Arginine Facilitates Inactivation of Enveloped Viruses

Arginine Facilitates Inactivation of Enveloped Viruses HISASHI YAMASAKI,1 KAZUKO TSUJIMOTO,1 A. HAJIME KOYAMA,1 DAISUKE EJIMA,2 TSUTOMU ARAKAWA3 1

Division of Virology, Department of Cellular and Molecular Medicine, Wakayama Medical University Graduate School of Medicine, Wakayama 641-8509, Japan 2

Applied Research Department, Amino Science Laboratories, Ajinomoto, Inc., Kawasaki, Kanagawa 210-8681, Japan

3

Alliance Protein Laboratories, Thousand Oaks, California 91360

Received 3 July 2007; revised 2 September 2007; accepted 14 September 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21224

ABSTRACT: Virus inactivation is a key step for the purification of pharmaceutical proteins derived from recombinant mammalian expression systems and conventionally done using low pH-treatment, which is often harmful to the proteins to be purified. This is particularly true for antibodies, because immunoglobulin proteins undergo conformational changes at acidic pH. We have been developing mild elution solvents using arginine for Protein-A chromatography to minimize the low pH-induced damages on the antibodies. Here we have tested the aqueous solutions containing arginine or butyroyl-arginine at or above pH 4.0 for their effects on virus inactivation, since these solvents are effective above pH 4.0 in elution of bound antibodies from Protein-A columns. When the virus was incubated on ice, 0.1 M sodium citrate was totally ineffective above pH 4.0, but aqueous solutions containing arginine above 0.35 M or butyroyl-arginine above 0.28 M showed extensive virus killing at or even above pH 4.0. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:3067–3073, 2008

Keywords: virus inactivation; low pH; arginine; butyroyl-arginine; monoclonal antibodies; viral safety

INTRODUCTION Monoclonal antibodies (mAbs) are currently developed at the unprecedented speed as the major pharmaceutical proteins.1,2 Viral safety is one of the major concerns for the purification and production of intravenous immunoglobulin G, mAbs and other recombinant proteins.3–7 Certain commercial purification modules, such as nanofiltration and low-pH inactivation,3–8 have been observed to reliably remove large enveloped viruses in the order of 104-fold. However, exposure

Correspondence to: A. Hajime Koyama (Telephone: 81-73441-0771; Fax: 81-73-441-0771) or Tsutomu Arakawa (Telephone: 805-388-1074; Fax: 805-388-7252; E-mail: [email protected] or [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 3067–3073 (2008) ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association

of mAbs to low pH causes their conformational changes, which are the major reason for antibody instability and aggregation.9–12 We have been developing new solvents useful for protein purification and analysis, based on unique properties of aqueous arginine solution. We showed that 0.3–2 M arginine can dissociate antibodies bound to Protein-A columns at or above pH 4.0, which minimizes their conformational changes.13,14 A conventional elution solvent, such as citrate and glycine, requires their pH to be at about 3.0–3.6.15,16 Although the aqueous arginine solutions eliminate the use of such low pH in the Protein-A chromatography step, mildly acidic pH may be insufficient for the virus inactivation, which makes mild arginine elution less attractive. In order to keep the pH above 4.0 throughout the process, the viral inactivation must be effective above this pH. We therefore tested the

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aqueous arginine solutions at or above pH 4 for their abilities to inactivate viruses.

poliovirus) or MDCK cells (influenza virus) as described previously.17–19

MATERIALS AND METHODS

Assay for Virucidal Activity

Materials

All the starting materials were stored on ice prior to the virus inactivation experiments. A 950 mL aliquot of the solvents to be tested was placed in 1.5 mL-plastic tube on ice and received 50 mL of virus preparations (approximately 108 or 109 plaque-forming units [PFU]/mL). This was immediately followed by vigorous mixing and the sample mixture was incubated at the indicated temperature (i.e., on ice or at 308C). At the indicated time points, aliquots of these virus samples were 100-fold diluted with Dulbecco’s phosphate-buffered saline without Caþþ and Mgþþ (PBS) containing 1% calf serum (for HSV-1 and poliovirus) or 0.1% BSA (for influenza virus). The viruses were further diluted with ice-cold PBS containing 1% calf serum or 0.1% BSA and the number of infectious virus in the treated preparation was measured by a plaque assay. All the experiments were done in duplicate or triplicate, as shown in the tables for some data. Others were omitted for brevity. There was little variation in the plaque assay, to the extent that the observed variation does not affect the conclusion.

