Efficiency of Atmospheric Pressure Nitrogen Gas Remote Plasma Sterilization and the Clarification of Sterilization Major Factors

Experiments reported here were conducted using atmospheric nitrogen gas remote plasma with a pulsed power source. The sterilization efficiency, major sterilization factors and most appropriate sterilization conditions were determined. By varying several factors such as hotplate temperature, relative humidity, water vapor supply location, etc., the most appropriate sterilization conditions were identified. The temperature of the hotplate was varied from 55°C to 75°C and with this 20°C increase in temperature, sterilization was completed in half the time. In this experiment, it was confirmed that the combined effect of a relative humidity (RH) of 0.5% and nitrogen gas was superior to the use of nitrogen gas alone. Furthermore, it was clarified that the optimal humidity was in the range of 0-5 % RH. When RHs of 0, 0.5 and 5% were tested, 0.5% RH was found to be optimal for sterilization. The location of the water vapor supply was changed relative to the hotplate, and use of the most remote port upstream of the reactor resulted in the most efficient sterilization. In addition, the results correlated with the amount of NO radicals generated. The NO radical is the precursor of OONO・(peroxynitrite anion radical). The sterilization factors associated with this experiment were NO radicals, H2O2, OH radicals, O2 ・(superoxide anion radicals) and OONO・only OONO・production correlated with sterilization efficiency. Therefore, OONO・is thought to be the major factor for nitrogen gas plasma sterilization. In addition, as already described, the highest sterilization efficiency was with 0.5% RH and the amount of OONO・produced correlated with the RH. These data support the idea that OONO・is the major contributing factor for nitrogen gas plasma sterilization. The D values for this experiment were approximately 10 min. *Corresponding author: Hideharu Shintani, Department of Science and Engineering, Chuo University, Kasuga Bunkyo 112-0003, Tokyo, Japan, Tel: +81338171733; E-mail: shintani@mail.hinocatv.ne.jp; hshintani@jcom.zaq.ne.jp Citation: Shintani, H., et al. Efficiency of Atmospheric Pressure Nitrogen Gas Remote Plasma Sterilization and the Clarification of Sterilization Major Factors. (2015) J Pharm Pharmaceutics 2(1): 2228. Efficiency of Atmospheric Pressure Nitrogen Gas Remote Plasma Sterilization and the Clarification of Sterilization Major Factors Hideharu Shintani1*, Naohiro Shimizu2, Shouji Tange2, Nobuo Takahashi2, Akikazu Sakudo3, Akira Mizuno4, Eiki Hotta5 Received date: April 17, 2015 Accepted date: November 18, 2015 Published date: November 24, 2015 DOI: 10.15436/2377-1313.15.004 Journal of Pharmacy &


Introduction
Several papers have been published on gas plasma sterilization [14,11,19,20]. The definition of sterilization can be found in the book published from NOVA [11]. The advantages of gas plasma are that sterilization with a sterility assurance level (SAL) of 10 -6 and material/functional compatibility can be attained without any difficulty. This is because the penetration depth of gas plasma sterilization is quite shallow (10-20 nm, [19]) and therefore only one layer of bioburden can be sterilized. The bioburden represents the type and number of viable microorganisms existing in/ on products. Most of the bioburden exists as one layer; therefore, deeper penetration capability is unnecessary for efficient sterilization.
In contrast, existing sterilization procedures including gamma-ray irradiation sterilization, electron-beam sterilization, moist heat sterilization, dry heat sterilization, ethylene oxide gas sterilization, hydrogen peroxide gas sterilization and so on, have the ability to penetrate deeper. Therefore, materials are easily sterilized using these methods and a SAL of 10 -6 can be attained; however, the sterilized products are useless due to degradation of the product material during the sterilization process, a phenomenon called failure to attain material/functional compatibility [16,17]. Good manufacturing practice (GMP) and sterilization guidelines require simultaneous attainment of both a SAL of 10 -6 and material/ functional compatibility, but this requirement is difficult to attain with the existing sterilization procedures. Therefore, sterilization procedures using stable gases that are safe to handle and that have shallow penetration depths are needed. As described previously, gas plasma sterilization has the characteristics necessary to meet these requirements [14,11,19,20] .
For the studies reported here, we used a remote type of nitrogen gas plasma sterilization procedure using a pulsed power supply of SIThy [13]. Several factors associated with the sterilization procedure were determined, and the main factors associated with sterilization were identified. In addition, appropriate sterilization conditions were identified and are reported herein.

