Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography

Area of Science:

• Biotechnology
• Chromatography
• Viral Vector Purification

Background:

• Size-exclusion chromatography (SEC) is a common method for purifying biological molecules.
• Traditional batch SEC processes often suffer from low productivity and product titer.
• Adenovirus serotype 5 (Ad5) purification requires efficient methods to remove impurities like DNA and host cell proteins (HCP).

Purpose of the Study:

• To develop and evaluate an efficient, quasi-continuous, two-column simulated moving-bed (SMB) SEC process for Ad5 purification.
• To assess the performance of the two-column SEC process in terms of virus recovery, impurity clearance, and productivity.
• To compare the two-column SEC process with traditional single-column batch chromatography.

Main Methods:

• A two-column, open-loop, quasi-continuous simulated moving-bed (SMB) process was designed for Ad5 separation.
• The operating cycle involved sequential elution and feeding steps across two columns.
• Impurity clearance (DNA and HCP) and virus recovery were experimentally measured during pilot-scale runs.

Main Results:

• The two-column SEC process achieved an 86% virus recovery with 90% DNA and 89% HCP clearance.
• Virus yield increased from 57% in batch chromatography to 86% with the two-column SEC process.
• Productivity was enhanced six-fold compared to the single-column batch system.

Conclusions:

• The innovative two-column SEC configuration effectively overcomes the limitations of low productivity and titer in traditional SEC.
• This quasi-continuous SMB process offers a significant advancement for Ad5 purification, improving yield and efficiency.
• The method demonstrates a highly favorable comparison against single-column batch chromatography for Ad5 purification.