Anion exchange (AEX) chromatography is a widely used analytical technique for separating empty (E) and full (F) adeno-associated virus (AAV) capsids. However, it sometimes fails to achieve baseline separation between E and F capsids in purified samples, and this challenge is even more pronounced in complex process samples. Moreover, AEX is typically serotype dependent, indicating that a specific method must be developed for each serotype. We propose several strategies that significantly enhance the separation of E and F, and in some instances, facilitate baseline separation while providing insights into additional AAV capsid subpopulations, including partially filled capsids, overfilled capsids, and aggregates. By using an AEX analytical Convective Interaction Media (CIM) monolithic column, we demonstrate how various organic solvents, their concentrations, the choice of Mg2+ counterions, and the pH of buffers affect the separation of E and F AAV capsids. These findings align with other orthogonal techniques and are incorporated into an enhanced chromatographic AEX method. The added value of this method lies in its rapid and enhanced characterization of a broad range of samples from various AAV serotypes and purities. It ultimately promotes the development of safer and more effective gene therapies.

The poly(A) tail of mRNA plays a vital role in mRNA transcript stability, translational efficiency, and immunogenicity. Co-transcriptionally polyadenylated in vitro transcribed (IVT) mRNAs typically contain poly(A) tails of 50–120 nucleotide tail length due to limitations in production of template pDNA with longer poly(A) sequences. In contrast, post-transcriptional enzymatic polyadenylation of mRNA with poly(A) polymerase (PAP) presents a modular alternative to increase the tail length. However, the lack of real-time control strategies for PAP-mediated tailing has limited its broader applicability in mRNA production. Here, we develop a methodology for controlling poly(A) tail length in post-transcriptional polyadenylation of mRNA that uses adenosine triphosphate (ATP) consumption measured at-line to predict the poly(A) tail length. We establish a novel analytical method based on monolith reverse-phase chromatography to validate the poly(A) predictions. We were able to produce longer poly(A) tails and accurately determine their length in 300–700 nt range. The resulting longer poly(A) tailed reporter mRNAs outperformed the encoded and shorter poly(A) tailed mRNAs in cell-based assays. This work presents a new strategy for controlled post-transcriptional polyadenylation using ATP consumption as a process control metric, an approach which may in future be expanded to other NTP-dependent enzymatic conversions.