1. Purification Platform for Poxviruses by Filtration Process

Poxviruses are potent viral vectors for versatile applications in gene therapy, cancer research and for novel vaccines against infectious diseases. The pharmaceutical model organisms Modified Vaccinia Ankara Virus (MVA) and Orf Virus (ORFV) are used to develop a platform technology for the purification of cell culture-derived poxviruses. To eliminate any specific adsorption process for the target virus, the classical adsorption chromatography step is replaced by filtration-based process units. After clarification and nuclease treatment of the virus harvest, the downstream process is based on tangential flow filtration (TFF) followed by flow-through polishing chromatography. The aim of the research project is to provide a productive and economic manufacturing process with high space-time yields, effective product separation from impurities, scalability up to production scale, and simplified transfer to a current Good Manufacturing Practice (cGMP) facility.

Literature and patents of the working group on the topic:

Eilts F, Labisch JJ, Orbay S, Harsy YMJ, Steger M, Pagallies F, et al. Stability studies for the identification of critical process parameters for a pharmaceutical production of the Orf virus. Vaccine. 2023;41(32):4731-42.

Eilts F, Harsy YMJ, Lothert K, Pagallies F, Amann R, Wolff MW. An investigation of excipients for a stable Orf viral vector formulation. Virus Res. 2023;336:199213.

Eilts F, Steger M, Pagallies F, Rziha HJ, Hardt M, Amann R, et al. Comparison of sample preparation techniques for the physicochemical characterization of Orf virus particles. J Virol Methods. 2022;310:114614.

Eilts F, Lothert K, Orbay S, Pagallies F, Amann R, Wolff MW. A Summary of Practical Considerations for the Application of the Steric Exclusion Chromatography for the Purification of the Orf Viral Vector. Membranes (Basel). 2022;12(11):1070.

Lothert K, Pagallies F, Eilts F, Sivanesapillai A, Hardt M, Moebus A, et al. A scalable downstream process for the purification of the cell culture-derived Orf virus for human or veterinary applications. J Biotechnol. 2020;323:221-30.

Lothert K, Pagallies F, Feger T, Amann R, Wolff MW. Selection of chromatographic methods for the purification of cell culture-derived Orf virus for its application as a vaccine or viral vector. J Biotechnol. 2020;323:62-72.

Wolff M, Opitz L, Reichl U, Method for the preparation of sulfated cellulose membranes and sulfated cellulose membranes. EU and USA patent EP 2144937 B1, US 8,173,021 B2. 2012.

Wolff MW, Siewert C, Lehmann S, Hansen SP, Djurup R, Faber R, et al. Capturing of cell culture-derived modified Vaccinia Ankara virus by ion exchange and pseudo-affinity membrane adsorbers. Biotechnol Bioeng. 2010;105(4):761-9.

Wolff MW, Siewert C, Hansen SP, Faber R, Reichl U. Purification of cell culture-derived modified vaccinia ankara virus by pseudo-affinity membrane adsorbers and hydrophobic interaction chromatography. Biotechnol Bioeng. 2010;107(2):312-20.

Post-Hansen S, Faber R, Reichl U, Wolff M, Gram AP Purification of Vaccinia viruses using hydrophobic interaction chromatography patent US 8,470,578 B2. 2010.

2. Continuous chromatographic Purification of Viral Vectors

Viral vectors are playing an increasingly important role in the development of vaccines, offering an efficient method for the delivery of genetic material into target cells. In cell-based production processes for the manufacture of gene therapeutics or viral vaccines, the subsequent downstream process often represents a significant bottleneck with considerable scope for optimization. In collaboration with our industry partner (Tosoh Bioscience), this project aims to improve the enhance the efficiency of the purification process by developing novel continuous chromatographic purification methods utilizing a range of model viruses, including Orf virus, Modified Vaccinia Virus Ankara (MVA) and Adeno-Associated Virus (AAV). The critical process parameters of the continuous chromatographic methods as well as the resins used, are optimized in terms of product yield and purity. This is done with a view to ensuring scalability, transferability to the production process and cost-effectiveness.

Literature of the working group on the topic:

Fortuna AR, Taft F, Villain L, Wolff MW, Reichl U. Continuous purification of influenza A virus particles using pseudo-affinity membrane chromatography. J Biotechnol. 2021;342:139-48.

Fischer LM, Wolff MW, Reichl U. Purification of cell culture-derived influenza A virus via continuous anion exchange chromatography on monoliths. Vaccine. 2018;36(22):3153-60.

