Comparison of sample preparation techniques for the physicochemical characterization of Orf virus particles

Eilts, Friederike; Steger, Marleen; Pagallies, Felix; Rziha, Hanns-Joachim; Hardt, Martin; Amann, Ralf; Wolff, Michael W.

doi:10.1016/j.jviromet.2022.114614

Cited by 6 publications

(9 citation statements)

References 41 publications

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“…SXC‐purified virus particles were analysed for their isoelectric point, as described previously, with minor modifications (Eilts, Steger, Pagallies et al, 2022). In brief, purified KHV, stored in PBS (Gibco), was diluted 1:5 with 0.1 M citrate (VWR Chemicals) phosphate (Carl Roth) buffer (CPB) (adjusted to 15 mS cm −1 with NaCl), or in PBS with 0.03% (v/v) polysorbate 20 (Carl Roth), both at pH values ranging from 3 to 11.…”

Section: Methodsmentioning

confidence: 99%

“…A possible purification method that meets the stated requirements is the steric exclusion chromatography (SXC). It is an economic, quick and easy‐to‐use method, which enabled high yields (>80%–90%) for previously published applications of (infectious) viral particles (Alvim et al, 2021; Eilts, Lothert, Orbay et al, 2022, Eilts, Steger, Pagallies et al, 2022; Gränicher et al, 2021; Labisch et al, 2022; Lee et al, 2012; Lothert, Offersgaard, Pihl et al, 2020, Lothert, Pagallies, Eilts et al 2020, Lothert, Sprick, Beyer et al, 2020; Marichal‐Gallardo et al, 2017, 2021). The impurity removal reaches 80%–100% for protein and >60% for host cell DNA without any enzymatic DNA digestion steps.…”

Section: Introductionmentioning

confidence: 99%

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Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography

Eilts,

Jordan,

Harsy

et al. 2023

Journal of Fish Diseases

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Koi herpesvirus (KHV) is the causative agent of a koi herpesvirus disease (KHVD) inducing high mortality rates in common carp and koi (Cyprinus carpio). No widespread effective vaccination strategy has been implemented yet, which is partly due to side effects of the immunized fish. In this study, we present an evaluation of the purification of infectious KHV from host cell protein and DNA, using the steric exclusion chromatography. The method is related to conventional polyethylene glycol (PEG) precipitation implemented in a chromatographic set‐up and has been applied for infectious virus particle purification with high recoveries and impurity removal. Here, we achieved a yield of up to 55% of infectious KHV by using 12% PEG (molecular weight of 6 kDa) at pH 7.0. The recoveries were higher when using chromatographic cellulose membranes with 3–5 μm pores in diameter instead of 1 μm. The losses were assumed to originate from dense KHV precipitates retained on the membranes. Additionally, the use of >0.6 M NaCl was shown to inactivate infectious KHV. In summary, we propose a first step towards a purification procedure for infectious KHV with a possible implementation in fish vaccine manufacturing.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography

Eilts,

Jordan,

Harsy

et al. 2023

Journal of Fish Diseases

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“…The latter preparation reached an infectious titer of 8 × 10 6 IU mL −1 . The methods were studied recently to generate comparable ORFV starting material ( Eilts et al., 2022c ). The ORFV titer was quantified by a flow cytometric assay described in Section 2.4.1 if not stated otherwise.…”

Section: Methodsmentioning

confidence: 99%

An investigation of excipients for a stable Orf viral vector formulation

Eilts,

Harsy,

Lothert

et al. 2023

Virus Research

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“…Inline mixing shortly before the column would then encourage the interaction of the target with the stationary phase, rather than with one another [1,18,31]. On the other hand, offline mixing and loading of the premixed solution via a loop worked well without any process impairments, such as low product recovery or pressure issues, in several studies [22][23][24]26,28,29,47].…”

Section: Mixing Approaches Of Peg With the Feed Solutionmentioning

confidence: 99%

Steric Exclusion Chromatography for Purification of Biomolecules—A Review

2023

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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.

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Comparison of sample preparation techniques for the physicochemical characterization of Orf virus particles

Cited by 6 publications

References 41 publications

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography

An investigation of excipients for a stable Orf viral vector formulation

Steric Exclusion Chromatography for Purification of Biomolecules—A Review

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