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Friday, March 22, 2013

Antibody Purification Methods

Antibody Prurification Methods

Antibody purification is a multistep process by which contaminants of source is removed and antibody with high purity is obtained. Antibodies are widely used as injectables and parenteral products for human use. Monoclonal antibodies dominate the antibody field. Before going to the antibody purification process, let’s see the contaminants of the antibody source.

Basic Structure of an Antibody

antibody structure

Antibody is a multi-chain protein, secreted in order to fight with the antigens which enters the body and thereby preventing possible infections. Generally an antibody is a polypeptide of four chains, having two identical light chains and two identical heavy chains. Antibodies are glycoproteins.

Possible contaminants:
Antibodies are produced in mice, rabbits, etc. Nowadays antibodies are expressed in cell culture with good yield. When antibodies are taken from animal source possible contaminants could be serum proteins such as albumin, transferrins and cell degradation products like DNA and cellular proteins. Currently serum free media for cell culture are developed which can ease the antibody purification process. With a combination of chromatographic steps and precipitation methods one can purify the antibody with good yield and resolution.

Purification Methods
The choice of a purification method is based on a these factors:
  1. Nature of antibody,
  2. Nature of feedstock,
  3. Scale of production,
  4. Economics – cost and other factors,
  5. Process Timings, and
  6. Desired purity.
As mention earlier antibody purification is multi step process, which mainly includes:
  • Sample Preparation
  • Capture
  • Initial Purification
  • Secondary Purification
  • Polishing / Formulation
Let’s go in detail on each of these steps for better understanding,

purification and characterization of antibody
Antibody Purification: Step 1: Sample Preparation

Sample prep or sample preparation is the initial step in which crude protein sample is conditioned or making it ready for the initial capture step. Generally this step involves changing pH or Ionic strength, dilution of the crude sample or addition of salts for the ionic strength. These above mentioned techniques may increase the cost in manufacturing so they may not be feasible in the large scale production process. So what is suitable in large scale antibody purification process is to use buffer exchange by size exclusion chromatography or to use ultrafiltration or diafiltration. Dialysis is one common method followed in lab scale level but in production scale it is not feasible.

Crude antibody sample need to be concentrated which is done either by centrifugation or Filtration, sometimes both the methods are combined to get faster results.
Antibody precipitation can be done to precipitate out, salts used for this purpose include ammonium sulphate, Poly ethylene glycol, etc. if the antibody is expressed in cell line media contaminants (dye – phenol red) need to be removed which in-turn can bind to the column and reduce the efficiency of the purification process.

Antibody Purification: Step 2: Capture
Capturing is the first major purification step in a process typically involves binding the antibody to chromatographic matrix, while impurities either flow through or are differentially eluted from the column. The process need to be optimized for better results, good yield and purity.

These are the various chromatographic techniques which are widely used for antibody purification.
  • Immunoaffinity
  • Immobilized Metal Affinity Chromatograhy (IMAC)
  • Ion – Exchange Chromatograhy (IEC)
  • Hydrophobic Interaction Chromatography (HIC)
  • Hydroxyapatite
  • Size – Exclusion chromatography (SEC)
All other techniques, except Hydroxyapatite chromatographic technique, are explained in the previous posts, so let’s look into the Hydroxyapatite chromatography.

In hyroxyapatite chromatography, interaction of the antibody with calcium and phosphate has a major role to play. Mostly elution is done using a phosphate buffer gradient. As this technique has the ability to bind to DNA and separate out idiotypes. Hydroxyapatite chromatography is used mostly in lab scale because only low flow rates can be used in this technique and resin cannot be reused. Due to these drawback hydroxyapatite chromatodraphic application is limited to lab scale purification.

Antibody Purification: Step 3: Secondary Purification
The secondary purification step is selected based on the nature and the optimization requirement of the crude antibody source, the initial capture step can often give purities in the range of 80 to 95%.  However, for higher purity grades in excess of 99% secondary purification is required. In addition to protein contaminants, other impurities such as DNA, endotoxins, viruses, and aggregates need to be removed. In such cases, a multistep procedure is almost inevitable. All the same techniques used for initial capture can be used for secondary purification. Indeed, many of the techniques, such as HIC and Hydroxyapatite chromatography, are used more often used as polishing steps than as initial capture steps.

Antibody Purification: Step 4:  Polishing / Formulation
Final polishing / formulation step can be considered as a part of purification in which it removes conditions that would impair the stability or utility of the antibody in its intended use. As the downstream processing rule, more no of steps results in more loss of the product. Final formulation may be as simple as a straightforward sterilization by membrane filtration through a sterile filter with pores 0.2 lm or less. Another relatively simple formulation step is adjusting the antibody concentration, either by dilution with buffer or by concentration on ultra filtration. In other circumstances, the buffer composition may need to be changed to achieve optimal stability of the antibody. For this purpose, SEC or diafiltration is widely used. A more complex formulation step would be the addition of excipients to confer stability. Finally, the antibody solution may need to be lyophilized/ Freeze dried to confer stability, and the liquid formulation may be changed to be compatible with the lyophilization process.
All the above mentioned steps need to be optimized to have a better yield, resolution and better stability.