Background: Process analytical technology (PAT) enhances product quality by monitoring and controlling critical quality attributes (CQAs), offering better insights into process performance. Raman spectroscopy is a promising PAT tool to support the development of control systems for continuous and automated processes. In this study, Raman spectroscopy was used to monitor in real-time ultra- and diafiltration (UF/DF) of adeno-associated virus (AAV) and lentiviral vector (LV). Method: The incorporation of a Raman probe in a tangential flow filtration (TFF) system, when operated in open loop, enabled spectral acquisition during a five-fold concentration of clarified bulk followed by a buffer exchange to PBS with five diafiltration volumes. Results: Principal Component Analysis reduced the dimensionality of the training set, and a Partial Least Squares model was developed for each parameter in each phase. After validation, Raman monitoring platforms showed good performance in predicting CQA of both viral vectors. Batch-to-batch variability was identified as the principal source of spectral variation, and its impact on UF/DF operation was observed. Discussion: This study emphasises the potential of Raman spectroscopy as a PAT for monitoring and control strategies in the downstream processing of viral vectors. Conclusions: These monitoring platforms enhance understanding of TFF’s operation and enable timely decision making by providing real-time information on multiple parameters.

Current lab-scale purification methods for ADDomer are labour-intensive, time-consuming and poorly scalable. The intracellular nature of ADDomer production further complicates downstream processing, requiring robust, scalable solutions for cell lysis and clarification. In the present work we focus on developing a scalable, GMP-compliant process for ADDomer purification. The workflow combines tangential microfiltration (TMF) using a 0.4 μm hollow fiber for cell retention, chemical lysis with 0.06 % Deviron® C16, and in-line Benzonase® treatment for DNA removal. Several capture approaches were explored, including anion and cation exchange, hydrophobic interaction and multimodal chromatography. Among these, the Sartobind® Q anion exchange membrane demonstrated superior performance, achieving ADDomer purity of ~ 83 % and recovery yields > 85 %. Ultrafiltration membranes of different chemistries (regenerated cellulose, RC, and polyethersulfone, PES) and cut-off (300 kDa, 500 kDa, 1 MDa) were evaluated for polishing and buffer exchange. The 1 MDa RC membrane enabled a recovery of ~ 87 % and purity > 97%. Final sterile filtration with a PES membrane preserved particle integrity, purity and achieved > 80 % recovery. Overall, the purification process herein established yielded ~ 47 mg of ADDomer particles per L of culture volume while removing > 97 % and > 99 % of total protein and dsDNA, respectively. In summary, this study showcases the implementation of a scalable and GMP-compliant purification platform for ADDomer, paving the way for the development of next-generation ADDomer-based vaccines and antivenoms.