Centrifuges
Centrifuges were the main separation equipment used for decades and are still used extensively today. The G-forces created by their spinning motion separates based on differences in density. The proteins that have precipitated have a higher density than the solubilized liquid plasma and remaining soluble proteins. The solid protein precipitates are retained within the centrifuge while the liquid plasma and remaining soluble proteins are allowed to pass through the centrifuge. Subcategories of centrifuges include multi-chamber bowls (used in large scale fractionation), tubular centrifuges (used in small-scale operations, such as a lab setting), and disc stack centrifuges (common in biomanufacturing but not widely used in plasma fractionation).
Depth Filtration
Another method of plasma protein separation is depth filtration. Depth filters are made up of an internal structure that creates a tortuous path, causing precipitated plasma proteins to become trapped or adsorbed within the filter structure. As for depth filtration, filter presses form the backbone of this category when applied to large scale plasma protein separation. Other forms of depth filtration that have seen application in plasma purification processes include cartridge filter housings and rotating disc filtration.
Chromatography
Packed bed chromatography is a well-established technique that is widely used in the downstream purification of plasma protein intermediates that have already been separated from the crude plasma by the previously mentioned separation techniques. Packed bed chromatography traditionally has not been well suited for crude plasma purification primarily due to the shear volume of proteins present, resulting in a required chromatography resin volume that is cost prohibitive for most large-scale manufacturers.
If anything stands a chance to replace the classic ethanol precipitation and mechanical separation approach in the future, it will likely be through technological advancements in large scale chromatography techniques. One of the more promising solutions is the use of expanded bed adsorption chromatography. This is still not a cost-effective solution for most large-scale plasma fractionators. Until then, ethanol fractionation and mechanical separation is here to stay.
Which separation method is best?
The first separation step of plasma fractionation, known as the cryoprecipitate step, is nearly always performed in a multi-chamber centrifuge. After that step, however, the process becomes much more variable in terms of common practices used in modern fractionation facilities.
The discussion below considers the pros and cons of two of the most common methods: using a multi-chamber bowl centrifuge versus a standard plate and frame filter press. These recommendations are based on using cold ethanol as the anti-solvent agent to precipitate the sought-after proteins such as immunoglobulins, albumin, and coagulation factors. Depending on the protein of interest, a different agent and/or separation method may be more suitable.
Capital costs
One of the common drivers for deciding which separation method to use is equipment capital cost. From an initial cost point-of-view, filter presses and centrifuges have similar price tags on a per unit basis. However, in a typical large-scale operation, only one filter press is typically needed while two to three centrifuges are required to process the same plasma volume.
Next, consider the cost of supporting equipment. A simple manual filter press does not require much supporting equipment beyond the usual clean utilities systems found in most GMP facilities, such as Clean-in-Place (CIP) systems. A centrifuge, however, often requires multiple dedicated cooling systems, as well as dedicated cleaning equipment in addition to CIP. Ensuring satisfactory plasma protein yield and quality requires very accurate temperature control throughout the separation process. The temperature setpoint varies depending on the target protein, but in some instances it can be as low as -10°C. The resultant required temperatures for some of the cooling systems can be as low as -35°C, making the cooling equipment used to achieve these very cold temperatures complex and expensive.
The standard plate and frame filter presses designed by most manufacturers for the plasma industry are manually indexed and cleaned, while the piping is cleaned with an automated CIP cycle. Chamber centrifuges have a large solid bowl that must be manually disassembled for cleaning. Some manufacturers rely on time- and labor-intensive manual cleaning while others use automated cleaning equipment, such as a Clean-Out-of-Place (COP) cabinet to wash the centrifuge bowl. There are further costs associated with the carts used to transport the bowl back-and-forth to the cleaning chamber. Factor in the need for multiple washers and carts for multiple centrifuges, and the cost quickly rises.
It is also worth noting, however, that the facility’s philosophy for handling of filter aid for filter press operations is an important consideration that can lead to additional equipment costs for the filter press option. Some facilities have procedures for manual weighing and manual addition of filter aid, in which case capital costs for filter aid equipment is minimal. Others may choose to adopt dispensing and pneumatic conveying systems that automatically transport filter aid to the required use points, substantially increasing equipment capital costs.