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Chromatography: Getting the Most out of Plasma Fractionation

Chromatography: Getting the Most out of Plasma Fractionation

Extracting High Value Components from Blood by Chromatography

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.

While modern plasma fractionation technology still relies on the original, ethanol-based protocol developed in the 1940s [4], chromatography-based methods are quickly growing in popularity. When added to a workflow, these methods improve purity and enable the production of new classes of 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.

1. 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.

Enter Edwin Cohn, a protein biochemist at the Harvard Medical School. With the support of the U.S. government, Cohn 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].

2. 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 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 that can be used to treat protein deficiencies 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 (like coagulation factors, anticoagulants, and protease inhibitors) can be preserved and isolated [4].

3. 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 comprises 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 and are anion exchangers. DEAE and ANX are weak anion exchangers, while Q is a strong anion exchanger. On the other hand, negatively charged groups like carboxymethyl (CM), methyl sulfonate (S), and sulfonyl (SP) are cation exchangers. Of these, 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 this 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. Both are available in 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.

emp BIOTECH offers a full suite of ion-exchange products for a variety of applications.

4. 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 can’t diffuse into 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 doesn’t chemically interact with the resin, buffer composition impacts resolution very little. SEC is useful in separating aggregates from dimers and monomers from larger molecular weight proteins, or complexes from smaller molecular weight proteins.

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.

5. 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 to implement the more effective chromatographic methods in pursuit of better purity and a diversified portfolio of therapeutic plasma products, with the end goal being better patient quality-of-life.


[1] Strengers, P. F. W. (2023). Challenges for plasma-derived medicinal products. Transfusion Medicine and Hemotherapy, 50(2), 116–122.

[2] Olaniyan, M. F., & Muhibi, M. A. (2023). Plasma-derived medicinal products in Nigeria. SN Comprehensive Clinical Medicine, 5(1).

[3] Burnouf, T. (2018). An overview of plasma fractionation. Annals of Blood, 3, 33–33.

[4] Burnouf, T. (2007). Modern Plasma Fractionation. Transfusion Medicine Reviews, 21(2), 101–117.

[5] Burnouf, T. (1995). Chromatography in plasma fractionation: Benefits and future trends. Journal of Chromatography B: Biomedical Sciences and Applications, 664(1), 3–15.

[6] Al-Haj Ibrahim, H. (2019). Introductory chapter: Fractionation. Fractionation.

[7] Featherstone, P. J., & Ball, C. M. (2023). The development of albumin solutions in the Second World War. Anaesthesia and Intensive Care, 51(4), 236–238.×231174704

[8] Kaur, J. (2023, May 8). Fibrinogen. StatPearls [Internet].

[9] Lopez, M. J. (2023, May 22). Thrombin. StatPearls [Internet].

[10] Azar, A. (2021, October 29). IGG deficiencies. Johns Hopkins Medicine.

[11]  Novaretti, M. C., & Dinardo, C. L. (2011). Immunoglobulin. Revista Brasileira de Hematologia e Hemoterapia, 33(5), 377–382.

[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.×1998001100003

[13] Harris, J. R. (1991). Blood separation and plasma fractionation. Wiley-Liss.

[14] Siew, A. (n.d.). Impurity testing of biologic drug products. BioPharm International.

[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.

Optimizing Use of Your Desalting Column

Optimizing Use of
Your Desalting Column

Desalting columns are an essential tool in the field of molecular biology, biochemistry, and protein research. They facilitate the removal of unwanted salts, small molecules, and other contaminants from a sample, purifying and concentrating target molecules in the process.

In this article, we will delve into the importance of optimizing desalting column for effective purification of samples. Desalting columns play a vital role in removing salts and other impurities, ensuring the delivery of high-quality samples for downstream applications.

