Mitigation of Virus Filter Fouling

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1		Sumith Ranil Wickramasinghe	--	1291	2022-04-24 06:15:09	\|
2	format correct	Conner Chen	Meta information modification	1291	2022-04-26 02:25:33	\|

Even though the support structure of the virus filter can function as an inline prefilter, significant fouling is often observed due to the product- and process-related foulants could be present in the feed stream. Standard practice involves the inclusion of a virus prefilter to remove these contaminants. Virus prefilters may rely on one or more mechanisms of action for the removal of foulants.

virus filtration

1. Introduction

Virus filtration is different from typical pressure-driven membrane filtration processes, as the filter is designed to obtain very high levels of removal of potential virus contaminants. Further, as it is impractical to validate that there is zero carryover of any trapped virus particles, reuse of the virus filter is impossible. Consequently, these are single-use devices. Virus filters are typically run in normal flow (dead end) mode, rather than tangential flow mode used for protein ultrafiltration, since normal flow is less complex and requires only a single pump.

The performance of virus filters is measured in terms of product recovery, log reduction value (LRV) of the virus (defined as the logarithm to base 10 of the ratio of the virus concentration in the feed to that in the permeate), and the productivity of the filter. Productivity is typically expressed as the volume of feed that can be processed per membrane area (L·m⁻²) before the filtrate flux has decreased to unacceptably low levels (for operation at constant transmembrane pressure). Since biopharmaceutical manufacturing operations are still essentially batch processes, the virus filter is often sized such that the entire batch can be processed in one shift.

Frequently, identifying a virus filter that meets the three performance requirements, product recovery, LRV, and productivity, is challenging and highly dependent on the feed stream and membrane properties. As the virus filtration step is located towards the end of the purification train, the product is highly purified and moderately concentrated ^[1]. Membrane fouling, which leads to compromised performance, is typically due to product- and process-related foulants rather than any rejected virus particles ^[1], since the concentration of virus particles in any process will be orders of magnitude less than that for the product.

In order to remove impurities and foulants, virus filtration membranes are sometimes designed with a reverse asymmetric structure ^[2]. In this case, the barrier layer faces away (downstream) while the more open support layer faces towards the feed stream ^[3]. The support layer can act as an inline prefilter that traps larger foulants and protects the tight barrier layer ^[4]. However, essentially symmetric membranes are also used industrially. The unique requirements of virus filtration are very different from typical pressure-driven membrane separation processes such as ultrafiltration. Identifying and sizing an appropriate virus filter is often particularly challenging.

2. Prefiltration before Virus Filtration

Even though the support structure of the virus filter can function as an inline prefilter, significant fouling is often observed due to the product- and process-related foulants listed above that could be present in the feed stream. Standard practice involves the inclusion of a virus prefilter to remove these contaminants. Virus prefilters may rely on one or more mechanisms of action for the removal of foulants.

A prefilter, often inline, is added upstream of a virus filter to increase permeate flux and productivity. The improvement in performance depends on the biotherapeutic product properties, prefilter material, and buffer conditions ^[1]. The mechanisms and conditions for foulant capture are different for different prefilters ^[1]. Table 1 gives a non-exhaustive list of common prefilters used to capture foulants and mitigate fouling of the virus filter. Size exclusion prefilters such as the 0.1- and 0.22-micron filters remove aggregates larger than the respective size cut-off of the prefilters. Ion exchange prefilters are more effective at low conductivity due to the reduction in electrostatic shielding.

Table 1. Commercially available prefilters, modes of action, and manufacturers ^[5].

