- Email: [email protected]
Enveloped virus flocculation and removal in osmolyte solutions
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Journal of Biotechnology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
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Short communication
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Enveloped virus flocculation and removal in osmolyte solutions
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Maria F. Gencoglu, Caryn. L. Heldt ∗ Department of Chemical Engineering, Michigan Technological University, 1400 Townsend Dr. Houghton, MI 49931, USA
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a r t i c l e
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a b s t r a c t
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Article history: Received 1 February 2015 Received in revised form 26 March 2015 Accepted 31 March 2015 Available online xxx
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Keywords: Sindbis virus Proline Mannitol High throughput screening Virus aggregation Vaccine purification
Our ability to reduce infectious disease burden throughout the world has been greatly improved by the creation of vaccines. However, worldwide immunization rates are low. The two most likely reasons are the lack of sufficient distribution in underdeveloped countries and the high cost of vaccine products. The high costs are due to the difficulties of manufacturing individual vaccine products with specialized purification trains. In this study, we propose to use virus flocculation in osmolytes, followed by microfiltration, as an alternative vaccine purification operation. In our previous work, we demonstrated that osmolytes preferentially flocculate a non-enveloped virus, porcine parvovirus (PPV). In this work we show that osmolytes flocculate the enveloped virus, Sindbis virus heat resistant strain (SVHR), and demonstrate a >80% removal with a 0.2 m microfilter membrane while leaving proteins in solution. The best osmolytes were tested for their ability to flocculate SVHR at different concentrations, pH and ionic strengths. Our best removal was 98% of SVHR in 0.3 M mannitol at a pH of 5. We propose that osmolytes are able to flocculate hydrophobic non-enveloped and enveloped virus particles by the reduction of the hydration layer around the particles, which stimulates virus aggregation. Now that we have demonstrated that protecting osmolytes flocculate viruses, it has the potential to be a future platform purification process for vaccines. © 2015 Published by Elsevier B.V.
Viral vaccines are the most effective method to prevent and control viral infections (Adida et al., 2013). Vaccines prevent around 2.5 millions deaths per year (WHO, 2013). However, vaccines are not able to offer global coverage, due to manufacturing limitations on the supply side (Adida et al., 2013). Once a vaccine is ready at lab scale, the manufacturing development time and the overall cost of the process needs to decrease in order to have sufficient supply and broad distribution. The ultimate goal would be a platform approach to vaccine manufacturing that would parallel the advances made in antibody purification and manufacturing (Shukla and Thommes, 2010). Downstream processes (DSP) account for 70% of the overall manufacturing cost in vaccine production (Morenweiser, 2005). The elevated costs in the DSP are mainly due to chromatography and nano- or ultrafiltration, each of which are regularly applied to vaccine manufacturing (Ray, 2011). Even though chromatography is the main unit operation for virus purification, viruses are large biomolecules that have limited diffusion in conventional resins and therefore have difficulties accessing the high internal surface area of the resins (Trilisky and Lenhoff, 2007). Another common method employed in virus purification is nano- or ultrafiltration. However,
∗ Corresponding author. Tel.: +1 906 487 1134; fax: +1 906 487 3213. E-mail address: [email protected] (Caryn.L. Heldt).
the efficiency of membranes can be affected by fouling (Bolton et al., 2006), leading to longer filtration time, high transmembrane pressure, and low flux through the membrane (Bolton et al., 2006; Morenweiser, 2005). Due to the limitations of chromatography and nano- or ultrafiltration, we propose to purify virus particles by flocculation in osmolytes, followed by microfiltration. Microfiltration, not typically used to retain viruses, would increase the flux and decrease the fouling as compared to nano- or ultrafiltration. Here, we demonstrate that a potential platform approach to vaccine purification is flocculation with osmolytes. Osmolytes are natural compounds found in the cells of many organisms. Their main function is to stabilize intracellular proteins and maintain cell volume when exposed to environmental stresses. There are two types of osmolytes, protecting and denaturing. Protecting osmolytes have the ability to stabilize proteins through the preferential exclusion of osmolytes from the protein surface, whereas denaturing osmolytes directly interact with the protein backbone (Street, 2007). Osmolytes are commonly used as excipients in pro- Q4 tein formulations (Roberts et al., 2013), making it likely that the osmolytes would not have to be removed to extremely low concentrations for formulation. In our previous work, we have shown that protecting osmolytes preferentially flocculate a model nonenveloped virus, porcine parvovirus (PPV). Our greatest removal were found with glycine, which demonstrated a 96% removal with a 0.2 m microfiltration filter while leaving model proteins, which
http://dx.doi.org/10.1016/j.jbiotec.2015.03.030 0168-1656/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Gencoglu, M.F., Heldt, Caryn.L., Enveloped virus flocculation and removal in osmolyte solutions. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.030
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Fig. 1. High-throughput screening of osmolytes as virus flocculants. A variety of osmolytes were compared to the positive control salts and PEG and the negative controls, Tris, and water. Flocculation and removal of SVHR was carried out in a 96-well filtration plate containing a 0.2 m micropore filter and centrifuged as described earlier (Gencoglu et al., 2014). 80% removal was used as the cut-off to pursue testing. % removal is defined in Eq. (1). All data points are the average of three separate experiments and the error bars represent the standard deviation.
