Protein Purification

Fractionation (partitioning) is the process of isolating, purifying and concentrating plasma proteins from human plasma. It involves changing the conditions (e.g., the temperature, alcohol concentration, the ionic strength or the pH) of the product intermediates so that proteins become insoluble and precipitate.

The CSL Behring plasma-derived proteins are produced using a modified Cohn or Kistler – Nitschmann fractionation. First the frozen plasma is thawed and pooled. The proteins, which are insoluble under cold conditions, namely, FVIII, vWF and FI (fibrinogen), are separated by centrifugation and remain in the cryoprecipitate.

Technician samples plasma fractionation solution
In the next step, the vitamin K dependant coagulation factors (FII, FVII, FIX and FX) are adsorbed on a chromatography resin from the supernatant (overlying liquid substance). With the addition of alcohol, factor XIII proteins precipitate and are removed. Immunoglobulins, Alpha-1 proteinase inhibitor and albumin are sequentially precipitated by changing the pH and increasing the alcohol concentration.

Fractionation includes the following processes:

  • Precipitation and adsorption
  • Depth filtration
  • Centrifugation
  • Chromatography

The first process, precipitation, is the formation of a solid in a solution during a chemical (or physical) reaction. The solid is filtered out. The selective precipitation of certain proteins is an effective way to separate proteins from each other. Adsorption occurs when a gas or liquid solute accumulates on the surface of a solid forming a molecular film (the adsorbate). Adsorptives are selectively transferred from the fluid phase to the surface of insoluble, rigid particles suspended in a vessel or packed in a column.

During the second process, depth filtration, the precipitate is held back from the supernatant. The third process, centrifugation, separates mixtures using centripetal force, while the fourth process, chromatography entails the separation of protein mixtures by passing a mixture of proteins dissolved in a mobile phase through a stationary phase, which separates and isolates the desired protein from other proteins in the mixture. This is typically done by ionic binding of the protein to a porous resin bead, and is recognized as a process which is gentle on the protein.

These purification processes have the potential to reduce the viral load. For increased safety, however, CSL Behring implements additional virus inactivation/elimination steps, including pasteurization and virus filtration.

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