Ask the Expert: A Platform for Purification and In-Process Analysis of Adenovirus Vectors

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Despite safety-related setbacks in the early 2000s, adenovirus (Ad) again is gaining traction as a vector for advanced therapies and vaccines. Thus, Ad production is accelerating. In November 2021, Hana Jug (project manager in process development for viral vectors and vaccines at BIA Separations, a Sartorius company) described how her company’s updated platform process for Ad vectors could enhance their purification, maximizing recovery of critical vaccine components. Jug also highlighted BIA’s abilities to supply chromatographic columns consistently and to support customer method development.

Jug’s Presentation
Jug highlighted the advantages of monolith chromatography for Ad purification. Monoliths contain interconnecting channels. Separation is flow independent therein, so eluent is processed quickly without generating turbulence and shear forces that could damage viral vectors. The channels permit no diffusion, and they lack dead-end pores, minimizing the amount of unrecoverable material. Thus, monolith columns feature high binding capacities and enable strong recoveries. They also show good scalability with minimal development requirements.

BIA’s initial Ad platform enabled high recovery of sufficiently pure viral vectors. It performed robustly with material from different cell lines used in gene therapy production. However, BIA determined that Ad processing could be enhanced through early management of host-cell chromatin.

When lysed, transfected cells release chromatin comprising positively charged histones and negatively charged DNA. Those molecules can bind with highly charged proteins. The resulting complexes can cause side effects in patients and must be removed. Doing so is difficult, however, because such complexes can present positive or negative charge depending on which surfaces are exposed.

BIA’s improved Ad platform minimizes chromatin complexing to enhance subsequent chromatography steps. At harvest, a cell pellet is diluted in buffer. Lysis is performed by adding sodium chloride and polysorbate 20 to concentrations of 0.5 M and 0.25%, respectively. Jug noted that the high salt concentration prevents formation of tightly bound chromatin complexes, facilitating nuclease digestion of DNA.

After a brief incubation, Kryptonase reagent (BIA Separations) is added. The material is incubated, clarified with coarse and fine filters, and subjected to tangential-flow filtration (TFF). After pH adjustment and filtration to remove aggregates, the material is loaded onto CIMmultus QA columns for anion-exchange (AEX) chromatography in bind–elute mode. Virions can be concentrated by TFF or purified further by another chromatography step.

New analytical methods can facilitate monitoring of impurity removal throughout the process. Knowing that PicoGreen dye binds with DNA, BIA developed a staining method that enables rapid DNA detection using in-process fractions. Samples are analyzed using a PATfix high-performance liquid chromatography (HPLC) system coupled with CIMac Adeno analytical columns. PATfix systems can also be equipped with multiangle light-scattering (MALS) detectors, enabling sensitive vector quantification and orthogonal confirmation of HPLC results.

Jug presented data from analyses of in-process fractions to demonstrate the platform’s capabilities. Lysate samples showed high levels of well-separated impurities, including DNA labeled with PicoGreen stain. Clarification, TFF, and filtration reduced those impurities significantly. Analysis of TFF retentate showed that small proteins and residual DNA largely had been removed. Eluate from the AEX step was pure and free from chromatin complexes.

Studies of loading capacity and breakthrough yielded similar results. BIA tested four samples representing harvests from an adherent cell line; a low-density, suspension-adapted cell line with a low multiplicity of infection (MoI); and two high-density, suspension-adapted cell lines with high MoIs. In all cases, the process removed >99.7% and >99.0% of host-cell DNA and proteins, respectively, with high Ad recoveries (>83%) from all four samples.

Those results indicate that early removal of chromatin from process lysate can improve chromatographic separation of Ad vectors in CIMmultus QA columns. Thus, BIA’s second-generation purification platform represents a promising approach to manufacturing Ad vectors.

Questions and Answers
Is BIA’s Ad platform serotype specific? It performs robustly across serotypes.

How can the platform’s loading capacity be improved? Proper sample preparation can increase capacity. The goal is to remove host-cell DNA before initiating chromatography. Operators should administer high concentrations of salt and ensure enzyme activity during lysate preparation. The filtration step after load preparation also helps to optimize capacity.

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