Thermo Fisher Scientific POROS CaptureSelect FcXP Affinity Resin User guide

Brand
Thermo Fisher Scientific
Model
POROS CaptureSelect FcXP Affinity Resin
Type
User guide
POROS™ CaptureSelect™ FcXP Anity Resin
Catalog NumbersA56249, A56251, A56252, A56253, and A56254
Pub.No. MAN0028745 Rev. A.0
WARNING! Read the Safety Data Sheets (SDSs)
and follow the handling instructions. Wear appropriate
protective eyewear, clothing, and gloves. Safety Data
Sheets (SDSs) are available from thermofisher.com/
support.
Product description
POROS™ CaptureSelect™ FcXP Anity Resin is designed for the
purification of recombinant human IgG, Fc fusion proteins, and
plasma‑derived IgG through binding of the CH3 domain of human
IgG. The FcXP ligand recognizes all human IgG subclasses (IgG1,
2, 3, and 4), and is designed to obtain increased binding capacity
(20–25 g IgG/L) at higher flow rates while maintaining elution
characteristics. The large pore structure of the POROS™ bead
enables ecient purification of larger biomolecules.
Contents and storage
POROS™ CaptureSelect™ FcXP Anity Resin
Cat. No. Amount Storage
A56249[1] 50mL Store at 2–8°C. Do not
freeze.
A56251[1] 250mL
A56252[2] 1,000mL
A56253[2] 5,000mL
A56254[2] 10,000mL
[1] For Research Use Only. Not for use in diagnostic procedures.
[2] For Research Use or Further Manufacturing. Not for diagnostic use or direct
administration into humans or animals.
Specifications
Characteristic Description
Support matrix Cross-linked poly(styrene-divinylbenzene)
Immobilized ligand Single‑domain [VHH] antibody fragment
Binding capacity ˃35 g/L resin[1]
Shipping solvent 18% (v/v) ethanol
Average particle size 50µm
Recommended
residence time
≥4 minutes
Mechanical resistance 100 bar (1,450 psi, 10 MPa)
pH Range (all ligands) 1–10
Ionic strength range 0 to 5 M, all common salts
Buer additives All common agents, including ethylene
glycol, and detergents
Agents that may degrade the protein ligand
are not recommended (for example, pH ˃10)
Solvents Water, ≤20% ethyl alcohol , ≤0.5 Macetic
acid, ≤0.5 Mcitric acid
Do not expose the resin to strong oxidizers
(such as hypochlorite), oxidizing acids (such
as nitric), strong reducing agents (such as
sulfite), acetone, or benzyl alcohol
Operating temperature 2–25°C
Do not freeze
[1] 5% breakthrough of Human IgG in PBS, pH7.5 at 300cm/hr in
4.6mmD x 200mmL column
USER GUIDE
For Research Use or Further Manufacturing. Not for diagnostic use or direct administration
into humans or animals.
Pack and qualify the column
Packing guidelines
• Resins are supplied as approximately 56% slurry in
18%ethanol.
• We recommend a compression factor in the range of
1.08–1.10 for lab scale columns (≤2.6cm i.d.).
• We recommend bed supports with a porosity of 10–12μm
for packing POROS™ CaptureSelect™ FcXP Anity Resin.
• We recommend a packing solution of 0.1M sodium chloride.
Prepare the slurry
1. Determine the required slurry volume.
Required slurry volume = Target packed bed volume ÷ slurry
concentration × compression factor
Example for packing a column with a target bed volume
25mL using a 56% slurry resin and a compression factor of
1.10:
Required slurry volume = 25mL ÷ 0.56 × 1.10 = 49.1mL
2. Mix the resin slurry until it is homogeneous, then transfer the
required slurry volume calculated in step1 to a graduated
cylinder.
3. Allow the resin to settle under gravity.
The settling time depends on the container dimensions,
packing solution, and slurry concentration. For example, a
~56% slurry sample requires approximately 4 hours to settle
at room temperature in a 100 mL graduated cylinder in
0.1MNaCl.
4. Carefully decant the supernatant, then replace the
supernatant with the same volume of the packing solution.
Do not disturb the bed.
Note: Some turbidity may be observed in the supernatant
as beads slough o the settled bed or come loose from the
carboy side walls. This is not problematic.
5. Resuspend the resin until it is homogenous.
6. Repeat step3 to step5 two more times to thoroughly
exchange into the packing solution.
For small resin volumes, see step7.
7. (Optional) For small resin volumes, instead of repeated
decantation and settling, exchange the resin into the packing
solution by using a vacuum membrane filter (0.2–0.45 mm)
or a sintered glass filter:
