Peter Czermak

• Professor Dr.-Ing. Dipl.-Ing.
• Professor (Full) at Mittelhessen University of Applied Sciences

Vibrio natriegens is widely used as a production host for biotechnological processes due to its superior maximum glucose consumption rate, high growth rate, and abundant ribosomes. Most bioprocesses also need a scalable biomass separation step. This can be achieved by cross-flow filtration with ceramic membranes, although the membrane pores are sus...

The presence of micropollutants in aquatic environments is an increasing global concern due to their persistence and potential harmful effects on aquatic organisms. Among the most concerning of these micropollutants are microplastics, pharmaceutical compounds, personal care products, and industrial chemicals, posing a significant threat to human he...

Vibrio natriegens is widely used as a production host for biotechnological processes due to its superior maximum glucose consumption rate, high growth rate, and abundant ribosomes. Most bioprocesses also need a scalable biomass separation step. This can be achieved by cross-flow filtration with ceramic membranes, although the membrane pores are sus...

Pharmaceutical compounds in aquatic environments are recognized as emerging contaminants due to their potential risks to living organisms. The growing accessibility, widespread use, and disposal of pharmaceuticals have led to a significant increase in their presence within wastewater streams. Due to their persistence and resistance to biodegradatio...

Oncolytic measles virus (MeV) is a promising anti-cancer treatment. However, the production of high titers of infectious MeV (typically 10⁷–10⁹ TCID50 per dose) is challenging because the virus is unstable under typical production conditions. The objective of this study was to investigate how the multiplicity of infection (MOI) and different media—...

Oncolytic measles virus (MeV) is a promising anti-cancer treatment. However, the production of high titers of infectious MeV (typically 107–109 TCID50 per dose) is challenging because the virus is unstable under typical production conditions. The objective of this study was to investigate how the multiplicity of infection (MOI) and different media...

The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up,...

Fructo-oligosaccharides (FOS) are often produced via enzymatic conversion in industry. Enzyme membrane bioreactor (EMBR) can enhance the catalytic efficiency by in-situ product removal. Its major bottleneck is the fouling caused by the deposition of enzyme on membrane surface. An EMBR system with a compact rotating disc device was established for c...

Yellow Fever (YF) is a severe disease that, while preventable through vaccination, lacks rapid intervention options for those already infected. There is an urgent need for passive immunization techniques using YF-virus-like particles (YF-VLPs). To address this, we successfully established a bioreactor-based production process for YF-VLPs, leveragin...

Among the variety of currently investigated oncolytic viruses, the excellent safety record, the natural oncotropism, and the possibilities of genetic engineering make the oncolytic Measles virus (oMeV) particularly promising for cancer (immuno-)virotherapy. Clinical trials of oMeV indicate an efficient and well-tolerated treatment. However, high do...

Ein unerlässlicher Bestandteil der modernen Medizin ist der Einsatz von pharmazeutischen Wirkstoffen. Ein Großteil der Pharmazeutika gelangt allerdings nach der Verabreichung durch Ausscheidungen in die kommunalen Kläranlagen. Dort können sie aufgrund ihrer schlechten biologischen Abbaubarkeit nicht umfassend eliminiert werden und gelangen über den...

Schistosomiasis is an infectious disease caused by blood flukes of the genus Schistosoma and affects approximately 200 million people worldwide. Since Praziquantel (PZQ) is the only drug for schistosomiasis, alternatives are needed. By a biochemical approach, we identified a tegumentally expressed aldehyde dehydrogenase (ALDH) of S. mansoni, SmALDH...

Mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) are considered a promising therapeutic tool in cell therapy due to their immunomodulatory, regenerative and angiogenic capabilities. However, there is a lack of process knowledge, particularly for a large-scale production of MSC-EV using fully controlled stirred tank bioreactor...

Intravenous lipid emulsions are biocompatible formulations used as clinical nutrition products and lipid-based delivery systems for sparingly soluble drugs. However, the particle-size distribution is associated with risks of embolism. Accordingly, the mean particle diameter (MPD) and particle-distribution tailing (characterized as the pFAT5 value)...

The good manufacturing practices (GMP) and process analytical technology (PAT) initiatives of the US Food and Drug Administration, in conjunction with International Council for Harmonisation (ICH) quality guidelines Q8, Q9, and Q10, ensure that manufacturing processes for parenteral formulations meet the requirements of increasingly strict regulati...

Planctomycetes such as Planctopirus limnophila offer a promising source of bioactive molecules, particularly when they switch from planktonic to sessile growth, but little is known about the corresponding biosynthetic gene clusters and how they are activated. We therefore screened for factors that promote sessile growth and biofilm formation to ena...