Experimental
In Figure 1, the schema of the experimental system is shown.
Remote gas plasma was utilized. The experimental system consists mainly of a humidity control device, plasma producer, and exhaust gas analyzer.
In Figure 2, a photograph of the plasma generator parts is presented and the experimental conditions are presented in Table  1. A detailed explanation of the experimental conditions can be found in the Master's thesis of Takuya Uyama (TISTech, 2015). Figure 3 presents the typical waveforms obtained when using the experimental conditions in Table 1.
The relative humidity (RH) was measured at the upper and lower sites of the reactor with BKPRECISION Ltd., 725 digital temperature/humidity sensors. Exhaust NOx gases were determined using a Shimadzu NOA-7000 analyzer and exhaust ozone gas was determined by using an EG-700EIII ozone monitor from Ebara Ltd.   Table 1.  Samples to be sterilized, including a biological indicator (BI), were placed on a hotplate (Figures 1 and 4). The distance between the reactor and hotplate was kept constant at 100 mm ( Figure 1). The temperature of the hotplate was varied from 55-75°C ( Figure 8).
Nitrogen gas was chosen for use in these sterilization studies because of its higher dissociation energy, which makes it relatively stable compared with other gases, and therefore it is inert and safe to handle ( Table 2).
The need for humidity in gas plasma sterilization has been reported [2,21]. A supply of water vapor was introduced at three locations as shown in Figures 4 and 5. The site of water vapor introduction was varied because water vapor can play a role in generating various reactive oxygen species that may function in the sterilization process. Location  was at the upper part of the reactor, location  was just below the reactor, and location  was just before the site of sample treatment. At location , NO radicals, which are actively involved in the sterilization process, may be produced [15,18]. In location , it can be speculated that N metastables or other reactive oxygen species (ROS) may be generated by reactions with water vapor. In location , short-lived OH radical may attack the biological indicator (BI) and result in its sterilization. The actual experimental set up with the different positions of water vapor introduction is shown in Figure 5.
Sterilization evaluation was confirmed by using a BI of Geobacillus stearothermophilus ATCC 7953 with 10 6 CFU (colony forming unit)/carrier, which was obtained from MESA Lab. The D value (decimal reduction value) was obtained by two methods, the fraction negative method and survivor curve method [3,4]. In the case of the fraction negative method, the BI was incubated using SCDB (soybean casein digest broth) liquid medium at 58°C for 2 days. The result was confirmed using a chemical indicator (CI, Figure 6). When the BI survived, the color changed to yellow, whereas when sterilization was successful, the color remained unchanged (purple). This is due to the production of organic acids (mostly citric acid) from the TCA cycle ( Figure 6). To generate the survivor curve, we used SCDA (soybean casein digest agar) solid medium. Ten-fold serial dilutions using SCDB were carried to achieve final plate counts of 30-300 CFU/plate as required in ISO 14161. Spores were retrieved from the BI carrier by using the procedures described in ISO 11737-1. According to ISO 11138-1, the D value must be obtained using both the fraction negative method and the survivor curve method, so we carried out both methods following the ISO 11138-1 requirement.
Surfaces of spores were observed by using scanning electron microscopy (SEM; S-5500 Hitachi technologies Ltd).
Several reactive oxygen species (ROSs) were analyzed by using emission spectrophotometric analyzers from Maya 2000 Pro (Ocean Optics Ltd.) ( Figure 7). A quartz window was incorporated into the reactor and analyses were conducted under the following conditions. The determination wavelength was 200-650 nm, grating was 600 lines/mm, entrance slit width was 10 μm, exposure time was 100 ms and analyses were repeated 5 times. Production of one type of ROS, H 2 O 2 , has been reported by nitrogen gas plasma sterilization [7,9,10], So we measured H 2 O 2 by using a chemical indicator (CI). The CI for H 2 O 2 analysis was from Quantofix Peroxide 25 (MACHEREY-NAGEL Ltd.) and the analysis range was 0-25 μg/mL.
All analyses described above are presented and explained in the Results and Discussion.

