Introduction Sino Biological Protein A Agarose Beads / Resin was produced by immobilizing recombinant protein A to 4% cross-linked agarose matrix. The recombinant protein A contains IgG binding domains, eliminating nonspecific binding sites and reducing steric hindrance.
Specifications Ligand:Recombinant protein A
Dynamic binding capacity:~30 mg human IgG/ml drained medium
Matrix:4% highly cross-linked agarose
Bead size:45–165 μm
Mean bead size:90 μm
Maximum linear flow rate:>150 cm/h
pH stability:
Long term:3–9
Short term: 2–10
Working: 2–9
Sanitization:Sanitize the packed column with 2% Hibitane/20% ethanol or with 70% ethanol
Storage:+4–8 °C in 20% ethanol
Experiment:
Column: 5x 45 mm, bed volume 0.9 ml
Resin:Protein A agarose resin
Binding Buffer:50 mM Tris, 100 mM NaCl, pH 8.0
Elution Buffer:100 mM glycin, 10 mM NaCl, pH 3.0
Sample:Human IgG, 2.2 mg/ml
Flow Rate:0.3 ml/min
Procedure:The column was equilibrated with biding buffer, then apply the sample at fow rate of 0.3 ml/min, when the flowthrough concentration reach 0.22 mg/min stop loading and wash the columm with biding buffer, when asorbency of 280 nm decrease to base line, elute with elution buffer.
Result:26.7 mg human IgG was recovered in eluant, Dynamic binding capacity of the colun is about 29.7 mg/ml 
Chromatography graph of measuring dynamic binding capacity
Recovery of monoclonal human antibody in recombinant cell culture
Column:5x 50 mm, bed volume 1 ml
Resin:Protein A agarose resin
Binding Buffer:50 mM Tris, 100 mM NaCl, pH 8.0
Elution Buffer:100 mM glycin, 10 mM NaCl, pH 3.0
Sample:CHO cell culture expressing monoclonal human antibody
Flow Rate:0.3 ml/min
Procedure:The column was equilibrated with biding buffer, then load cell culture that contain known amount of antibody, after washing, elute the antibody and calculate the recovery.
Result:when load 30 mg antibody/ml medium, the recover of antibody is 99%
| Antibody loaded mg | recovery % | | 30 | 99 | | 35 | 96.6 | | 40 | 87.5 |
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Repeated Cycle Usage
Repeated usage in practical manufacturing:
The feedstock used was the supernatant of 293 cell line expressing various humanized monocolnal antibody, human monoclonal antibody or protein-Fc chimera. The volume of culture varies from 1000ml to 2000ml.
Column:bed volumn of 20 ml with dimention of 26x 100 mm
Flow rate:7 ml/min
Binding buffer:50 mM Tris, 100 mM NaCl, pH 8.0
Elution buffer:100 mM glycerin, 10 mM NaCl, pH 3.0
CIP buffer:50 mM NaOH, 1 M NaCl
Purification cycle:
• 5 cv binding buffer wash the column
• 5 cv elution buffer
• 2 cv binding buffer
• 3 cv CIP buffer
• 3 cv binding buffer
Measure column capacity:
Comlumn capacity was tested every 15 practical purification cycle by applying 600 mg of a humanized monoclonal antibody with concentration of 1 mg/ml, and measure the recovery of the antibody. The procedure is the same with purification cycle. All capacity test experiment use the same antibody.
Recovery at the loading of 30 mg/ml medium after repeated usage cycles 
SDS-PAGE of monoclonal antibody purified from 105th cycle purification usage 
HPLC-SEC of monoclonal antibody purified from 105th cycle purification usage
CIP Protocol
The most common CIP procedure for Protein A resin is washing the resin with 2 column volumes of 6 M guanidine hydrochloride to remove precipitated or denatured substances, followed by re-equilibrating with at least 5 column volumes of binding buffer.
