Capturing of cell culture-derived modified Vaccinia Ankara virus by ion exchange and pseudo-affinity membrane adsorbers

This review covers the structure and function of heparin and heparan sulfate glycosaminoglycans. Their chemical structures are discussed, including recently developed methods for sequencing picomole to nanomole quantities of heparin- and heparan sulfate-derived oligosaccharides. The biosynthesis of heparin and heparan sulfate is reviewed as it relates to their diverse and varied structures, and their biological activities and functions are discussed.

Baby hamster kidney cells (BHK), a well-characterized, easily maintained cell line, supported MVA growth and as proficient expression of the E. coli lacZ reporter gene as the highly efficient CEF, whereas other cell lines were non-permissive or allowed only very limited MVA replication. Importantly, no virus production occurred in patient-derived infected primary human cells. These results emphasize the safety and now improved accessibility of MVA for the development of expression vectors and live recombinant vaccines. Diseases such as HIV/AIDS, tuberculosis, malaria and cancer are prime targets for prophylactic or therapeutic vaccination, but have proven partially or wholly resistant to traditional approaches to vaccine design.

The process involves a number of complex tasks and unit operations ranging from selection of host cells and virus strains for the cultivation in bioreactors to the purification and formulation of the final product. For the majority of cell culture-derived products, efforts focused on maximization of bioreactor yields, whereas design and optimization of downstream processes were often neglected. Owing to this biased focus, downstream procedures today often constitute a bottleneck in various manufacturing processes and account for the majority of the overall production costs.

The most efficient media, a polymer and a cellulose membrane, have been further characterized by protein and host cell DNA measurements. Separations based on the polymer matrix and the cellulose membrane removed contaminating DNA to 0.2 and 1%, respectively. Total protein contents were decreased to 50 and 31%, respectively. The EEL-membrane showed the highest influenza virus binding capacity. These characteristics demonstrate that EEL affinity chromatography is a promising candidate for capturing influenza viruses from MDCK cell culture broths in addition to currently applied chromatographic media.

Population balance modeling to improve influenza vaccine production

The development of efficient purification trains is restricted by limited analytical methods, as well as by the complexity of large viruses, as well as the high variability in starting material from cell culture. Vaccinia virus holds great potential as an oncolytic and immunotherapeutic vaccine against a broad spectrum of cancers. In this work, monolith-based capture and polishing chromatographic steps for vaccinia virus Lister strain has been developed. Virus produced in CV-1 cells was harvested and passed through a 0.8μm pre-filter before loading onto CIEX, AIEX and HIC CIM monoliths.

The chromatographic separation characteristics of reinforced cellulose membranes and different matrices such as agarose, cellulose, polymer and glass particles with immobilized EEL have been determined. Results obtained were compared to affinity matrices, which are currently used in large-scale vaccine manufacturing. Mass balances for the viral membrane protein hemagglutinin showed that EEL affinity chromatography results in higher recoveries than conventional processes using Cellufine sulphate and heparinized agarose.

Cutting Edge: Mucosal Application of a Lyophilized Viral Vector Vaccine Confers Systemic and Protect…

  • Most importantly, a moderate to high level of CSE significantly increases the chance of responding in both T-CBT and FtF-CBT.
  • Compared to the centrifugation method, use of the novel magnetic sulfated cellulose particles reduced the influenza A virus particle purification time from 3.5 h to 30 min before mass spectrometry analysis.
  • Heparan sulphate preparations of low sulphate content (0.5 and 0.7 sulphate groups/disaccharide unit) failed to show any significant interaction with HSV.

Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1—-4)-D-GlcNAc and delta UA(1—-4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer.

However, efforts are currently underway to optimize these processes focusing, for example, on ion exchange or affinity based membrane adsorption chromatography. This review covers the main aspects relevant for the downstream processing of egg and mammalian cell culture-derived whole influenza viruses. Efforts to make vaccines against infectious diseases and immunotherapies for cancer have evolved to utilize a variety of heterologous expression systems such as viral vectors. These vectors are often attenuated or engineered to safely deliver genes encoding antigens of different pathogens. Adenovirus and poxvirus vectors are among the viral vectors that are most frequently used to develop prophylactic vaccines against infectious diseases as well as therapeutic cancer vaccines.

Even when the mice were inoculated with MVA intracranially, they were not affected. Significant protection against a lethal dose of an orthopoxvirus was obtained in mice following immunization with the Lister strain, while larger doses and repeated vaccination procedure, were required with MVA. The Lister virus stock applied in Israel, was found to be heterogeneous in its plaque morphology. Two variants isolated from it, showed significant attenuation for mice, when inoculated intranasally and intracranially, as compared to a third variant and to the unpurified stock of the virus. The complete genomic DNA sequence of the highly attenuated vaccinia strain modified vaccinia Ankara (MVA) was determined.

Heparin is a highly sulfated linear polysaccharide that was discovered in 1916 as an anticoagulant and has been used clinically since 1935 (see ref. 1 for review). The anticoagulant activity of heparin results from its binding to the serine protease inhibitor, antithrombin III (ATIII) (2, 3).

Thus, the attenuated phenotype of MVA is the result of numerous mutations, particularly affecting the host interactive proteins, including the ankyrin-like genes, but also involving some structural proteins. It has been suggested that heparan sulphate has a receptor function in the initial phase of the attachment of herpes simplex virus (HSV) to cells.

This mini-review describes the trends and processes in large-scale production of adenovirus and poxvirus vectors to meet the needs of clinical applications. We briefly describe the general principles for the production and purification of adenovirus and poxvirus viral vectors. Currently, adenovirus and poxvirus vector manufacturing methods rely on well-established cell culture technologies. Several improvements have been evaluated to increase the yield and to reduce the overall manufacturing cost, such as cultivation at high cell densities and continuous downstream processing.

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