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Chromatography in blood processing



Chromatographic techniques have been used in blood processing and purification since the 1980's. It has emerged as an effective method of purifying blood components for therapeutic use.

Additional recommended knowledge

Contents

Human blood plasma

The goal of plasma purification and processing is to extract specific materials that are present in blood, and use them for restoration and repair. There are several components that make up blood plasma, one of which is the protein albumin. Albumin is a highly water-soluble protein with considerable structural stability. It serves as a transportation device for materials such as hormones, enzymes, fatty acids, metal ions, and medicinal products. It is also used for therapeutic purposes, being essential in restoration and maintenance of circulating blood volume in imperative situations such as severe trauma or surgery. With little room for error, extremely pure samples that are lacking impurities needs to be at hand in good amount.

Development of chromatography

Traditionally, the Cohn process incorporating cold ethanol fractionation has been used for albumin purification. However, chromatographic methods for separation started being adopted in the early 1980’s. Developments were ongoing in the time period between when Cohn fractionation started being used, in 1946, and when chromatography started being used, in 1983. In 1962, the Kistler & Nistchmann process was created which was a spinoff of the Cohn process. Chromatographic processes began to take shape in 1983. In the 1990’s, the Zenalb and the CSL Albumex processes were created which incorporated chromatography with a few variations.

The general procedure followed when using chromatography was:

  • The plasma would be buffer exchanged through column filtration and/or diafiltration
  • One or more column chromatography steps
  • A round of gel filtration chromatography

This is a more efficient alternative than the Cohn process for four main reasons:

  • Smooth automation and a relatively inexpensive plant was needed
  • Easier to sterilize equipment and maintain a good manufacturing environment
  • Chromatographic processes are less damaging to the albumin protein.
  • A more successful albumin end result can be achieved

Compared with the Cohn process, the albumin purity went up from about 95% to 98% using chromatography, and the yield increased from about 65% to 85%. Small percentage increases make a difference in regard to sensitive measurements like purity. There is one big drawback in using chromatography, which has to do with the economics of the process. Although the method was efficient from the processing aspect, acquiring the necessary equipment is a big task. Large machinery is necessary, and for a long time the lack of equipment availability was not conducive to its widespread use. The components are more readily available now but it is still a work in progress.

Bridging Methods

Integrating traditional and modern methods is a useful way to process albumin.

There are three main steps that combine Cohn fractionation with chromatography.

  • Factors I, II, and III are removed via cold ethanol fractionation.
  • DEAE–Sepharose fast flow ion exchange and CM–Sepharose fast flow chromatography procedures are run
  • Gel filtration on Sephacryl S-200 HR is run

The result is albumin with 9% lower aluminum levels with a processing time that is almost twice as fast.

Although it was hard to make chromatographic processing methods widely adopted, global expansion is a work in progress. Various blood components must be readily available at various medical treatment centers around the world. The Institute of Transfusion Medicine in Skopje, Macedonia is a plasma fractionation center in the Balkans. Their modernized albumin purification process consists of five steps:

  • Starting material is plasma that has been pretreated by centrifugation
  • A round of gel filtration is run on Sephadex G-25 Coarse
  • Ion exchange on DEAE Sepharose CL-6B is run to bind the albumin to the column
  • Albumin is eluted with a sodium acetate buffer
  • Final polishing with gel filtration on Sephacryl S-200 HR

The end result is a highly pure and safe batch of albumin that is 100% non-pyrogenic, sterile, and free of active HIV virus. The product purity is greater than 98% and the protein content is about 50.33 g/L. The next goal is to further modernize the facility by scaling down production costs and increasing capacity.

Non-chromatographic processing methods

Other plasma processing methods exist, however they are not as versatile for the purpose of plasma processing.

  • Two-stage liquid extraction is done using polyethylene glycol (PEG)-phosphate aqueous two-phase systems, with a PEG-rich top layer and a phosphate-rich bottom layer. Although this method is somewhat useful for protein recovery, it does not work as well for the recovery of other blood components.
  • Membrane fractionation has the advantage of very minimal unwanted protein loss yet high removal of pathological plasma components. This method incorporates processes such as thermofiltration and applying pulsate flow. The latest two-stage membrane system utilizes a high flow recirculation circuit that is effective for removal of LDL cholesterol. This procedure is useful when dealing with patients that have clogged arteries and other cardiovascular problems involving cholesterol.
  • Batch adsorption is only useful when dealing with smaller samples of plasma, typically 200 mL or less. When large amounts of plasma are processed, there are larger elution volumes which require larger membranes for concentration, which leads to most of the target protein being lost on the membrane.

References

  • New Century Pharmaceuticals, Inc.
  • British Journal of Anaesthesia
  • Scientific Electronic Library Online
  • Journal of Biological Chemistry
  • PubMed by National Library of Medicine and NIH
  • General Electric: Healthcare
  • Production of Human Albumin by Plasma Fractionation
 
This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Chromatography_in_blood_processing". A list of authors is available in Wikipedia.
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