a. Transfer the required volume of resin slurry to the top of
a bottle-top filter.
b. Use a vacuum to filter out the storage solution.
c. Resuspend the resin cake to the starting resin slurry
volume with packing solution. Mix with a plastic or
rubber spatula.
Do not grind the resin bed or tear the filter membrane.
d. Repeat substep7b and substep7c two more times for
a total of three exchanges.
8. Determine the slurry concentration after the last buer
exchange and settling.
The slurry concentration is defined as the ratio of
gravity‑settled volume to total slurry volume. The
recommended slurry concentration for packing is 30–70%.
9. Confirm there is sucient slurry volume needed to pack the
target bed volume based on the slurry concentration and
compression factor.
For flow packing, see “Flow packing” on page2.
For axial compression packing, see “Axial compression packing”
on page3.
Flow packing
1. Ensure the column outlet is closed.
2. Mix the resin slurry until it is homogeneous.
3. Pour the required slurry volume into the column.
Note: For pilot scale columns, mix the slurry directly inside
the column using a plastic paddle. During mixing, keep the
paddle on top of the settled resin.
4. Connect the column to the top adapter.
5. Open the column outlet, then apply flow at 100–300cm/hr
until a stable bed is formed.
Some turbidity may be observed in the euent as the
packing starts. Turbidity will clear as packing proceeds and
1–2 bed volumes of packing buer pass through the column.
6. Lower the top adapter toward the bed, leaving a gap of
1–2cm.
7. Apply flow at 500–1,000 cm/hr until the pressure drop
across the column is stable. Increase the flow rate until a
pressure drop ≥3 bar is reached.
Record the bed height.
8. Lower the top adapter by 1–2mm below the bed height
recorded in step7.
9. (Optional) As an alternative to step7 and step8, if a
pressure drop ≥3bar cannot be achieved by applying flow
(for example, due to system pump limitations), lower the top
adapter to the target bed height based on the target bed
height and compression factor.
10. Condition the packed bed by applying flow at the highest
processing flow rate in upflow for 1–2 CV.
11. Repeat step10 in the downflow direction.
12. Perform a column qualification test. See “Qualify the
column” on page3.
2POROS™ CaptureSelect™ FcXP Anity Resin User Guide
Axial compression packing
1. Pour the resin slurry into the column.
2. Mix the slurry directly inside the column (for example, pilot
scale columns) using a plastic paddle. During mixing, keep
the paddle on top of the settled resin.
For larger axial compression columns (for example,
˃20 cm i.d.) the resin slurry is typically transferred with aid of
the automation of the column hardware. In these cases, use
a column filling speed of 200–300 cm/hr.
3. Pack the column using a packing speed of 60cm/hr for the
top adapter and a packing factor of 1.15–1.20. Under these
conditions the packing factor and compression factor are
the same for POROS™ resins.
4. Flow condition the packed bed by applying flow at the
highest processing flow rate in downflow for 1–2 CV.
5. Perform a column qualification test. See “Qualify the
column” on page3.
Qualify the column
To qualify the integrity of a packed column, determine HETP
(height equivalent to a theoretical plate) and asymmetry using
a non-binding analyte (a “plug”). Degas solutions before use to
avoid gassing out during operation.
Recommended column qualification conditions
Parameter Recommendation
Flow rate 100cm/hr
Equilibration buer 0.1 M sodium chloride
Plug solution 1 M sodium chloride
Plug volume 2–4% of column volume
Setting specifications
Qualification results depend on several factors, including the:
• Test solutions
• Flow rate
• Column/system hold-up volume
After you define a column qualification procedure for a specific
setup, the results can be used as reference and should be kept
constant when comparing results.
Tests with special solute samples under defined conditions are
useful for comparing the bed integrity before use. These tests
have limits in predicting the performance of the column with
real samples under real conditions (1). Qualification acceptance
criteria should be based on the process performance with scale-
down models rather than theoretical values for small molecule
