Integrating affinity chromatography in the platform process for Adenovirus purification

Abstract

Adenoviral vectors (AdVs) are gaining prominence in cancer therapy and vaccine development, posing the need for a modern AdV manufacturing platform. Current AdV purification by ion-exchange chromatography indeed struggles to achieve the products yield and purity of processes that employ affinity technologies. Addressing these challenges, this study presents the first affinity-based process that delivers high product yield and clearance of host cell proteins and DNA (HCPs and hcDNA) in two chromatography steps. The affinity capture utilizes resins functionalized with peptide ligands that target AdV hexon proteins (AEFFIWNA and TNDGPDYSSPLTGSG), and provide high capacity (>5{middle dot}1010 vp per mL of resin) and yield under mild elution conditions ([~]50% at pH 8.0). Peptide-functionalized adsorbents prepared using different matrices (polymethylmethacrylate vs. agarose) were initially tested to compare the purification performance. AEFFIWNA-SulfoLink resin was selected for its yield of cell- transducing AdVs ([~]50%) and removal of HCPs and hcDNA (144-fold and 56-fold). Similarly, TNDGPDYSSPLTGSG-Toyopearl(R) resin afforded [~]50% yield and >50-fold reduction of con- taminants. Additional gains in product purity were achieved by optimizing the washing step, which removed free hexon proteins and additional HCPs. All peptide-functionalized resins maintained their purification performance for ten cycles upon regeneration at pH [~]2.0. The purification process was assembled to include clarification, affinity capture in bind-and-elute mode using AEFFIWNA-SulfoLink resin, and polishing in flow-through mode using mixed- mode resins. The optimized process provided a yield [~]50% of cell-infecting units (IFU) and a product titer [~]107 IFU/mL, along with residual HCP and hcDNA levels ([~]10 ng/mL and 44 ng per dose, respectively) that meet clinical requirements.