ProA Application Note:
Syringe Columns for Antibody Purification
Introduction
Small volume protein purification remains a bottleneck to life science research. New tools are needed to simplify the procedures, increase throughput, and drive down costs. Gravity columns offer users the most flexibility but are inconvenient to pack and maintain. These columns should not be allowed to run dry, so the user is at the mercy of watching the columns drip. Magnetic beads are a popular choice for purification of antibodies but are limited by capacity and the incubation time for a binding process that relies upon Brownian Motion. Furthermore, magnetic beads are a single use system, which drives up the cost. Spin columns are convenient and easy to use but sacrifice performance. And the single use design of spin columns is also an expensive choice. Here, syringe columns are presented as a suitable choice for performance and speed with the benefit of being reusable. Syringe columns are a suitable alternative to plate-based, gravity, magnetic bead and FPLC applications because of intuitive procedures. These syringe columns purify samples quickly without sacrificing performance. In addition, syringe columns are reusable making them very cost effective.
Materials and Methods
Lyophilized IgG from human serum (Sigma Aldrich, I4505) was brought into solution using TBS (Quality Biological, 35058-101) to a final concentration of about 1 mg/mL. 1 mL sample was processed through the 5 mL syringe column packed with 100 µL of Protein A resin. The syringe column was fitted with a luer lock needle and was equilibrated by aspirating and dispensing 1 mL of TBS. After equilibration, 200 µL of air was used to blow out the interstitial space. The column captured the IgG from the 1 mg/mL sample using 8-16 cycles of aspirate and dispense. After the final dispense, 200 µL of air was used to blow out the syringe column. The syringe column was washed with 1 mL TBS using 2 aspirate and dispense cycles. This was followed by 2 aspirate and dispense cycles in 140 mM NaCl (prepared by diluting a 5M solution with water, Sigma Aldrich, 5150). After the last blow out, 500 µL of air was aspirated and dispensed from the syringe column. The IgG was eluted in two sequential fractions of 400 µL elution buffer (Cytiva, 2890359). This was followed by an addition of 100 µL of Neutralization buffer (Cytiva, 2890359) to each elution fraction.
The flow through and eluted IgG were analyzed by the NanoDrop OneC (Thermo Fisher Scientific, ND-ONE-C).
Results
Highly efficient binding and recovery of IgG using Syringe Columns (figure 1) were formed.
The flow through of the sample was monitored using the NanoDrop after 8, 12 and 16 aspirate and dispense cycles. Most of the IgG was captured after 8 cycles (Table 1). Increasing to 16 cycles saturated the column with 74% captured IgG. Complete elution was achieved using 8 aspirate and dispense cycles (Table 1). While the columns were saturated at 74% binding, the complete mass of loaded IgG was recovered.
Discussion
Syringe columns offer an attractive choice for routine protein purification. The syringe-based format is intuitive and easy to use. These offer high performance and are cost effective. 740 µg of human IgG was loaded and recovered using the 5 mL syringe column packed with 100 µL of protein A resin. This calculates to a dynamic binding capacity of 7.4 mg/mL.
Conclusion
Syringe columns purify samples easily and quickly without sacrificing performance. Reusability of these syringe columns makes purification cost effective and thus are great alternative to other purification methods & technologies.
Following elution, the following steps are recommended for optimal preservation of the syringe column for a subsequent purification. Aspirate and dispense 1 mL of elution buffer such as 50 mM Phosphate pH 2.5. Repeat for 2 cycles. Next, equilibrate using 2 cycles of 1 mL TBS buffer. Aspirate 200 uL of a preservation solution such as 20% ethanol. Cap the syringe columns and store at 4 degrees C. Syringe columns can be stored indefinitely with this method.