Plasma contains over 120 different proteins that help with everything from blood clotting to immunity, making it the largest and most diverse set of proteins in the human body [1]. Several of these, when purified and concentrated, become life-saving therapeutics. These plasma-derived medicinal products (PDMPs) are critical to the treatment of many diseases, including bleeding disorders and immune deficiencies [2].

While other protein therapeutics, like insulin, are now produced through recombinant protein expression, PDMPs still need to be processed from donated plasma [3]. The separation, purification, and concentration of PDMPs is called plasma fractionation. 

Modern plasma fractionation technology still relies on the original, ethanol-based protocol, which was developed in the 1940s [4]. However, the industry is slowly shifting to chromatography-based methods, which improve purity and enable the production of new therapeutics [5]. 

Two such methods are ion-exchange chromatography (IEC) and size-exclusion chromatography (SEC). In this article, we will explore these developments in plasma fractionation technology.

What is Plasma Fractionation?

Any process in which a mixture of liquids, gases, or solids is separated into its components is called “fractionation” [6]. So, plasma fractionation is simply the separation of the various components of blood plasma, many of which are highly therapeutic. 

Plasma fractionation was first developed in anticipation of World War II. People knew that most combat-related fatalities occurred not from direct physical damage, but from blood loss. In the spring of 1940, as another global conflict loomed, it became clear that a way to quickly treat blood loss would be necessary. 

Albumin was identified as a promising blood substitute, being easier to ship and less prone to spoilage than whole blood, but scientists still needed to figure out how to efficiently isolate it from plasma. 

Edwin Cohn, a protein biochemist from Harvard Medical School, took on this problem at the request of the U.S. government. He eventually developed a novel technique that separates plasma into five major fractions, with the fifth fraction containing the albumin [7].

What would later be known as the Cohn process used successive steps at defined ethanol concentrations, which were associated with shifts in pH and temperature, to selectively precipitate proteins. The precipitates were then separated by centrifugation or filtration [4]. 

Thanks to its scalability [7], the Cohn process enabled the production of enough albumin to keep up with the demands of World War II. Eventually, the process revealed other plasma proteins with therapeutic properties: fibrinogen, thrombin, and immunoglobulin G (IgG) [7]:

• Fibrinogen: prevents and treats acute bleeding in trauma patients, or patients with congenital fibrinogen deficiency [8].
• Thrombin: a blood coagulant used to control and minimize blood loss during surgical procedures [9].
• Immunoglobulin G (IgG): the main class of antibody. Used to treat primary immunodeficiency [10] and is currently the most widely used blood product [11]. 

Chromatography in Plasma Fractionation

Because the Cohn process is both inexpensive and scalable [12], it is still the backbone of most modern plasma fractionation technologies [4]. It does, however, have a few disadvantages:

First, the Cohn process still yields a relatively impure product: for example, it can only extract albumin to a purity of 95% [13]. Impurities can negatively impact the stability and efficacy of protein therapeutics [14], and cause side effects in patients [5].

Another, and perhaps more important, drawback is that there are several plasma derivatives that the Cohn method can’t isolate at all. Because of its low specificity and selectivity, the classic method can’t isolate components found only in low concentrations [15]. The Cohn process can’t produce more fragile proteins (like coagulation factors, protease inhibitors, or anticoagulants) either, because of the harsh conditions (low pH, and the denaturing effects of ethanol) during fractionation [5]. 

To address these issues, Chromatographic techniques were introduced in the early 1980s. Compared to the Cohn process, these methods boast an improved purity profile and greater recovery [3]. Chromatography also expands the repertoire of what can be purified from plasma: while ethanol fractionation is mostly used to isolate albumin, newer plasma products like coagulation factors, anticoagulants, and protease inhibitors are largely extracted using chromatographic techniques [3].

This is possible because chromatography is so precise: most methods can capture even trace proteins [15]. And because techniques like ion-exchange chromatography (IEC) capture proteins at native pH and ionic strength, even fragile proteins can be preserved and isolated [4]. 

Ion-exchange Chromatography for Plasma Fractionation

Ion-exchange chromatography (IEC) is a method that separates proteins based on net charge. An ion-exchange resin functions as the stationary phase and is composed of charged functional groups covalently bound to an insoluble matrix.  There are two types of ion-exchange resins:

• In cation-exchange resins, the functional groups bound to the matrix are negatively charged, so the resin exchanges positively charged proteins.
• In anion-exchange resins, the functional groups bound to the matrix are positively charged, so the resin exchanges negatively charged proteins.