Relationship between Sterilization Efficiency and Hotplate Temperature
As shown in Figure 8, it was determined that the higher the temperature, the greater the sterilization efficiency. From Figure 8, it is seen that sterilization can be completed in 240 min, 150 min and 120 min at 55°C, 65°C and 75°C, respectively. These results indicate that 75°C is the best temperature for sterilization because an increase of 20°C from 55°C to 75°C resulted in a sterilization time that was half as long, and it is expected that the target materials including the BI are tolerant to this temperature.

Relationship between Sterilization Efficiency and Relative Humidity
As shown in Figure 9, the optimum relative humidity (RH) was determined. A RH of 0.5% was the most appropriate for sterilization. This result has been confirmed in another experiments with consistent results.

Determination of NOx and O 3 (ozone) as exhaust gases
NOx and ozone were determined and their amounts were less than 0.6 ppm and 0.04 ppm, respectively. Since the amounts generated were so low, it can be concluded that these ROSs do not contribute to nitrogen gas plasma sterilization.

Relationship between Sterilization Efficiency and Water Vapor Supply Location
As shown in Figure 10, water vapor supply locations  and  in     The tube on the left indicates survival of the BI (acid produced) and that on the right is sterilized (color is unchanged). Figure 4 were superior to that of  with respect to sterilization efficiency. The reason  in Figure 4 was inferior to the others was likely due to N metastables or OH radicals being inactivated before reaching the BI target. Location in Figure 4 represents the shortest distance between the water vapor supply and the site of sterilization, whereas location  in Figure 4 was the most remote, but the abundantly produced NO radicals are the precursors of OONO • -(peroxynitrite anion radicals), which are the real sterilization factors described later. Measurement of NO radicals was conducted using Figures 7 and 14. NO radical detection using CI was also reported by Shintani et al., 2014. Additional results supported the conclusion that water vapor supply location , rather than location  resulted in the best sterilization efficiency.

D Value (Decimal Reduction Value) Determination by the Fraction Negative Method
The D value was determined by the Stumbo Murphy Cochran Procedure, one of the fraction negative methods (ISO 14161).
The results are summarized in Table 3. The D value was the lowest at a RH of 0.5% (~ 8.7 min). The others were approximately 10 min, indicating that a RH of 0.5% resulted in the most efficient sterilization. The D value was determined under the following conditions: hotplate temperature, 75°C, RH, 0.5% or 0%, and water vapor supply location  or  as shown in Figures 11 and 12.
From data in Figure 11, it can be concluded that the use of a RH of 0.5% was superior to 0% RH, as the D was approximately 10 min. Results presented in Figure 12 indicate that water vapor supply location  was superior to location , and the D value was approximately 10 min. These data are consistent with the D values obtained by the fraction negative method. The D value from the survivor curve method was determined using a regression line with a coefficient of correlation of greater than 0.8 as required in ISO 11138-1. Figure 13 shows the SEM observation of spores. Compared with the untreated control (left), sterilized spores showed no shrinkage, but some roughness of the surface was observed for spores that were treated for 30 min (middle). However, roughness did not always increase with increasing treatment time up to 90 min (right), indicating that roughness is a temporary rather than permanent phenomenon. It therefore appears that SEM observation does not provide any useful information regarding the success of the sterilization process. Nitrogen gas plasma does not cause any etching in contrast to O 2 gas plasma [20,21].

