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How to implement a risk-based approach to eliminate viruses using orthogonal technologies.
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.
Today's downstream processing operations generally focus on two main areas: the initial recovery phase, when bulk purity is achieved, and the subsequent polishing phase, which adds safety through orthogonal strategies for impurity and pathogen clearance.1 Commercial manufacturing of therapeutic antibodies requires robust and reliable processes that are economical and deliver high yields of a product that is pure and safe for human use. One factor that poses a constant threat to product safety is the presence of viruses in the finished product. Virus contamination of products derived from human or animal cells can have disastrous clinical consequences causing diseases ranging from common colds and influenza, to acquired immune deficiency syndrome (AIDS), hepatitis, herpes, measles, and poliomyelitis. Some viruses like Epstein-Barr, human papillomavirus, and retroviruses are even oncogenic, causing the insertion of cancer-causing genes into cellular genomes.2 It is essential to review both the short- and long-term consequences of viral contaminants existing in biopharmaceutical products. In this context, it is worthwhile to understand some important aspects of current and state-of-the-art methods for inactivating and eliminating viruses from process streams that generate products intended for use by humans.
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.
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
The robust and reliable capability to eliminate viruses must be demonstrated by a risk-based approach.10 Today's requirements demand a statistically independent combination of methods (orthogonal technologies) for removing enveloped and nonenveloped viruses based on the different physical principles of removal and inactivation, and yet are complementary to each other.11–12
Several methods can be used for virus clearance in bioprocessing. These include inactivation methods such as solvent and detergent (SD) or chemical treatments, low pH, microwave heating, adsorption by chromatography, and removal by mechanical or molecular sieving using normal and tangential-flow filtration methods. The first three of these, treatments with solvents and detergents, low pH, or microwave heating, all have significant limitations in their ability to inactivate small nonenveloped viruses. SD treatments were commonly used for plasma proteins and were considered the gold standard for inactivating enveloped viruses.13 It has been shown that SD treatments of a recombinant protein can completely and rapidly inactivate enveloped viruses like PI-3, XMuLV, IBR, and MCF.14 However, small nonenveloped viruses are not being eliminated substantially by this virus-clearance technology. Low pH inactivation of murine retroviruses is reported to be highly dependent on time, temperature, pH, and relatively independent of the recombinant protein type or conductivity conditions outlined.15 Heating is not considered one of the most reliable methods for virus inactivation because of the variation in stability of each viral genome to heat or temperature.
Chromatography and filtration, on the other hand, are widely accepted methods for virus adsorption and removal respectively and act as orthogonal techniques in the viral-clearance platform. Membrane chromatography, a relatively newer technique gaining prominence in biomanufacturing, has proven to be efficient in removing small nonenveloped viruses.16 Ion-exchange membrane adsorbers, with ligand–virus-binding properties similar to those of anion-exchange (AEX) chromatography, have the disposable option as an added advantage. This not only reduces capital costs but also eliminates post-use cleaning, sterilization, validation, and risk of carry-over contamination, thereby simplifying adsorptive virus clearance.17,18
Efficient clearance data between 4.41 log10 and 6.67 log10 for MVM has been determined for membrane chromatography.19 Additional studies have demonstrated that membrane chromatography meets and exceeds viral-clearance performance of Q resin chromatography.20 Clearance capabilities of Sartobind Q for nonenveloped viruses have been shown to be between 3.56 log10 for MVM and more than 6.92 log10 for PPV.21 It has been demonstrated that the platform tested membrane chromatography, has a process capacity greater than 3,000 g MAb/m2 or 10.7 kg MAb/L with a LRV >5 for four model viruses.17 Mass balance in viral-clearance study is another important parameter to demonstrate efficient virus removal by membrane adsorbers. A 100% recovery was demonstrated for PRV, Reo-3, and MVM, when the membrane was stripped with 1-M NaCl, illustrating efficient charge capture for the three model viruses while high salt treatment of the membrane showed 70% recovery for MuLV.16 The virus-clearance capability of such technology has been presented in Tables 1a and 1b.
