Chromatography in blood processing
Encyclopedia
Chromatographic techniques
have been used in blood processing and purification since the 1980s. It has emerged as an effective method of purifying blood components for therapeutic use.
is the liquid component of blood, which contains dissolved proteins, nutrients, ions, and other soluble components. In whole blood, red blood cells, leukocytes, and platelets are suspended within the 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.
incorporating cold ethanol fractionation
has been used for albumin purification. However, chromatographic methods
for separation started being adopted in the early 1980s. 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 1990s, the Zenalb and the CSL Albumex processes were created which incorporated chromatography with a few variations.
The general approach to using chromatography for plasma fractionation for albumin is:
The recovered purified material is formulated with combinations of sodium octanoate and sodium N-Acetyl tryptophanate and then subjected to viral inactivation procedures, including pasteurisation at 60°C.
This is a more efficient alternative than the Cohn process for four main reasons:
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.
There are three main steps that combine Cohn fractionation with chromatography.
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:
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 g/L.
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
have been used in blood processing and purification since the 1980s. It has emerged as an effective method of purifying blood components for therapeutic use.
Human blood plasma
Blood plasmaBlood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
is the liquid component of blood, which contains dissolved proteins, nutrients, ions, and other soluble components. In whole blood, red blood cells, leukocytes, and platelets are suspended within the plasma. The goal of plasma purification and processing
Blood plasma fractionation
Blood plasma fractionation refers to the general processes of separating the various components of blood plasma, which in turn is a component of blood obtained through blood fractionation.-Blood plasma:...
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
Human serum albumin
Human serum albumin is the most abundant protein in human blood plasma. It is produced in the liver. Albumin constitutes about half of the blood serum protein...
. 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 processCohn process
The Cohn process is a series of purification steps with the purpose of extracting albumin from blood plasma. The process is based on the differential solubility of albumin and other plasma proteins based on pH, ethanol concentration, temperature, ionic strength, and protein concentration. Albumin...
incorporating cold ethanol fractionation
Fractionation
See also: Fractionated spacecraftFractionation is a separation process in which a certain quantity of a mixture is divided up in a number of smaller quantities in which the composition changes according to a gradient. Fractions are collected based on differences in a specific property of the...
has been used for albumin purification. However, chromatographic methods
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
for separation started being adopted in the early 1980s. 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 1990s, the Zenalb and the CSL Albumex processes were created which incorporated chromatography with a few variations.
The general approach to using chromatography for plasma fractionation for albumin is:
- Recovery of Supernatant I
- Delipidation
- Anion exchange chromatography
- Cation exchange chromatography
- Gel filtration chromatography.
The recovered purified material is formulated with combinations of sodium octanoate and sodium N-Acetyl tryptophanate and then subjected to viral inactivation procedures, including pasteurisation at 60°C.
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.
- SepharoseSepharoseSepharose is a tradename for a crosslinked, beaded-form of a polysaccharide polymer material extracted from seaweed. It is crosslinked through lysine side chains. Iodoacetyl functional groups can be added to selectively bind cysteine side chains and this method is often used to immobilize peptides....
fast flow ion exchange and Sepharose fast flow chromatography procedures are run - Gel filtration 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
Skopje
Skopje is the capital and largest city of the Republic of Macedonia with about a third of the total population. It is the country's political, cultural, economic, and academic centre...
, Macedonia
Republic of Macedonia
Macedonia , officially the Republic of Macedonia , is a country located in the central Balkan peninsula in Southeast Europe. It is one of the successor states of the former Yugoslavia, from which it declared independence in 1991...
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 centrifugationCentrifugationCentrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture...
- A round of gel filtration is run
- Ion exchangeIon exchange chromatographyIon-exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the...
on DEAEDEAEDiethylaminoethyl cellulose is a positively charged resin used in ion exchange chromatography, a type of column chromatography, used in protein and nucleic acid purification/separation...
Sepharose is run to bind the albumin to the column - Albumin is eluted with a sodium acetate bufferBuffer solutionA buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a...
- Final polishing with gel filtration
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 g/L.
Non-chromatographic processing methods
Other plasma processing methods exist, but generally do not provide the resolution or purity of chromatographic methods:- Two-phase liquid extraction may be performed using polyethylene glycolPolyethylene glycolPolyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
(PEG)-phosphate Aqueous two-phase systemsAqueous two phase systemAqueous biphasic systems or aqueous two phase systems are clean alternatives for traditional organic-water solvent extraction 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 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. It may prove useful for patients that have clogged arteries and other cardiovascular problems involving cholesterol.
- Batch adsorption, e.g. onto ion exchange media, is only useful when dealing with smaller samples of plasma, typically 200 mL or less. Batch adsorption recovers the product in a larger volume of elution buffer than does column chromatography or frontal chromatography, and the resulting more dilute product requires concentration, typically on a membrane system, which can lead to loss of product by irreversible adsorption to the membrane.