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HPLC Columns

TSKgel Protein A-5PW

The TSKgel Protein A-5PW column has been designed for the rapid separation and robust quantification of a variety of antibodies. Monoclonal antibodies from harvested cell culture media can be captured and accurately quantitated in less than 2 minutes per injection. The column can be used for more than 2,000 injections without regeneration or cleaning.

Packed with hydroxylated methacrylic polymer beads, the TSKgel Protein A-5PW column is designed with a high degree of crosslinking, which allows high flow rate for chromatography while still maintaining chromatographic efficiency, peak width and resolution. The recombinant protein A ligand is a code-modified hexamer of the C domain. An enhanced rProtein A ligand is bound to the TSKgel 5PW base bead via multipoint attachment resulting in excellent base stability in 0.1 mol/L NaOH.

 

The wide range loading capacity of the TSKgel Protein A-5PW column can accurately determine the titer of mAb at various stages of cell culture media processing. The low level of protein A leaching makes this column a good candidate for small scale purification of mAbs for initial characterization.

 

 
P/NDescriptionParticle Size Housing MaterialID (mm)Length (cm)Price ($) 
23483TSKgel Protein A-5PW20PEEK4.63.5950
        

Product Attributes

Pore size (mean): 100 nm
Particle size: 20 µm
pH stability: 2.0 - 12.0
Exclusion limit: 1,000 kDa
Ligand: Recombinant protein A, hexamer of C domain


Affinity for Various Antibodies

Species Subclass Protein A ligand of Protein A-5PW Native
Protein A
Human IgG1
IgG2
IgG3
IgG4
+++++
+++++
-
+++++
++++
++++
-
++++
Mouse
IgG1
IgG2a
IgG2b
IgG3
++++
+++++
+++++
++++
+
++++
+++
++
Rat IgG1
IgG2a
IgG2b
IgG2c
++++
-
+++
++++
-
-
-
-
Goat
IgGS ++++
-
Chicken IgY - -
Rabbit
IgG +++++ ++++

         

Rapid Separation of IgG from Impurities

ProA_Protein-A-5PW_Fig1_PO44.png

Column: TSKgel Protein A-5PW, 20 µm,
4.6 mm ID × 3.5 cm

Binding buffer: 20 mmol/L sodium phosphate buffer, pH 7.4
Elution buffer: 20 mmol/L sodium phosphate buffer, pH 2.5
Stepwise gradient: 0 - 0.5 min: binding buffer
0.5 - 1.1 min: elution buffer
1.1 - 2.0 min: binding buffer
Flow Rate: 2 mL/min
Detection: UV @ 280 nm
Sample: 20 µL of CHO cell culture supernatant spiked with polyclonal IgG (0.5 mg/mL)

 

For additional applications, please see our applications database.

 

                  

Durability and Dynamic Range

ProA_Protein-A-5PW_Fig2_PO44.png

The TSKgel Protein A-5PW column was subjected to a linearity analysis test. Purified IgG was initially injected onto the column with subsequent injections of IgG made at different volumes. The column was then used up to 2,009 injections without being cleaned. A linearity analysis test was then repeated. No significant change in the calibration curve for IgG was seen. The column still maintained its high loading capacity with an excellent linearity (R2=0.9999).

 

Wide Range of Loading Concentrations of Purified IgG

ProA_Protein-A-5PW_Fig2_AN96.png
ProA_Protein-A-5PW_Fig3_AN96.png

 

Column: TSKgel Protein A-5PW, 20 µm,
4.6 mm ID × 3.5 cm (PEEK)

Binding and washing buffer: 20 mmol/L sodium phosphate buffer,
pH 7.4
Elution buffer: 20 mmol/L sodium phosphate buffer,
pH 2.5
Note: IgG can also be eluted with 12 mmol/L HCL, 20-100 mmol/L citric acid, pH 2.5-3.5, 20-100 mmol/L glycine, pH 2.5-3.5, 5-10% acetic acid
Stepwise gradient: 0 - 0.5 min: binding buffer
0.5 - 1.1 min: elution buffer
1.1 - 2.0 min: binding buffer
Flow rate: 2 mL/min
Detection: UV @ 280 nm
Sample: IgG

 

Leaching Analysis

Resin Concentration
(mg/mL)
Protein A
(ppm)
TSKgel Protein A-5PW 0.538 2.3
Competitor Protein A
0.508 10.8

 

An analysis of an IgG sample was run on both a TSKgel Protein A-5PW, 20 μm, 4.6 mm ID × 3.5 cm column and a similar competitive 20 μm protein A, 4.6 mm ID × 5 cm column. The table shows that the protein A ligand from the TSKgel Protein A-5PW column has 2.3 ppm of Protein A leaching, which is many folds lower than the competitor’s protein A column. This data suggests that the coupling process of protein A onto the TSKgel 5PW base bead is better than its competitor’s coupling process.

 

 

Varying Flow Rates to Capture IgG

 

ProA_Protein-A-5PW_Fig5_AN96.png

Column: TSKgel Protein A-5PW, 20µm,
4.6 mm ID × 3.5 cm (PEEK)

Binding and washing buffer: 20 mmol/L sodium phosphate buffer,
pH 7.4
Elution buffer: 20 mmol/L sodium phosphate buffer,
pH 2.5
Note: IgG can also be eluted with 12 mmol/L HCL, 20-100 mmol/L citric acid, pH 2.5-3.5, 20-100 mmol/L glycine, pH 2.5-3.5, 5-10% acetic acid
Stepwise gradient: 0 - 0.5 min: binding buffer
0.5 - 1.1 min: elution buffer
1.1 - 2.0 min: binding buffer
Flow rate: 2 mL/min
Detection: UV @ 280 nm
Sample: IgG

 

Code #DescriptionLiterature Type
AN96 Fast monoclonal antibody titer determination with TSKgel Protein A-5PW columnApplication Note
DS1242 TSKgel Protein A-5PW ProductsOperating Conditions and Specifications
IM01 TSKgel Column Instruction ManualInstruction Manual
LCAT06A TSKgel HPLC/UHPLC Columns 2017 Product Guide -- Protein A ColumnsProduct Catalog
PO44 TSKgel Protein A-5PW ColumnProduct Overview