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mAB Analytic via Protein A Columns
Analysis of Immunoglobulins (Antibodies)
Affinity Chromatography with Protein A
Immunoglobulins (antibodies) are proteins that are produced in humans and animals in response to certain substances (antigens). These include immunoglobulin G (IgG) and monoclonal antibodies (mAb's), which are of increasing importance in today's medical research and applications.
The analysis or purification of antibodies is often carried out by protein A affinity chromatography, as protein A has the ability to bind many of the immunoglobulins reversibly. Under physiological conditions, protein A and its recombinant derivatives bind to the Fc part of the antibodies and their aggregates. The immunoglobulins are eluted by lowering the pH value.
The main field of application of protein A affinity chromatography media is capturing* during the purification process of antibodies. In industry, this is carried out in large self-packed columns. Protein A HPLC columns are used, among other things, to determine the concentration of mAb's during cell line selection or in the upstream of bioprocess optimisation. Furthermore, small amounts of immunoglobulins can also be purified for analytical purposes.
[English:] Figure 1: [English:] Schematic illustration of an antibody
Technical Data
Manufacturers of Protein A Affinity Chromatography Columns and Bulk Material are:
1. Tosoh Bioscience
Tosoh Bioscience offers the following analytical HPLC column and bulk materials for Protein A affinity chromatography:
TSKgel Protein A-5PW is an analytical affinity HPLC column for reproducible and robust antibody analyses.
| Base material | Polymethacrylate |
| Particle Size | 20 µm |
| Pore Size | 100 nm |
| Ligand | Recombinant Protein A, Hexamer of c Domain |
| pH stability | 2.5 - 12.0 |
| Ligand Density | 0.9 - 1.0 g/l gel |
| Exclusion Limit | 1,000 kDa |
| Dimension | 4.6 x 35 mm (PEEK) |
| Part-Number | 0023483 |
Features:
- Fast separations possible (1-2 min/analysis)
- Long service life: > 2000 injections
- High sensitivity for the determination of mAb titres
- High stability in alkaline for effective CIP cycles
Applications:
- Tosoh TSKgel Protein A-5PW Fast mAb Titer Determination Applications
- Tosoh TOYOPEARL AF-rProtein A HC-650F Improve Performance of the Protein A Capturing Step Applications
- Tosoh TOYOPEARL AR-rProtein A Increased productivity for downstream processin of biotherapeutics Applications
- Tosoh TOYOPEARL AR-rProtein A HC-650F Protein A chromatography - The process economics driver in mab manufacturing Applications
TOYOPEARL AF-rProtein A HC-650F is a high-performance material for bioprocess chromatography with an exceptionally high dynamic binding capacity. These materials are also available as pre-packed ToyoScreen and Minichrom columns and as RoboColumns.
| Base material | Polymethacrylate |
| Particle Size | 45 µm |
| Pore Size | 100 nm |
| Ligand | Recombinant Protein A, Hexamer of c Domain |
| pH stability | 2.0 - 13.0 |
| DBC** | >50 g/L at 2 min Residence time >65 g/L at 5 min Residence time |
| Part-Number | 10 mL → 0023425 25 mL → 0023426 100 mL → 0023427 |
Characteristics:
- Very high dynamic binding capacity for human IgG
- Binding capacity increases with antibody titre up to 10 g/L
- Fast binding kinetics and moderate elution conditions
- Optimised alkali stability
- Minimal leaching
Applications:
- Tosoh TSKgel Protein A-5PW Reproducible and Robust Antibody Titer Analysis Applications
- Tosoh TOYOPEARL AF-rProtein A HC-650F Improve Performance of the Protein A Capturing Step Applications
- TOSOH Comparison of Toyopearl AF-rProtein a HC-650F binding capacity at various bed heights and constant linear velocity
2. Shodex - AFpak APA-894
| Base material | Polyhydroxymethacrylate |
| Particle Size | 18 µm |
| Ligand | Protein A |
| pH stability | 2.0 - 10.0 |
| Ligand Density | 4 mg/g gel |
| Binding Capacity | 13 mg IgG Human/g resin |
| Dimension | 8.0 x 50 mm |
| Part-Number | F7118946 |
Features:
- Stable polymer-based packing material for fast analyses
- Minimal leaching of functional groups for reproducible results
3. Thermo Fisher Scientific - MAbPac Protein A
| Base Material | Polydivinyl-benzene Core with hydrophilic surface |
| Particle Size | 12 µm |
| Ligand | Recombinant Protein A |
| pH-Stability | 2.5 - 7.5 |
| Binding Capacity | ~3.5 mg IgG/g resin |
| DBC** | 100 µg IgG/column at 2 mL/min flow rate |
| Dimension | 4.0 x 35 mm (PEEK) |
| Part-Number | 082539 |
Features:
- Efficient column with low back pressure
- Linear over a wide concentration range (0.025 to 5 mg/mL) without carryover
- Excellent sample recovery
Applications:
4. Sepax - MAbPurix
Sepax MAbPurix is available both in packed columns and as bulk material.
| Base Material | 4% cross linked Agarose |
| Particle Size | 45 - 165 µm |
| Ligand | Recombinant Protein A |
| pH stability | 3.0 - 10.0 (Working range) 2.0 - 11.0 (For CIP Cycles) |
| DBC** | 33 mg human IgG/mL Resin |
| Protein A Leaching | ≤ 10 ng/mg IgG |
| Caustic Stability in 0.1 N NaOH | 100 cycles |
| Dimension | 6.6 x 150 mm (Glass) → P/N 281085-6615 15 x 150 mm (Glass) → P/N 281085-15015 7.0 x 25 mm (Plastic) → P/N 281085-70025 16 x 25 mm (Plastic) → P/N 281085-160025 |
Characteristics:
- Very low protein A leaching
- High stability in alkaline medium
- High sample recovery and reproducibility
Applications:
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