tracers. Qualification acceptance criteria should also take into
consideration scale-up limitations.
(1) PDA Technical Report 14. Validation of Column-Based
Chromatography Processes for the Purification of Proteins.
Chromatography condition optimization
Equilibration/binding conditions guidelines
50 mM Tris-HCl pH7.5, 125mM NaCl or PBS pH7.0–7.5 is a
good starting buer for equilibration. The pH of the equilibration
buer and load sample must be in the range of 6–8.
Wash conditions guidelines
After the load, wash unbound material from the column with the
equilibration buer. Generally, a 5–10CV wash is sucient to
remove all unbound proteins from the column. Samples with high
impurity levels may require a longer wash to return to a stable
baseline.
Washing with a secondary or intermediate wash can increase
impurity removal and make impurity clearance more predictable
especially when there is interaction with the protein of interest
and the impurity. High‑salt washes such as 1 M NaCl, varying pH
either up or down, and the use of additives such as amino acids,
glycols, or chaotropic agents can be used for washing to improve
purity.
Elution conditions guidelines
20 mM acetic acid, pH 4.0–4.5.
Regeneration conditions guidelines
After the wash step remove the impurities bound by the resin
with a low pH solution such as 0.1M phosphoric acid (pH 1.7).
Generally, 3–5CV is sucient to regenerate the resin.
Resin cleaning and storage
Column cleaning and lifetime study considerations
To avoid contaminant buildup and to ensure long column lifetime,
strip the column every cycle and clean the column in place (CIP)
with a stronger solution, if needed, after 10–20 cycles.
In any cleaning method, reversing the flow direction is
recommended to help flush out particulates and to prevent
contamination of the lower part of the bed. Also, slow the flow
rate to give several minutes' exposure to the regeneration solution
at each step of the cleaning protocol, for example 3–5CV at
4–6 minutes residence time.
The resin is acid stable and has limited caustic stability. For
cleaning, use solutions in the range of pH 2–10. Test cleaning
solutions in this order, then optimize cleaning based on results:
• 0.1–0.5 M citric acid
• 0.5–1.0 M acetic acid
• 0.5 M phosphoric acid
• 2 M urea
• 2 M guanidine hydrochloride
• 20% ethanol (with or without acid)
• 20% isopropanol (with or without acid)
POROS™ CaptureSelect™ FcXP Anity Resin User Guide 3
• 10–20 mM NaOH
After cleaning the resin, re-equilibrate the resin with a neutral pH
buer such as PBS, pH 7.5 or store in buered ethanol.
Resin storage guidelines
• Store bulk resin at 2°C to 8°C. Do not freeze.
• Store packed columns at 2°C to 8°C (long-term) or
room temperature (short-term) after cleaning (see “Column
cleaning and lifetime study considerations” on page3) in a
neutral‐pH solution with a bacteriostatic agent such as 0.1M
sodium phosphate (pH7.0) with 20% ethanol.
Note: Changing storage temperature from room temperature to
refrigerated temperature can aect packed bed stability and buer
outgassing.
Troubleshooting
Observation Possible cause Recommended action
High backpressure Presence of any amount of ethanol
(shipping/storage solution) in the
slurry or in the column
Fully exchange the ethanol before packing. Typically, this requires
three exchanges.
Compromised flow path:
• Compressed sanitary gaskets
• Closed, partially closed, or
blocked inlet and outlet valves
on the column
• Improperly functioning valves
on the chromatography system
• Blocked inline filters
• Characterize the pressure of the entire chromatography system
with no column in place, the system and empty column with the
column outlet plumbed directly to waste, and the system and
empty column with the column outlet plumbed back into the
skid.
• Ensure that the entire flow path is clear.
• Change the inline filters.
Bed supports were clogged • Change or clean the bed support.
• Run the column in upflow for 3CVs, then downflow again.
Observe if there is a change in pressure.
Improperly scaled chromatography
systems, including small-diameter
tubing anywhere in the system and
operating at the high end of the
system range
Verify that the skid pump and tubing diameters are scaled
appropriately for the column operation and replace as needed.