Prefilter	Material	Mechanism of Action	Manufacturer
Planova 75 N	Regenerated cellulose	Size exclusion, removal of small aggregates	Asahi Kasei Bioprocess
Bottle top 0.1/0.22 µm	Polyethersulfone	Size exclusion, removal of large aggregates	Multiple
Pegasus Protect	Nylon	Size exclusion, removal of large aggregates	Pall
Sartobind Q	Quaternary ammonium ligands	Anion exchange	Sartorius AG
Sartobind S	Sulfonic acid ligands	Cation exchange	Sartorius AG
Sartobind phenyl	Phenyl ligands	Hydrophobic interaction	Sartorius AG
Viresolve Pro Shield	Surface modified PES	Size exclusion, ion exchange (cation)	MilliporeSigma
Viresolve Pro Shield H	Surface modified PES	Size exclusion, hydrophobic interaction	MilliporeSigma
Viresolve Prefilter	Diatomaceous earth, cellulose fibers, and a cationic imine binder	Cation exchange, size exclusion, hydrophobic interaction, ion exchange	MilliporeSigma

The Sartobind S and Q are commonly used as polishing steps, usually before the virus filter. They are run as a separate unit operation (not inline) and can also provide significant virus clearance by adsorption. Brown et al. indicate that virus filter throughput may be increased by adsorptive ion exchange membrane prefiltration ^[6]. Consequently, if an ion exchange polishing step is used just before the virus filtration step, it may lead to higher fluxes and productivity during virus filtration. However, if the prefiltration step is not conducted in line, the improvement in performance of the virus filter will depend on the hold time between the two unit operations and the product properties under the buffer conditions of interest.

Wickramasinghe et al. opined that trace amounts of aggregates that have a diameter less than 50 nm play a significant role in virus filtration membrane fouling ^[1]. These small aggregates with diameters less than 50 nm cannot be removed by 0.1 μm or 0.22 μm size exclusion filters but can block the virus filter pores. Virus filtration membranes typically have a pore size around 20 nm at the separation-active layer. Soluble aggregates (20–50 nm) can be removed using adsorptive prefilters (cation exchange, anion exchange, multimodal) to prevent fouling of virus filters. Adsorptive prefilters have been shown to bind aggregates, thereby reducing subsequent fouling of virus filters. Adsorptive prefilters work well for product oligomers in the 600–1500 kDa range, which cannot be removed by 0.22-µm size exclusion prefilters. Ion exchange prefilters have shown great potential in clearing aggregates for effective downstream processing operations ^[7].

Endotoxins can be removed using hydrophobic prefilters, which bind the phosphorylated lipid moiety, or using anion exchange prefilters to capture the polysaccharide moiety ^[8]^[9]^[10]. Anion exchange membranes work well for endotoxin removal due to the positively-charged ligands binding with the negatively-charged endotoxin (isoelectric point = 1–4).

Hydrophobic prefilters require a moderate/high salt content (ionic strength) to reduce the product’s solvation layer, enabling exposed hydrophobic patches to adsorb on the hydrophobic prefilter. Hydrophobic interaction prefilters can be effective in removing product variants with different hydrophobicity, as well as some of the more hydrophobic product aggregates.

Ion exchange prefilters are helpful in the downstream removal of HCPs due to the pI difference between mAbs and most HCPs. DNA is strongly negatively charged in aqueous solution and can be effectively removed using anion exchange membranes during polishing operations ^[10]^[11].

Multimodal prefilters are useful to filter out foulants that cannot be removed by ion exchange, size exclusion, or hydrophobic interaction-based prefilters alone. These multimodal prefilters include the three prefilters from MilliporeSigma, as shown in Table 4.

3. Mitigation of Virus Filter Fouling Using Process Parameters

Monoclonal antibody properties are highly dependent on the buffer conditions and excipients that are part of the formulation. Excipients are non-drug substance components of the formulation. During high-throughput screening of mAbs for optimum buffer conditions, a specific buffer type and composition may be found to inhibit aggregation and mitigate fouling of virus filters. Phosphate, acetate, and tris buffers may work for some biomolecules, while viscosity-inhibiting buffers may be preferred for highly concentrated mAbs. Arginine reduces mAb monomeric self-association, non-specific membrane interactions, and mAb aggregation ^[12]. Excipients such as histidine and arginine help to marginally improve the stability of monomeric species during formulation ^[13]^[14]. These excipients can result in cost reduction for virus filter consumables but may require further removal before drug substance delivery to the patient.

