Tangential-Flow Electrophoresis in Plasma Fractionation

An Effective Separation of Human Serum Albumin

The human plasma constitutes 55% of our blood and is the largest component. This light yellow liquid is a complex mixture of biomolecules including serum albumin, immunoglobulins, coagulation factors, and others. These protein biomolecules are important, but are often present at low levels or lacking in affected patients with certain life-threatening conditions. Over the decades, medical researchers have learned to extract these valuable proteins using a number of purification methods. 

Plasma fractionation can be traced back to the middle of the 20th century when Edwin Cohn of Harvard University developed the first industrial process to purify proteins from crude human plasma. The Cohn fractionation method is widely used until today, as is a combination of several column chromatography separations.

Processing standards have improved further with orthogonal viral inactivation and removal methods, in order to produce high-quality plasma-derived therapeutics.  

Current Methods of Plasma Fractionation

The Cohn fractionation method uses a protein precipitation approach based on cold ethanol. Essentially, different plasma proteins are precipitated by an increasing volume percentage of ethanol under –5 °C, with a decreasing pH from 7 (like pure water) to <5 (slightly acidic). Plasma proteins precipitate out in stages before further polishing with ion-exchange chromatography columns.

 This method, however, has several scalability issues:

1. Current methods of fractionation like (like Cohn’s) require a large volume of solvents.
2. High-energy expenditures for maintaining sub-zero processing temperatures, making this method somewhat costly to set up and maintain.
3. Due to high investment and operating costs, most developing countries are unable to establish their own local fractionation plants to ensure sustainable supplies to local/regional patients.
4. Costs of transporting and shipping frozen plasma products further complicate delivery, as protein products are heat-labile (change or broken down in the presence of heat).

On another note, logistics are easier to manage if a fractionation plant is located closer to patients in regional hospitals and other relevant health institutions. A local fractionation plant also could provide the advantage of curtailing a potential epidemic by providing a supply of purified hyper-immune antibodies in a short period.

Membrane Electrophoresis: Enter PRIME

To address these challenges, researchers have developed a new plasma fractionation approach based on membrane electrophoresis. The goal is to provide a practical and cost-effective method to separate proteins from human plasma. This separation technique is known as preparative isolation using membrane electrophoresis (PRIME).

The operating principle of PRIME is by electrophoretic migration of charged proteins through a polyacrylamide membrane with different pore sizes optimized for a range of biomolecules.

Here’s how it works:

1. Each protein has an isoelectric point (pI).
2. By changing buffering salts and their concentration, you can change the charge of target proteins accordingly.
3. A buffer with a pH greater than that of the protein’s pI negatively charges that protein; a buffer with a pH lower than that of a protein’s pI positively charges that protein; a buffer with the same pH as the pI will neutralize the protein of interest.
4. Therefore, the PRIME process can separate proteins with different pI values.
5. Positively charged molecules are attracted toward the negative electrode, whereas negatively charged molecules migrate toward the positive electrode through the pores on the separation membrane.

The PRIME process also includes a co-separation based on molecular size by using membranes with predefined or specific molecular weight cutoffs (MWCOs). Two types of membranes are used: restriction membranes and separation membranes.

Various benefits of the PRIME system includes:

1. It does not involve large consumption of flammable solvents, nor does it require subzero temperatures. Hence, it is safer and more energy efficient than conventional fractionation methods used in the blood-plasma industry.
2. With different combinations of pore size as well as a buffer system, the system can be applied to isolate a broad range of therapeutic biomolecules.
3. Studies have shown that the system also can perform a partial virus-removing step by trapping larger viral particles in the crude feed stream while allowing target therapeutic proteins to cross the separation membrane into harvest. The operating principle here is similar to that of typical nano-filtration.
4. In addition, it does not rely on trans-membrane pressure for protein transfer. The crossflow or tangential-flow system used during the process reduces contamination, resulting in purer protein products.

In developmental runs, this preparative purification system can process high protein concentrations in crude feeds with satisfactory process yields — paired with good purity of a target product. In other words, the PRIME unit could achieve high process yields (>90%) under sub-optimized conditions. 

In fact, under ideal conditions, even a small area of membrane should be able to handle the maximum amount of feed load in terms of volume and protein mass.

PRIME technology is a relatively new protein purification process that combines elements of ultra-filtration (UF) or diafiltration (DF) with electrophoresis to offer a practical alternative to the growing demand for downstream processing of new and challenging biomolecules.

Hence, PRIME offers one of the most promising methods of protein purification to date. It is safer, energy-efficient and flexible for processing a wide range of therapeutic products.

Source:

Ng Kheng Tee, Benjamin Lim, Smitha Kenchath, Wong Tee Wee. “Tangential-Flow Electrophoresis: A New Plasma Fractionation Technique Analyzed for the Separation of Human Serum Albumin from Human Plasma.” BioProcess International, 14 Feb. 2019, bioprocessintl.com/downstream-processing/separation-purification/tangential-flow-electrophoresis-investigation-of-factors-involved-in-an-effective-separation-of-human-serum-albumin-from-human-plasma/.