Specific focus on the patent literature
In this paper, virus purification and removal by ultrafiltration are reviewed with specific focus on the patent literature. Membrane ultrafiltration is a pressure driven process which has a wide spectrum of industrial applications but is most attractive for sensitive biological streams carrying molecules like proteins. This process has thus naturally been adopted for the removal of viruses from blood and biopharmaceutical streams as well as for virus removal from drinking water. Safety regulations and associated penalties provide further incentives for limiting virus titers in such bioprocessing. Virus ultrafiltration aims at flux improvement, higher efficiency of removal and elongated filter life. In this regard, the proper choice of membrane material and technique is essential. Recent patents show strives in two directions namely inventions on membrane material design and filtration configuration and operation (methods). The diversity of biological fluids is seen to be a continuous challenge for researchers aiming for generic filtration methods for virus removal.
Keywords: Virus removal; Virus clearance; Virus concentration; Ultrafiltration; membrane; Pharmaceutical.
Purity, safety and efficacy are important objectives in biopharmaceutical operations. Membrane filtration is an attractive method playing an increasing role towards achieving the said objectives alongside economical and reliable production [1- 3]. Various researchers have reviewed membrane filtration operations for micro-organisms separation covering the different size scales, thus discussing microfiltration (MF) [4,6,8,9], ultrafiltration (UF) [4,5,7,8,9], nanofiltration (NF) [9,10] and reverse osmosis (RO) . Although the development of laboratory scale UF dates back to 1960s , its actual exploration as colloidion membranes was much earlier dating back to 1900s and was later investigated by Bechhold . With the continuous development and improvement of membranes, UF is considered to be more preferable and largely replacing size-exclusion chromatography for removing a wide range of biological and non-biological particulates from solutions . UF can obtain high rejection values with the appropriate membrane and operating conditions . The removal of microorganisms like bacteria and mycoplasma from biological suspensions was conventionally done by filtration [5, 16]. With the progress in filtration technologies this has been extended to virus removal [7, 17].
In the context of virus UF, which is the focus of this paper, the size differences between the retained and rejected entities and the membrane pore size becomes an important factor . Hepatitis B, Hepatitis C, human immunodeficiency virus, parvovirus and cytomegalovirus are of greatest concerns to human health. They are different and offer diversity in being enveloped, non-enveloped, ribonucleic acid, deoxyribonucleic acid viruses that follow a broad spectrum of sizes [8,19,20,21]. Contaminations by these viruses [22,23] have been reported in various cell cultures, vaccines and biotherapeutic products. In this paper we focus on virus removal from biological fluids by UF and review the related developments in the recent patent literature.
Principles of ultrafiltration and virus removal
Ultrafiltration is a membrane separation technique used for the separation, from fluids, of extremely small particles at the submicron range and for the concentration of dilute solutions of biomolecules 1,24, 25,26]. Particle size plays a major role apart from particle shape and charge, during separation. Particles larger than the membrane pores will be retained on the membrane side forming a concentrated suspension known as the retentate. The stream passing through the membrane and carrying particles smaller than the membrane pores is the permeate stream. Ultrafiltration is considered to be a far gentler process to the particles being processed in comparison with non-membrane processes like chromatography, dialysis or solvent extraction [2, 27].
The removal of virus particles from fluids can be accomplished by two UF methods namely Dead-end Filtration (DEF) or tangential flow filtration (TFF) (Fig. (1)). In DEF (or otherwise called direct flow filtration) systems, the membrane is a flat or pleated sheet with pore sizes normally ranging between 50-500 kDa. This is quite an easier system to operate while being cost effective. The disadvantage of this system is that the filter life is relatively shorter. In tangential flow (or otherwise called cross-flow) filtration systems, the fluid is passed across the membrane surface reducing gel layer formation by the flow shear action and thus resulting in relatively longer membrane life. In TFF, the membrane can be a flat or hollow fibre while the pore sizes range between 70-300 kDa. Such membranes can be made from natural or synthetic materials. These materials could be regenerated from cellulose fibre, poly acrylonitrile, polysulfone, polyethersulfone, polyamide, polyimide, cellulose acetate or polyacrylamide.
