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Process development for cross‐flow diafiltration‐based VLP disassembly: A novel high‐throughput screening approach
Author(s) -
Hillebrandt Nils,
Vormittag Philipp,
Dietrich Annabelle,
Wegner Christina H.,
Hubbuch Jürgen
Publication year - 2021
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27868
Subject(s) - diafiltration , downstream processing , capsid , chemistry , virus like particle , cross flow filtration , downstream (manufacturing) , chromatography , biophysics , nanotechnology , recombinant dna , materials science , biochemistry , biology , membrane , operations management , economics , gene , microfiltration
Virus‐like particles (VLPs) are particulate structures, which are applied as vaccines or delivery vehicles. VLPs assemble from subunits, named capsomeres, composed of recombinantly expressed viral structural proteins. During downstream processing, in vivo ‐assembled VLPs are typically dis‐ and reassembled to remove encapsulated impurities and to improve particle morphology. Disassembly is achieved in a high‐pH solution and by the addition of a denaturant or reducing agent. The optimal disassembly conditions depend on the VLP amino acid sequence and structure, thus requiring material‐consuming disassembly experiments. To this end, we developed a low‐volume and high‐resolution disassembly screening that provides time‐resolved insight into the VLP disassembly progress. In this study, two variants of C‐terminally truncated hepatitis B core antigen were investigated showing different disassembly behaviors. For both VLPs, the best capsomere yield was achieved at moderately high urea concentration and pH. Nonetheless, their disassembly behaviors differed particularly with respect to disassembly rate and aggregation. Based on the high‐throughput screening results, a diafiltration‐based disassembly process step was developed. Compared with mixing‐based disassembly, it resulted in higher yields of up to 0.84 and allowed for integrated purification. This process step was embedded in a filtration‐based process sequence of disassembly, capsomere separation, and reassembly, considerably reducing high‐molecular‐weight species.