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are likely less hydrophobic, in solution (Gencoglu et al., 2014). In this work we demonstrate that protecting osmolytes also flocculate an enveloped virus and allow removal with a microfiltration membrane. This further validates our hypothesis that osmolyte flocculation could create a platform purification process for viral products. Sindbis virus heat resistant strain (SVHR), our model enveloped virus, is a single-stranded, RNA, icosahedral virus, with a diameter between 60 and 70 nm (Norkin, 2010). A variety of osmolytes, such as dihydrate, glycine (GLY), d-alanine (ALA), betaine (BET), l-proline (PRO), trimethylamine N-oxide (TMAO), urea, sucrose (SUC), d-(+)-trehalose dehydrate (TREH), d-(+)-raffinose pentahydrate (RAF), and d-mannitol (MAN) were screened to demonstrate their ability to flocculate SVHR and the subsequent removal of the virus with a 0.2 m pore-sized filter (Fig. 1). Polyethylene glycol (PEG) and the salts magnesium sulfate (M. SUL), and magnesium chloride (M. CHL) were selected as positive controls since salts and PEG have been used to precipitate filamentous bacteriophages (Branston et al., 2012). Tris(hydroxymethyl)aminomethane hydrochloride (TRIS) and water (H2 O) were used as negative controls. Osmolytes, such as proline, betaine, sucrose, trehalose, raffinose, and mannitol were able to flocculate the enveloped virus SVHR, and showed a high removal (<80%) at all concentrations tested. Percent removal is defined in Eq. (1),
% Removal = 1 −
Cf Ci
× 100
(1)
where cf is the concentration of infectious virus or protein after filtration, and ci is the concentration of infectious virus or protein before filtration. Infectious titer was determined by the MTT assay, as described previously (Mi et al., 2014). In our previous work we found that the protecting osmolytes, including glycine and mannitol, have the ability to flocculate the non-enveloped virus PPV (Gencoglu et al., 2014). Due to the fact that a variety of protecting osmolytes flocculate viruses, we propose that this could be used as a novel process for vaccine purification. The osmolytes urea and glycine showed >80% virus removal at low concentrations, but at high concentrations, they inactivated SVHR (>1 LRV) (see Table S1). Virus inactivation was confirmed by testing the virus titer of each flocculating solution prior to
Fig. 2. pH effect on SVHR flocculation with a micropore filter. The pH of the osmolyte solutions was adjusted by the addition of HCl or NaOH and filtered as described in Fig. 1. % Removal is defined in Eq. (1). All data points are the average of three separate experiments and the error bars represent the standard deviation.
