The application of Process Analytical Technology (PAT) principles for manufacturing of biotherapeutics proffers the prospect of ensuring consistent product quality along with increased productivity as well as substantial cost and time savings. Although this paradigm shift from a traditional, rather rigid manufacturing model to a more scientific, risk-based approach has been advocated by health authorities for almost two decades, the practical implementation of PAT in the biopharmaceutical industry is still limited by the lack of fit-for-purpose analytical methods. In this regard, most of the proposed spectroscopic techniques are sufficiently fast but exhibit deficiencies in terms of selectivity and sensitivity, while well-established offline methods, such as (ultra-)high-performance liquid chromatography, are generally considered as too slow for this task. To address these reservations, we introduce here a novel online Liquid Chromatography (LC) setup that was specifically designed to enable real-time monitoring of critical product quality attributes during time-sensitive purification operations in downstream processing. Using this online LC solution in combination with fast, purpose-built analytical methods, sampling cycle times between 1.30 and 2.35 min were achieved, without compromising on the ability to resolve and quantify the product variants of interest. The capabilities of our approach are ultimately assessed in three case studies, involving various biotherapeutic modalities, downstream processes and analytical chromatographic separation modes. Altogether, our results highlight the expansive opportunities of online LC based applications to serve as a PAT tool for biopharmaceutical manufacturing.

Recombinant adeno-associated viruses (rAAVs) are promising gene delivery vectors in the emerging field of in vivo gene therapies. To ensure their consistent quality during manufacturing and process development, multiple analytical techniques have been proposed for the characterization and quantification of rAAV capsids. Despite their indisputable capabilities for performing this task, current analytical methods are rather time-consuming, material intensive, complicated, and costly, restricting their suitability for process development in which time and sample throughput are severe constraints. To eliminate this bottleneck, we introduce here an affinity-based high-performance liquid chromatography method that allows the determination of the capsid titer and the full/empty ratio of rAAVs within less than 5 min. By packing the commercially available AAVX affinity resin into small analytical columns, the rAAV fraction of diverse serotypes can be isolated from process-related impurities and analyzed by UV and fluorescence detection. As demonstrated by both method qualification data and side-by-side comparison with AAV enzyme-linked immunosorbent assay results for rAAV8 samples as well as by experiments using additional rAAV2, rAAV8, and rAAV9 constructs, our approach showed good performance, indicating its potential as a fast, simple and efficient tool for supporting the development of rAAV gene therapies.

Recombinant adeno-associated virus (rAAV) vectors are prominent vectors for in-vivo gene therapies. However, achieving consistent product quality is a major challenge due to the inherent complexity of their structure and the associated production process. During manufacturing and storage, virus particles are exposed to a variety of stresses, which can have a direct impact on product attributes, thereby potentially also affecting safety and efficacy. Current understanding of stress-induced degradation for rAAVs is still fragmented, both on a mechanistic level and regarding the identification of the most appropriate analytical tools to detect the underlying changes. To address these gaps, the impact of different stress conditions, namely freeze-thaw, elevated temperature, high/low pH and light exposure, was tested on two different serotypes. For this, a comprehensive panel of orthogonal analytical methods was applied to identify significant changes in capsid and genome titer, full-to-total ratio, content of aggregates, payload integrity and post-translational modifications. By correlating this extensive dataset with potency results, this study provides detailed insights into the degradation behavior of the employed rAAVs and the suitability of state-of-the-art analytical techniques to indicate the stability of the product. Overall, substantial differences in stress responses were observed between the two serotypes investigated, and changes in transduction efficiency were mostly represented inadequately at the structural or molecular level by the analytical methods employed. Based on these findings, this study serves as a framework for refining the analytical control strategy of rAAV-based gene therapies and for developing robust, stability-indicating assays.