Virus‐like particle preparation is improved by control over capsomere‐DNA interactions during chromatographic purification

Abstract

Expression of viral capsomeres in bacterial systems and subsequent in vitro assembly into virus‐like particles is a possible pathway for affordable future vaccines. However, purification is challenging as viral capsomeres show poor binding to chromatography media. In this study, the behavior of capsomeres in unfractionated bacterial lysate was compared with that for purified capsomeres, with or without added microbial DNA, to better understand reasons for poor bioprocess behavior. We show that aggregates or complexes form through the interaction between viral capsomeres and DNA, especially in bacterial lysates rich in contaminating DNA. The formation of these complexes prevents the target protein capsomeres from accessing the pores of chromatography media. We find that protein–DNA interactions can be modulated by controlling the ionic strength of the buffer and that at elevated ionic strengths the protein–DNA complexes dissociate. Capsomeres thus released show enhanced bind‐elute behavior on salt‐tolerant chromatography media. DNA could therefore be efficiently removed. We believe this is the first report of the use of an optimized salt concentration that dissociates capsomere–DNA complexes yet enables binding to salt‐tolerant media. Post purification, assembly experiments indicate that DNA–protein interactions can play a negative role during in vitro assembly, as DNA–protein complexes could not be assembled into virus‐like particles, but formed worm‐like structures. This study reveals that the control over DNA–protein interaction is a critical consideration during downstream process development for viral vaccines.

Process development for cross‐flow diafiltration‐based VLP disassembly: A novel high‐throughput screening approach

“…It is important to mention that the feed A260/A280 in Table 1 is calculated based on the peak areas of HMWS, VLPs, and dimers while it was 0.64 for the VLP peak, suggesting that the depleted nucleic acids were mainly associated with (or bound to) the HMWS. This was also observed in a recent study with murine polyomavirus VLPs (Gerstweiler et al, 2021). Both VLPs used in this study lack the C‐terminal protamine‐like region of the wild‐type HBcAg, which reduces packaging of nucleic acids (Crowther et al, 1994; Zlotnick et al, 1997).…”

Development of purification process for dual‐function recombinant human heavy‐chain ferritin by the investigation of genetic modification impact on conformation

“…Ion exchanger has been commonly applied to separate protein and nucleic acids based on the differences of their binding strengths [22]. In this work, an anion exchanger, Q FF, was selected, because of the protein stability on acid condition and the isoelectric point (pI) of HFn and modified HFns.…”

An integrated and continuous downstream process for microbial virus‐like particle vaccine biomanufacture

“…In batch processing, the use of salt‐tolerant mixed‐mode resins with a previous flow‐through step allows processing at elevated salt concentrations, which suppress VP1‐DNA aggregation, and therefore leads to better recovery. The salt‐tolerant mixed‐mode resin furthermore allows an integration of the two‐unit operations without buffer adjustment in between and enables a wide design space (Gerstweiler et al, 2021b, 2021c). The continuous process described in this study, developed by building on these batch studies, produces VLPs of a constant good quality, removes DNA and most contaminants, is scalable and can act as a platform technology for the development for new continuous production pathways of vaccines and biopharmaceuticals other than monoclonal antibodies.…”

“…The process couples a flow through Capto™ Q chromatography step followed by a bind-elute multimodal (Capto™ MMC) PCC process with subsequent in-line assembly of VLPs. It has been previously shown that nonpurified VP1 capsomeres form soluble aggregates with microbial DNA at low buffer salt concentrations, hindering purification (Gerstweiler et al, 2021b). In batch processing, the use of salt-tolerant mixed-mode resins with a previous flow-through step allows processing at elevated salt concentrations, which suppress VP1-DNA aggregation, and therefore leads to better recovery.…”