Emission spectrum analysis
In Figure 14, the emission spectrum at a RH of 0.5% is shown. By using the equipment shown in Figure 7, the emission spectrum can be obtained. NO radicals, N 2 second positives and N 2 + were detected. However, no OH radicals were detected at 310 nm, indicating that OH radicals are not major contributors to nitrogen gas plasma sterilization.

Emission intensity depending on RH at 258.55 nm
A wavelength of 258.55 nm was used for the detection of NO radicals as shown in Figure 14. In Figure 15, the relationship between emission intensity at 258.55 nm and relative humidity is presented. As shown in Table 3, sterilization efficiency was optimal at a RH of 0.5%, indicating that the NO radical itself does not function as a sterilization factor because results in Figure 15 do not indicate that 0.5% RH was optimal. In addition, 258.55 nm is in the UV-C range and UV-C is thought to be effective for sterilization of microorganisms by causing thymine dimer formation. However, no role for UV-C in sterilization could be demonstrated in previous studies using E. coli and Bacillus atrophaeus ATCC 9372 [6,1].

H 2 O 2 (hydrogen peroxide) determination
H 2 O 2 formation was analyzed using a CI from Macherey-Nagel Ltd., and the relationship between H 2 O 2 concentration and humidity is presented in Figure 16. As shown in Table 3, sterilization efficiency was optimal at a RH of 0.5%, indicating that H 2 O 2 or OH radicals from H 2 O 2 do not correlate with the RH tendency; therefore H 2 O 2 or OH radicals do not appear to be major contributors in nitrogen gas plasma sterilization.

Formation of superoxide anion radicals (O 2 • -)
Superoxide anion radicals (O 2 • -) were speculated to be produced at the reactor site and reach the treatment location as shown in Figure 17. NO• and O 2 •were also speculated to be produced even when the water vapor was introduced at location 3, the lowest part in Figure 4, because sterilization was successful at this location.
Measurement of O 2 •was not successful; therefore, its effect on gas plasma sterilization remains uncertain, but it can be speculated that O 2 •supports the production of peroxynitrite anion radicals (ONOO• -) as shown in Figure 18.

Formation of OH radicals (HO •)
OH radicals may be formed by the reaction shown in Figure 18 and/or from H 2 O 2 . However, as shown in Figure 14 and mentioned in section of 8, OH radicals were not detected and therefore OH radicals do not appear to be major contributors to nitrogen gas plasma sterilization.

Evaluation of peroxynitrite anion radical (ONOO • -) Formation
Peroxynitrite anion radicals (ONOO• -) can be formed from NO radicals + superoxide anion radicals (O 2 • -) (Figure 18, Nova E and Parola M, 2008). The reaction in Figure 18 will occur just at the upper layer of bacteria (Figure 19), indicating that NO radicals and O 2 •migrate from the reactor site to the treatment site and react as shown in Figure 18 to produce OONO• -. Figure 12. Relationship between exposure time and colony forming unit at water vapor supply locations  and , with a RH of 0.5% and a temperature of 75 o C Location  was found to be the most appropriate. The left panel is the control, the middle is after a 30 min treatment and the right is after a 90 min treatment. Roughness did not always increase with increasing treated time, indicating roughness is not always a permanent factor. Reactor O 2 •-produced in the reactor migrates to the treatment location and reacts with NO•, producing ONOO•-just before reaching the BI (Figures 18 and 19).

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Peroxynitrite anion radicals were detected by using aminophenyl fluorescein (APF) reagent as shown in Figure 20 [12]. The relationship between the peroxynitrite anion radical (ONOO• -) concentration and relative humidity is presented in Figure 21. As shown in Table 3, sterilization efficiency was optimal at a RH of 0.5%, indicating that the peroxynitrite anion radical concentration correlates with the RH level. Based on this finding it can be speculated that peroxynitrite anion radicals function as a major sterilization factor in nitrogen gas plasma sterilization. In addition, please refer to the footnote of Figure 20 for further clarification of ONOO•as a major sterilization factor.