A second orthogonal technique in the virus-clearance platform is nanofiltration that has traditionally been accepted as a robust method for virus clearance.22 This is the most expensive downstream step, accounting for up to 40% of costs, and is the natural target for optimization.1 Initially, virus removal by filtration was found to be highly dependent on size of the virus, and less dependent on parameters like buffer composition, process time, protein type, and pressure.23 Earlier studies have shown the principal feasibility of PP7, a small nonenveloped 25 nm bacteriophage, to act as a model virus for small, nonenveloped viruses.24 The ability of commonly available nanofilters to retain bacteriophage has been clearly demonstrated in recent studies.25 Virus spiking trials using 20-nm retentive virus removal filters have also shown to clear both large and small viruses (Tables 2a and 2b).26,27
The evaluation of a virus filter should not be limited only to its capacity. An ideal virus filter should retain all viruses and allow high protein transmission while maintaining a high flow rate without significant virus breakthrough. Unexpected virus passage during a virus-clearance step is undesirable from a good manufacturing practice (GMP) and validation standpoint.28–30 Virus passage through the filter could occur for many reasons, including accumulation of aggregates, high spike concentrations, and other impurities that may block the filter or result in a breakthrough.31 This is a serious safety concern that must be minimized. Although contaminants and other various parameters may be the main cause of filter breakdown, some nanofilters still efficiently remove viruses at high LRVs even when experiencing high flow decay. Earlier, detailed analysis of the retention characteristics of PP7 by a PESU-based 20-nm nanofilter underlined the principal capability of nanofiltration to act as a robust and effective virus removal step independent of flow decay or the nature of product being filtered (Figure 1).31 Additionally, a recent report outlined the various titer reduction capabilities of virus retentive nanofilters.32 The study showed that not all filters tested for their LRVs versus flow decay profile experienced a significant loss of titer reduction with increasing flow decay. To ensure the highest level of viral safety of biopharmaceuticals, it is important to understand and predict the efficiency of virus removal steps while also realizing that small virus-retentive filters should not be viewed as absolute in their capacity to clear viruses.
Figure 1. Log-reduction value (LRV) versus flow decay using monoclonal antibodies (MAbs) at 8 mg/mL in 20 mM Tris, 25 mM NaCl, at 7.2 pH, 2 bar, and upstream titer of 7.3 x 107/mL. All samples prefiltered through 0.1 mm membrane.31
In this article, we propose implementing ultraviolet C (UVC) as a third orthogonal technology for virus clearance in the downstream purification process. UVC, newly developed as a virus-inactivation technology, targets small, nonenveloped viruses and offers another robust method for removing adventitious viruses. In UVC radiation, low-dose radiation at 254 nm destroys the viral nucleic acid while maintaining the structural and functional integrity of the protein of interest. IgG losses are <5%.33 The efficiency of viral inactivation and product recovery is sensitive to the viscosity and absorption coefficient of the protein solution and its residence time in the radiation chamber.34 However, further studies are on to demonstrate the effect of UVC, if any, on the protein-folding characteristics, disulphide bonds, glycosylation, and phosphorylation patterns of the protein of interest.
When evaluating the reliability and robustness of nanofiltration, membrane chromatography, and UVC technologies as orthogonal steps in virus clearance, the inclusion of single-use disposables to the above platform is also gaining prominence. In addition to reduced capital costs, disposables provide the flexibility and ease of operation in process optimization and early stages of manufacturing. Further, disposables eliminate cleaning, sterilization, process validation, and the risk of carryover contamination in viral-clearance studies. Various scenarios presented so far have provided evidence that the disposable option for both nanofiltration and membrane chromatography has proven to be technically possible, and furthermore, cost effective.32,35 To underline such statement, cost-model scenarios have been developed and are used to help evaluate the economic justification of sourcing disposable technologies.36
Striking an optimum balance between ensuring high pathogen safety, achieving maximum product recovery, and meeting adequate regulatory expectations is a big challenge for the biopharmaceutical industry. Nanofiltration, membrane chromatography, and UVC, together used as a technology platform for virus clearance by removal, adsorption, and inactivation, provide robust and efficient clearance capability for all viruses with major focus on small, nonenveloped viruses such as PPV or MVM. Driven by regulatory guidance, technologies with the capability to remove or inactivate small, nonenveloped viruses should be implemented from an early stage into the downstream process of a biopharmaceutical to fulfill virus-clearance expectations. Furthermore, the disposable option, when integrated into this technology platform, offers higher flexibility in manufacturing, increased ease and speed of operation, and eliminates the risk of carry-over contamination. The present age recognizes several paradigm shifts in virus and contaminant clearance, which are scalable, economical, and orthogonal. Together with these, the trend toward disposables is a clear implication to combine steps and orthogonal strategies for every objective in bioseparation to realize the industry goals for yield and quality.
SUMA RAY, PhD, is a process development scientist, viral clearance and cell line development, Global Purification Technologies Group, and KLAUS TARRACH is a senior product manager of purification technologies, both at Sartorius Stedim Biotech, Goettigen, Germany, +49.551.308.3959, firstname.lastname@example.org
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