High-throughput characterization of virus-like particles by interlaced size-exclusion chromatography
Introduction
In recent years, promising prophylactic and therapeutic vaccination prospects for public health threats have arisen from the development of virus-like particles (VLPs). VLPs are protein assemblages which are produced by recombinant expression of viral structural proteins [1], [2]. Thus, the structure of highly pathogenic viruses such as HIV [3], Influenza [4] and Ebola [5] can be mimicked or tailor-made nanocarriers for antigenic epitope presentation [6], [7], [8], [9] can be designed. Due to production in genetically modified organisms, the analysis of product-related and process-related impurities is important during development and manufacturing of VLP vaccines [10], [11]. Process-related impurities such as host cell proteins (HCPs) and DNA can be rapidly assessed by methods standardized in the biopharmaceutical industry for therapeutic proteins [12]. In contrast, quantitative analysis of product-related impurities such as aggregates is more challenging and mostly tailor-made for each vaccine due to the large size and complexity of VLPs. Traditionally, VLP characterization is often done by transmission electron microscopy (TEM) requiring high investment costs, extensive sample and instrument preparation work, and specialized staff. A rapid technology for VLP characterization is dynamic light scattering (DLS) [13], [14], [15]. The method allows the determination of hydrodynamic particle diameters by measuring the fluctuations of light scattering from particles in solution. However, DLS is less sensitive in resolving aggregates [16]. Currently used quantitative methods for VLP aggregates are asymmetrical flow field-flow fractionation (AF4) [17], [18], [16], disc centrifugation particle size analysis [19], electrospray differential mobility analysis [18], and size-exclusion chromatography (SEC) [20]. These techniques are very time-consuming with analysis times ranging from 30 to 60 min per sample [17], [18], [16], [20]. SEC is the most widely used technique for aggregate quantification in the biopharmaceutical industry [21]. SEC methods have been successfully applied for process monitoring of capsomere vaccines [22], recombinant fusion protein vaccines [15], and human hepatitis B virus surface antigen (HBsAg) VLPs [23], [24], [25], [26]. Recently, rapid size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) methods have been realized for monoclonal antibodies by performing interlaced sample injections with analysis times of 2–6 min [27], [28]. Fig. 1 shows a schematic drawing of the principle of interlaced SEC (iSEC) methods. The ‘information phase’ (green) in a SEC run is the time range including the elution of relevant species (aggregates, monomer). The longest phase in a classical single injection SEC method run is the ‘lag phase’ (blue), which is the time range from injection to elution of the first species. The ‘hold- up’ (blue) phase refers to the time from the end of the information phase to the column's void time defined by the elution of small molecules such as salts. In order to reduce the total analysis time of SEC methods without changing the performance of ‘information phases’, the ‘lag phase’ can be eliminated by injecting subsequent samples prior to the complete elution of previous sample components. This operation is referred to as interlaced injection mode.
In this work, we present the development and application of an iSE-UHPLC method for recombinant protein-based VLPs. The feasibility of the assay was evaluated for human papilloma (HPV) VLPs [29], human enterovirus 71 (EV71) VLPs [30], murine polyomavirus (MuPyV) VLPs [7], human B19 parvo (B19 VP1/VP2) VLPs [31]., and chimeric hepatitis B core antigen (HBcAg) VLPs [8]. Two case studies are presented for the application of the iSE-UHPLC during downstream process development and stability studies. The designed method allows a rapid assessment of VLP dispersity and is well-suited for high-throughput pharmaceutical process development of VLPs.
Section snippets
Disposables
For precipitation screenings, sample storage, fractionation by FPLC and UHPLC, 350 μL-polypropylene plates (Greiner Bio-One, Kremsmünster, Austria) were used. Stability studies with HPV VLPs were performed in 1.5 mL-polypropylene Eppendorf® Safe-Lock Tubes (Eppendorf, Hamburg, Germany). Frozen VLPs were thawed and centrifuged in the same tubes at 18,000 × g and 4 °C for 10 min.
Chemicals and buffers
For the SE-UHPLC method, K2HPO4 was obtained from VWR BDH Prolabo (Radnor, Pennsylvania, USA). MOPS was purchased from Carl
Development of an interlaced SEC-UHPLC method
In the International Conference on Harmonization (ICH) guideline Q2 [36] on the validation of analytical procedures, it is suggested to verify among others the specificity, precision, linearity, and robustness of novel methods for biopharmaceutical products. The initial objective was therefore to identify an analytical column separating VLPs and VLP aggregates in order to develop a specific analytical method. A pre-selection of columns was done based on pore sizes (>300 Å) and published SEC
Conclusion and outlook
In this work, we report the development and application of a high-throughput analytical tool for the characterization of virus-like particles. Recently, the relevance of SE-HPLC methods for viral vaccines has been highlighted by Yang et al. [20]. Using a low-dispersion UHPLC system, we identified an SRT 1000 column as optimal SEC column for quantification of VLP aggregates at low buffer consumption and high flow rates. The implementation of an interlaced SE-UHPLC procedure allowed the
Acknowledgements
The authors gratefully acknowledge material supply by Diarect AG, Merck & Co, Sentinext Therapeutics, BioNTech Protein Therapeutics, Tosoh Bioscience and financial support from the German Federal Ministry of Education and Research (Grant agreement 0315640B). In addition, we would like to thank Mohammad Fotouhi from the KIT Laboratory for Electron Microscopy for acquisition of TEM micrographs, Thiemo Huuk for his support regarding UHPLC methods, Ozan Ötes for performing part of the experimental
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