Affinity and Pseudo-Affinity Membrane Chromatography for Viral Vector and Vaccine Purifications: A Review.

Several chromatographic approaches have been established over the last decades for the production of pharmaceutically relevant viruses. Due to the large size of these products compared to other biopharmaceuticals, e.g., proteins, convective flow media have proven to be superior to bead-based resins in terms of process productivity and column capacity. One representative of such convective flow materials is membranes, which can be modified to suit the particular operating principle and are also suitable for economical single-use applications. Among the different membrane variants, affinity surfaces allow for the most selective separation of the target molecule from other components in the feed solution, especially from host cell-derived DNA and proteins. A successful membrane affinity chromatography, however, requires the identification and implementation of ligands, which can be applied economically while at the same time being stable during the process and non-toxic in the case of any leaching. This review summarizes the current evaluation of membrane-based affinity purifications for viruses and virus-like particles, including traditional resin and monolith approaches and the advantages of membrane applications. An overview of potential affinity ligands is given, as well as considerations of suitable affinity platform technologies, e.g., for different virus serotypes, including a description of processes using pseudo-affinity matrices, such as sulfated cellulose membrane adsorbers.

Evaluating Novel Quantification Methods for Infectious Baculoviruses.

Accurate and rapid quantification of (infectious) virus titers is of paramount importance in the manufacture of viral vectors and vaccines. Reliable quantification data allow efficient process development at a laboratory scale and thorough process monitoring in later production. However, current gold standard applications, such as endpoint dilution assays, are cumbersome and do not provide true process analytical monitoring. Accordingly, flow cytometry and quantitative polymerase chain reaction have attracted increasing interest in recent years, offering various advantages for rapid quantification. Here, we compared different approaches for the assessment of infectious viruses, using a model baculovirus. Firstly, infectivity was estimated by the quantification of viral nucleic acids in infected cells, and secondly, different flow cytometric approaches were investigated regarding analysis times and calibration ranges. The flow cytometry technique included a quantification based on post-infection fluorophore expression and labeling of a viral surface protein using fluorescent antibodies. Additionally, the possibility of viral (m)RNA labeling in infected cells was investigated as a proof of concept. The results confirmed that infectivity assessment based on qPCR is not trivial and requires sophisticated method optimization, whereas staining of viral surface proteins is a fast and feasible approach for enveloped viruses. Finally, labeling of viral (m)RNA in infected cells appears to be a promising opportunity but will require further research.

An investigation of excipients for a stable Orf viral vector formulation.

The Orf virus (ORFV) is a promising candidate for vector vaccines as well as for immunomodulatory and oncolytic therapies. However, few publications are available on its infectivity degradation or on suitable additives for prolonging its viral stability. In this study, the non-supplemented ORFV itself showed a very high stability at storage temperatures up to 28 °C, with a linear titer loss of 0.10 log infectious particles per day at 4 °C over a period of five weeks. To prolong this inherent stability, thirty additives, i.e., detergents, sugars, proteins, salts, and buffers as well as amino acids, were tested for their time- and temperature-dependent influence on the ORFV infectivity. A stabilizing effect on the infectivity was identified for the addition of all tested proteins, i.e., gelatine, bovine serum albumin, and recombinant human serum albumin (rHSA), of several sugars, i.e., mannitol, galactose, sucrose, and trehalose, of amino acids, i.e., arginine and proline, of the detergent Pluronic F68, and of the salt Na2SO4. The infectivity preservation was especially pronounced for proteins in liquid and frozen formulations, sugars in frozen state, and arginine und Pluronic in liquid formulations at high storage temperatures (37 °C). The addition of 1% rHSA with and without 5% sucrose was evaluated as a very stable formulation with a high safety profile and economic validity at storage temperatures up to 28 °C. At increased temperatures, the supplementation with 200 mM arginine performed better than with rHSA. In summary, this comprehensive data provides different options for a stable ORFV formulation, considering temperature, storage time, economic aspects, and downstream processing integrity.

Evaluation of restricted access media for the purification of cell culture-derived Orf viruses.