3. Purification of Plasma Proteins using continuous Chromatography

The composition of blood is comprised of two primary elements: the cellular component and the blood plasma. The latter consists of a variety of dissolved substances, including nutrients, hormones, and proteins such as antibodies, albumin, and coagulation factors. Plasma proteins play a pivotal role in a multitude of biological functions and are employed in research, diagnostic applications and therapeutics, with some still being purified directly from blood plasma. This includes the use of plasm proteins in therapeutic applications, for which the purification process must adhere to the requisite regulatory standards. The purification of individual proteins from plasma represents a significant challenge, largely due to the inherent complexity and limited availability of this biological fluid. To date, the classic cold fractional ethanol precipitation (Cohn fractionation) and batch chromatography techniques have been employed. Continuous chromatography represents a further development of batch chromatography whereby the feed material is continuously applied and separated. This technique allows for a more efficient use of the column capacity, is easily scalable and thus saves costs by reducing buffer consumption or shortening process times. Furthermore, it allows for a more efficient use of the plasma starting material.

The objective of tis project is to develop continuous chromatography methods for the purification of defined plasma proteins.

4. Biotechnological Products derived from Insect Cell Culture

Insect cell cultures have become a vital tool in the field of biotechnology, with applications in the production of recombinant proteins, virus-like particles, and the study of viral infections. Moreover, they are utilized in a variety of genetic and cellular analyses.

Insect cells are eukaryotic, which implies that they contain the necessary cellular machinery for proper folding, assembly, and post-translational modifications of the expressed recombinant protein. In contrast, prokaryotic systems in general lack these capabilities, which often impairs the activity of the target protein. Insect cells offer several advantages over other eukaryotic cells, including ease of genetic manipulation, a favorable safety profile, and high expression levels compared to those of mammalian cells. The combination of these properties underscores the broad applicability of insect cell culture in a variety of biotechnological fields beyond pharmaceutical biotechnology. However, in other contexts, the economic viability of insect production processes is constrained by high costs associated with the cell culture media. As the regulatory requirements in other areas of biotechnology are significantly less stringent, the costs of the media used for the cultivation of the production cells can be considerably reduced by using a bioeconomic approach.

Biological insecticides are defined as a naturally occurring products derived from living organisms or their decomposition products. They are utilized as an environmentally benign approach to friendly control insect pests, eschewing the use of deleterious chemicals.  One example of a biological insecticide is a virus that infects and kills specific insects, a viral insecticide. These viruses are typically innocuous to humans and other insects, offering targeted pest control without significantly impairing the biodiversity of insects.

The objective of this project is to develop and optimize insect cell media for S2-cells in terms of cost-effectiveness, with a view to using it for the production of insect viruses that can be employed as viral insecticides as an alternative to chemical insecticides with a broad spectrum of activity.

The media to be developed for the production of viral insecticides represent a significant economic challenge due to the price pressure of the classical chemical insecticides, suggesting that the insect culture media to be developed could be used in the future for the production of a wide range of non-pharmaceutical biotechnology products.

5. Adeno-Associated-Virus project in Cooperation with Sartorius Stedim Biotech GmbH

Context

• Adeno-associated viruses (AAV) are widely used viral vectors for in vivo gene therapy.

Objective

• AAV purification and analytics are challenging. Therefore, the project's scope is to establish novel, simplified, and more efficient purification and analytical methods.

Methods

• Filtration
• Chromatography (capture, polishing)
• Several analytical techniques including dPCR, BLI, flow cytometry, live-cell imaging, and many more.
• Cell culture (shake flask and bioreactor-scale AAV production)

Literature of the working group on the topic:

Meierrieks F, Kour A, Patz M, Pflanz K, Wolff MW, Pickl A. Unveiling the secrets of adeno-associated virus: novel high-throughput approaches for the quantification of multiple serotypes. Mol Ther Methods Clin Dev. 2023;31:101118.

Meierrieks F, Pickl A, Wolff MW. A robust and efficient alluvial filtration method for the clarification of adeno-associated viruses from crude cell lysates. J Biotechnol. 2023;367:31-41.

Meierrieks F, Weltken A, Pflanz K, Pickl A, Graf B, Wolff MW. A Novel and Simplified Anion Exchange Flow-Through Polishing Approach for the Separation of Full From Empty Adeno-Associated Virus Capsids. Biotechnology Journal. 2024;19(10):e202400430.