Optimizing Desalting Column:
Factors to Consider

The following factors should be considered to optimize the
performance of your desalting column:

  1. Sample Preparation: Prior to using the desalting column, ensure that your sample is prepared correctly. This includes selecting the appropriate buffer, pH, and sample concentration. The buffer should be compatible with both the target molecule and the desalting column matrix. Additionally, the pH should be maintained within the optimal range for the target molecule’s stability.
  2. Column Selection: Choose the right desalting column for your specific application. EMP Biotech offers desalting columns covering a wide range of sample volumes. Selecting the appropriate column ensures optimal workflow, purification, and recovery of your target molecule.
  3. Equilibration: Before loading your sample onto the desalting column, it is essential to equilibrate the column with the desired buffer. Proper equilibration removes any preservatives and ensures that the column matrix is in the optimal condition for sample separation. Follow the manufacturer’s guidelines for equilibrating your EMP Biotech desalting column.
  4. Sample Loading: Load your sample onto the desalting column according to the manufacturer’s instructions. Be mindful of the sample volume, as overloading the column can lead to inadequate separation and reduced recovery of the target molecule. For optimal results, ensure that the sample volume does not exceed the recommended loading capacity for your specific desalting column.
  5. Elution: Elute your sample from the desalting column following the manufacturer’s recommended elution protocol. Proper elution techniques ensure the efficient recovery of your target molecule and minimize the presence of contaminants. Be sure to collect the eluted fractions in appropriate collection tubes or vessels to prevent cross-contamination.
  6. Monitoring and Evaluation: Monitor the elution process by measuring the absorbance of the collected fractions at an appropriate wavelength. This will help you track the elution profile of your target molecule and identify the fractions containing the purified molecule. Evaluate the performance of your desalting column by assessing the purity and recovery of the target molecule in the eluted fractions.

Optimizing Desalting Column:
Tips and Best Practices

To further enhance the performance and efficiency of your desalting column, consider the following tips and best practices:

    1. Maintain optimal storage conditions: Store your desalting column according to the manufacturer’s recommendations. This will help preserve the column’s performance and extend its lifespan.
    2. Clean the column, if necessary: If the desalting column’s performance declines, consider cleaning the column following the manufacturer’s guidelines. Proper cleaning can restore the column’s performance and prolong its life.
    3. Monitor the column’s performance over time: Regularly assess the performance of your desalting column by evaluating the purity and recovery of your target molecule. This will help you identify any potential issues and take appropriate action to maintain optimal column performance.


emp BIOTECH desalting columns are valuable tools for the purification and concentration of target molecules in various research applications.

By considering factors such as sample preparation, column selection, equilibration, sample loading, elution, and monitoring, you can optimize the use of your desalting column and achieve consistent, high-quality results. Incorporating tips and best practices such as proper storage, column cleaning, buffer optimization, and gradient elution can further enhance the performance and efficiency of your desalting column, ensuring the success of your biomolecule purification.

Are you purifying proteins or DNA?

Are you purifying proteins or DNA?

Read emp BIOTECH’s latest brochure
on Biomolecule Purification

If you already know emp BIOTECH’s desalting columns, or their SMART Chromatography™ technology, you might not be aware that emp also manufactures a range of resins, available in bulk quantities, that can be applied to a number of purification challenges.

The latest catalogue covers emp’s range in:

  • Affinity chromatography
  • Ion exchange (IEX) purification
  • Hydrophobic interaction chromatography (HIC)
  • Activated resins – for attaching your “favourite” molecules

…and of course, emp BIOTECH’s SEC columns, resins etc. are also covered.


Biomolecule Purification, Catalog 09/2023

New: TurboTag™ Protein/Antibody Labeling Kits

New: TurboTag™ Protein/Antibody Labeling Kits

emp BIOTECH’s popular protein labelling kits have
now been renamed as TurboTag™ precision protein labeling kits.

The kits will now only be available in a 3-reaction size. Everything else remains the same:

  • 10 minute labelling protocol, from start to finish
  • Optimized protocols for 1 mg and 100 µg of protein
  • Over 35 different fluorescent and non-fluorescent labels to choose from


TurboTag™ Labeling Kits, Flyer 09/2021

Find all products

Search by TurboTag

Watch our video to get an overview
of TurboTag™ technology.

Open the box, label the protein and
purify – all in 10 minutes.