Ultrafiltration is receiving increased attention to solve issues related to disease virus contamination . The removal of viruses is dependent on the fluid in which it is suspended in, the virus size and the size of other particles in the fluid . Table 1 presents a summary of some important viruses, their sizes, typical UF membrane parameters, the type of fluid carrying the viruses as well as log reduction values (LRV)1. The LRV is known to be a strong function of the membrane configuration , the virus particle size as well as the concentration [31, 32]. In general, variability in membrane configurations provides variable filtration performance. Two examples include the use of cylindrical ceramic membrane of TFF type for MLV titers that lead to a multi-fold increase in concentration  and the study by Bohonak and Zydney (2005)  that demonstrated how normalised flux declined by nearly 50% depending on the orientation of the membrane and because of submicron particle fouling. Demonstrating the hydraulic characteristics of some commercial virus filtration membranes, that same study  also showed reduced permeability as a result of membrane compaction and improved flux due to time-dependent wetting of the membrane pores.
1 Log reduction value or LRV is otherwise known as virus retention. This is the logarithmic of the ratio of virus concentration in the feed to that in the permeate. Throughput is defined as the volumetric permeate flow per membrane area. The virus filtration membranes are generally rated based on the LRV and throughput.
Safety Considerations and Regulatory Requirements
Safety of biotechnology products from viral contamination receives high priority in the production processes of the biotechnology and food industries. Safety and regulatory requirements work to control virus contamination of industries’ marketed products. The flow diagram shown in Fig. (2) illustrates the stages of processing a product would go through in order to assure quality control as well as almost virus free product output. The potential sources of virus contamination are either from the master cell bank or adventitious viruses introduced during production. The presence of virus particles in the process streams is assessed before a virus clearance step is carried out. The configuration and operation of the virus clearance is dictated by the biology of the virus(es) which accordingly can be inactivated (by heat, radiation or chemical treatment) or removed by UF (or by chromatographic means). The production process is evaluated by the use of relevant viruses, specific/non-specific model viruses or virus-like nanoparticles. The effectiveness of the virus removal procedures is judged based on factors like LRV taking into account variations in process parameters and selectivity amongst other factors. The final product would be expected to contain less than one virus particle per one million doses.
Bioproducts manufacturers typically consult with regulatory bodies to meet set guidelines on UF-based virus clearance operations. Regulatory requirements can be very stringent as typified by the following :
- Manufacturers are required to assess clearance of viruses at each and every stage of production in their manufacturing process before marketing.
- The European (Germany and France) regulatory agencies require that manufacturing processes be evaluated to clear non-enveloped parvoviruses in addition to retroviruses.
- The US Food and Drug Administration (FDA) requires manufacturers to exhibit the capability for viral clearance with one relevant retrovirus before starting further phase studies.
Accelerated developments are taking place in the manufacturing methods of UF membranes, their configurations and material makeup towards achievement of appropriate retention values, throughput and permeability for virus removal. A patent review covering inventions on the use of UF for virus removal is summarised in Table 2. High retention values were obtained with porous polyvinylidene fluoride membranes [40,41] and cellulosic membranes . Defect free cellulosic UF membranes made from microporous polymeric substrates, were capable of maintaining desirable flux values as well as good retention performance . Moya’s patent  discusses membranes useful for removing virus from the protein solution, where the method for making membranes includes an additional step of autoclaving in boiling water and/or steam. This provides a caustic resistant, hollow fiber or sheet type which is hydrophobic polysulfone composite polymer membrane having its surface rendered hydrophilic with a hydroxyalkyl cellulose capable of achieving higher LRV value and throughput.
