The main aim of DSP is to eliminate the impurities, which can be divided into

two subgroups: process-related and product-related impurities [1]. Product-

related contaminants are related to variants that differ from the desired product,

such as dimers, aggregates, empty capsids, product with heterogeneity of post-

translational modifications (glycosylation, phosphorylation and acylation).

Process-related contaminants include all the potential ones added or derived

from the manufacturing process during up- or downstream, such as host cell

protein (HCP), host cell DNA (HCDNA), cell culture media components,

endotoxins, anti-foam reagents, stabilizers, excipients, proteases, and nucleases,

which do not have properties comparable to those of the desired product with

respect to activity, efficacy, and safety. Residual host cell DNA, HCP, and en-

dotoxins are the impurities more strictly controlled by the authorities. The

maximum residual amounts of host-cell DNA depend on the cellular production

platform. Nevertheless, it should be between 100 pg/dose to 10 ng/dose of sizes

not above 200 base pairs [2]. Regarding HCP, no strict limitation have been

established by regulatory authorities. However, most therapeutic proteins re-

viewed by FDA have been reported to contain ELISA-based host cell protein

level between 1–100 ppm [3]. Also, a recommendation of endotoxin level below

20 EU/mL for recombinant sub-unit vaccines and below 10 EU/mL for genetic

vectors is suggested; although higher endotoxin levels may be acceptable in

certain cases [4].

Finally, the goal is to obtain a product with high purity, potency, and quality that

can meet the stringent guidelines of the regulatory authorities, the U.S. Food and

Drug Administration (FDA) and the European medicines Agency (EMA) [5,6].

7.1.2

TRADITIONAL METHODS FOR VIRUS PURIFICATION

Complexity of the purification processes holds in the fact that viruses are quite

large when compared with other biomolecules present in the harvested cell lysate

such as proteins, peptides, sugars, and nucleic acids. For example, proteins ty-

pically range from 0.005 to 0.006 × 10⁶ Da, whereas viruses are around 5 × 10⁶

Da. They present sizes up to 1,000 nm, although the most common viruses

produced have sizes between 20–400 nm [7]. This is why the first purification

strategies implemented in vaccine manufacturing were based on virus size and

density properties. Ultracentrifugation and density gradient centrifugation, later

combined with filtration techniques, were the first methods to be used for the

purification of viral particles.

Density gradient centrifugation, either used with sucrose, caesium chloride (CsCl),

or iodixanol gradients, is a well-known and established traditional purification tech-

nique. It is generally used to purify limited amounts of bulk for preclinical applica-

tions. These gradients were used to process many viral particle preparations, including

adenovirus [8], AAVs [9], and influenza [10], but also several other viral particles

[11]. Figure 7.2 shows a typical result of CsCl purification of adeno- (AdVs) and

adeno-associated virus (AAVs) with their respective analyses of the final purified

viruses by transmission electron microscopy (TEM) images. A purification strategy

Downstream processing

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