L-Arginine

hydrochloride (simply described as arginine) and L-butyroyl-arginine (butyroyl-arginine) were obtained from Ajinomoto Co., Inc. (Kawasaki, Japan) Butyroyl-arginine has a modification at the amino group of arginine and hence has no net charge at neutral pH. Aqueous solutions containing arginine were prepared by dissolving arginine hydrochloride and adjusting the pH to the indicated values with HCl. Arginine provides sufficient buffer action at the concentration and pH tested. For comparison, 0.7 M arginine/acetate was prepared for stronger buffer action around pH 4.0. The pH of butyroyl-arginine was also adjusted with HCl. To mimic the antibody purification process, a mAb at 5 mg/mL was included in these solvents. Cells and Viruses Vero and MDCK cells were grown in Eagle’s minimum essential medium (MEM) containing 10% newborn calf serum. Herpes simplex virus type-1/strain F (HSV-1), poliovirus type 1/Sabin strain, and influenza virus A/Aichi (H3N2) were used throughout the experiments. Both poliovirus and HSV-1 were propagated in Vero cells in MEM supplemented with 0.5% fetal bovine serum. Influenza virus was propagated in MDCK cells in the medium supplemented with 0.1% bovine serum albumin (BSA) and acetylated trypsin (4 mg/mL). The viruses were stored at
808C until use. The amount of virus was measured by a plaque assay on Vero cells (for HSV-1 and

RESULTS AND DISCUSSION Low pH treatment below pH 3.5 is generally an effective means of virus inactivation against enveloped viruses and hence was compared here with the new solvent system containing arginine. The virus first chosen is the enveloped DNA virus, herpes simplex virus type-1 (HSV-1). The virus was incubated with the solvent, listed in Table 1,

Table 1. Inactivation of HSV-1 by Various Solvents Solvent

pH

Relative Virus Yield

Log Reduction Value

PBS control 0.1 M sodium citrate 0.1 M sodium citrate 0.1 M sodium citrate 1 M arginine 0.7 M arginine/20 mM acetate 0.7 M butyroyl-arginine

7.4 4.3 4.0 3.5 4.3 4.0 4.0

1 0.816/0.884 0.032/0.046 2  10
6>/4  10
6> 2  10
6> 2  10
6> 2  10
6>

0 0.1/0.1 1.5/1.3 >5.7/>5.4 >5.7 >5.7 >5.7

These solvents in this table and Tables 2 and 3 and figures contained mAb at 5 mg/mL. HSV-1 virus was incubated for 60 min on ice. For certain cases, duplicate data are shown. Log reduction value (LRV) ¼ log(YieldPBS/Yieldsolvent). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008

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for 60 min on ice as described in the previous section. The virus inactivation in Table 1 is expressed as a relative number of surviving virus after the treatment with the respective solvent to that with PBS or more conventional log of reduction value (LRV), as calculated by  log reduction value ðLRVÞ ¼ log

YieldPBS Yieldsolvent



where YieldPBS is the virus yield in PBS (i.e., 1) and Yieldsolvent is the virus yield in the indicated solvents. It is noted that there is essentially no virus inactivation (killing) when incubated for 60 min in PBS on ice. There is extensive virus inactivation under the standard condition, that is, in 0.1 M citrate, pH 3.5 (line 4 in Tab. 1). It is evident that the virus yield is identical, within experimental error, for this standard condition and 0.7 M arginine, pH 4.0 (line 4), both leading to 10
6 surviving virus numbers (LRV of 5.7) of the PBS-control. Essentially an identical result was observed with 0.7 M butyroyl-arginine at pH 4.0 (last line), indicating that these high pH solvents confer virus inactivation achieved by pH 3.5 using citrate. Even at higher pH of 4.3, 1 M arginine resulted in the virus inactivation of the same magnitude (line 5). These virus-killing activities of arginine or butyroyl-arginine are much higher than the virus killing achieved by 0.1 M citrate at pH 4.0 and 4.3, which was almost negligible under the conditions tested. These results demonstrate that arginine and butyroyl-arginine enhance the virus inactivation at the pH, at which acidic pH alone is normally ineffective (i.e., above pH 4.0). However, both arginine and butyroyl-arginine became nearly ineffective above pH 5.0, as shown in Figure 1. At pH 4.5, butyroyl-arginine was still

