Removing viral contaminants from animal cell-culture derived biologicals is a major challenge of downstream purification because it involves laborious and time-consuming techniques that result in increased manufacturing costs. Updated regulatory guidelines demanding higher safety margins and enforcing good manufacturing practices are leading to tighter specifications. This stresses the need to implement robust and efficient orthogonal strategies for virus clearance to meet the requirements of a virus-clearance approach based on risk assessment. Such technologies can involve virus removal by nanofiltration, inactivation by ultraviolet C (UVC), and adsorption by membrane chromatography. Additionally, this three-tier platform should be characterized by using disposables to meet the flexibility and low capital requirements needed in early-stage process development. All of these new paradigms in virus clearance are scalable, economical, orthogonal, and disposable.
Viruses are composed of small amounts of DNA or RNA, encapsulated by a protein coat, and may be enclosed in an envelope made of proteins, carbohydrates, and lipids. Viruses exploit the enzymes and other host-cell machinery to replicate themselves. The viral nucleic acid can be single- or double-strand DNA or RNA. A single virion is a completely developed virus particle made of 1–50% nucleic acid and 50–99% proteins or glycoproteins and lipids. Virions range from about 15 to 450 nm in size.
Regulatory Requirements Regarding Viral Inactivation and Clearance
Viral contamination is a risk to all biotechnology products derived from cell lines of human or animal origin. Contamination of a product with endogenous viruses from cell banks, or adventitious viruses from personnel can have serious clinical implications.3 To ensure maximum viral safety, the ICH Q5A regulatory guideline mandates that manufacturers of therapeutic biological products for human use implement adequate technologies in their manufacturing process and demonstrate the capability of their processes to remove or inactivate known or adventitious contaminants based on a process-specific virus clearance strategy.3 According to the latest draft on regulatory guidance from the European Agency for Evaluation of Medicinal Products (EMEA), potential contaminants may be enveloped or nonenveloped, small or large, DNA or RNA, labile or resistant viruses.4
Viral safety of licensed biological products must be assured by three complementary approaches: (i) thorough testing of the cell line and all raw materials for viral contaminants, (ii) assessing the capacity of downstream processing to clear infectious viruses, and (iii) testing the product at appropriate steps for contaminating viruses.3 The first study is required before Phase 1 clinical trials, in which the process should be evaluated for inactivation or removal of an enveloped and a small nonenveloped virus and at least two orthogonal steps should be used for achieving the same.3–4 A second and more complex study is then conducted before manufacturing Phase 2/3 materials to provide evidence of the effective and adequate clearance of relevant and known viruses, as well as the removal of a range of novel and unpredictable viruses.5 A viral clearance study with at least four viruses for late stage is state-of-the-art and log-reduction values (LRV) of four or higher are perceived as robust and effective safety measures.1 At least one of these clearance steps evaluated in a validation study must be effective against nonenveloped viruses, such as porcine parvovirus (PPV), canine parvovirus (CPV), or minute virus of mice (MVM). As for enveloped retroviruses, although no cases of infection or transmission of Chinese hamster ovary (CHO) cell-related type A and C virus particles have been reported so far, retrovirus-like particles theoretically pose safety concerns to humans because of their morphological and biochemical resemblance to tumorigenic retroviruses, and therefore, have to be completely removed or inactivated.6–9