Integrating Affinity Chromatography in the Platform Process for Adenovirus Purification.

Area of Science:

• Biotechnology
• Biopharmaceutical Manufacturing
• Viral Vector Technology

Background:

• Adenoviral vectors (AdVs) are crucial for gene therapy, cancer treatment, and vaccine development.
• Current AdV purification methods, primarily ion-exchange chromatography, face challenges in achieving optimal yield and purity.
• There is a growing need for advanced AdV manufacturing platforms to meet increasing demand.

Purpose of the Study:

• To develop and validate a novel affinity-based purification process for adenoviral vectors (AdVs).
• To enhance product yield and purity compared to existing chromatographic methods.
• To ensure efficient clearance of host cell proteins (HCPs) and host cell DNA (hcDNA).

Main Methods:

• Development of affinity chromatography using peptide ligands (AEFFIWNA and TNDGPDYSSPLTGSG) targeting AdV hexon proteins.
• Evaluation of different resin matrices (polymethylmethacrylate and agarose) for peptide-functionalized adsorbents.
• Optimization of washing steps to improve impurity removal.
• Assembly of a multi-step purification process including clarification, affinity capture, and mixed-mode polishing.

Main Results:

• The affinity process achieved high capacity (> >5·10¹⁰ vp/mL) and yield (~50%) under mild elution conditions.
• Selected peptide-functionalized resins demonstrated significant clearance of HCPs (144-fold) and hcDNA (56-fold).
• The optimized process yielded ~50% infectious units (IFU) with residual HCP and hcDNA levels meeting clinical standards.

Conclusions:

• The developed affinity-based purification process offers a significant advancement for AdV manufacturing.
• This method provides high yield and purity, effectively removing critical impurities.
• The process is robust, scalable, and meets the stringent requirements for clinical applications of AdVs.