A Summary of Practical Considerations for the Application of the Steric Exclusion Chromatography for the Purification of the Orf Viral Vector

Abstract

Steric exclusion chromatography (SXC) is a promising purification method for biological macromolecules such as the Orf virus (ORFV) vector. The method’s principle is closely related to conventional polyethylene glycol (PEG) precipitation, repeatedly implementing membranes as porous chromatographic media. In the past decade, several purification tasks with SXC showed exceptionally high yields and a high impurity removal. However, the effect of varying process parameters, on the precipitation success and its limitations to SXC, is not yet well understood. For this reason, the precipitation behavior and SXC adaptation for ORFV were investigated for the PEG/ORFV contact time, the membranes pore size, and the type and concentration of ions. All three parameters influenced the ORFV recoveries significantly. A small pore size and a long contact time induced filtration effects and inhibited a full virus recovery. The application of salts had complex concentration-dependent effects on precipitation and SXC yields, and ranged from a complete prevention of precipitation in the presence of kosmotropic substances to increased efficiencies with Mg2+ ions. The latter finding might be useful to reduce PEG concentrations while maintaining high yields. With this knowledge, we hope to clarify several limitations of SXC operations and improve the tool-set for a successful process adaptation.

Scaling Up of Steric Exclusion Membrane Chromatography for Lentiviral Vector Purification

“…Infectious LV recovery was significantly higher ( p ≤ 0.01) when internal mixing was performed ( Figure 3 C) than when external mixing was performed (73.94 ± 12.13% and 24.53 ± 13.43%, respectively). These findings support the results of Labisch et al and Eilts et al, in which the same effect was observed for other module sizes [ 10 , 13 ]. Moreover, a significantly higher amount of LVs (38.05 ± 12.37%) was lost in the flow through ( p ≤ 0.05) when LVs were loaded after external mixing.…”

“…Since the average pore diameter remains unchanged, the flow velocity inside the pores decreases and falls below the optimal flow velocity inside the stationary phase to achieve efficient LV capture during loading and release during elution. Another possible explanation, recently discussed in [ 13 ], is that a limited spontaneous encounter for the LV and the stationary phase could lead to a less efficient depletion interaction. In our case, a certain flow rate through the membrane pores might be necessary to increase the probability of an encounter between the LVs and the stationary phase.…”

“…A study on the use of steric exclusion chromatography (SXC) for LV purification was recently published [ 10 ]. A variety of viruses have been previously purified by SXC, including baculovirus [ 11 ], Orf virus [ 12 , 13 ], AAV [ 14 ], and influenza A virus [ 15 ]. SXC is a gentle purification method offering high potential for the purification of large, enveloped, fragile viral vectors as it does not require any chemical interaction between the target species and the stationary phase and preserves viral infectivity.…”

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography

“…Similar findings were reported before for Orf virus (Eilts, Lothert, Orbay et al, 2022) and for latex particles (Eilts, Steger, Lothert & Wolff, 2022b). However, the final precipitate sizes were expected to vary between the static experiments and the dynamic SXC runs.…”

“…Time-dependent size measurements of the KHV-containing cell culture supernatant were conducted via dynamic light scattering, adapted from Eilts, Lothert, Orbay et al (2022). Therefore, clarified virus suspension was mixed in equal parts with CPB (15 mS cm −1 ) of a defined pH (4-7.4) and PEG-6000-concentration (Carl Roth) (0%-12%).…”