filtration and comparing to the virus titer of the water control solution. Osmolytes, such as d-arginine (ARG) and l-serine (SER), and the salt ammonium sulfate (A. SUL) were also found to inactivate SVHR (>1 LRV) (see Table S1). Urea at high concentrations has been shown to inactivate enveloped viruses, such as Sindbis, herpes simplex-1 and vaccinia (Roberts and Lloyd, 2007), likely due to the compound destabilizing viral proteins. Arginine has been shown to inactivate other enveloped viruses, such as the influenza virus and the herpes simplex virus (Yamasaki et al., 2008). It has been suggested that arginine binds to proteins without denaturing them (Yamasaki et al., 2008) and likely interacts with the lipid membrane (McCue et al., 2014). In this study, we focused on flocculants that demonstrated high removal without inactivating SVHR. The osmolytes betaine, mannitol and proline were used to study the effect of pH on SVHR flocculation (Fig. 2). These osmolytes were selected because they represented three different catagories of osmolytes, n-oxide, sugar/polyol and amino acid, respectively. Osmolyte solution pH was adjusted to change the overall charge of the virus. SVHR has been shown to be stable in the pH range of 5–8 (Roberts, 2008), so a pH range of 5.5–8.5 was selected in this study. It was confirmed that virus inactivation did not occur, as stated earlier. The highest removal occurred as the pH neared the isoelectric point (pI) of SVHR, which has been found to be ∼4.2
Please cite this article in press as: Gencoglu, M.F., Heldt, Caryn.L., Enveloped virus flocculation and removal in osmolyte solutions. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.030
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Fig. 3. SVHR and protein removal with a micropore filter. Samples prepared and filtered as described in Fig. 1. Protein sample absorbance before and after filtration was measured on a Synergy Mx microplate reader at 280 nm. All data points are the average of three separate experiments and the error bars represent the standard deviation.
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optimized case was able to remove 98.1% of SVHR in 0.3 M mannitol. In our previous studies, mannitol was able to aggregate and flocculated a non-enveloped virus, PPV (85% removal) (Gencoglu et al., 2014). We hypothesize that mannitol has the ability to control water molecules around hydrophobic enveloped and nonenveloped virus, leading to a decreased hydration layer around the virus and increasing the hydrophobic interactions between virus particles. Virus flocculation with mannitol, followed by micro- or ultrafiltration, has the potential to become a platform process for virus purification. Future work will look at optimization of filter pore size for improved vaccine purification and/or virus removal along with a demonstration of purification and not just removal of the virus. Uncited reference
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(Dalrymple et al., 1976). At pH values close to the pI of the virus, the overall charge of the virus becomes neutral and the tendency for virus aggregation increases. As the pH value was increased, the negative charge on the virus increased, and virus aggregation decreased, likely due to an increase in the electrostatic repulsion between virus particles (Fig. 2). In our previous work, we showed that changes in pH for the zwitterionic osmolytes glycine and alanine could increase the charge–charge repulsion of PPV particles. This behavior was not seen with the sugar and sugar alcohol osmolytes, likely because neutral molecules are able to promote virus flocculation by possible hydrogen-bonding interactions and not charge–charge interactions (Gencoglu et al., 2014). Zwitterion and neutral osmolytes appear to have the same effect on virus aggregation as a function of pH for a non-enveloped (Gencoglu et al., 2014) and an enveloped virus. The zwitterion, proline and the neutral molecule, mannitol were used to investigate the ionic strength effect on SVHR flocculation. For both mannitol and proline, virus removal decreased at high ionic strength (Fig. S1). It is likely that the strength of electrostatic interactions and hydrogen bonding interactions are not appreciably affected by low concentration of the weak chaotropic agent, NaCl. The possible mechanisms for this are discussed in the Supplementary information. The model proteins bovine serum albumin (BSA) and lysozyme were used to demonstrate the preferential flocculation of osmolytes with SVHR (Fig. 3). The osmolytes mannitol and proline at 0.3 M and the salt magnesium sulfate at 3.0 M were able to flocculate SVHR particles. BSA and lysozyme removal was very low with osmolytes (<23%) as compared to