The resin-bound charged groups can be classified by charge and strength. Groups like Diethylaminoethyl (DEAE), Diethylaminopropyl (ANX), and Quaternary amine (Q) are positively charged anion exchangers; while the DEAE and ANX are weak anion exchangers, Q is a strong anion exchanger. On the other hand, groups like Carboxymethyl (CM), Methyl sulfonate (S), and Sulfonyl (SP) are negatively charged cation exchangers. SP is a strong cation exchanger, while CM and S are weak cation exchangers. 

The charge of the protein depends on the pH of the environment: if it is above the protein’s pI, the protein will be negatively charged; on the other hand, if the environment’s pH is below the pI, the protein will be positively charged.

IEC is a flexible, low-cost technique that has found places both upstream and downstream of a purification scheme: it can be used both to extract proteins from crude plasma and to eliminate unwanted proteins in a polishing step [5]. This has made it the most widely used chromatographic technique in plasma fractionation [15].

Because many plasma proteins are negatively charged at biological pH, anion exchangers are commonly used to purify plasma proteins. You Do Bio’s partners at emp BIOTECH offer a full range of ion exchange products, including DEAE and Q anion-exchange resins. DEAE is available bound to two different solid phases: Zetarose and Zetadex.

Based on porous and stable cross-linked beaded agaroses, Zetarose offers a robust platform for both small and large-scale applications and is utilized for a wide variety of separation techniques. Zetadex, on the other hand, is a beaded composite material comprised of ultrapure cross-linked dextran. It exhibits high selectivity, superb resolution, low non-specific adsorption, and robust chemical stability.

Size-exclusion Chromatography for Plasma Fractionation

Size-exclusion chromatography (SEC), also known as gel filtration, is a method that separates different molecules according to their size. In SEC, molecules enter a porous matrix (or “gel”) that lacks reactivity or adsorptive properties. Within the matrix, separation occurs when smaller molecules diffuse into the beads through the pores and are slowed down, while larger molecules do not enter the pores and elute first. 

Because the SEC matrix is inert, this method is the mildest of all the chromatography techniques. Additionally, because the sample does not bind to the resin (unlike in IEC), buffer composition does not impact resolution.  

In plasma fractionation, SEC is often used as a terminal polishing step to remove protein contaminants from a pre-purified protein mixture. emp BIOTECH offers SEC resins in a range of molecular weight cut-offs and materials. Not only are SEC resins available in Zetarose and Zetadex, but they are also available in DeXtra, an agarose-dextran composite.   

Conclusion

Plasma fractionation is critical to the treatment of many life-threatening diseases. While it has historically been a conservative field, in that it has relied on the same core methodology for over 70 years, it is perhaps high time implement the more effective chromatographic methods in pursuit of better purity and a diversified portfolio of therapeutic plasma products.

References:

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[2] Olaniyan, M. F., & Muhibi, M. A. (2023). Plasma-derived medicinal products in Nigeria. SN Comprehensive Clinical Medicine, 5(1). https://doi.org/10.1007/s42399-023-01527-8 

[3] Burnouf, T. (2018). An overview of plasma fractionation. Annals of Blood, 3, 33–33. https://doi.org/10.21037/aob.2018.05.03 

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[8] Kaur, J. (2023, May 8). Fibrinogen. StatPearls [Internet]. https://www.ncbi.nlm.nih.gov/books/NBK537184/ 

[9] Lopez, M. J. (2023, May 22). Thrombin. StatPearls [Internet]. https://www.ncbi.nlm.nih.gov/books/NBK557836/ 

[10] Azar, A. (2021, October 29). IGG deficiencies. Johns Hopkins Medicine. https://www.hopkinsmedicine.org/health/conditions-and-diseases/igg-deficiencies

[11]  Novaretti, M. C., & Dinardo, C. L. (2011). Immunoglobulin. Revista Brasileira de Hematologia e Hemoterapia, 33(5), 377–382. https://doi.org/10.5581/1516-8484.20110102 

[12] Tanaka, K., Shigueoka, E. M., Sawatani, E., Dias, G. A., Arashiro, F., Campos, T. C. X. B., & Nakao, H. C. (1998). Purification of human albumin by the combination of the method of cohn with liquid chromatography. Brazilian Journal of Medical and Biological Research, 31(11), 1383–1388. https://doi.org/10.1590/s0100-879×1998001100003 

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[14] Siew, A. (n.d.). Impurity testing of biologic drug products. BioPharm International. https://www.biopharminternational.com/view/impurity-testing-0 

[15] Johnston, A., & Adcock, W. (2000). The use of chromatography to manufacture purer and safer plasma products. Biotechnology and Genetic Engineering Reviews, 17(1), 37–70. https://doi.org/10.1080/02648725.2000.10647987