Volker Sandig

New avian suspension cell lines provide production of influenza virus and MVA in serum-free media: Studies on growth, metabolism and virus propagation

Few suspension cells can be used for vaccine manufacturing today as they either do not meet requirements from health regulatory authorities or do not produce high virus titres. Two new avian designer cell lines (AGE1.CR and AGE1.CR.pIX) that have been adapted to grow in suspension in serum-free medium were evaluated for their potential as host cells for influenza and modified vaccinia Ankara (MVA, wild type) vaccine production. Their metabolism was studied during growth in static (T-flasks) and dynamic cultivation systems (roller bottles, stirred tank reactor, wave bioreactor). High cell concentrations up to 5.8x10(6)cells/mL were obtained with doubling times of 23h for AGE1.CR and 35h for AGE1.CR.pIX, respectively. Both viruses were produced to high titres (3.5 logHA/100 microL for influenza virus, 3.2x10(8)pfu/mL for MVA). Hence, the CR cell lines are an appropriate substrate for pharmaceutical influenza and MVA production.

Production of non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase

All IgG-type antibodies are N-glycosylated in their Fc part at Asn-297. Typically, a fucose residue is attached to the first N-acetylglucosamine of these complex-type N-glycans. Antibodies lacking core fucosylation show a significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and an increased efficacy of anti-tumor activity. In cases where the clinical efficacy of an antibody is to some extent mediated by its ADCC effector function, afucosylated N-glycans could help to reduce dose requirement and save manufacturing costs. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate here that heterologous expression of the prokaryotic enzyme GDP-6-deoxy-d-lyxo-4-hexulose reductase within the cytosol can efficiently deflect the fucose de novo pathway. Antibody-producing CHO cells that were modified in this way secrete antibodies lacking core fucose as demonstrated by MALDI-TOF mass spectrometry and HPAEC-PAD monosaccharide analysis. Engineering of the fucose de novo pathway has led to the construction of IgGs with a strongly enhanced ADCC effector function. The method described here should have broad practical applicability for the development of next-generation therapeutic antibodies.

An avian cell line designed for production of highly attenuated viruses

Several viral vaccines, including highly promising vectors such as modified vaccinia Ankara (MVA), are produced on chicken embryo fibroblasts. Dependence on primary cells complicates production especially in large vaccination programs. With primary cells it is also not possible to create packaging lines for replication-deficient vectors that are adapted to proliferation in an avian host. To obviate requirement for primary cells permanent lines from specific tissues of muscovy duck were derived (AGE1.CR, CS, and CA) and further modified: we demonstrate that stable expression of the structural gene pIX from human adenovirus increases titers for unrelated poxvirus in the avian cells. This augmentation appears to be mediated via induction of heat shock and thus provides a novel cellular substrate that may allow further attenuation of vaccine strains.

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis.

For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines.

Glycan analysis in cell culture-based influenza vaccine production: Influence of host cell line and virus strain on the glycosylation pattern of viral hemagglutinin

Mammalian cell culture processes are commonly used for production of recombinant glycoproteins, antibodies and viral vaccines. Since several years there is an increasing interest in cell culture-based influenza vaccine production to overcome limitations of egg-based production systems, to improve vaccine supply and to increase flexibility in vaccine manufacturing. With the switch of the production system several key questions concerning the possible impact of host cell lines on antigen quality, passage-dependent selection of certain viral phenotypes or changes in hemagglutinin (HA) conformation have to be addressed to guarantee safety and efficiency of vaccines. In contrast to the production of recombinant glycoproteins, comparatively little is known regarding glycosylation of HA, derived from mammalian cell cultures. Within this study, a capillary DNA-sequencer (based on CGE-LIF technology), was utilized for N-glycan analysis of three different influenza virus strains, which were replicated in six different cell lines. Detailed results concerning the influence of the host cell line on complexity and composition of the HA N-glycosylation pattern, are presented. Strong host cell but also virus type and subtype dependence of HA N-glycosylation was found. Clear differences were already observed, by N-glycan fingerprint comparison. Further structural investigations of the N-glycan pools revealed that host cell dependence of HA N-glycosylation was mainly related to minor variations of the (monomeric) constitution of single N-glycans. To some extent, shifts in the N-glycan pool composition regarding the proportion of different N-glycan types were observed. In contrast to this, a principal switch of the N-glycan type attached to HA was observed when comparing different virus types (A and B) and subtypes (H1N1 and H3N2).

N-Glycosylation and Biological Activity of Recombinant Human Alpha1-Antitrypsin Expressed in a Novel Human Neuronal Cell Line

