Affinity Chromatography

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Affinity Chromatography

• Audrey Cheung
• Michael Kwong
• Marc-Andre Lynch

Image source Bio-Rad Laboratories
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Function in process

• Removal of contaminant proteins
• Utilizes affinity of protein-A for the Mab to
  purify the product.
• High efficiency 90
• Protein-A effective for monoclonal antibody
  purification.

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How it works

1. Equilibrate add mixture to batch to bind target
   molecule (Mab)
2. Wash out mixture rid of impurities
3. Add wash buffer
4. Remove wash buffer
5. Add elution buffer
6. Remove elution buffer containing target molecule
7. Regeneration of original conditions

Image sourceWikipedia
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Alternatives

• Protein G can have higher affinity for certain
  molecules however it is generally more expensive.
• Protein L - used for purifying monoclonal
  antibodies from impurities. Can be ordered as
  ready-to-use prepacked devices for isolation and
  purification of immunoglobulin.
• Melon Gel Support - binds non-antibody serum
  proteins (ex. albumin and transferrin) while
  allowing the antibody to flow through in a mild
  buffer. Offers a gentle way to purify antibodies
  from serum.

Source www.pierce.net
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Current design

• Parameters of interest for optimization Volume
  load, mass load, flow rate, resolution, recovery,
  bed configuration, purity and cost.

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Parameter investigation

• Difficult to vary the yield likely due to an
  already high affinity for Mab.
• Inverse relationship between batch time and
  linear velocity.
• Effect of linear velocity on Mab/acetic acid
  ratio found to be negligible.
• Increase in column height resulted in slight
  increase in batch time.
• Increase in height decreased Mab/acetic acid
  ratio consistent with theory that columns tend
  to be shorter and wider in scale-up process.