Recently, multimodal chromatography using restricted access media (RAM) for the purification of nanoparticles, such as viruses has regained increasing attention. These chromatography resins combine size exclusion on the particle shell and adsorptive interaction within the core. Accordingly, smaller process-related impurities, for example, DNA and proteins, can be retained, while larger product viruses can pass unhindered. We evaluated a range of currently available RAM, differing in the shells' pore cut-off and the core chemistry, for the purification of a cell culture-derived clarified model virus, namely the Orf virus (ORFV). We examined impurity depletion and product recovery as relevant criteria for the evaluation of column performance, as well as scale-up robustness and regeneration potential for evaluating a multiple use application. The results indicate that some columns, for example, the Capto Core, enable both a high DNA and protein removal, while others, for example, the Monomix Core 60 (MC60), are more suitable for DNA depletion. Furthermore, column regeneration is facilitated by using columns with larger shell pores (5000 vs. 700 kDa) and weaker binding interactions (anion exchange vs. multimodal). According to these findings, the choice of RAM resins should be selected according to the respective feed sample composition and the planned number of application cycles.

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

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.

Quantification methods for viruses and virus-like particles applied in biopharmaceutical production processes.

INTRODUCTION: Effective cell-based production processes of virus particles are the foundation for the global availability of classical vaccines, gene therapeutic vectors, and viral oncolytic treatments. Their production is subject to regulatory standards ensuring the safety and efficacy of the pharmaceutical product. Process analytics must be fast and reliable to provide an efficient process development and a robust process control during production. Additionally, for the product release, the drug compound and the contaminants must be quantified by assays specified by regulatory authorities. AREAS COVERED: This review summarizes analytical methods suitable for the quantification of viruses or virus-like particles. The different techniques are grouped by the analytical question that may be addressed. Accordingly, methods focus on the infectivity of the drug component on the one hand, and on particle counting and the quantification of viral elements on the other hand. The different techniques are compared regarding their advantages, drawbacks, required assay time, and sample throughput. EXPERT OPINION: Among the technologies summarized, a tendency toward fast methods, allowing a high throughput and a wide applicability, can be foreseen. Driving forces for this progress are miniaturization and automation, and the continuous enhancement of process-relevant databases for a successful future process control.

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

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.

Upstream and Downstream Processes for Viral Nanoplexes as Vaccines.

The increasing medical interest in viral nanoplexes, such as viruses or virus-like particles used for vaccines, gene therapy products, or oncolytic agents, raises the need for fast and efficient production processes. In general, these processes comprise upstream and downstream processing. For the upstream process, efficiency is mainly characterized by robustly achieving high titer yields, while reducing process times and costs with regard to the cell culture medium, the host cell selection, and the applied process conditions. The downstream part, on the other hand, should effectively remove process-related contaminants, such as host cells/cell debris as well as host cell DNA and proteins, while maintaining product stability and reducing product losses. This chapter outlines a combination of process steps to successfully produce virus particles in the controlled environment of a stirred tank bioreactor, combined with a platform-based purification approach using filtration-based clarification and steric exclusion chromatography. Additionally, suggestions for off-line analytics in terms of virus characterization and quantification as well as for contaminant estimation are provided.

Selection of chromatographic methods for the purification of cell culture-derived Orf virus for its application as a vaccine or viral vector.

In recent years, the Orf virus has become a promising tool for protective recombinant vaccines and oncolytic therapy. However, suitable methods for an Orf virus production, including up- and downstream, are very limited. The presented study focuses on downstream processing, describing the evaluation of different chromatographic unit operations. In this context, ion exchange-, pseudo-affinity- and steric exclusion chromatography were employed for the purification of the cell culture-derived Orf virus, aiming at a maximum in virus recovery and contaminant depletion. The most promising chromatographic methods for capturing the virus particles were the steric exclusion- or salt-tolerant anion exchange membrane chromatography, recovering 84 % and 86 % of the infectious virus. Combining the steric exclusion chromatography with a subsequent Capto™ Core 700 resin or hydrophobic interaction membrane chromatography as a secondary chromatographic step, overall virus recoveries of up to 76 % were achieved. Furthermore, a complete cellular protein removal and a host cell DNA depletion of up to 82 % was possible for the steric exclusion membranes and the Capto™ Core 700 combination. The study reveals a range of possible unit operations suited for the chromatographic purification of the cell culture-derived Orf virus, depending on the intended application, i.e. a human or veterinary use, and the required purity.

