amount of DNA present in the final product. Also, DNA removal early in the

process will prevent the formation of complexes with the virus [44]. Nuclease

treatment, such as Benzonase®, is one option frequently used to reduce the DNA

content. However, there have been emerging alternative options, such as Denarase®

and, more recently salt activated nuclease (SAN), that showed improved results in

AAV production when compared with the other two commercially available en-

donucleases [45]. Oxford Biomedica developed a novel strategy that overcomes the

nuclease addition step using secreted nucleases (SecNuc®) in co-production with

viral vector manufacturing. Although it is a challenging step, these alternatives can

overcome the challenges by reducing the overall manufacturing costs. The nuclease

addition step can be performed before or after the clarification step as it is an

additional impurity that should be removed during the next purification steps.

Notwithstanding, the industry witnessing the end of Benzonase’s patent and the

appearance of several competitors will certainly cause a substantial decrease in

nuclease price in the future. Other more conventional and cheaper alternatives for

host-cell DNA removal have been proposed. As examples, selective precipitation

and flocculation can replace nuclease addition and compensate for the higher costs

through the process [2]. Kröber et al. developed a method for DNA precipitation

with polyethylamine (PEI) together with a specific unit operation which allowed to

reach a 500-fold decrease of host-cell DNA at a pilot scale [46]. This technique for

DNA clearance could also work with single-use centrifugation, allowing low-speed

and consequent high-titer viruses.

7.2.2

INTERMEDIATE PURIFICATION––VIRUS CONCENTRATION AND

CHROMATOGRAPHY

Viral vaccine manufacturing requires a combination of several downstream pur-

ification strategies to achieve the desired purity and virus concentration. After clar-

ification, the intermediate purification strategy is highly dependent on the final

vaccine specifications. To move away from the ultracentrifugation techniques, the

knowledge acquired with chromatography in protein purification has been adapted to

viral particles. However, classical chromatography devices and matrices were initially

developed for smaller biomolecules, such as proteins. Thus, there has been a need for

evolution of these processes, to be applied to more complex products like viruses [47].

Furthermore, to reach the required virus dose, concentration methods are ne-

cessary. The intermediate purification of viral vaccines usually comprises a con-

centration using membrane filtration processes and/or a chromatography method.

Currently, ultrafiltration (UF) is the key technique for the concentration and dia-

filtration of viral particles, either for laboratory or large-scale bioprocesses. It allows

the removal of low molecular weight impurities, the reduction of the volumetric

volume, and enables buffer exchange of viral solution. This operation is a pressure-

driven separation method, with transmembrane pressure (TMP), feed, retentate, and

permeate flux as well as the molecular weight cut-off (MWCO) as key parameters.

The MWCO is the mean pore size of a normal distribution, which depends on

the membrane material and the manufacturer. For virus purification, 100−750 kDa

pore size range is the typical range that gives reasonable recoveries (70–85%). The

184

Bioprocessing of Viral Vaccines