For strongly bound hydrophobic proteins, lipoproteins and lipids, wash the resin with a non-ionic detergent, for instance, 0.1%. Triton X-100, at 37 °C. Immediately re-equilibrate with at least 5 column volumes of binding buffer.
If the above methods are still not effective to remove the impurities, wash the medium with 5 column volumes of 50 mM NaOH, 1 M NaCl solution, Re-equilibrate with at least 5 column volumes of binding buffer.
Protein A residual
The resin has very low ligand leakage.
Protein A residual in 10 different monoclonal antibodies
Protein A residual in 10 monoclonal antibodies randomly taken from manufactural purification. The feedstock used was the supernatant of 293 cell line expressing various humanized monocolnal antibody, human monoclonal antibody or protein-Fc chimera. The volume of culture loaded varies from 1000 ml to 2000 ml.
Stability Study The IgG binding capacity and recovery was maintained after seven days storage at room temperature in solutions list below:
3 M NaSCN
6 M guanidine-HCl
8 M urea
0.1 M glycine, pH 3.0
70% ethanol.
Recovery of antibody at the loading burden of 30 mg/ml medium after storage in varous solutions.
Column Regeneration
Regeneration step should be operated after elution to remove the residue proteins and impurities on the resin for repeated use. Wash the resin with 2–3 bed volumes of regeneration buffer followed by re-equilibration with 2–3 bed volumes of binding buffer. Sometimes the regeneration buffer is the same with the elution buffer, such as 0.1 M glycine buffer pH 3.0 or 0.1 M citric acid pH 3.0. When the elution buffer has a milder condition to avoid aggregation or degradation, the regeneration buffer is different, with a lower pH or containing different salts. But this procedure does not guarantee removing all kinds of impurities like denatured proteins or lipids. In this case, cleaning in place procedure is indispensable.
Storage For storage, keep the medium at 2°C to 8°C in a suitable bacteriostat, e.g. 20% ethanol and/or 0.02% sodium azide. Notice: The resin must not be frozen.
IgG binding of protein A, G, and L
Species | | Protein A | Protein G | Protein L | Human | IgG | +++ | +++ | +++ | | IgG1 | ++++ | ++++ | ++++ | | IgG2 | ++++ | ++++ | ++++ | | IgG3 | - | +++ | +++ | | IgG4 | ++++ | ++++ | ++++ | | IgA | + | - | +++ | | IgA1 | + | - | +++ | | IgA2 | + | - | +++ | | IgD | - | - | +++ | | IgE | ++ | - | +++ | | IgM | + | - | +++ | Rabbit | IgG | +++ | +++ | + | Cow | IgG | + | +++ | - | | IgG1 | + | +++ | - | | IgG2 | +++ | +++ | - | Cat | IgG | +++ | + | ? | Horse | IgG | ++ | ++++ | ? | Goat | IgG | + | ++ | - | | IgG1 | + | +++ | - | | IgG2 | +++ | +++ | - | Guinea-pig | IgG1 | ++ | + | ? | | IgG2 | ++ | + | ? | Sheep | IgG | + | ++ | - | | IgG1 | + | ++ | - | | IgG2 | +++ | +++ | - | Dog | | ++ | + | ? | Pig | | +++ | ++ | +++ | Rat | IgG | + | ++ | +++ | | IgG1 | - | + | +++ | | IgG2a | - | ++++ | +++ | | IgG2b | - | ++ | +++ | | IgG2c | ++ | ++ | +++ | | IgG3 | + | ++ | ? | Mouse | IgG | ++ | ++ | +++ | | IgG1 | + | ++++ | +++ | | IgG2a | ++++ | ++++ | +++ | | IgG2b | +++ | +++ | +++ | | IgG3 | ++ | +++ | +++ | | IgM | - | - | +++ | Chicken | IgY | - | - | - | Monkey(rhesus) | IgG | ++++ | ++++ | ? | Hamster | | + | ++ | +++ | Koala | | - | + | ? | Llama | | - | + | ? |
Strong binding ＋＋, medium interaction ＋, weak or no interaction －. |
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