Hemicelluloses are an abundant biopolymer resource with interesting properties for applications in coatings and composite materials. The objective of this investigation was to identify variables of industrially relevant extraction processes that increase the purity of hemicelluloses extracted from fruit residues. Our main finding is that extraction...

Background Vibrio natriegens is a Gram-negative bacterium that offers a greater metabolic capacity than Escherichia coli for the production of recombinant proteins. This potential includes a low minimum doubling time of 7 min and a high maximum glucose uptake rate of 3.9 g*g⁻¹*h⁻¹. We therefore tested the ability of V. natriegens to produce the ins...

Background Spodoptera frugiperda (Sf-9) cells are often used to produce recombinant proteins by transient expression. The culture medium contributes to the efficiency of protein production, but the effect of modern insect cell culture media on Sf-9 cells has not been evaluated for different transient expression systems. Accordingly, we compared the...

The transient transfection of mammalian cells is a rapid and versatile platform for the manufacture of recombinant proteins, but industrial processes depend on reliable scalability and efficient conversion from adherent to suspension cell cultures. Here we describe the optimized transfection of HEK 293 T cells in both culture formats. DMEM was the...

Nutrient-rich side streams can be used in fermentation media to produce biomaterials, biofuels and other high-value products, contributing to the circular bioeconomy. However, the efficient valorization of side streams requires media optimized for the fermentation organism and the desired product. In an exemplary process, we investigated statistica...

The antibiotic resistance crisis has prompted research into alternative candidates such as antimicrobial peptides (AMPs). However, the demand for such molecules can only be met by continuous production processes, which achieve high product yields and offer compatibility with the Quality-by-Design initiative by implementing process analytical techno...

Measles virus (MV) is an important representative of a new class of cancer therapeutics known as oncolytic viruses. However, process intensification for the downstream purification of this fragile product is challenging. We previously found that a mid-range molecular weight cut-off (300 kDa) is optimal for the concentration of MV. Here, we tested c...

Aureolysin, a secreted metallopeptidase (MP) from the thermolysin family, functions as a major virulence factor in Staphylococcus aureus. No specific aureolysin inhibitors have yet been described, making this an important target for the development of novel antimicrobial drugs in times of rampant antibiotic resistance. Although small-molecule inhib...

Non-ribosomal peptide synthases (NRPSs) generate versatile bioactive peptides by incorporating non-proteinogenic amino acids and catalyzing diverse modifications. Here, we developed an efficient downstream process for the capture, intermediate purification and polishing of a rhabdopeptide (RXP) produced by the NRPS VietABC. Many typical unit operat...

Fructo-oligosaccharides (FOS) are prebiotic sugar substitutes that can be produced from sucrose using fructosyltransferases (FTases). However, the economic value of this process is limited by inefficient product purification and enzyme reusability. In this study, enzyme-free FOS preparations were produced by immobilizing the FTase on resin carriers...

Background Planctomycetes is a phylum of biofilm-forming bacteria with numerous biosynthetic gene clusters, offering a promising source of new bioactive secondary metabolites. However, the current generation of chemically defined media achieves only low biomass yields, hindering research on these species. We therefore developed a chemically defined...

Cancer therapy with oncolytic measles virus (MV) requires high titers of pure, infectious virus particles (~10⁸ times more particles per dose than the measles vaccine). A gentle but efficient downstream purification process is therefore essential. Here we investigated the suitability of ion-exchange chromatography for the purification of MV using r...

Background The heterologous expression of parasitic proteins is challenging because the sequence composition often differs significantly from host preferences. However, the production of such proteins is important because they are potential drug targets and can be screened for interactions with new lead compounds. Here we compared two expression sy...

The valorization of agro-industrial residues using yeasts as biocatalysts requires efficient methods for biomass separation. Filtration with ceramic membranes is suitable for this task, however, the challenge of flux decline and the unavoidable cleaning must be taken into account. We investigated the filtration of fermentation broth and its compone...

Background Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited...

The characterization of membrane fouling provides valuable information about the performance and operational range of filtration processes. The range of operational parameters for the purification and concentration of protein aggregates from cell lysates by ultrafiltration is determined by evaluating the filtration resistances. We therefore investi...

Probiotics are microbes that promote health when consumed in sufficient amounts. They are present in many fermented foods or can be provided directly as supplements. Probiotics utilize non-digestible prebiotic oligosaccharides for growth in the intestinal tract, contributing to a healthy microbiome. The oligosaccharides favored by probiotics are sp...

Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) are gaining importance as so-called advanced therapy medicinal products. However, the manufacturing of such products is challenging due to their complexity and sensitivity to intrinsic and environmental parameters, which determine their therapeutic functionality. MSCs respond stro...