Figure 1. Effect of pH on virus inactivation by 0.7 M arginine/20 mM acetate, 0.7 M butyroyl-arginie/20 mM acetate and 0.1 M citrate. DOI 10.1002/jps

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effective and 0.1 M citrate has completely lost the activity. The sensitivity of influenza virus, another enveloped virus, was tested against the acidic solvents described above. Hemagglutinin (HA) spike proteins of influenza virus are known to undergo a conformational change and unstable at the acidic pH.20 This acid-induced conformational change of HA proteins is essential for the virus infectivity; following the adsorption of virus particle to the cell surface receptor, the virus particle is endocytosed into the endosome where acidic pH causes transient conformational changes of the HA proteins and leads to the fusion of viral envelope with the endosomal membrane.21 Table 2 summarizes the virus inactivation data when the virus was incubated on ice for 30 or 60 min. As shown in Table 2, this virus appears to be more sensitive than HSV-1 to citrate at pH 4.3 (0.051 or 0.077 virus yield vs. 0.816 for HSV-1), in agreement with the instability of HA proteins at mildly acidic endosomal pH as described above. However, this virus is resistant to exposure to the lower pH. While, upon incubation for 30–60 min on ice, the results with 0.1 M citrate at pH 4.0 (line 3) were similar to those of HSV-1, this virus shows a higher virus yield (less killing) in 0.1 M citrate, pH 3.5, by about 1000-fold (compare line 4 of Tab. 2 with line 4 of Tab. 1). This acid-resistance may have resulted from the fact that influenza virion has only two envelope proteins (i.e., HA and NA spike proteins) while the envelope of HSV-1 contains numerous glycoproteins, such as gB, gC, gD, gE, gH, gI, and so on. There appear to be little differences between 30 and 60 min incubation on ice, indicating that virus inactivation occurs must faster than this time scale. When compared with these effects of citrate, arginine shows a higher virus inactivation than does 0.1 M citrate. At pH 4.3, 1 M arginine (line 5) was about 13-fold more effective than 0.1 M citrate (at pH 4.3). At pH 4.0, 0.7 M arginine (line 6) was about 100–150-fold more effective than 0.1 M citrate (line 3). Even 0.35 M arginine was equally effective to 0.7 M arginine at pH 4.0. To our surprise, arginine at these pHs is more effective than 0.1 M citrate, pH 3.5. Butyroyl-arginine at 0.35 and 0.7 M was also highly effective against influenza virus at pH 4.0. At this pH, NaCl at 0.7 M was also more effective than 0.1 M citrate, suggesting contribution of ionic strength on inactivation of influenza virus. However, NaCl is less effective than arginine, indicating that a factor(s) other than JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008

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Table 2. Inactivation of Influenza Virus by Various Solvents Relative Virus Yield (LRV) Solvent

pH

30 min On Ice

60 min On Ice

PBS 0.1 M sodium citrate 0.1 M sodium citrate 0.1 M sodium citrate 1 M arginine 0.7 M arginine/20 mM acetate 0.35 M arginine/20 mM acetate 0.7 M butyroyl-arginine 0.35 M butyroyl-arginine/20 mM acetate 0.7 M NaCl/20 mM acetate

7.4 4.3 4.0 3.5 4.3 4.0 4.0 4.0 4.0 4.0

1 (0) 0.051 (1.3) 0.031 (1.5) 0.0075 (2.1) 0.0039 (2.4) 0.0002, one plaque (3.7)

