Sepapure protein A FPLC column

Product catalog summary

Specifications
Sepapure Protein A columns are designed for antibody purification using Protein A – Fc region interaction. They are compatible with low-pressure FPLC systems and should be operated below 3 bar. The column housing is made of polypropylene with polyethylene frits, and it comes in 1 and 5 mL bed volumes. The resin is Sepapure Protein A FF, with a base matrix of 4% cross-linked agarose and a ligand density of 6 mg/mL. The static antibody binding capacity is over 30 mg/mL human IgG, and the flow rate exceeds 300 cm/hr. The pH stability ranges from 2 to 10 short-term and 3 to 9 long-term.

Buffers
The columns are compatible with most aqueous phase chromatography buffers. Suggested buffers include PBS, pH 7.4 for equilibration and wash, and 0.1 M glycine or citric acid, pH 3.0 for elution.

Preparing the Column
Before use, remove the 20% ethanol storage buffer by flushing with 3 to 5 column volumes (CVs) of binding buffer. Equilibrate with 3 to 10 CVs of binding buffer to stabilize pH, conductivity, and UV280 signals.

Sample Preparation
Use clarified samples with the columns. Most antibodies bind in the pH range 7.0 – 7.4. Avoid high NaCl concentrations to prevent non-specific binding.

Antibody Purification
Load samples at approximately 1 CV/min. Adjust flow rates based on binding kinetics and sample viscosity. Wash the column with binding buffer until the UV280 trace stabilizes. Elute proteins using the selected buffer, collecting all fractions for analysis. Neutralize acid-labile antibodies immediately after elution.

Column Regeneration
Post-elution, wash with 3 to 5 CVs of elution buffer and equilibration buffer. For persistent contaminants, use 6 M Guanidine HCl or 0.1% Triton-X100, followed by equilibration buffer. NaOH can be used for short periods for cleaning.

Column Storage
Store columns in PBS with 20% ethanol at +4°C. Do not freeze.

Catalog excerpts

Sepapure protein A FPLC column Short guide

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Note: For your own safety, read the instructions and observe the warnings and safety information on the device and in the instructions. Keep the instructions for future reference. KNAUER Technical Support: Version information: Phone: +49 30 809727-111 (9-17h, Central European Time) Fax: +49 30 8015010 E-Mail: [email protected] Languages: German, English KNAUER Wissenschaftliche Geräte GmbH Hegauer Weg 38 14163 Berlin Germany Phone: +49 30 809727-0 Fax: +49 30 8015010 Internet: www.knauer.net E-Mail: [email protected] Article number: V6011 Version number: 1.0 Last update: 2019/01/29 The information...

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Table of Contents 1. Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 Sepapure FPLC columns short guide, V6011

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Specifications Definition Sepapure Protein A columns are designed for the purification of antibodies and antibody fragments via the Protein A – Fc region interaction. The columns are designed to be used with low-pressure FPLC-type automated purification systems and operated below 3 bar (0.3 MPa, 2.96 atm). Hardware specifications Column housing Polyethylene (nominal 20 µm porosity) Resin specifications Resin name Base matrix material 4% cross-linked, beaded agarose Mean bead diameter Ligand density Static antibody binding capacity Flow rate pH stability (with metal ion loaded) 2 – 10 (short term)...

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Sepapure columns are designed to be used with most aqueous phase chromatography buffers. A suggested buffer system is shown below, although other buffers may be used. Please check the resin specifications for further details. Equilibration buffer Wash buffer Elution buffer 0.1 M glycine, pH 3.0 or 0.1 M citric acid, pH 3.0 2. Preparing the column Sepapure Protein A columns are supplied with 20% ethanol as the storage buffer. This must be removed prior to purification. Process Remove the end-plugs and connect the column to the control system, taking care to avoid introduction of air into the system....

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3 Elute the protein using the selected elution buffer. A stepwise or linear gradient may be used to determine the precise elution point of the target antibody. Antibodies can be eluted in a pH range starting from 5.5 and decreasing to as low as 3.0. Strongly bound antibodies may require an elution pH of between 2.0 and 3.0. Initially, all elution fractions should be collected for further analysis. Buffer exchange and/or desalting might be required following elution and we recommend Sepapure Desalting Columns (010X460SPZ and 020X460SPZ) for this purpose. Acid-labile antibodies should be neutralised...

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Latest KNAUER instructions online: https://www.knauer.net/en/Support/User-manuals KNAUER Wissenschaftliche Geräte GmbH Hegauer Weg 38 14163 Berlin Phone: +49 30 809727-0 Fax: +49 30 8015010 E-Mail: [email protected] Internet: www.knauer.net

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