The enzymatic conversion of sucrose to fructo-oligosaccharides (FOS) catalyzed by Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus , under free condition was studied. A mathematical analysis of the transfructosylation reactions was carried out to estimate the dynamic and steady-state performance of an enzyme membrane...

de Im Rahmen dieser Forschungsarbeit wurde ein effizientes membranbasiertes Verfahren zur Rückgewinnung von gereinigtem Ligninsulfonat (LS) aus der Dünnlauge des chemischen Holzaufschlusses mithilfe von keramischen Ultrafiltrationsmembranen entwickelt. Das Ziel dabei war, die Ligninsulfonate von niedermolekularen organischen Verunreinigungen (Zucke...

Appropriately designed and operated enzyme membrane bioreactors (EMBRs) can fully utilize the activity of free, soluble enzymes while maximizing enzyme reusability and simultaneously removing the enzyme‐free product in situ. A filtration model system was applied to determine critical EMBR design factors suitable for continuous enzymatic production...

The enzymatic production of prebiotic fructo-oligosaccharides (FOS) from sucrose involves fructosyltransferases (FFTs) and invertases, both of which catalyze forward (transferase) and reverse (hydrolysis) reactions. FOS yields can therefore be increased by favoring the forward reaction. We investigated process conditions that favored transferase ac...

The discovery ofthe genome-editing tool CRISPR-Cas9 is revolutionizing the world ofgene therapy and will extend the gene therapy product pipeline. While applying gene therapy products, the main difficulty is an efficient and effective transfer of the nucleic acids carrying the relevant information to their target destination, the nucleus ofthe cell...

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...

Several vaccines are already produced using the baculovirus expression vector system (BEVS). This chapter describes methods for generating recombinant baculoviral DNA (also called bacmid) for cultivating Spodoptera frugiperda Sf-9 cells and producing a baculovirus stock from the recombinant bacmid and for producing a protein-based vaccine with the...

Fructo-oligosaccharides (FOS) are low-calorie sweeteners that can be synthesized by the transfructosylation of sucrose using enzymes known as fructosyltransferases. However, enzymatic conversion is inhibited by the accumulation of glucose as a byproduct, which limits the conversion rate and yield. We therefore developed a semi-continuous production...

The oil and gas industry generates a large volume of contaminated water (produced water) which must be processed to recover oil before discharge. Here, we evaluated the performance and fouling behavior of commercial ceramic silicon carbide membranes in the treatment of oily wastewaters. In this context, microfiltration and ultrafiltration ceramic m...

Oncolytic viruses (including measles virus) offer an alternative approach to reduce the high mortality rate of late-stage cancer. Several measles virus strains infect and lyse cancer cells efficiently, but the broad application of this therapeutic concept is hindered by the large number of infectious particles required (10⁸–10¹² TCID50 per dose). T...

Short-chain prebiotic fructo-oligosaccharides (FOS) produced by enzymatic conversion from sucrose often contains high concentration of monosaccharides as byproducts. In addition to conventional physical/chemical purification processes, microbial treatment is an alternative method to remove these byproducts. We used Bacillus coagulans to reduce the...

The production of biopharmaceuticals in cell culture involves stringent controls to ensure product safety and quality. To meet these requirements, quality by design principles must be applied during the development of cell culture processes so that quality is built into the product by understanding the manufacturing process. One key aspect is proce...

The therapeutic use of oncolytic measles virus (MV) for cancer treatment requires >10⁸ infectious MV particles per dose in a highly pure form. The concentration/purification of viruses is typically achieved by tangential flow filtration (TFF) but the efficiency of this process for the preparation of MV has not been tested in detail. We therefore in...

Fructo-oligosaccharides (FOS) are linear fructans comprising 2–5 fructose units linked to a terminal glucose residue. They are widely used as food and feed additives due to their sweetness, low calorific value, and prebiotic properties. Here we describe the synthesis of FOS catalyzed by a cell-free crude enzyme solution containing recombinant fruct...

The optimization of recombinant protein production in bacteria is an important stage of process development, especially for difficult-to-express proteins that are particularly sensitive or recalcitrant. The optimal expression level must be neither too low, which would limit yields, nor too high, which would promote the formation of insoluble inclus...

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 transf...

Oncolytic measles virus (MV) is a promising treatment for cancer but titers of up to 1011 infectious particles per dose are needed for therapeutic efficacy, which requires an efficient, robust, and scalable production process. MV is highly sensitive to process conditions, and a substantial fraction of the virus is lost during current purification p...

Fructo-oligosaccharides (FOS) are prebiotic low-calorie sweeteners that are synthesized by the transfer of fructose units from sucrose by enzymes known as fructosyltransferases. If these enzymes generate β-(2,6) glycosidic bonds, the resulting oligosaccharides belong to the neoseries (neoFOS). Here, we characterized the properties of three differen...