Relation between sterilization efficiency (%) and relative humidity (RH%)
The relationship between sterilization efficiency and relative humidity combined with several ROSs is presented in Figure 22.
The results indicate that sterilization efficiency coincides with the tendency of peroxynitrite anion radical (ONOO• -) formation; therefore, peroxynitrite anion radicals (ONOO• -) are thought to be the major factor of nitrogen gas plasma sterilization. Other factors such as NO radicals, H 2 O 2 , OH radicals or O 2 •-do not co-incide with the % RH (Figures 14-16). Peroxynitrite anion radicals (ONOO• -) react with tyrosine, causing nitration at the p site and with DNA bases, especially guanine, causing nitration (-NO 2 ) and hydroxylation (-OH), which results in transcription failure.

Conclusion
The experiments reported here were conducted to identify the nitrogen gas plasma sterilization factor(s) and the appropriate sterilization conditions. By varying hotplate temperature, relative humidity (RH) and water vapor supply location, sterilization efficiency was confirmed. In addition, SEM observation of spore surfaces, emission spectrophotometric analysis, and determination and evaluation of peroxynitrite anion radicals (ONOO• -) were conducted to determine which ROSs contribute to nitrogen gas plasma sterilization.
The sterilization times at 55°C, 65°C and 75°C were 240 min, 150 min, and 120 min, respectively, indicating that at higher hotplate temperatures, the sterilization periods were shorter. Increasing the temperature by 20°C reduced the sterilization period by half.   The sterilization efficiency was improved by using a combination of water vapor and nitrogen gas. Relative humidity (RH) was changed from 0.0% RH, 0.5% RH and 5% RH and the D values under these conditions were 10.71 min, 8.66 min and 10.07 min, respectively, indicating that the optimum RH is 0.5%. In order to identify the sterilization factors, the water vapor supply location was varied. The results indicate that the active species were relatively long-lived because the most efficient location was the most remote from the reactor.
SEM observation indicated that there was no significant difference in the appearance of control and treated spores, and no etching occurred. Treated spores seemed to have increased roughness compared with control spores, but this roughness did not always increase with increasing sterilization time, so roughness is not always an indication of sterilization. The reason has not been clarified, so no ROSs can be confirmed from SEM observation.
By attaching a quartz window to the reactor, it was possible to carry out emission spectrophotometric analysis. Based on the emission spectrum at a RH of 0.5%, NO radicals, N 2 second positives and N 2 + were detected ( Figure 14). In this experiment, NO radicals, which are detected at 258.55 nm in the UV-C range, increased with increasing relative humidity ( Figure 15). This indicates that the tendency of NO radical formation does not coincide with that of the sterilization tendency as shown in Figure 22. This result indicates that NO radicals do not participate directly as a major factor in nitrogen gas plasma sterilization. ROSs such as NO radicals, H 2 O 2 , OH radicals, O 2 • -(super oxide anion radicals) or ONOO• -(peroxynitrite anion radicals) were compared for their contribution to sterilization ( Figure 22). The RH tendency coincided with that of OONO• - (Figures 21 and  22); therefore, we conclude that OONO•may be the major sterilization factor in nitrogen gas plasma sterilization.
Based on the experimental conditions for nitrogen gas plasma sterilization, the water vapor supply position was best at location  (furthest from the reactor; Figure 4) and the humidity was optimal at 0.5% RH (Figure 9). Hotplate temperature was optimal at 75°C ( Figure 8). Together these results indicate that higher temperature and optimum RH at 0.5% were the best when using position  for the water vapor supply (Figures 10, 11 and 12). All results support the concluding data summarized in Figure 22.
We reported the original description of ONOO• -(peroxynitrite anion radical), and demonstrated that ONOO•is the major factor in nitrogen gas plasma sterilization. In contrast to ONOO• - (Figures 21 and 22), other ROSs do not have identical tendencies with respect to RH (Figure 9, 15 and 16) and only ONOO• presents an identical tendency to the RH. Therefore, ONOO• can be defined as the major sterilization factor in nitrogen gas plasma sterilization. Normalized values [-]