7.2.3

POLISHING

A GMP compliance purification process often includes one or more final steps for

minor impurities removal, named polishing steps. Besides of removal of trace

impurities, the aim of this part of the process is also to ensure the final product

formulation, stabilization, and sterilization. This is challenging since the impurities

still present at this stage are very closely related to the product of interest, such as

aggregates and wrongly assembled particles. Consequently, they are more difficult

to remove. Thus, the resolution is especially important to guarantee a successful

separation, so chromatography techniques are the workhorses at this step. Size

exclusion chromatography (SEC) is the most used technique to remove low mo-

lecular weight impurities, where the virus elutes in the void volume, while the

smaller impurities elute afterward. UF/DF (TFF) can be also used for the same

purpose since both methods rely on size differences. However, SEC has limitations

in scale-up, since the load should not exceed the 10% of column bed volume. Both

techniques can be used for buffer exchange since the final formulation is a crucial

factor to guarantee not only the particles’ stability but also a proper immune re-

sponse. The formulation and stabilization field is gaining special attention as a

research field of interest since the use of adjuvants contributes to an improved

immune response [61–63].

Adsorptive chromatography can be also used for the polishing step, but is

usually preferably operated in negative mode. Ion-exchange chromatography

(IEC) is also a widely used technique for resolving host cell protein-virus particle,

host cell DNA-virus, or even damaged from intact viral particles. There are

several described processes in the literature, using SEC followed by IEC as a

polishing step [32,64]. Multi-modal or mixed-mode (MM) resins are a novel

technology, combining simultaneously various types of interaction in the same

chromatographic media, such as ionic interaction, hydrogen bonding, and hy-

drophobic interaction. One well-known example of mixed-mode media is ceramic

hydroxyapatite, which has already proven its worth for virus purification [65].

Another example of MM is Capto Core 700 resin, which plays with both binding

property and size exclusion. This resin has been used in the negative mode for the

polishing of biotherapeutics [13,54,66].

Sterilizing filtration is usually the final process, where the product is filtered

through a 0.2 μm filter for the final removal of bioburden. This step is exclusively

dependent on the particle size, so particles larger than 200 nm cannot be sterile-

filtered. This is the case for Vaccinia virus and Poxvirus, which impose to work in a

closed-system process to guarantee sterility or bioburden control since the begin-

ning of the process. The membrane material, similar to the other filtration techni-

ques already described in this chapter, is one of the essential parameters which

should be optimized to avoid non-specific interaction between the viral particles and

the membrane matrix. Another critical parameter is the transmembrane pressure

(TMP), which can increase uncontrollably at constant flux, due to filter clogging.

There is still some limitation in sterile filtration in viral-based bioprocesses, since

either by virus adsorption to the filter or presence of aggregates that decrease the

total product recovery [67].

Downstream processing

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