1 (0) 0.077 (1.1) 0.085 (1.1) 0.0129 (1.9) 0.0042 (2.4) 0.00072 (3.1) 0.00027 (3.6) 0.000001>(>6) 0.000185 (3.7) 0.0083 (2.1)

0.0002, one plaque (3.7)

the ionic strength plays a role in inactivation of influenza virus: note that arginine is mostly monovalent at the pH range examined and hence has a nearly identical ionic strength to NaCl at the same molar concentration. On the contrary, butyroyl-arginine is a zwitter-ion (zero net charge) at this pH range. In addition to the effect on HSV-1 and influenza virus, these solvents were tested on nonenveloped virus, poliovirus, which is also often used as model virus for virus clearing testing. Virion of poliovirus has a very simple structure, consisting of one genomic RNA and four capsid proteins. None of these solvents in Table 1 was effective against this virus under the same incubation conditions (data not shown), consistent with the highly resistant nature of this virus against low pH.22,23 Generally those viruses (e.g., poliovirus), which are transmitted by fecal excretion and oral adsorption, are resistant to environmental stresses including acidic pH.24 Increasing the incubation temperature to 308C resulted in an identical outcome, that is, essentially no virus killing with these solvents. It is evident that a stronger condition, for example, more chaotropic solvents, higher temperature, or longer incubation time,

would be required for the inactivation of this virus, as has been observed with harsh solvents, which would also destroy proteins.23 In the plaque assay used here (Tabs. 1–3), a small amount of ingredients present in the solvents (Tab. 1), such as arginine, butyroylarginine or citrate, will be introduced into the cell monolayer during the step of virus adsorption, due to dilution of the incubated virus samples. Although highly unlikely because of extensive dilution, there may be possible interference by these compounds with the process of the plaque formation, resulting in a false positive of virus killing observed with HSV-1 and influenza virus as described above. However, such possibility is eliminated from the observation that the presence of the ingredient at this concentration in the inoculums did not affect the plating efficiency of the viruses (data not shown). Next, the effects of high salt concentration were tested with NaCl against HSV-1, since relatively high concentrations of arginine and butyroylarginine were used and hence the observed virus inactivation in these solvents could merely be due to high osmolality. However, as shown in Table 3, the results confirm the specific virus killing effects

Table 3. Inactivation on of HSV-1 by Arginine and NaCl Solvent

pH

Relative Virus Yield (LRV), Experiment 1

PBS control 1 M NaCl/20 mM acetate 0.7 M NaCl/20 mM acetate 0.35 M arginine/20 mM acetate 0.35 M butyroyl-arginine/20 mM acetate 0.7 M arginine/20 mM acetate 0.1 M arginine/20 mM acetate

7.4 4.3 4.0 4.0 4.0 3.5 3.5

1 (0) 1.05/0.851 (0/0.07) 0.159/0.189 (0.80/0.72) 5.7  10
5/4.0  10
5 (4.2/4.4) 3  10
6>/5  10
6> (>5.5/>5.3) 3  10
6> (>5.5) 3  10
6> (>5.5)

HSV-1 virus was incubated for 60 min on ice. For certain cases, duplicate data are shown. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008

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of arginine and the arginine derivative. There is essentially no virus inactivation by NaCl at 0.7–1.0 M at the pH (lines 2 and 3), at which arginine and butyroyl-arginine showed extensive virus killing; the difference in magnitude is in the order of 105–106-fold (5–6-fold larger LRV). Table 3 also shows that arginine and butyroylarginine were effective at 0.35 M at pH 4.0 against HSV-1, although 0.35 M arginine appears to be slightly less effective than 0.7 M arginine (LRV of 4.22 vs. 5.7 for 0.7 M arginine). Comparison of 0.35 M and 0.7–1 M arginine (Tabs. 1 and 3) suggests that the effects of arginine level off around 0.35–0.7 M and butyroyl-arginine may still be effective below 0.35 M (LRV of 5.52 vs. 5.7 at 0.7 M). At pH 3.5, 0.1–0.7 M arginine is extremely effective (Tab. 3), but without merit, since 0.1 M citrate was also effective at this pH. At least there is no adverse effect of arginine at pH 3.5 on virus inactivation. The time course of virus inactivation was examined using two solvents, that is, 0.7 M arginine/20 mM sodium acetate and 0.1 M sodium citrate, both at pH 4.0. The results are shown in Figure 2, in which virus yield is plotted as a function of incubation time (i.e., the length of exposure of the HSV-1 in these two solvents prior to plaque assay). The virus inactivation was fast