Peptides and proteins containing disulfide bonds can be produced in Escherichia coli by targeting the oxidizing periplasm, co-expressing isomerases or chaperons, refolding from inclusion bodies, or by using redox-engineered E. coli strains. Thus far, protein expression in glutathione reductase and thioredoxin reductase deficient (Δgor ΔtrxB) E. col...

de Die Abtrennung von Wasser aus Dieselkraftstoff ist aus sicherheitstechnischen, ökologischen sowie ökonomischen Gründen sehr wichtig, da es andernfalls u. a. zu einer schlechteren Verbrennungseffizienz und Motorproblemen kommen kann. Zudem lässt sich aus Ultra‐low‐sulfur‐Diesel (ULSD) das freie Wasser mit den klassischen Abscheidesystemen nur unz...

Oncolytic Measles virus is a promising candidate for cancer treatment, but clinical studies have shown that extremely high doses (up to 10¹¹ TCID50 per dose) are required to effect a cure. Very high titers of the virus must therefore be achieved during production to ensure an adequate supply. We have previously shown that Measles virus can be produ...

Several new classes of biologicals offer hope that we may soon be able to address some of our most intractable health challenges. These products include antimicrobial peptides and bacterial toxins for the treatment and prevention of infectious diseases and advanced therapy medicinal products (gene and cell therapy, including viruses) that may turn...

The Cry4AaCter tag is a pull-down tag which promotes the formation of inclusion bodies (IBs) that can be resolubilized in an alkaline buffer. Here, we used the Cry4AaCter tag to create a platform for the production of antimicrobial peptides (AMPs) in Escherichia coli featuring a uniform resolubilization process independent of the peptide fused to t...

Objectives The objective of this study was to investigate the therapeutic potential of the insect metalloproteinase inhibitor (IMPI) from Galleria mellonella, the only known specific inhibitor of M4 metalloproteinases. Methods The fusion protein IMPI‐GST (glutathione‐S‐transferase) was produced by fermentation in Escherichia coli and was tested fo...

The industrial-scale production of fructo-oligosaccharides from sucrose requires large quantities of the enzyme fructosyltransferase. An Aspergillus terreus fructosyltransferase was therefore expressed in Kluyveromyces lactis GG799 and secreted into the medium. K. lactis was cultivated in shaking flasks at 30°C and 250 rpm using either rich or chem...

The generation of monoclonal cell lines is an important early process development step for recombinant protein production. Although single-cell cloning is an established method in mammalian cell lines, straightforward protocols are not yet available for insect cells. We describe a new method for the generation of monoclonal insect cells without usi...

In Escherichia coli, the production of active disulfide bonds is achieved by expression into the oxidizing periplasm, by refolding from inclusion bodies, by co-expression of a sulfhydryl oxidase and a disulfide bond isomerase, or by expression into a genetically engineered oxidizing cytoplasm. Thus, engineered strains with oxidizing cytoplasm were...

The cover image, by Tanja A. Grein et al., is based on the Article High titer oncolytic measles virus production process by integration of dielectric spectroscopy as online monitoring system, DOI: 10.1002/bit.26538.

tandard production processes for antimicrobial peptides (AMPs) comprise of batch cultivations in upstream and chromatographic purification in downstream. In spite of limitations in scale-up and high costs, chromatography still remains industrial gold standard. To overcome this bottleneck, alternative downstream strategies using aggregating tags and...

The production of recombinant proteins in bioreactors requires real-time process monitoring and control to increase process efficiency and to meet the requirements for a comprehensive audit trail. The combination of optical near-infrared turbidity sensors and dielectric spectroscopy provides diverse system information because different measurement...

Diabetes is a prominent health problem caused by the failure of pancreatic beta cells. One therapeutic approach is the transplantation of functional beta cells, but it is difficult to generate sufficient beta cells in vitro and to ensure these cells remain viable at the transplantation site. Beta cells suffer from hypoxia, undergo apoptosis, or are...

A novel Dye-decolorizing peroxidase from the basidiomycete Pleurotus sapidus was screened for dyedecolorizing peroxidase activity with 2,2‘-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid), Remazol Brilliant Blue R and Guaiacol. Additionally, the catalytic efficiency on degrading β-carotene into volatile products, and the catalyst storage stabili...

Short-chain fructo-oligosaccharides (FOS) are considered as low-calorie carbohydrates with prebiotic function. They can be produced from sucrose by fructosyltransferase activity, resulting in a mixture of saccharides with different chain lengths. Current practice for carbohydrate analysis involves the use of time-costly and off-line chromatographic...