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A simplified ATP method for the rapid control of cell viability in a freeze-dried BCG vaccine
Journal of Microbiological Methods 130 (2016) 48–53
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
A simplified ATP method for the rapid control of cell viability in a freeze-dried BCG vaccine Natalia N. Ugarova a,b,⁎, Galina Yu. Lomakina a,b, Yulia Modestova a,b, Sergey V. Chernikov c, Natalia V. Vinokurova c, Elena V. Оtrashevskaya c, Vyacheslav Y. Gorbachev c a b c
Faculty of Chemistry, Lomonosоv Moscow State University, Moscow 119991, Russia LLC Lumtek, Vorobievy Gory 1/77, Moscow 119992, Russia FSUC SIC Microgen, MOH RF, 10, Vtoroi Volkonsky per., Moscow 127473, Russia
a r t i c l e
i n f o
Article history: Received 12 July 2016 Received in revised form 24 August 2016 Accepted 28 August 2016 Available online 29 August 2016 Keywords: ATP assay BCG viability Colony forming units Freeze-dried BCG vaccine Tuberculosis
a b s t r a c t We propose a simple and cost-effective ATP method for controlling the specific activity of a freeze-dried BCG vaccine. A freeze-dried BCG vaccine is reconstituted with 1 ml saline and incubated for 15 min at room temperature and then for 1 h at 37 °C. The vaccine is then treated with apyrase to remove extracellular ATP. After that, the cells are lysed with DMSO and the ATP content in the lysate is measured by the bioluminescence method. To implement the method, we developed a kit that requires no time-consuming preparation before the analysis. We demonstrated the linear relationship between the experimental values of the specific activity (106 CFU/mg) and intracellular ATP content (ATP, pmol/mg) for different batches of the studied BCG vaccines; the proportionality coefficient was К = 0.36 ± 0.02. We proposed a formula for calculating the specific activity from the measured content of intracellular ATP (ATP, pmol/mg). The comparison of the measured and calculated values of the specific activity (106 CFU/mg) shows that these values are similar; their differences fall within the allowable range of deviations for the specific activity values of the BCG vaccine. © 2016 Published by Elsevier B.V.
1. Introduction The vaccine based on an attenuated strain of freeze-dried viable bacteria Mycobacterium bovis (BCG vaccine) is widely used for vaccination against tuberculosis (Ho et al., 2008; Jensen et al., 2008; Knezevic and Corbel, 2006); it is also used as an immuno-therapeutic agent against bladder cancer (Brandau and Suttmann, 2007). Hundreds of millions of the vaccine doses are produced annually in different countries all over the world (WHO, 2004). One of the key characteristics of the BCG vaccine is the specific activity of freeze-dried М. bovis BCG bacteria. The cell viability of the BCG vaccine is traditionally characterized by a number of colony-forming units per 1 ml of the vaccine suspension (CFU/ml) or per 1 mg of biomass (CFU/mg). Cell viability of the vaccine is generally determined by plate counting. The mycobacterial cells grow very slowly, so three to five weeks are usually required to complete the testing of the vaccine samples. Moreover, the CFU assay is variable and often not reproducible because of the difficulties associated with the cultivation of mycobacteria: these organisms are prone to adhesion and aggregation and the composition of their cultivation medium is very complex.
⁎ Corresponding author at: Faculty of Chemistry, Lomonosоv Moscow State University, Leninskie Gory 1/3, Moscow 119991, Russia. E-mail address: [email protected] (N.N. Ugarova).
http://dx.doi.org/10.1016/j.mimet.2016.08.027 0167-7012/© 2016 Published by Elsevier B.V.
In the last decade, an alternative method for the rapid quantitative determination of viable cells in BCG vaccine was proposed and approved by the World Health Organization (WHO). This method is based on the bioluminescence assay of intracellular adenosine-5′-triphosphate (ATP) (Ho et al., 2008; Jensen et al., 2008; Knezevic and Corbel, 2006). The bioluminescence method makes it possible to determine the amount of intracellular ATP rapidly. The intracellular ATP content is the main indicator of cell viability. When a cell dies, the production of ATP stops and the intracellular ATP content rapidly decreases by tens and hundreds of times. The intracellular ATP content is an indicator of the metabolic status of cells; it depends on the nature and size of cells, their energy state, and the external conditions (Lomakina et al., 2015). Bioluminescence ATP assay is based on the ability of the firefly luciferase enzyme to catalyze the oxidation of D-luciferin by oxygen in the presence of a magnesium salt and ATP. The formation of the reaction product, oxyluciferin, is accompanied by the emission of visible light. If the contents of luciferase, D-luciferin, and the magnesium salt in the reaction mixture are fixed, the light intensity is proportional to the ATP content in the analyzed sample. Thus, the viability of cells in the sample can be determined using the bioluminescence method (Ugarova et al., 1987; Ugarova, 1993, Lundin, 2000). Since 1970s, the bioluminescence method has been used by many authors to determine the activity of BCG cells and to study their properties in different systems (Crispen, 1971; Gheorghiu and Lagranderie,