Figure 2. Time course of HSV-1 inactivation. Immediately after mixing the virus with the indicated solvents, the virus was incubated for 60 min on ice. Circle, PBS; triangle, 0.7 M arginine/20 mM acetate (pH 4.0); closed triangle, 0.1 M citrate (pH 4.0). DOI 10.1002/jps

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in 0.7 M arginine, with the virus yield of 10
5-fold within 2 min of incubation on ice and reaching the level below the detection at 5 min. On the contrary, the virus inactivation was slow in 0.1 M citrate (pH 4.0), only 30-fold decrease in the yield at 60 min of incubation. It is evident that arginine lowers the activation energy required for virus inactivation. The fact that the virus yield was greater at 2 min incubation than the later time points confirms no adverse effects of solvent, which was introduced into the plaque assay upon the addition of the virus, consistent with the observed absence of virus inactivation for poliovirus (as described above). To further clarify the effective concentration of arginine or butyroyl-arginine, the stock solutions at pH 4.0 were serially diluted and lower concentrations tested for the virus inactivation. Figure 3 plots the LRV value against the concentration of arginine (open circle) and butyroylarginine (black square). At this pH, butyroylarginine was still highly effective at 0.28 M (LRV ¼ 6) followed by a dose-dependent activity loss, while arginine lost the virus killing activity dose-dependently below 0.35 M. A virus spike experiment also showed the virus inactivation effects of arginine (data not shown). HSV-1 was spiked into the loading sample (the same mAb as used above) and loaded on to Protein-A columns. While a majority of HSV-1 flowed through the column, the virus did bind to the column, most likely by hydrophobic interaction.25 The bound virus slowly dissociated from the column during washing and elution, but was inactivated upon elution of the bound mAb in 1 M arginine at pH 4.0. How do arginine and butyroyl-arginine enhance virus inactivation? Virus inactivation by low

Figure 3. Concentration dependence of virus inactivation by arginine and butyroyl-arginine at pH 4.0. Open circle, arginine; black square, butyroyl-arginine. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 8, AUGUST 2008

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pH treatment is believed to be due to acid-induced conformational changes of the components of viral particles. Low pH treatment has been shown to retain the density of the particle, an indication that the particle composition is intact.5 Both virus particle aggregation and morphology changes have been implicated as the mechanism of low pH-induced virus inactivation.3 The same author showed that the low pH caused damages on capsid and membrane structure. Therefore, as the pH is increased, the virus inactivation by acid becomes less effective. To our surprise, the aqueous arginine and butyroyl-arginine solutions resulted in extensive virus killing at pH, at which acid alone (e.g., using citrate) was ineffective (Tab. 1). We are now investigating the mechanism, by which these compounds enhance acid-induced virus inactivation. At this point, we know that arginine binds to proteins, but without denaturing or destabilizing them, although exact sites of arginine binding is not clear.26–30 Arginine also binds to phosphate groups on membrane lipids.31 Although no binding data are available for butyroyl-arginine, it is likely that it also binds to the proteins and lipids, perhaps more strongly due to its hydrophobic moiety. How these properties of arginine and butyroyl-arginine play a role in virus inactivation must await further studies. As a final remark, we have done virus inactivation experiments in the presence of antibodies, since this step is usually performed on the Protein-A eluate. We have shown that the antibodies are stable above pH 4.0 in the absence and presence of arginine and arginine or butyroylarginine are effective for elution of the bound antibodies from Protein-A columns.12–14 Although the virus inactivation by these reagents may be further enhanced as the pH is lowered below 4.0, the stability of antibodies will also be compromised.

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