Steric Exclusion Chromatography for Purification of Biomolecules—A Review

Abstract

Steric exclusion chromatography (SXC) is a purification method that is based on steric exclusion effects from the surface of the target and a hydrophilic stationary phase after the addition of polyethylene glycol (PEG), which leads to an association of the target with the stationary phase without direct binding, such as covalent, electrostatic, and hydrophilic/hydrophobic interactions. The gentle nature of the method has led to an increased focus on sensitive targets such as enveloped viruses with potential for other sensitive entities, e.g., extracellular vesicles and virus-like particles. SXC is related to PEG-mediated protein precipitation, but investigation of further process parameters was crucial to gain a better understanding of the SXC method. After explaining mechanistic fundamentals and their discovery, this review summarizes the findings on SXC from its first reference 11 years ago until today. Different applications of SXC are presented, demonstrating that the method can be used for a wide variety of targets and achieves high recovery rates and impurity removal. Further, critical process parameters for successful process implementation are discussed, including technical requirements, buffer composition, and scalability.

Impact of the Membrane Structure of the Stationary Phase on Steric Exclusion Chromatography (SXC) of Lentiviral Vectors

“…Potentially unbound antibodies are removed in the flow-through due to the large size difference of IgG (15.5 × 8.5 × 4.0 nm [20] and LVs (80-120 nm diameter [5]. The pronounced size difference allows for the selective retention of the larger LV particles, as discussed in more detail elsewhere [8,12]. Therefore, it can be assumed that unbound antibodies are not retained and efficiently removed in the flow through fraction.…”

“…Five layers of a Hydrosart membrane (type I, 2.2 µm pore size) and the Whatman RC membrane (1 µm pore size), which are the two membranes predominantly used for SXC [8], were used to purify clarified LVs, using three different PEG concentrations. The highest infectious LV recovery of 73-79% was achieved with the Hydrosart membrane in combination with a 12.5% or 15% PEG buffer (Figure 4).…”

“…Due to the increasing demand for cell and gene therapies, cost-effective methods are needed to ensure affordability and accessibility for patients. Steric exclusion chromatography (SXC) is a potential alternative to commonly used chromatography methods such as ion exchange or affinity chromatography, particularly for enveloped viral vectors [8]. As this method does not require any chemical interaction between the target species and the stationary phase, it can be considered a platform technology [9].…”

“…A detailed description of the SXC purification principle can be found elsewhere [10][11][12]. SXC offers a high potential for viral vector purification and other large sensitive biomolecules [8,13]. The mild process conditions, the high product recovery yields, and the low cost of the method show promising incentives to further develop and understand the method.…”

“…The mild process conditions, the high product recovery yields, and the low cost of the method show promising incentives to further develop and understand the method. A considerable number of studies have been performed in recent years, demonstrating applicability for various targets using different stationary phases [8]. The underlying mechanism of SXC is based on PEG-mediated depletion interaction of the target with the stationary phase [12].…”