magnesium sulfate (>60%). This indicates that salts flocculate proteins as well as viruses, while osmolyte flocculation is specific to SVHR particles, making this a potential method to purify virus particles from protein contaminants. Moreover, virus flocculation and consequently high virus removal, was effective at much lower concentrations for osmolytes as compared to the salts tested (Figs. 1 and 3). Salt flocculation occurs due to changes in the Debye length as a function of ionic strength. When the salt concentration reaches a critical concentration, both proteins and virus particles aggregate. Osmolyte flocculation has now been shown to be specific to SVHR and PPV virus particles (Gencoglu et al., 2014) demonstrating this as a strong candidate for future platform approaches for vaccine purification. Virus purification is essential in vaccine manufacturing processes. Current unit operations, such as chromatography and nanoor ultrafiltration need to be replaced if we hope to reduce the overall manufacturing costs of current and future vaccines. In this high throughput study, we have shown that the osmolytes proline and mannitol were able to aggregate and flocculate SVHR and demonstrate a high virus removal (>80%), with a 0.2 m filter. Our
Acknowledgements The authors would like to thank the Department of Chemical Q6 Engineering at Michigan Technological University, the MTU-REF Research Seed Grant, the Biotechnology Research Center at Michigan Technological University and the National Science Foundation (NSF) (CBET-1159425) for funding. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jbiotec.2015.03.030. References Adida, E., Dey, D., Mamani, H., 2013. Operational issues and network effects in vaccine markets. Eur. J. Oper. Res. 231 (2), 414–427, http://dx.doi.org/10.1016/ j.ejor.2013.05.034. Bolton, G., LaCasse, D., Kuriyel, R., 2006. Combined models of membrane fouling: development and application to microfiltration and ultrafiltration of biological fluids. J. Membr. Sci. 277 (1), 75–84, http://dx.doi.org/10.1016/ j.memsci.2004.12.053. Branston, S., Stanley, E., Keshavarz-Moore, E., Ward, J., 2012. Precipitation of filamentous bacteriophages for their selective recovery in primary purification. Biotechnol. Prog. 28 (1), 129–136, http://dx.doi.org/10.1002/btpr.705. Dalrymple, J.M., Schlesinger, S., Russell, P.K., 1976. Antigenic characterization of two Sindbis envelope glycoproteins separated by isoelectric focusing. Virology 69 (1), 93–103, http://dx.doi.org/10.1016/0042-6822(76)90197-5. Gencoglu, M.F., Pearson, E., Heldt, C.L., 2014. Porcine Parvovirus flocculation and removal in the presence of osmolytes. J. Biotecnol. 186, 83–90, http://dx.doi.org/10.1016/j.jbiotec.2014.06.011. McCue, J.T., Selvitelli, K., Cecchini, D., Brown, R., 2014. Enveloped virus inactivation using neutral arginine solutions and applications in therapeutic protein purification processes. Biotechnol. Prog. 30 (1), 108–112, http://dx.doi.org/ 10.1002/btpr.1816. Mi, X., Vijayaragavan, K.S., Heldt, C.L., 2014. Virus adsorption of water-stable quaternized chitosan nanofibers. Carbohydr. Res. 387, 24–29. Morenweiser, R., 2005. Downstream processing of viral vectors and vaccines. Gene Ther. 12, S103–S110, http://dx.doi.org/10.1038/sj.gt.3302624. Norkin, L.C., 2010. Virology: Molecular Biology and Pathogenesis. ASM Press. Ray, S., 2011. Challenges and trends in vaccine manufacturing. Biopharm. Int. 24 (10), S3-+. Roberts, C.J., Nesta, D.P., Kim, N., 2013. Effects of temperature and osmolytes on competing degradation routes for an IgG1 antibody. J. Pharm. Sci. 102 (10), 3556–3566, http://dx.doi.org/10.1002/jps.23668. Roberts, P.L., 2008. Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII. Biologicals 36 (5), 330–335, http://dx.doi.org/ 10.1016/j.biologicals.2008.06.002. Roberts, P.L., Lloyd, D., 2007. Virus inactivation by protein denaturants used in affinity chromatography. Biologicals 35 (4), 343–347, http://dx.doi.org/ 10.1016/j.biologicals.2007.02.005. Shukla, A.A., Thommes, J., 2010. Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends Biotechnol. 28 (5), 253–261, http://dx.doi.org/10.1016/j.tibtech.2010.02.001.
Please cite this article in press as: Gencoglu, M.F., Heldt, Caryn.L., Enveloped virus flocculation and removal in osmolyte solutions. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.030
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Street, T.O., Bolen, D.W., Rose, G.D., 2006. A molecular mechanism for osmolyteinduced protein stability. PNAS 103 (38), 13997–14002, http://dx.doi.org/ 10.1073/pnas.0606236103. Trilisky, E., Lenhoff, A., 2007. Sorption processes in ion-exchange chromatography of viruses. J. Chromatogr. A 1142 (1), 2–12, http://dx.doi.org/10.1016/ j.chroma.2006.12.094.
