Native elution in immunoaffinity chromatography of viruses – a step toward high-purity virus particle purification (CROSBI ID 643237)
Prilog sa skupa u zborniku | sažetak izlaganja sa skupa | domaća recenzija
Podaci o odgovornosti
Brgles, Marija ; Sviben, Dora ; Forčić, Dubravko ; Halassy, Beata
engleski
Native elution in immunoaffinity chromatography of viruses – a step toward high-purity virus particle purification
Whole viral particles are the active principles in prophylactic (attenuated viruses) and therapeutic vaccines (viral vectors). The manufacturing process of viral particles consists of upstream (USP, virus in vivo production in bioreactors) and downstream (DSP, virus purification) part. The immunogenicity and stability of viruses throughout the entire production chain should be maintained. The main aim of DSP is to eliminate contaminants, either process related or product related (host cell proteins, DNA, free proteins, aggregated and empty capsids, host cell exosomes). Downstream processing of viruses has currently been a bottleneck of virus manufacturing, and accounts for up to 70% of overall production costs. Immunoaffinity chromatography is one of the most powerful techniques in the protein purification. High specificity and high affinity of antigen-antibody interaction enables high purification (up to 1000x) and concentration in a single step. However, the drawback of immunoaffinity is that the high affinity interaction between antigen and antibody can be disrupted only under harsh elution conditions that inevitably disturb also intramolecular non-covalent interaction, disordering the protein conformation, and consequently function. Particularly viruses are especially sensitive to elution condition effective in immunoaffinity chromatography, so the immunoaffinity chromatography has not been used in purification of viable viruses. Here we describe the novel principle of native elution, i.e. effective elution of viruses under native, physiological conditions that maintains virus stability and infectivity. It is based on elution of antigen in immunoaffinity chromatography by different amino acid solutions of high molarity, under physiological pH (7.2-7.4). The mechanism of antigen (virus) desorption from antibody is competition. Using attenuated mumps virus strain, as a model virus, we were able to elute 49±16.5% (n=10) infective virus particles using 0.75 M Arg/0.75 M imidazole as eluting agent and 68±13.5% (n=12) infective virus particles using 0.75 M Arg/0.75 M Ser. Moreover, we were able to demonstrate that native elution was effective in separation of infective from non- infective particles. Since amino acids have already been experimentally demonstrated to have virus stabilizing features, a combination of high specificity exhibited by immunoaffinity chromatography and efficient elution with pH neutral, stabilizing solution opens new possibilities for commercial use.
Immunoaffinity chromatography ; Native elution ; Virus ; Monolith
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Podaci o prilogu
38-38.
2016.
objavljeno
Podaci o matičnoj publikaciji
2016 Annual Meeting of the Croatian Immunological Society : Abstract Book
Kelava, Tomislav ; Markotić, Antonio ; Šućur, Alan
Zagreb: Hrvatsko imunološko društvo
Podaci o skupu
Annual meeting of the Croatian Immunological Society 2016
predavanje
14.10.2016-15.10.2016
Ogulin, Hrvatska