ORCID Profile
0000-0002-2421-5503
Current Organisation
University of Adelaide
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Publisher: Elsevier BV
Date: 02-2021
Publisher: Wiley
Date: 19-02-2021
DOI: 10.1002/BIT.27687
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.
Publisher: Elsevier BV
Date: 11-2023
Publisher: Wiley
Date: 10-05-2022
DOI: 10.1002/BIT.28118
Abstract: In this study, we present the first integrated and continuous downstream process for the production of microbial virus‐like particle vaccines. Modular murine polyomavirus major capsid VP1 with integrated J8 antigen was used as a model virus‐like particle vaccine. The integrated continuous downstream process starts with crude cell lysate and consists of a flow‐through chromatography step followed by periodic counter‐current chromatography (PCC) (bind‐elute) using salt‐tolerant mixed‐mode resin and subsequent in‐line assembly. The automated process showed a robust behavior over different inlet feed concentrations ranging from 1.0 to 3.2 mg ml −1 with only minimal adjustments needed, and produced continuously high‐quality virus‐like particles, free of nucleic acids, with constant purity over extended periods of time. The average size remained constant between 44.8 ± 2.3 and 47.2 ± 2.9 nm comparable to literature. The process had an overall product recovery of 88.6% and a process productivity up to 2.56 mg h −1 ml resin −1 in the PCC step, depending on the inlet concentration. Integrating a flow through step with a subsequent PCC step allowed streamlined processing, showing a possible continuous pathway for a wide range of products of interest.
Publisher: Wiley
Date: 12-07-2022
DOI: 10.1002/BIT.28107
Publisher: Elsevier BV
Date: 02-2021
Publisher: Elsevier BV
Date: 03-2022
DOI: 10.1016/J.CHROMA.2022.462884
Abstract: Fluctuations of the inlet feed stream concentration are a challenge in controlling continuous multi-column counter current chromatography systems with standard methods. We propose a new control strategy based on calculated product column breakthrough from UV sensor signals by neglecting an impurity baseline and instead using the impurity to product ratio. This calculation is independent of the inlet feed concentration. In-silico simulation showed that the proposed method can calculate the product column breakthrough perfectly even with fluctuating and highly unstable inlet feed concentration during a loading cycle. Applying the proposed method to control a three column periodic counter current chromatography process with fluctuating inlet feed concentration resulted in constant column loading in each cycle, while using the standard method failed to do so. Unavoidable band broadening caused by diffusion and dispersion has been identified as an inherent limiting factor for accurate calculation of column breakthrough comparing inlet and outlet UV signals. The proposed advanced calculations increase the robustness of periodic counter current chromatography and extend the capability to process unstable inlet streams.
Publisher: Wiley
Date: 16-03-2021
DOI: 10.1002/BIT.27398
No related grants have been discovered for Lukas Gerstweiler.