This entry is adapted from 10.3390/bioengineering9040155

References

Wickramasinghe, S.R.; Namila; Fan, R.; Qian, X. Virus Removal and Virus Purification. In Current Trends and Future Developments on (Bio-) Membranes; Elsevier: Amsterdam, The Netherlands, 2019; pp. 69–96.
Zhang, D.; Patel, P.; Strauss, D.; Qian, X.; Wickramasinghe, S.R. Modeling flux in tangential flow filtration using a reverse asymmetric membrane for Chinese hamster ovary cell clarification. Biotechnol. Prog. 2021, 37, e3115.
Hoffmann, D.; Leber, J.; Loewe, D.; Lothert, K.; Oppermann, T.; Zitzmann, J.; Weidner, T.; Salzig, D.; Wolff, M.; Czermak, P. Chapter 5—Purification of New Biologicals Using Membrane-Based Processes. In Current Trends and Future Developments on (Bio-) Membranes; Basile, A., Charcosset, C., Eds.; Elsevier: Amsterdam, The Netherlands, 2019; pp. 123–150.
Wickramasinghe, S.R.; Stump, E.D.; Grzenia, D.L.; Husson, S.M.; Pellegrino, J. Understanding virus filtration membrane performance. J. Membr. Sci. 2010, 365, 160–169.
Johnson, S.A.; Chen, S.; Bolton, G.; Chen, Q.; Lute, S.; Fisher, J.; Brorson, K. Virus filtration: A review of current and future practices in bioprocessing. Biotechnol Bioeng 2021, 119, 743–761.
Brown, A.; Bechtel, C.; Bill, J.; Liu, H.; Liu, J.; McDonald, D.; Pai, S.; Radhamohan, A.; Renslow, R.; Thayer, B.; et al. Increasing parvovirus filter throughput of monoclonal antibodies using ion exchange membrane adsorptive pre-filtration. Biotechnol. Bioeng. 2010, 106, 627–637.
Yigzaw, Y.; Hinckley, P.; Hewig, A.; Vedantham, G. Ion exchange chromatography of proteins and clearance of aggregates. Curr. Pharm. Biotechnol. 2009, 10, 421–426.
Zhang, M.; Zhang, L.; Cheng, L.-H.; Xu, K.; Xu, Q.-P.; Chen, H.-L.; Lai, J.-Y.; Tung, K.-L. Extracorporeal endotoxin removal by novel l-serine grafted PVDF membrane modules. J. Membr. Sci. 2012, 405–406, 104–112.
Ritzén, U.; Rotticci-Mulder, J.; Strömberg, P.; Schmidt, S.R. Endotoxin reduction in monoclonal antibody preparations using arginine. J. Chromatogr. B 2007, 856, 343–347.
Saraswat, M.; Musante, L.; Ravida, A.; Shortt, B.; Byrne, B.; Holthofer, H. Preparative purification of recombinant proteins: Current status and future trends. Biomed. Res. Int. 2013, 2013, 312709.
Miesegaes, G.R.; Lute, S.C.; Read, E.K.; Brorson, K.A. Viral clearance by flow-through mode ion exchange columns and membrane adsorbers. Biotechnol. Prog. 2014, 30, 124–131.
Arakawa, T.; Tsumoto, K.; Nagase, K.; Ejima, D. The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography. Protein Expr. Purif. 2007, 54, 110–116.
Philo, J.S.; Arakawa, T. Mechanisms of protein aggregation. Curr. Pharm. Biotechnol. 2009, 10, 348–351.
Jabra, M.G.; Tao, Y.; Moomaw, J.F.; Yu, Z.; Hotovec, B.J.; Geng, S.B.; Zydney, A.L. pH and excipient profiles during formulation of highly concentrated biotherapeutics using bufferless media. Biotechnol. Bioeng. 2020, 117, 3390–3399.

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Sumith Wickramasinghe

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1. Introduction

2. Prefiltration before Virus Filtration

3. Mitigation of Virus Filter Fouling Using Process Parameters

References