WHO, 2013. Global Vaccine Action Plan 2011–2020. Yamasaki, H., Tsujimoto, K., Koyama, A.H., Ejima, D., Arakawa, T., 2008. Arginine facilitates inactivation of enveloped viruses. J. Pharm. Sci. 97 (8), 3067–3073, http://dx.doi.org/10.1002/jps.21224.
Please cite this article in press as: Gencoglu, M.F., Heldt, Caryn.L., Enveloped virus flocculation and removal in osmolyte solutions. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.030
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ARTICLE IN PRESS
BIOTEC 7074 1–4
Journal of Biotechnology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
1
Short communication
2
Enveloped virus flocculation and removal in osmolyte solutions
3
Q1
4
Maria F. Gencoglu, Caryn. L. Heldt ∗ Department of Chemical Engineering, Michigan Technological University, 1400 Townsend Dr. Houghton, MI 49931, USA
5
6 21
a r t i c l e
i n f o
a b s t r a c t
7 8 9 10 11 12
Article history: Received 1 February 2015 Received in revised form 26 March 2015 Accepted 31 March 2015 Available online xxx
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Keywords: Sindbis virus Proline Mannitol High throughput screening Virus aggregation Vaccine purification
Our ability to reduce infectious disease burden throughout the world has been greatly improved by the creation of vaccines. However, worldwide immunization rates are low. The two most likely reasons are the lack of sufficient distribution in underdeveloped countries and the high cost of vaccine products. The high costs are due to the difficulties of manufacturing individual vaccine products with specialized purification trains. In this study, we propose to use virus flocculation in osmolytes, followed by microfiltration, as an alternative vaccine purification operation. In our previous work, we demonstrated that osmolytes preferentially flocculate a non-enveloped virus, porcine parvovirus (PPV). In this work we show that osmolytes flocculate the enveloped virus, Sindbis virus heat resistant strain (SVHR), and demonstrate a >80% removal with a 0.2 m microfilter membrane while leaving proteins in solution. The best osmolytes were tested for their ability to flocculate SVHR at different concentrations, pH and ionic strengths. Our best removal was 98% of SVHR in 0.3 M mannitol at a pH of 5. We propose that osmolytes are able to flocculate hydrophobic non-enveloped and enveloped virus particles by the reduction of the hydration layer around the particles, which stimulates virus aggregation. Now that we have demonstrated that protecting osmolytes flocculate viruses, it has the potential to be a future platform purification process for vaccines. © 2015 Published by Elsevier B.V.
Viral vaccines are the most effective method to prevent and control viral infections (Adida et al., 2013). Vaccines prevent around 2.5 millions deaths per year (WHO, 2013). However, vaccines are not able to offer global coverage, due to manufacturing limitations on the supply side (Adida et al., 2013). Once a vaccine is ready at lab scale, the manufacturing development time and the overall cost of the process needs to decrease in order to have sufficient supply and broad distribution. The ultimate goal would be a platform approach to vaccine manufacturing that would parallel the advances made in antibody purification and manufacturing (Shukla and Thommes, 2010). Downstream processes (DSP) account for 70% of the overall manufacturing cost in vaccine production (Morenweiser, 2005). The elevated costs in the DSP are mainly due to chromatography and nano- or ultrafiltration, each of which are regularly applied to vaccine manufacturing (Ray, 2011). Even though chromatography is the main unit operation for virus purification, viruses are large biomolecules that have limited diffusion in conventional resins and therefore have difficulties accessing the high internal surface area of the resins (Trilisky and Lenhoff, 2007). Another common method employed in virus purification is nano- or ultrafiltration. However,
∗ Corresponding author. Tel.: +1 906 487 1134; fax: +1 906 487 3213. E-mail address: [email protected] (Caryn.L. Heldt).