A TFF system has been considered by van Reiss [44, 45] to separate species of interest in the range 1 to 1000 kDa MWCO (mostly targeting biological fluids) from mixtures. The flux is found to be dependent on transmembrane pressure until the pressure reaches a transition point beyond which this dependency drops off. This patented filter is claimed to be capable of filtering similar sized specimens. Osbourn et al’s  method employs filtration aid together with low concentration of metal ions in place of nucleases for commercial scale purification of encapsulated viruses from cell culture. In these methods, UF plays an important role in separating viruses from DNA and RNA species present in the cell culture. The filtration is aided not by enzymatic nucleases but rather by filtration aids such as diatomaceous earth. This offers advantages for commercial scale purification of encapsulated viruses particularly as specification tests that would have to be designed to demonstrate the removal (i.e. absence) of the nuclease(s) in the final product would be eliminated. Similarly, a tangential flow system with the hollow type UF membrane is recommended as the preferable and robust method for virus concentration ; Coffey’s improved method for virus purification from cell culture involves a simple extraction step in which a detergent is directly added to the cell culture where the resulting viruses are suitable for clinical administration to mammals, this being a major step towards cell therapy . Tullis  utilized hollow fiber membranes (200-500 nm pore size) for virus (110 nm dia) removal from the blood stream. The retention level of min 3 LRV of virus removal from protein solutions was achieved using normal flow or DEF filtration but with one or more UF membranes [50,51,52]. Winge’s  virus filtration method for solutions containing macromolecules suggests that the DEF technique offers economic advantages due to simpler equipment, simpler operating procedures and reduction in the loss of macromolecules. It facilitated the virus filtration process in terms of residence time reduction and optimized yield in comparison with the TFF as DEF involves placing the macromolecule containing solution in a pressure vessel prior to filtration and pressing the solution through the membrane with the aid of a pressure source.
The method of using multilayered membranes [54-56] with at least one membrane being oriented tight side down stream showed satisfactory levels of virus removal but the protein passage and flux were low. Such multilayered membranes can be UF membranes supported with monomer surface coating or two anisotropic membranes with one membrane juxtaposed with at least one other membrane so that substantially all the skin surface of one membrane is in intimate contact with all of the skin of the other membrane. Other such membranes can be comprised of a tight side with porous support which is characterized by having asymmetric structure free of macro-voids when exposed to protein solution, exhibiting low protein binding and these are normally used to filter viruses from protein streams. In order to increase the filtration rate during the UF of viruses, the viruses to be removed are increased in size by incubation with a high molecular weight receptor, binding preferably a specific antibody so that the separation effect is improved and large pore diameter membranes can be chosen .
From this patent study, it is evident that based on the size of the particles to be separated from the fluid, a suitable membrane and membrane configuration should be selected in tandem to obtain the required retention performance. As such there is a body of knowledge in the patent literature that one ought to consult in the process of choosing a more suitable filtration setup. It can be seen that there is an art at play here where the skilled artisan may select through experimental campaigns the optimal combination of membrane, membrane configuration, operating conditions, processing steps, and so on. Thus well-defined deterministic knowledge for virus removal by UF is still outstanding. Having said that, there is a wide scope of research to be done in the area of virus removal by UF. This provides ample opportunities for scientific inventors in this field.
Current & Future Developments
The removal of viruses by UF methods could potentially play a key role in combating a wide range of diseases. Further, virus removal is an essential step in purification of water for drinking and in the production of virus-free biopharmaceuticals as well as virus drug vectors. Virus removal research focuses on membrane material design, membrane system configuration and operation aiming for improved flux values and elongated filter life. This research area is expanding and currently receiving more attention due to safe drinking water becoming ever so scarce and to the rapid growth observed in the biopharmaceuticals and drug delivery industry sectors amongst other factors. We attempted in this paper to review the recent patents on the use of ultrafiltration in virus removal. A silver bullet solution was not exactly found, suggesting that there are challenges to overcome related to the regulatory requirements, membrane design, process operation, and the appropriate and integrated selection of virus filters and configurations. There is hence ample opportunities for researchers and inventors in this overly knowhow driven field. Future progress will require an interdisciplinary approach to the problem by teams of scientists and engineers particularly teams comprising members from the fields of biology, materials, fluids and process systems. Process modeling  will be an important avenue towards understanding and optimising designs and operations [37-39]. Complex models can be developed aided by the high computation power that is continuously increasing. Models that account for virus-membrane stochastic interactions as well as fluid dynamics will provide opportunities for improved UF designs and operation.
- MLV Murine Lukemia Virus
- HIV Human Immunodeficiency Virus
- PVDF Poly Vinylidene Fluoride
- PBS Phosphate Buffered Saline
- FCS Fetal Calf Serum
- HSA Human Serum Albumin
- MWCO Molecular Weight Cut Off
- TFF Tangential Flow Filtration
- DFF Direct Flow Filtration
- DEF Dead End Filtration
- LRV Log Reduction Value
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