Figure 1. Frequently, two polishing steps are used to remove the remaining impurities and product variants/aggregates [17]^[14]. Resin- or membrane-based chromatography (ion exchange or hydrophobic interaction chromatography) is frequently used. The polishing steps remove impurities such as DNA, host cell proteins (HCP), and product aggregates ^[2]. Typically, all streams and buffers which enter the purification process are passed through sterilizing grade (0.22 μm pore size) filters to reduce bioburden. As shown in Figure 1, the virus filtration step is typically located near the end of the purification train. The product is relatively concentrated and highly purified. High product concentrations can lead to compromised performance due to product aggregation and increased adsorption to the virus filter membrane. A final ultrafiltration/diafiltration step is used to concentrate the product and place it in the formulation buffer needed for stability during shipping/storage and delivery to the patient. The final 0.22 µm pore size filter is used to ensure sterility of the product and is often part of the final fill-finish operation.

2.2. Viruses, Virus Clearance, and Virus Filters

Many mammalian cell lines produce endogenous retrovirus-like particles ^[2]. These particles are typically around 80–100 nm in size. Clearance can be achieved by inactivation and/or physical removal from the process stream [12,18,19]^[15][16][17]. During purification, manufacturers of mammalian cell-derived biotherapeutics must demonstrate that the process will yield a final product containing no more than one virus particle in a million doses. Estimates of the number of virus particles in a single dose equivalent from the bioreactor could be as high as 10¹⁰–10¹⁵ retrovirus-like particles per mL ^[2]. Removal of adventitious viruses such as parvovirus is also required. These much smaller viruses are around 20 nm in size. In the past, filters targeted for retrovirus and parvovirus removal were included in the purification train ^[3]. Recent studies show that virus clearance filters designed to provide clearance of smaller parvovirus can be used to clear much larger retroviruses simultaneously [20]^[18]. Table 2 shows some viruses that are employed for validation studies in biomanufacturing. The enveloped retroviruses are typically larger than the non-enveloped parvoviruses. Consequently, virus filtration membranes that are validated for removal of parvovirus are also effective at clearing retrovirus from the product.