the efficiency of membranes can be affected by fouling (Bolton et al., 2006), leading to longer filtration time, high transmembrane pressure, and low flux through the membrane (Bolton et al., 2006; Morenweiser, 2005). Due to the limitations of chromatography and nano- or ultrafiltration, we propose to purify virus particles by flocculation in osmolytes, followed by microfiltration. Microfiltration, not typically used to retain viruses, would increase the flux and decrease the fouling as compared to nano- or ultrafiltration. Here, we demonstrate that a potential platform approach to vaccine purification is flocculation with osmolytes. Osmolytes are natural compounds found in the cells of many organisms. Their main function is to stabilize intracellular proteins and maintain cell volume when exposed to environmental stresses. There are two types of osmolytes, protecting and denaturing. Protecting osmolytes have the ability to stabilize proteins through the preferential exclusion of osmolytes from the protein surface, whereas denaturing osmolytes directly interact with the protein backbone (Street, 2007). Osmolytes are commonly used as excipients in pro- Q4 tein formulations (Roberts et al., 2013), making it likely that the osmolytes would not have to be removed to extremely low concentrations for formulation. In our previous work, we have shown that protecting osmolytes preferentially flocculate a model nonenveloped virus, porcine parvovirus (PPV). Our greatest removal were found with glycine, which demonstrated a 96% removal with a 0.2 m microfiltration filter while leaving model proteins, which
http://dx.doi.org/10.1016/j.jbiotec.2015.03.030 0168-1656/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Gencoglu, M.F., Heldt, Caryn.L., Enveloped virus flocculation and removal in osmolyte solutions. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.030
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Fig. 1. High-throughput screening of osmolytes as virus flocculants. A variety of osmolytes were compared to the positive control salts and PEG and the negative controls, Tris, and water. Flocculation and removal of SVHR was carried out in a 96-well filtration plate containing a 0.2 m micropore filter and centrifuged as described earlier (Gencoglu et al., 2014). 80% removal was used as the cut-off to pursue testing. % removal is defined in Eq. (1). All data points are the average of three separate experiments and the error bars represent the standard deviation.
68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91
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are likely less hydrophobic, in solution (Gencoglu et al., 2014). In this work we demonstrate that protecting osmolytes also flocculate an enveloped virus and allow removal with a microfiltration membrane. This further validates our hypothesis that osmolyte flocculation could create a platform purification process for viral products. Sindbis virus heat resistant strain (SVHR), our model enveloped virus, is a single-stranded, RNA, icosahedral virus, with a diameter between 60 and 70 nm (Norkin, 2010). A variety of osmolytes, such as dihydrate, glycine (GLY), d-alanine (ALA), betaine (BET), l-proline (PRO), trimethylamine N-oxide (TMAO), urea, sucrose (SUC), d-(+)-trehalose dehydrate (TREH), d-(+)-raffinose pentahydrate (RAF), and d-mannitol (MAN) were screened to demonstrate their ability to flocculate SVHR and the subsequent removal of the virus with a 0.2 m pore-sized filter (Fig. 1). Polyethylene glycol (PEG) and the salts magnesium sulfate (M. SUL), and magnesium chloride (M. CHL) were selected as positive controls since salts and PEG have been used to precipitate filamentous bacteriophages (Branston et al., 2012). Tris(hydroxymethyl)aminomethane hydrochloride (TRIS) and water (H2 O) were used as negative controls. Osmolytes, such as proline, betaine, sucrose, trehalose, raffinose, and mannitol were able to flocculate the enveloped virus SVHR, and showed a high removal (<80%) at all concentrations tested. Percent removal is defined in Eq. (1),
% Removal = 1 −
Cf Ci
× 100
(1)
where cf is the concentration of infectious virus or protein after filtration, and ci is the concentration of infectious virus or protein before filtration. Infectious titer was determined by the MTT assay, as described previously (Mi et al., 2014). In our previous work we found that the protecting osmolytes, including glycine and mannitol, have the ability to flocculate the non-enveloped virus PPV (Gencoglu et al., 2014). Due to the fact that a variety of protecting osmolytes flocculate viruses, we propose that this could be used as a novel process for vaccine purification. The osmolytes urea and glycine showed >80% virus removal at low concentrations, but at high concentrations, they inactivated SVHR (>1 LRV) (see Table S1). Virus inactivation was confirmed by testing the virus titer of each flocculating solution prior to
Fig. 2. pH effect on SVHR flocculation with a micropore filter. The pH of the osmolyte solutions was adjusted by the addition of HCl or NaOH and filtered as described in Fig. 1. % Removal is defined in Eq. (1). All data points are the average of three separate experiments and the error bars represent the standard deviation.
