Affinity resin is developed and established based on the principle of specific adsorption between biomolecules and other ligand molecules, aiming to purify target molecules through the specific adsorption between ligands on the resins and the target molecules. Protein A is a golden staphylococcal cell wall protein with a molecular weight of 42 kDa. It contains five structural domains that specifically bind to the Fc region of antibody IgG molecules.
By coupling modified Protein A ligands onto cross-linked agarose, arProtein A Focurose HR resin has been successfully developed. It is primarily used for the separation and purification of ascites fluid, cell culture supernatant, monoclonal antibodies, polyclonal antibodies, and Fc-tagged proteins derived from serum. With advantages such as high loading capacity, stable alkali resistance, excellent purification effect, and good reproducibility, arProtein A Focurose HR resin can significantly reduce the costs of customer affinity research and production by directly replacing other resins.
Features
Fast and simple (typically achieves purity of 95% or higher with a single purification step).
High loading capacity, fast flow rate, good alkali resistance, and easy to scale up.
Low ligand leaching rate and good reproducibility.
arProtein A Focurose HR Performance Parameters
Resin | Highly cross-linked 6% agarose |
Particle size range | 45-165µm |
Average particle size (D50) | 75±5µm |
Binding capacity | ~60mg (human IgG) /mL; Test parameters: 5-minute retention time, column height 10cm |
pH stability | 3-12 (long-term) 2-14 (short-term) |
Operating temperature | 4℃-40℃ |
Recommended retention time | 4-6 minutes, and the specific retention time can be adjusted according to project requirements |
Recommended flow rate | 200-300 cm/h (for a column height of 20cm) |
Operating pressure | ≤0.5MPa |
Storage solution | 20% ethanol or 2% benzyl alcohol |
Storage conditions | 2℃-8℃ |
Note:
1. The binding capacity of the resin may vary depending on the sample source and subtype variations.
2. Prolonged immersion of the resin in the elution solution can result in ligand hydrolysis, ultimately affecting the resin's loading capacity.
DBC Loading at Different Retention Times
When the retention time is ≥5 minutes, the loading capacity shows a slow increasing trend. To ensure experimental time and loading capacity, it is recommended to set the retention time as 5 minutes. At this retention time, the packing material loading is above 60 g/L.
Alkali Resistance Test
1. Static Alkali Resistance Test
Soaking in 0.1M NaOH for 1440 hours does not affect the DBC loading.
Soaking in 0.5M NaOH and 1M NaOH for 168 hours results in a decrease in DBC loading of less than 20%.
2. Dynamic Alkali Resistance Test
After 200 cycles in 0.1M NaOH, the DBC remains essentially unchanged. After 100 cycles in 0.5M NaOH, the DBC maintains 80% of its initial value.
Pressure and Flow Rate Test
Using a 1.6cm diameter chromatography column, under the conditions of a column height of 26cm and a flow rate of 500cm/h, the pressure remains around 0.25Mpa, significantly below the maximum pressure tolerance of the packing material at 0.50Mpa.
Under the same column height and linear flow rate, our research-grade column exhibits comparable backpressure performance to the production-grade column.
Purification Performance
1. Load Stability
In 160 purification experiments, the decrease in DBC loading was within 15%, indicating that the loading capacity of the packing material can be consistently maintained above 50g/L in 160 experiments, satisfying the majority of production requirements.
2. Purity Enhancement
In 200 validations, the SEC purity remained above 95%, indicating strong stability of the packing material in terms of purity enhancement.
3. Yield
In 200 validations, the yield did not show significant reduction throughout the entire lifespan, with a decrease of less than 10%.
4. HCP Residuals
In 200 validations, HCP remained below 700ppm, demonstrating excellent stability in HCP removal capability for the packing material.
5. Ligand Leaching Rate
In 200 monoclonal antibody lifespan validations, the residual Protein A ligand was consistently below 10ppm.
Frequently Asked Questions and Solutions
Issue | Possible Causes | Solutions |
Target protein does not bind or has low binding capacity during purification | 1. Overloading of sample | Reduce the sample load |
2. Sample flow rate is too fast | Reduce the sample flow rate and load the sample according to a retention time of 4-6 minutes | |
3. Protein or lipids aggregate in the resin, affecting binding | Efficiently clean the resin or replace with a new resin | |
4. Weak binding between target molecule and resin | Confirm the source and subtype of IgG and select the appropriate resin | |
5. Inappropriate choice of buffer during purification | Confirm the pH of the sample, and for weakly binding target molecules, increase the pH (8-9) and salt concentration (1-3M NaCl) to enhance the binding between the sample and resin | |
Not collecting the target protein during elution or collecting only a small amount of the target protein. | 1. Target protein does not bind to the resin or has low binding capacity | First, confirm if the target protein binds to the resin |
2. Unsuitable elution conditions | Optimize elution conditions to increase elution strength | |
3. Insufficient elution time | Lower the flow rate and extend the retention time of the elution buffer | |
4. Elution volume is too small | Increase elution volume, generally recommended 3CV | |
Target protein purity is low | 1. Unreasonable sample pre-processing | Based on the sample source, it is recommended to perform filtering or dilution before column loading |
2. The sample has high viscosity | Dilute the sample appropriately with a balancing solution to reduce sample viscosity and concentration | |
3. Incomplete removal of impurities | Optimize the washing conditions to remove impurities | |
4. Impurities such as proteins or lipids aggregate and precipitate in the resin | Clean the resin promptly and effectively | |
5. Degradation of the target substance | Determine the possibility of degradation of the target substance in process conditions or components. Pay attention to the storage conditions of the sample before and after purification | |
6. Poor column packing | Reload or use new packing material | |
7. Microbial growth in the resin | After using the resin, clean and disinfect it promptly, and store the resin properly | |
Decrease in resin loading | 1. Too fast sample flow rate | Reduce the sample flow rate |
2. Aggregation of proteins or lipids in the resin resulting in decreased loading |
Clean the resin promptly | |
3. Gradual detachment of ligands | Optimize process conditions and replace with new resin | |
Peak fronting | Resin is packed too tightly or the solid-to-liquid ratio is high | Adjust column parameters and repack the column |
Peak tailing | Resin is packed too loosely or the solid-to-liquid ratio is low | Adjust column parameters and repack the column |
Cracks or dryness in the column bed | Leakage or introduction of large air bubbles | Check for leaks or bubbles in the tubing and repack the column if necessary |
Slow liquid flow | 1. Aggregation of proteins or lipids | Clean the resin or membrane promptly |
2. Microbial growth in the separation column | All reagents used must be filtered and degassed. The sample must be centrifuged or filtered before applying it to the column |
Order information
Product | Specification | Item number |
arProtein A Focurose HR | 25mL | HQ320827025M |
arProtein A Focurose HR | 100mL | HQ320827100M |
arProtein A Focurose HR | 500mL | HQ320827500M |
arProtein A Focurose HR | 1L | HQ320827001L |
arProtein A Focurose HR | 5L | HQ320827005L |
arProtein A Focurose HR | 20L | HQ320827020L |
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