Two-Column MAB Purification Protocol

Two-Column – Protein A and Desalting MAB Purification Protocol

Sample Preparation

  1. Remove cells and debris using centrifugation or depth filtration
  2. Filter sample through a 0.45 µm filter
  3. Adjust pH to 6–8 and conductivity to match binding buffer (equivalent to 150 mM NaCl)

Buffer Preparation

  1. Dilute 10× Binding Buffer (PBS) and 2× Elution Buffer (Glycine pH 3.0) with distilled water
  2. Filter all diluted buffers through a 0.45 µm filter
Note: Neutralization Buffer (1 M Tris, pH 8.0) does not require dilution

Column Preparation

  1. Remove the stopper and snap-off from the Protein A column
  2. Attach threaded fittings (for chromatography system) or Luer adaptors (for syringe use)
  3. Prime all connections with water before attachment to prevent air introduction
  4. Flush columns with 5 column volumes (CV) of distilled water to remove the 20% ethanol storage solution
Refer to Table 1 for the recommended flow rates and volumes.

Purification Methods

Method 1: Continuous Process

Best for: High throughput applications and syringe-based flow systems
  1. Equilibrate both Protein A and Desalting columns with 5 CV of Binding Buffer
  2. Load sample onto the Protein A column
  3. Wash the Protein A column with 5 CV of Binding Buffer
  4. Connect the Desalting column to the outlet of the Protein A column
  5. Elute the Protein A column and load the Desalting column simultaneously (see volume and flow rate recommendations)
  6. Disconnect the Protein A column and connect the flow directly to the Desalting column inlet
  7. Elute the Desalting column
Refer to Table 1 for the recommended flow rates and volumes.

Method 2: Non-Continuous Process

Best for: Maximizing yield and initial optimization studies
  1. Equilibrate both Protein A and Desalting columns with 5 CV of Binding Buffer (see flow rate recommendations)
  2. Load sample onto the Protein A column
  3. Wash with 5 CV of Binding Buffer
  4. Prepare fraction collection tubes by adding 0.1 CV of Neutralizing Buffer to each tube
  5. Elute the Protein A column with Elution Buffer, collecting 1.0 CV fractions
  6. Analyze fractions or use the A280 chromatogram to identify peak location
  7. Pool appropriate fractions for loading onto the Desalting column
  8. Load pooled fractions onto the Desalting column
  9. Elute the Desalting column
Refer to Table 1 for the recommended flow rates and volumes.
Step Column 25 mg Scale 50 mg Scale 250 mg Scale
Flow Rate (mL/min) Volume (mL) Flow Rate (mL/min) Volume (mL) Flow Rate (mL/min) Volume (mL)
Equil/Wash Prot A 0.5 2.5 1.0 5.0 5.0 25
Desalt 2.5 50 5.0 100 25 500
Load Prot A 0.25 NA 0.5 NA 2.5 NA
Desalt 0.5 2.0 1.0 4.0 5.0 20
Elute Prot A 0.25 2.0 0.50 4.0 2.5 20
Desalt 0.5 2.5 1.0 5.0 5.0 25
Table 1 – Recommended Flow Rates and Volumes for 2-Step Purification Process
orld!