filtration and comparing to the virus titer of the water control solution. Osmolytes, such as d-arginine (ARG) and l-serine (SER), and the salt ammonium sulfate (A. SUL) were also found to inactivate SVHR (>1 LRV) (see Table S1). Urea at high concentrations has been shown to inactivate enveloped viruses, such as Sindbis, herpes simplex-1 and vaccinia (Roberts and Lloyd, 2007), likely due to the compound destabilizing viral proteins. Arginine has been shown to inactivate other enveloped viruses, such as the influenza virus and the herpes simplex virus (Yamasaki et al., 2008). It has been suggested that arginine binds to proteins without denaturing them (Yamasaki et al., 2008) and likely interacts with the lipid membrane (McCue et al., 2014). In this study, we focused on flocculants that demonstrated high removal without inactivating SVHR. The osmolytes betaine, mannitol and proline were used to study the effect of pH on SVHR flocculation (Fig. 2). These osmolytes were selected because they represented three different catagories of osmolytes, n-oxide, sugar/polyol and amino acid, respectively. Osmolyte solution pH was adjusted to change the overall charge of the virus. SVHR has been shown to be stable in the pH range of 5–8 (Roberts, 2008), so a pH range of 5.5–8.5 was selected in this study. It was confirmed that virus inactivation did not occur, as stated earlier. The highest removal occurred as the pH neared the isoelectric point (pI) of SVHR, which has been found to be ∼4.2
Please cite this article in press as: Gencoglu, M.F., Heldt, Caryn.L., Enveloped virus flocculation and removal in osmolyte solutions. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.030
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Fig. 3. SVHR and protein removal with a micropore filter. Samples prepared and filtered as described in Fig. 1. Protein sample absorbance before and after filtration was measured on a Synergy Mx microplate reader at 280 nm. All data points are the average of three separate experiments and the error bars represent the standard deviation.
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optimized case was able to remove 98.1% of SVHR in 0.3 M mannitol. In our previous studies, mannitol was able to aggregate and flocculated a non-enveloped virus, PPV (85% removal) (Gencoglu et al., 2014). We hypothesize that mannitol has the ability to control water molecules around hydrophobic enveloped and nonenveloped virus, leading to a decreased hydration layer around the virus and increasing the hydrophobic interactions between virus particles. Virus flocculation with mannitol, followed by micro- or ultrafiltration, has the potential to become a platform process for virus purification. Future work will look at optimization of filter pore size for improved vaccine purification and/or virus removal along with a demonstration of purification and not just removal of the virus. Uncited reference
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(Dalrymple et al., 1976). At pH values close to the pI of the virus, the overall charge of the virus becomes neutral and the tendency for virus aggregation increases. As the pH value was increased, the negative charge on the virus increased, and virus aggregation decreased, likely due to an increase in the electrostatic repulsion between virus particles (Fig. 2). In our previous work, we showed that changes in pH for the zwitterionic osmolytes glycine and alanine could increase the charge–charge repulsion of PPV particles. This behavior was not seen with the sugar and sugar alcohol osmolytes, likely because neutral molecules are able to promote virus flocculation by possible hydrogen-bonding interactions and not charge–charge interactions (Gencoglu et al., 2014). Zwitterion and neutral osmolytes appear to have the same effect on virus aggregation as a function of pH for a non-enveloped (Gencoglu et al., 2014) and an enveloped virus. The zwitterion, proline and the neutral molecule, mannitol were used to investigate the ionic strength effect on SVHR flocculation. For both mannitol and proline, virus removal decreased at high ionic strength (Fig. S1). It is likely that the strength of electrostatic interactions and hydrogen bonding interactions are not appreciably affected by low concentration of the weak chaotropic agent, NaCl. The possible mechanisms for this are discussed in the Supplementary information. The model proteins bovine serum albumin (BSA) and lysozyme were used to demonstrate the preferential flocculation of osmolytes with SVHR (Fig. 3). The osmolytes mannitol and proline at 0.3 M and the salt magnesium sulfate at 3.0 M were able to flocculate SVHR particles. BSA and lysozyme removal