Turbid column euent after >3CVs
during packing
Column bed supports were too
large for the resin (>12µm frit)
Use standard 10–12 µm bed supports.
Compromised flow adaptor seal,
improperly assembled flow adaptor,
or defective flow adaptor.
Take the adapter apart, inspect all parts, and replace as needed.
Column qualification — high
asymmetry
Column was insuciently
compressed; that is, the column
was not packed at a high enough
flow rate/pressure.
• Pack at a higher flow rate/pressure.
• The top adapter position may need to be better seated in the
packed resin bed to ensure that a headspace does not form.
The system and plumbing allowed
for dilution of the salt plug.
• Characterize a salt plug through the chromatography system at
the qualification flow rate to understand how the plug moves
through the system with no packed column in line.
• Verify that the plumbing throughout the system (pre- and
post‑column) is consistent and that areas for dilution are
minimized.
• Verify that there is no air under the distributor.
Salt injection method was not
optimized.
Verify that the desired amount of salt is loaded by checking the peak
height and width. Ensure that the injection is consistent and applied
as close to the column inlet as possible to minimize dilution from
the system. The injection method should be well-described in your
operating procedures to maintain reproducibility.
Column qualification – low
asymmetry
Column was overpacked or packed
inconsistently
Repack the column following the recommended procedure.
Water was used as the mobile
phase
Add some salt to the mobile phase to reduce the charge interaction
between the salt and the bead.
Column was not equilibrated long
enough with sodium chloride before
salt injection
Equilibrate ³ 4 CVs if the packing solution is dierent from the
qualification mobile phase.
4POROS™ CaptureSelect™ FcXP Anity Resin User Guide
Observation Possible cause Recommended action
Column qualification – low plates or
high HETP
The system and plumbing allowed
for dilution of the pulse solution.
• Characterize a salt pulse injection through the chromatography
system at the qualification flow rate with no packed column in
line.
• Verify that the plumbing throughout the system (pre- and post-
column) is consistent and that areas for dilution are minimized.
• Verify that there is no air under the column flow distributor.
Details: Decreased performance:
• Increased elution peak volume
• Decreased binding capacity
• Decreased recovery
• Increased pressure drop
• Trace or “ghost” peaks during
blank runs
Insucient or unoptimized cleaning
leading to precipitation of product or
impurities, irreversible binding of lipid
material, or other impurities.
Change and improve the cleaning regime.
Support
For service and technical support, go to thermofisher.com/poros
or call toll-free in US: 1.800.831.6844.
For the latest service and support information at all locations, or
to obtain Certificates of Analysis or Safety Data Sheets (SDSs;
also known as MSDSs), go to thermofisher.com/support or
contact your local Thermo Fisher Scientific representative.
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their
products as set forth in the Life Technologies' General Terms
and Conditions of Sale at www.thermofisher.com/us/en/home/
global/terms-and-conditions.html. If you have any questions,
please contact Life Technologies at www.thermofisher.com/
support.
Life Technologies Corporation | 2 Preston Court | Bedford, Massachusetts 01730 USA
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
Revision history:Pub.No.MAN0028745
Revision Date Description
A.0 6 February 2023 New document for POROS™ CaptureSelect™ FcXP Anity Resin.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
©2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
thermofisher.com/support|thermofisher.com/askaquestion
thermofisher.com
6 February 2023
  • Page 1 1
  • Page 2 2
  • Page 3 3
  • Page 4 4
  • Page 5 5

Thermo Fisher Scientific POROS CaptureSelect FcXP Affinity Resin User guide

Brand
Thermo Fisher Scientific
Model
POROS CaptureSelect FcXP Affinity Resin
Type
User guide
Download PDF
report

Ask a question and I''ll find the answer in the document

Finding information in a document is now easier with AI

Related papers

Other documents