A scalable downstream process for the purification of the cell culture-derived Orf virus for human or veterinary applications

Lothert, Keven; Pagallies, Felix; Eilts, Friederike; Sivanesapillai, Arabi; Hardt, Martin; Moebus, Anna; Feger, Thomas; Amann, Ralf; Wolff, Michael W.

doi:10.1016/j.jbiotec.2020.08.014

Cited by 24 publications

(31 citation statements)

References 48 publications

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“…The occurrence of CPS in the flow-through fraction was diminished by increased PEG 8000 concentrations and reduced pH values. For these conditions, the slope of the pressure increase was considerably steeper than reported for biological nanoparticles using the SXC [ 1 , 10 , 16 , 17 ]. We propose two reasons for this behavior: (1) the approximated two-dimensional surface charge distribution of the hard latex spheres is less complex than the three-dimensional characteristics of so-called soft particles, e.g., viruses [ 26 , 27 , 28 , 29 , 30 ], which additionally have an amphoteric character.…”

Section: Discussionmentioning

confidence: 66%

“…CP particles with 190 nm mean diameter, were purchased with two types of surface functionalization, either sulfate-or hydroxyl-groups, CPS and CPH, respectively (Polybead, Polysciences Europe, Hirschberg a. d. Bergstrasse, Germany). Size distribution and charge measurements were performed with a Zetasizer Nano ZS90 and the corresponding Zetasizer software (version 7.13) (both Malvern Panalytical, Malvern, UK) according to Lothert et al [ 16 ] with the deviation of the pre-set “polystyrene latex”. CP with a final concentration of 10 10 particles mL −1 were prepared in 0.1 M citrate phosphate buffer (CPB), the conductivity (15 mS cm −1 ) adjusted with NaCl.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, pressure limits are of utmost importance for the stationary phase as well as for the LC system. In the past, convective media, such as monoliths [ 1 , 2 , 13 , 14 ] and membranes [ 15 , 16 , 17 , 18 , 19 ], were applied to counteract diffusion limitations and clogging caused by the virus particles’ large size. As a secondary effect, the high viscosity of the PEG solutions was easier to handle with these stationary phases.…”

Section: Introductionmentioning

confidence: 99%

“…As a secondary effect, the high viscosity of the PEG solutions was easier to handle with these stationary phases. Last, in-line mixing, representing the blending in the mixer and tubes of the LC immediately before loading onto the stationary phase, was proposed to reduce a possible pressure increase [ 1 , 18 ], but several authors worked on a laboratory scale with off-line mixing without describing such limitations [ 10 , 15 , 16 , 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

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The Suitability of Latex Particles to Evaluate Critical Process Parameters in Steric Exclusion Chromatography

et al. 2022

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The steric exclusion chromatography (SXC) is a rather new method for the purification of large biomolecules and biological nanoparticles based on the principles of precipitation. The mutual steric exclusion of a nonionic organic polymer, i.e., polyethylene glycol (PEG), induces target precipitation and leads to their retention on the chromatographic stationary phase. In this work, we investigated the application of latex particles in the SXC by altering the particle’s surface charge as well as the PEG concentration and correlated both with their aggregation behavior. The parameters of interest were offline precipitation kinetics, the product recovery and yield, and the chromatographic column blockage. Sulfated and hydroxylated polystyrene particles were first characterized concerning their aggregation behavior and charge in the presence of PEG and different pH conditions. Subsequently, the SXC performance was evaluated based on the preliminary tests. The studies showed (1) that the SXC process with latex particles was limited by aggregation and pore blockage, while (2) not the aggregate size itself, but rather the aggregation kinetics dominated the recoveries, and (3) functionalized polystyrene particles were only suitable to a limited extent to represent biological nanoparticles of comparable size and charge.

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Section: Discussionmentioning

confidence: 66%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Suitability of Latex Particles to Evaluate Critical Process Parameters in Steric Exclusion Chromatography

et al. 2022

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“…The unique characteristics of antimicrobial, anti-inflammatory, and antineoplastic activities have made lysozyme a suitable candidate for food preservation, home appliances, and aseptic and therapeutic uses. − This protein is commercially separated from chicken egg white, which contains about 3.5% (w/w) lysozyme. , The conventional strategies used to separate and purify lysozyme include chromatography, precipitation, membrane filtration, and centrifugation. − However, the commonly used downstream processes are accompanied by extra steps for the efficient removal of impurities and product purification, which result in time-consuming and high-cost procedures. , Thus, to overcome these problems, the development of simple, fast, cost-effective, and efficient alternative technologies for the separation and purification of biological products is still of crucial importance. , Among various adsorbent materials, magnetic particles, particularly magnetite (Fe 3 O 4 ) nanoparticles, have been of utmost interest for bioseparation applications due to their easy and inexpensive synthesis approaches, low toxicity, superparamagnetic properties, and rapid magnetic separation from the corresponding media. − …”

Section: Introductionmentioning

confidence: 99%

Effect of the Structure of Magnetic Nanocomposite Adsorbents on the Lysozyme Separation Efficiency

Esmaeilnejad-Ahranjani

Maboudi

Arpanaei

2022

ACS Appl. Bio Mater.

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In this study, we prepared various anionic magnetic adsorbents through the carboxyl functionalization of core/shell-structured Fe3O4/SiO2 (FS) particles by either succinic anhydride (FSC), low-molecular-weight (MW 1800) polyacrylic acid (PAA) (FSP1), or high-molecular-weight (MW 100,000) PAA (FSP2), and then, investigated the effect of the structure of adsorbents and operational parameters on their performance for the lysozyme separation. The type and size of functional molecules have significant effects on the surface concentration of functional carboxyl groups onto the adsorbent particles (increase in the order of FSP2 > FSP1 > FSC), and consequently on the adsorption efficiency (AE) (∼100, 98, and 62%, respectively) and adsorption capacity (AC) (∼1000, 980, and 621 mg·g–1, respectively) of the adsorbents. However, the loss of the antibacterial activity of separated lysozyme molecules due to the molecular conformational change increased in the order of FSP2 > FSP1 = FSC, as compared to the free lysozyme. The application of basic buffer solutions for the elution of adsorbed enzyme molecules resulted in more adverse effects on the enzyme activity. The obtained results recommend that FSP1 can be used as a suitable anionic adsorbent for the isolation of positively charged proteins, owing to its high adsorption capacity, excellent reusability, and structural stability, as well as the high purity, structural stability, and activity recovery of the isolated proteins.

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Protein adsorption on core‐shell resins for flow‐through purifications: Effect of protein molecular size, shape, and salt concentration

Wang²,

Winters³

et al. 2022

Biotechnology Progress

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This work addresses the functional properties of the core‐shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose‐based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.

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A scalable downstream process for the purification of the cell culture-derived Orf virus for human or veterinary applications

Cited by 24 publications

References 48 publications

The Suitability of Latex Particles to Evaluate Critical Process Parameters in Steric Exclusion Chromatography

The Suitability of Latex Particles to Evaluate Critical Process Parameters in Steric Exclusion Chromatography

Effect of the Structure of Magnetic Nanocomposite Adsorbents on the Lysozyme Separation Efficiency

Protein adsorption on core‐shell resins for flow‐through purifications: Effect of protein molecular size, shape, and salt concentration

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