Human alpha‐1‐antitrypsin (A1AT) is a protease inhibitor that is involved in the protection of lungs from neutrophil elastase enzyme that drastically modifies tissue functioning. The glycoprotein consists of 394 amino acids and is N‐glycosylated at Asn‐46, Asn‐83, and Asn‐247. A1AT deficiency is currently treated with A1AT that is purified from human serum. In view of therapeutic applications, rA1AT was produced using a novel human neuronal cell line (AGE1.HN®) and we investigated the N‐glycosylation pattern as well as the in vitro anti‐inflammatory activity of the recombinant glycoprotein. rA1AT (300 mg/L) was biologically active as analyzed using elastase assay. The N‐glycan pool, released by PNGase F digestion, was characterized using 2D‐HPLC, MALDI‐TOF mass spectrometry, and by exoglycosidase digestions. A total of 28 N‐glycan structures were identified, ranging from diantennary to tetraantennary complex‐type N‐glycans. Most of the N‐glycans were found to be (α1–6) core‐fucosylated and part of them contain the Lewis X epitope. The two major compounds are a monosialylated diantennary difucosylated glycan and a disialylated diantennary core‐fucosylated glycan, representing 25% and 18% of the total N‐glycan pool, respectively. Analysis of the site‐specificity revealed that Asn‐247 was mainly occupied by diantennary N‐glycans whereas Asn‐46 was occupied by di‐, and triantennary N‐glycans. Asn‐83 was exclusively occupied by sialylated tri‐ and tetraantennary N‐glycans. Next, we evaluated the anti‐inflammatory activity of rA1AT using A1AT purified from human serum as a reference. rA1AT was found to inhibit the production of TNF‐α in neutrophils and monocytes as commercial A1AT does. Biotechnol. Bioeng. 2011;108:2118–2128.

Establishment of fruit bat cells (Rousettus aegyptiacus) as a model system for the investigation of filoviral infection.

Background The fruit bat species Rousettus aegyptiacus was identified as a potential reservoir for the highly pathogenic filovirus Marburg virus. To establish a basis for a molecular understanding of the biology of filoviruses in the reservoir host, we have adapted a set of molecular tools for investigation of filovirus replication in a recently developed cell line, R06E, derived from the species Rousettus aegyptiacus. Methodology/Principal Findings Upon infection with Ebola or Marburg viruses, R06E cells produced viral titers comparable to VeroE6 cells, as shown by TCID50 analysis. Electron microscopic analysis of infected cells revealed morphological signs of filovirus infection as described for human- and monkey-derived cell lines. Using R06E cells, we detected an unusually high amount of intracellular viral proteins, which correlated with the accumulation of high numbers of filoviral nucleocapsids in the cytoplasm. We established protocols to produce Marburg infectious virus-like particles from R06E cells, which were then used to infect naïve target cells to investigate primary transcription. This was not possible with other cell lines previously tested. Moreover, we established protocols to reliably rescue recombinant Marburg viruses from R06E cells. Conclusion/Significance These data indicated that R06E cells are highly suitable to investigate the biology of filoviruses in cells derived from their presumed reservoir.

Metabolite channeling and compartmentation in the human cell line AGE1.HN determined by 13C labeling experiments and 13C metabolic flux analysis

This study focused on analyzing active pathways and the metabolic flux distribution in human neuronal AGE1.HN cells that is a desirable basis for a rational design and optimization of producing cell lines and production processes for biopharmaceuticals. (13)C-labeling experiments and (13)C metabolic flux analysis were conducted using glucose, glutamine, alanine and lactate tracers in parallel experiments. Connections between cytosolic and mitochondrial metabolite pools were verified, e.g., flux from TCA cycle metabolite (13)C to glycolytic metabolites. It was also found that lactate and alanine are produced from the same pyruvate pool and that consumed alanine is mainly directly metabolized and secreted as lactate. Activity of the pentose phosphate pathway was low being around 2.3% of the glucose uptake flux. This might be compensated in AGE1.HN by high mitochondrial malic enzyme flux producing NADPH. Mitochondrial pyruvate transport was almost zero. Instead pyruvate carbons were channeled via oxaloacetate into the TCA cycle which was mainly fed via α-ketoglutarate and oxaloacetate during the investigated phase. The data indicate that further optimization of this cell line should focus on the improved substrate usage which can be accomplished by an improved connectivity between glycolytic and mitochondrial pyruvate pools or by better control of the substrate uptake.

A chemically defined production process for highly attenuated poxviruses.

Highly attenuated poxviruses are promising vectors for protective and therapeutic vaccines. These vectors do not replicate in human cells and can therefore be safely given even to immunocompromised recipients. They can accommodate very large inserts and provide strong stimulation of the immune system against the vectored antigen. Disadvantages include that very high numbers of infectious units are required per dose for full efficacy. Because they are difficult to produce, improved cellular substrates and processes are urgently needed to facilitate programs intended to reach a large number of vaccinees. We have developed a fully scalable and very efficient chemically-defined production process for modified vaccinia Ankara (MVA), canarypox (CNPV, strain ALVAC) and fowlpox viruses (FPV) based on a continuous cell line.

Cell lines from the Egyptian fruit bat are permissive for modified vaccinia Ankara

Abstract Bats are reservoir hosts for a spectrum of infectious diseases. Some pathogens (such as Hendra, Nipah and Marburg viruses) appear to use mainly fruit bats as reservoir. We describe designed immortalization of primary fetal cells from the Egyptian fruit bat (Rousettus aegyptiacus) to facilitate isolation and characterization of pathogens associated with these mammals. Three cell lines with different properties were recovered and successful immortalization was confirmed by continuous cultivation for over 18 months. Surprisingly, the cell lines are fully permissive for a highly attenuated poxvirus, modified vaccinia Ankara (MVA). MVA is a safe and well characterized vaccine vector that cannot replicate in most mammalian cells. High permissivity of Rousettus cell lines could justify testing bats for susceptibility to MVA as a replication competent vector with low zoonotic potential to induce herd immunity in bat colonies against viruses causing rabies or haemorrhagic fevers.