monoclonal antibodies (mAbs). Chromatography is based on different interactions

between the target viral particle and components placed on the stationary phase.

These interactions rely on the surface properties of the viral particle (adsorptive

chromatography) or their size (size exclusion chromatography). In the case of ad-

sorptive chromatography, the viral particles’ solution is passed through a solid

phase (stationary phase) coated with functional groups that capture the viral particle

(also known as bind-and-elude mode or positive chromatography) or capture the

impurities (also known as flow-through mode or negative chromatography). This

depends on the global charge of the virus and the operation mode. In this case,

cationic or anionic exchangers can be used. Ion exchange chromatography (IEX) is

the most used chromatographic technique in vaccines and viral vectors manu-

facturing, exploring the reversible interaction between a charged particle surface

and an oppositely charged matrix. Besides IEX, there are other interactions which

have been exploited such as affinity (AC), hydrophobic interaction (HIC), mixed-

mode (MMC), and size exclusion (SEC). Affinity chromatography, used for long

time in protein purification, has recently gained special prominence in the complex

biotherapeutics field due to their specificity separation, high degree of purity

and recovery, and consequent contribution to the reduction of necessary unit op-

erations [52,53].

The flow-through mode chromatography avoids some of the issues addressed

with other chromatography separation methods, being transversal for different

biological systems. Harsh elution conditions may be omitted and, consequently, the

risk of immunogenicity/infectivity loss and aggregation is also reduced [54]. This

strategy has been recently evaluated for the purification of hepatitis C virus-like

particles. In this process, the product is recovered in the flow-through, avoiding

harsh elution conditions and the risk of virus’s disassembly [37].

The stationary phase can be physically structured as packed beds, membrane

adsorbers, and monoliths, as shown in Figure 7.5. The most commonly used are

packed beds, which consist of beads physically packed into a chromatographic

column. They were extensively used for protein separation, but viruses are gen-

erally larger than proteins, between 20 nm to 400 nm, resulting in low binding

capacities [55]. Given the high interest in developing purification processes for viral

particles, there are already some novel optimized resins suitable for the purification

of large molecules such as POROS® (ThermoFisher Scientific) and NUVIA HP-Q®

(Bio-Rad). These beads are especially suitable for large molecules, due to their

rigidity, robustness, and increased surface area. However, these resins largely de-

pend on the size of the target molecule, especially in the case of a virus. They might

be ideal for small viruses (20–30 nm) such as AAV or poliovirus but they show

limitations for larger viruses (above 80–100 nm), which is the case for the majority

of oncolytic viruses.

Chromatography on convective flow devices such as monoliths, nanofibers, and

membrane adsorbers has been emerging as an efficient alternative to conventional

ones. As depicted in Table 7.2, membrane chromatography has several advantages

over packed-bed chromatography. Owing to their different architecture, mass

transport through the pores/channels takes place mainly by convection overcoming

virus particle diffusion issues faced by the traditional packed-bed chromatography.

186

Bioprocessing of Viral Vaccines