was very low with osmolytes (<23%) as compared to magnesium sulfate (>60%). This indicates that salts flocculate proteins as well as viruses, while osmolyte flocculation is specific to SVHR particles, making this a potential method to purify virus particles from protein contaminants. Moreover, virus flocculation and consequently high virus removal, was effective at much lower concentrations for osmolytes as compared to the salts tested (Figs. 1 and 3). Salt flocculation occurs due to changes in the Debye length as a function of ionic strength. When the salt concentration reaches a critical concentration, both proteins and virus particles aggregate. Osmolyte flocculation has now been shown to be specific to SVHR and PPV virus particles (Gencoglu et al., 2014) demonstrating this as a strong candidate for future platform approaches for vaccine purification. Virus purification is essential in vaccine manufacturing processes. Current unit operations, such as chromatography and nanoor ultrafiltration need to be replaced if we hope to reduce the overall manufacturing costs of current and future vaccines. In this high throughput study, we have shown that the osmolytes proline and mannitol were able to aggregate and flocculate SVHR and demonstrate a high virus removal (>80%), with a 0.2 m filter. Our
Acknowledgements The authors would like to thank the Department of Chemical Q6 Engineering at Michigan Technological University, the MTU-REF Research Seed Grant, the Biotechnology Research Center at Michigan Technological University and the National Science Foundation (NSF) (CBET-1159425) for funding. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jbiotec.2015.03.030. References Adida, E., Dey, D., Mamani, H., 2013. Operational issues and network effects in vaccine markets. Eur. J. Oper. Res. 231 (2), 414–427, http://dx.doi.org/10.1016/ j.ejor.2013.05.034. Bolton, G., LaCasse, D., Kuriyel, R., 2006. Combined models of membrane fouling: development and application to microfiltration and ultrafiltration of biological fluids. J. Membr. Sci. 277 (1), 75–84, http://dx.doi.org/10.1016/ j.memsci.2004.12.053. Branston, S., Stanley, E., Keshavarz-Moore, E., Ward, J., 2012. Precipitation of filamentous bacteriophages for their selective recovery in primary purification. Biotechnol. Prog. 28 (1), 129–136, http://dx.doi.org/10.1002/btpr.705. Dalrymple, J.M., Schlesinger, S., Russell, P.K., 1976. Antigenic characterization of two Sindbis envelope glycoproteins separated by isoelectric focusing. Virology 69 (1), 93–103, http://dx.doi.org/10.1016/0042-6822(76)90197-5. Gencoglu, M.F., Pearson, E., Heldt, C.L., 2014. Porcine Parvovirus flocculation and removal in the presence of osmolytes. J. Biotecnol. 186, 83–90, http://dx.doi.org/10.1016/j.jbiotec.2014.06.011. McCue, J.T., Selvitelli, K., Cecchini, D., Brown, R., 2014. Enveloped virus inactivation using neutral arginine solutions and applications in therapeutic protein purification processes. Biotechnol. Prog. 30 (1), 108–112, http://dx.doi.org/ 10.1002/btpr.1816. Mi, X., Vijayaragavan, K.S., Heldt, C.L., 2014. Virus adsorption of water-stable quaternized chitosan nanofibers. Carbohydr. Res. 387, 24–29. Morenweiser, R., 2005. Downstream processing of viral vectors and vaccines. Gene Ther. 12, S103–S110, http://dx.doi.org/10.1038/sj.gt.3302624. Norkin, L.C., 2010. Virology: Molecular Biology and Pathogenesis. ASM Press. Ray, S., 2011. Challenges and trends in vaccine manufacturing. Biopharm. Int. 24 (10), S3-+. Roberts, C.J., Nesta, D.P., Kim, N., 2013. Effects of temperature and osmolytes on competing degradation routes for an IgG1 antibody. J. Pharm. Sci. 102 (10), 3556–3566, http://dx.doi.org/10.1002/jps.23668. Roberts, P.L., 2008. Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII. Biologicals 36 (5), 330–335, http://dx.doi.org/ 10.1016/j.biologicals.2008.06.002. Roberts, P.L., Lloyd, D., 2007. Virus inactivation by protein denaturants used in affinity chromatography. Biologicals 35 (4), 343–347, http://dx.doi.org/ 10.1016/j.biologicals.2007.02.005. Shukla, A.A., Thommes, J., 2010. Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends Biotechnol. 28 (5), 253–261, http://dx.doi.org/10.1016/j.tibtech.2010.02.001.
Please cite this article in press as: Gencoglu, M.F., Heldt, Caryn.L., Enveloped virus flocculation and removal in osmolyte solutions. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.030
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Please cite this article in press as: Gencoglu, M.F., Heldt, Caryn.L., Enveloped virus flocculation and removal in osmolyte solutions. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.030
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