Cryo‐EM Analysis of Adenovirus Vector Reveals Purification Conditions Leading to Loss of Viral Fiber Proteins and Reduced Infectivity

ABSTRACT Vaccines and gene therapies require efficient delivery of genetic material to host cells. Viral vectors leverage viruses' ability to enter host cells to achieve this. Following the SARS‐CoV‐2 vaccine and novel gene therapies that utilize adenovirus vectors, there has been an increase in the demand for large‐scale production of vectors. A benefit of adenovirus vectors is that they can act as a “plug and play” tool for any future emerging diseases for the development of vaccines. A key obstacle was the heterogeneity that is found in the final product. This can reduce the functionality of the vector and affect immune responses. A possible origin of heterogeneity is the downstream processing, specifically the bind and elute steps of anion exchange chromatography (AEX), which is widely used at scale. To better understand the heterogeneity that AEX could elicit on the adenovirus capsid, cryogenic‐electron tomography was used to assess the capsid integrity after a variety of chromatography conditions in comparison to a cesium chloride (CsCl) gradient ultracentrifugation purified control. To investigate the effect of absorption time during chromatography, AEX conditions with a 5, 10, and 20 min on‐resin absorption periods were evaluated. Following AEX binding at pH 6.5, samples showed a reduction from 17% in the CsCl purified control to 10% infectious particles. When examined using electron microscopy, the virions exhibited aggregation and a decrease from 70% to 54% in fiber occupancy. After a 20 min AEX hold at pH 6.5 there was almost complete loss of infectivity and fiber occupancy decreased from ~70% to below 30%. Samples run at pH 8 did not show the same loss in infectivity when held bound for 20 min. This suggests that purification conditions can cause removal of the fiber protein, which was likely causing the decrease in infectivity, due to the fiber's role in cell entry. We hypothesize that fiber removal was caused by a combination of hold time and weakened binding between the fiber and penton base due to the low pH conditions that are often used during AEX. This work demonstrates a link between fiber loss and reduced adenovirus vector functionality and how purification conditions can impact vector functionality. Through studying the relationship between structure and function, we can hopefully minimize loss of infectivity during purification by optimizing processing conditions, reduce manufacturing cost and improve the quality of adenovirus vaccines and gene therapies.