Membrane-based steric exclusion chromatography for the purification of a recombinant baculovirus and its application for cell therapy.

The continuously increasing potential of stem cell treatments for various medical conditions has accelerated the need for fast and efficient purification techniques for individualized cell therapy applications. Genetic stem cell engineering is commonly done with viral vectors like the baculovirus. The baculovirus is a safe and efficient gene transfer tool, that has been used for the expression of recombinant proteins for many years. Its purification has been based mainly on ion exchange matrices. However, these techniques impair process robustness, if different genetically modified virus particles are applied. Here, we evaluated the membrane-based steric exclusion chromatography for the purification of insect cell culture-derived recombinant Autographa californica multicapsid nucleopolehydroviruses for an application in cell therapy. The method has already proven to be a powerful tool for the purification of Influenza A virus particles, using cellulose membranes. Aside from the aforementioned cellulose, we evaluated alternative stationary phases, such as glass fiber and polyamide membranes. The highest dynamic binding capacitiy was determined for cellulose with 5.08E + 07 pfu per cm² membrane. Critical process parameters were optimized, using a design of experiments (DoE) approach. The determined process conditions were verified by different production batches, obtaining a mean virus yield of 91% ± 6.5%. Impurity depletion was >99% and 85% for protein and dsDNA, without nuclease treatment. Due to the method's specificity, its application to other baculoviruses, with varying surface modifications, is conceivable without major process changes. The physiological buffer conditions enable a gentle handling of the virus particles without decreasing the transduction efficacy. The simple procedure with sufficient impurity removal enables the substitution of time-consuming ultra centrifugation steps and can serve as a first process unit operation to obtain higher purities.

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

The large demand for safe and efficient viral vector-based vaccines and gene therapies against both inherited and acquired diseases accelerates the development of viral vectors. One outstanding example, the Orf virus, has a wide range of applications, a superior efficacy and an excellent safety profile combined with a reduced pathogenicity compared to other viral vectors. However, besides these favorable attributes, an efficient and scalable downstream process still needs to be developed. Recently, we screened potential chromatographic stationary phases for Orf virus purification. Based on these previous accomplishments, we developed a complete downstream process for the cell culture-derived Orf virus. The described process comprises a membrane-based clarification step, a nuclease treatment, steric exclusion chromatography, and a secondary chromatographic purification step using Capto® Core 700 resin. The applicability of this process to a variety of diverse Orf virus vectors was shown, testing two different genotypes. These studies render the possibility to apply the developed downstream scheme for both genotypes, and lead to overall virus yields of about 64 %, with step recoveries of >70 % for the clarification, and >90 % for the chromatography train. Protein concentrations of the final product are below the detection limits, and the final DNA concentration of about 1 ng per 1E + 06 infective virus units resembles a total DNA depletion of 96-98 %.

Development of a downstream process for the production of an inactivated whole hepatitis C virus vaccine.

There is a large unmet need for a prophylactic hepatitis C virus (HCV) vaccine to control the ongoing epidemic with this deadly pathogen. Many antiviral vaccines employ whole viruses as antigens. For HCV, this approach became feasible following the development of infectious cell culture systems for virus production. However, the lack of efficient downstream processes (DSP) for HCV purification poses a roadblock for the development of a whole virus vaccine. Using cell culture-derived genotype 1a HCV we developed a scalable and efficient DSP train, employing commonly used clarification and ultrafiltration techniques, followed by two membrane-based chromatography steps. For virus capture, steric exclusion chromatography using cellulose membranes was established, resulting in a virtually complete virus recovery with > 99% protein and 84% DNA depletion. Virus polishing was achieved by sulphated cellulose membrane adsorbers with ~ 50% virus recovery and > 99% protein and 90% DNA depletion. Additional nuclease digestion resulted in 99% overall DNA depletion with final DNA concentrations of 2 ng/mL. Process results were comparable for cell culture-derived HCV of another major genotype (5a). This study provides proof-of-concept for establishment of an efficient and economically attractive DSP with potential application for production of an inactivated whole virus vaccine against HCV for human use.