steps, see 4.2.4.
4.1. Recommendations for Sample Preparation
Note: It is not necessary to include the second elution step for the same process
condition in the mass balance calculation as the amount of proteins recovered in
this fraction is most of the time not significant.
5. Recommendations for Screening / Optimization Conditions
It is recommended to proceed to Design of Experiments (DoE) plan for screening chromatography sorbents and process conditions. The main parameters to test are buffer formulation,
pH and conductivity at binding, loading, washing and elution steps.
5.1. On MEP, HEA and PPA HyperCel Mixed-Mode Sorbents
MEP, HEA and PPA HyperCel sorbents are mixed-mode sorbents designed for protein
capture and impurity removal. They operate on the basis of a mixed-mode mechanism
where both hydrophobic and ionic effects are predominant and provide new selectivity,
orthogonal to conventional chromatography steps.
Mixed-mode separation performance is influenced by several factors, including pH and
salt concentration of buffers used. For example, addition of salt in binding buffer may
enhance binding in some cases. Washing steps (with or without salt) also influence the
final result, allowing to remove specific family of impurities.
General guidelines for screening using the AcroPrep ScreenExpert Plate for mixed-mode
including MEP, HEA and PPA HyperCel sorbents are suggested in Table 5.
Table 5
Guidelines for HyperCel Mixed-Mode Sorbents
12-Run1 Screening on MEP, HEA, and PPA HyperCel Sorbents
Conditions 1
Conditions 2
Binding
PBS, pH 7.4
20 mM Na phosphate, Tris, pH 8.5
pH 7.4 + 1 M NaCl
Tris, pH 8.5
+ 1 M NaCl
Load
~50 – 500 μg
protein per well
~50 – 500 μg
protein per well
Add 1 M NaCl
~50 – 500 μg
protein per well
Adjust at pH 8.5
and add 1 M NaCl
Wash
PBS, pH 7.4
20 mM Na phosphate, Tris, pH 8.5
pH 7.4 + 1 M NaCl
Optional
washes
50 mM Na phosphate, 20 mM Na phosphate, 50 mM Na phosphate, 50 mM Tris, pH 8.5,
pH 6.0
pH 7.4, then 50 mM
pH 7.0
then 50 mM Na
Na phosphate, pH 6.0
phosphate, pH 7.0
Elution 1
100 mM Na acetate, pH 5.0
Elution 2
100 mM Na acetate, pH 4.0
Elution 3
100 mM Na acetate, pH 3.0
4.2.3. Fraction Collection
4.1.1. Sample Load
An appropriate volume of sample should be prepared depending on load volume
and the number of wells to process. It is recommended to prepare an excess
volume of sample, especially when a robotic system is used. Prior to loading, the
pH and conductivity of the sample loaded should be adjusted and be the same as
the equilibration buffer. Prepare as many sample aliquots as there are of binding
conditions to test.
Since unclarified sample may clog the membrane at the bottom of the wells, it is
necessary to filter the sample through a 0.2 μm filter. Pall recommends Acrodisc®
PF Syringe Filter 0.8/0.2 μm Supor low protein binding membrane (part number
4187).
4.1.2. Conductivity and pH
They can be adjusted by dilution of the sample in a buffer of appropriate conductivity and pH. Alternatively, the sample can be buffer-exchanged by diafiltration
using AcroPrep Advance 96-Well Filter Plate with an Omega™ membrane 10 to
100 K MWCO depending on the size of the biomolecules of interest.
4.1.3. Loading Volume
Typically 100 to 400 μL. Do not load less than 100 μL of sample per well (a
minimum ratio sample/sorbent of 2 in the well is required to get an acceptable
mixing during sample incubation), it may be necessary to dilute the sample
before sample loading. In any case, the maximum working volume of supernatant
recommended is 500 μL.
4.1.4. Protein Capacity Per Well to Be Evaluated First Before Screening
Before screening, it is recommended to evaluate first, under favorable pH and
conductivity conditions for binding, the maximum amount of protein to be loaded
on a single well without detecting any target protein in the flow through. This value
should not be exceeded during the screening tests in order to limit any risk of data
mis-interpretation.
2
Step Number
– If sample load is 200 μL, use 200 μL elution buffer, unless sample is very
diluted. In this case, use only 100 μL to concentrate the eluate 2-fold.
USD 2880
The fractions are collected in a standard 96-well receiver plate after each step of
the chromatography sequence using vacuum aspiration at -0.3 to -0.5 bar g for
30 to 40 seconds or using centrifugation for 1 to 2 minutes at ~500 x g. The
collection plate should be protected with an adhesive foil or a cover, and stored
at 2 to 8 °C until subsequent analysis to avoid evaporation and/or sample
degradation. A new collection plate should be used before each step of the
chromatography sequence.
Note: When plates are processed using a robotic system, it is recommended to
include in the program a movement of the robotic arm that will gently move the
AcroPrep ScreenExpert Plate 1 cm above the vacuum manifold and drop it to
eliminate any remaining drops at the outlets of the plate.
1
Tris, pH 8.5
+ 1 M NaCl
5.1.1. Protein Binding
u
Standard conditions: PBS pH 7.4 for equilibration and binding, no salt addition.
u
Alternative binding conditions (if binding of the target protein needs to be
strengthened):
– 20 mM Na phosphate, pH 7.4 + 1 M NaCl: adjust sample conductivity.
– 50 mM Tris-HCl, pH 8.5: adjust sample pH to 8.5.
– 50 mM Tris-HCl, pH 8.5 + 1 M NaCl: adjust sample pH and conductivity.
Plate Equilibration
If most of the target protein remains in the flowthrough, 0.5 M ammonium
sulfate may be used as a replacement of sodium chloride.
Pall recommends carrying out three successive equilibration steps with 400 μL
of equilibration buffer since it has been demonstrated that it enables to reach the
desired pH and conductivity (i.e. of the equilibration buffer).
Work below capacity for first trials (to avoid overloading). Optimal binding
capacity may be determined once process conditions have been optimized.
Sample Loading and Incubation
5.1.2. Washing
Note: Below 100 μL, the sample / sorbent volume ratio is below 2 and the mixing
of the sorbent + sample may not be satisfying to reach the equilibrium. Above
500 μL, weeping issues may occur through the fluid directors at the bottom of
the plate. If the sample loaded has a low concentration, it is possible to load
2 or 3 times the same amount of sample. In that case, plate processing will be
longer as sample incubation steps will be multiplied.
3
~50 – 500 μg
protein per well
Adjust at pH 8.5
Conditions 4
12-run screening = 4 conditions x 3 sorbents. It is strongly recommended to run several replicates
for each condition.
4.2.4. Standard Process Steps
– Load a volume of sample typically comprised between 100 μL and 400 μL per
well. In any case, do not go above 500 μL.
Conditions 3
u
Standard conditions wash: PBS, pH 7.4.
u
Optional wash (to remove contaminants):
– Decrease conductivity (only if binding buffer contained salt) while maintaining
the same pH.
– Decrease pH (avoid pH lower than pH 5.5 as desorption of the target protein
may occur) while maintaining the same salt concentration.
– Optimization may lead to an additional washing step combining a decrease
of both pH and conductivity.
USD 2880
www.pall.com/biopharm
4