Process monitoring framework for cross‐flow diafiltration‐based virus‐like particle disassembly: Tracing product properties and filtration performance

Abstract

Virus-like particles (VLPs) are an emerging biopharmaceutical modality with great potential as a platform technology. VLPs can be applied as gene therapy vectors and prophylactic or therapeutic vaccines. For non-enveloped VLPs, recombinant production of the protein subunits leads to intracellular self-assembly. The subsequent purification process includes VLP dis-and reassembly which aim at removing encapsulated impurities and improving particle properties. Filtration-based separation and processing has proven successful for VLPs but requires large product quantities and laborious experiments in early development stages. Both challenges can be tackled by implementation of process analytical technology (PAT) to efficiently obtain extensive process information. In this study, an existing PAT setup was extended to comprehensively monitor the diafiltration-based disassembly of hepatitis B core antigen (HBcAg) VLPs. Process-related signals were monitored in-line, while product-related signals, such as ultraviolet light (UV) spectra as well as static and dynamic light scattering (SLS and DLS), were monitored on-line. The applicability of the sensors for disassembly monitoring was evaluated under varying processing conditions. HBcAg VLP subunit concentrations were accurately predicted based on UV data using ordinary and partial least squares regression models (Q 2 from 0.909 to 0.976). DLS data were used for aggregation monitoring while the SLS intensity qualitatively reflected the disassembly progress.

Raman-based PAT for VLP precipitation: systematic data diversification and preprocessing pipeline identification

“…The PLS models may also use other correlative information available in the Raman spectra (Santos et al, 2018;Goldrick et al, 2020) which is not necessarily specific for the precipitation of VLPs such as the turbidity or precipitant markers. Nevertheless, the comparison with MLR as a benchmark is a useful strategy as in less complex cases, the relevant information may be reliably contained in a few individual variables (Hillebrandt et al, 2022).…”

“…Besides the optical spectroscopic techniques ultraviolet-visible (UV/Vis) and infrared (IR) spectroscopy, Raman spectroscopy coupled with chemometrics has found extensive applications in monitoring various processes for biopharmaceutical products, including raw material testing (Li et al, 2010), cell culture (Abu-Absi et al, 2011;Berry et al, 2015;Golabgir and Herwig, 2016), chromatography (Feidl et al, 2019;Rolinger et al, 2021;Wang et al, 2023), filtration (Rolinger et al, 2023), freezing (Roessl et al, 2014;Weber and Hubbuch, 2021), or formulation (Wei et al, 2022). For VLPs in particular, recent studies demonstrated the real-time monitoring of a baculovirus cultivation for the production of rabies VLPs (Guardalini et al, 2023a;b; as well as the cross-flow filtrationbased polishing operations such as dis-and reassembly of the HBcAg-VLPs (Rüdt et al, 2019;Hillebrandt et al, 2022). Despite the broad applicability of spectroscopic methods in biopharmaceutical processing, their application to precipitation processes is rather unexplored.…”

Size-selective downstream processing of virus particles and non-enveloped virus-like particles

“…VLP dis- and reassembly were investigated in the frame of DF providing a scalable process and the opportunity to improve VLP yield by controlled buffer exchange ( Liew et al, 2012 ; Hillebrandt et al, 2021 ). Furthermore, process monitoring approaches were developed to monitor product and process properties during DF-based VLP dis- and reassembly ( Rüdt et al, 2019 ; Hillebrandt et al, 2022 ).…”