Introduction: The development of novel complex biotherapeutics led to new challenges in biopharmaceutical industry. The potential of these particles has been demonstrated by the approval of several products, in the different fields of gene therapy, oncolytic therapy, and tumor vaccines. However, their manufacturing still presents challenges related to the high dosages and purity required. Areas covered: The main challenges that biopharmaceutical industry faces today and the most recent developments in the manufacturing of different biotherapeutic particles are reported here. Several unit operations and downstream trains to purify virus, virus-like particles and extracellular vesicles are described. Innovations on the different purification steps are also highlighted with an eye on the implementation of continuous and integrated processes. Expert opinion: Manufacturing platforms that consist of a low number of unit operations, with higheryielding processes and reduced costs will be highly appreciated by the industry. The pipeline of complex therapeutic particles is expanding and there is a clear need for advanced tools and manufacturing capacity. The use of single-use technologies, as well as continuous integrated operations, are gaining ground in the biopharmaceutical industry and should be supported by more accurate and faster analytical methods.

Currently, marketed influenza vaccines are only efficient against homologous viruses, thus requiring a seasonal update based on circulating subtypes. This constant reformulation adds several challenges to manufacturing, particularly in purification due to the variation of the physicochemical properties of the vaccine product. A universal platform approach capable of handling such variation is therefore of utmost importance. In this work, a filtration‐based approach is explored to purify influenza virus‐like particles. Switching from adsorptive separation to size‐based purification allows overcoming the differences in retention observed for different influenza strains. The proposed process employs a cascade of ultrafiltration and diafiltration steps, followed by a sterile filtration step. Different process parameters are assessed in terms of product recovery and impurities’ removal. Membrane chemistry, pore size, operation modes, critical flux, transmembrane pressure, and permeate control strategies are evaluated. After membrane selection and parameter optimization, concentration factors and diafiltration volumes are also defined. By optimizing the filtration mode of operation, it is possible to achieve product recoveries of approximately 80%. Overall, the process time is decreased by 30%, its scalability is improved, and the costs are reduced due to the removal of chromatography and associated buffer consumptions, cleaning, and its validation steps.

Introduction: Oncolytic virus therapy is currently considered as a promising therapeutic ap-proach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer prepara-tions. The addition of this surfactant in the list of substances with the very high concern of the Registra-tion, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives.Methods: In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant – Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorb-ate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impu-rities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell pro-teins and ds-DNA was 99.9% and 97.1%, respectively.Results & Conclusion: The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the devel-oped process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.

Virus‐like particles (VLPs) are becoming established as vaccines, in particular for influenza pandemics, increasing the interest in the development of VLPs manufacturing bioprocess. However, for complex VLPs, the analytical tools used for quantification are not yet able to keep up with the bioprocess progress. Currently, quantification for Influenza relies on traditional methods: hemagglutination assay or Single Radial Immunodiffusion. These analytical technologies are time‐consuming, cumbersome, and not supportive of efficient downstream process development and monitoring. Hereby we report a label‐free tool that uses Biolayer interferometry (BLI) technology applied on an Octet platform to quantify Influenza VLPs at all stages of bioprocess. Human (α2,6‐linked sialic acid) and avian (α2,3‐linked sialic acid) biotinylated receptors associated with streptavidin biosensors were used, to quantify hemagglutinin content in several mono‐ and multivalent Influenza VLPs. The applied method was able to quantify hemagglutinin from crude samples up to final bioprocessing VLP product. BLI technology confirmed its value as a high throughput analytical tool with high sensitivity and improved detection limits compared to traditional methods. This simple and fast method allowed for real‐time results, which are crucial for in‐line monitoring of downstream processing, improving process development, control and optimization.