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1979; Beckers et al., 1985; Chen et al., 1989). It was shown that there is a correlation between the intracellular ATP content and the number of viable cells in liquid and freeze-dried preparations of the BCG vaccine (Askgaard et al., 1995; Janaszek et al., 1987; Prioli et al., 1985; Shi et al., 1989). However, it was only in 2008 that the paper was published describing the validation of the ATP method for the rapid assessment of the viability of freeze-dried BCG vaccine preparations (Jensen et al., 2008). In this variant of the method, the BCG vaccine is reconstituted using a full mycobacterial growth medium (the Dubos medium with Tween and bovine albumin) and incubated for 24 h at 37 °C to restore the metabolic activity of the cells and, consequently, increase the intracellular ATP content. The intracellular ATP is then extracted by boiling the vaccine suspension in an EDTA-containing Tris-acetate buffer solution after which the intracellular ATP content is determined by the bioluminescence ATP assay. The cell viability is then estimated by the correlation between the ATP content and the CFU values. The developed method has reduced the total duration of analysis from 30 days to 36 h. The authors showed that the method is characterized by a good reproducibility, robustness, accuracy, linearity, and high sensitivity. On the initiative of the WHO, the developed method was tested in six laboratories in different countries. However, only three laboratories managed to obtain the same results because the conditions, the testing procedures, the reagent base, and the equipment varied somewhat in different laboratories (Ho et al., 2008). Kolibab et al. (2012) later modified the protocol proposed in (Jensen et al., 2008) by eliminating the timeconsuming incubation step: BCG vaccines were reconstituted in diluted Sauton's SSI media and incubated for 30 min in the presence of apyrase at room temperature. The aim of the present study was to develop a simplified ATP method suitable for use in the factory for the rapid control of the specific activity (viability) of the production batches of a freeze-dried BCG vaccine. 2. Materials and methods 2.1. BCG samples We used a BCG vaccine containing viable mycobacteria of a BCG Mycobacterium bovis production strain, substrain BCG-1 (Russia), freezedried in a 1.5% sodium glutamate solution and sealed in evacuated vials. Each vial contained 0.5 mg BCG cells and 3 mg sodium glutamate. In several experiments, a liquid BCG vaccine was assayed before its freeze-drying.
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suspension (sample no. 1 and no. 2). The samples were gently mixed and incubated at room temperature for 10 min without stirring. 2.3.2. ATP extraction and measurement of bioluminescence signal for the resulting extracts The first test tube (sample no. 1) containing the apyrase-treated vaccine suspension was supplied with 1 ml DMSO and mixed. After 1 min of incubation, 100 μl ATP-reagent was added to 20 μl of the ATP extract in DMSO and the bioluminescence signal was measured in a LUM-1 luminometer (Lumtek, Russia). Each measurement was repeated at least twice and the arithmetic mean of the two repeats was calculated. If the difference between the two repeats exceeded 20%, the measurement was performed for a third time. Similarly, the measurements were conducted for the sample no. 2 from the same vaccine vial. The arithmetic mean value of the bioluminescence signal was determined for the analyzed vial (Ivial). 2.3.3. Measurement of bioluminescence signal for the ATP-control and calculation the ATP content (ATP, pmol/mg) Two test tubes were supplied with the mixture of 100 μl of the reconstituted ATP-control solution, 20 μl saline, and 1 ml DMSO. The resulting solution was mixed and the bioluminescence signal for the ATP-control was measured as described above for the BCG vaccine. The arithmetic mean value of the bioluminescence signal for the ATPcontrol (Icontrol) was calculated; the value was then used to calculate the intracellular ATP content in a sample of the dry BCG vaccine using Formula (1). ðATP Þ; pmol ¼ ðATP Þcontrol
I vial ; pmol I control
ð1Þ
Here, (ATP)control is the content of the ATP-control in the initial saline solution, pmol; Icontrol is the mean value of the bioluminescence signal for the ATP-control; and Ivial is the mean value of the bioluminescence signal for the vial. The intracellular ATP content in 1 mg of the BCG vaccine was calculated by Formula (2) using the obtained value of the ATP content in the vial (pmol) and the mass of BCG cells in the vial (0.5 mg): ðATP Þ; pmol=mg ¼
ðATP Þ; pmol BCG mass; mg
ð2Þ
2.2. Analytical reagents
2.4. Determination of CFU
For this study, we developed a reagent kit based on the ATP-reagent (Lumtek, Russia). This kit contained freeze-dried ATP-reagent, ATP-control and apyrase; liquid sterile deionized water; saline (0.9% NaCl in water); and dimethylsulfoxide (DMSO). The ATP-reagent (Ugarova et al., 2009) comprised of the thermostable firefly luciferase Luciola mingrelica (Koksharov and Ugarova, 2011), luciferin, buffer components, and stabilizers was reconstituted with 4 ml of deionized water 1 h before the experiment. The ATP-control, 10 pmol, was reconstituted in 1 ml saline. The apyrase was also reconstituted in saline; the resulting solution contained 5 U/ml apyrase. The reconstituted solutions were stored at room temperature and used within one working day.
Six BCG vials were reconstituted in saline to a concentration of 0.5 mg BCG/ml. The BCG suspensions from the vials were pooled and diluted in saline. Three serial dilutions of the suspension were inoculated onto Löwenstein Jensen medium and incubated at 37 °C for four weeks before plate counting and calculating the number of CFU (106 CFU/mg) (WHO, 1977).
2.3. Determination of intracellular ATP 2.3.1. Reconstitution of freeze-dried BCG vaccine and degradation of extracellular ATP A vial containing freeze-dried vaccine was reconstituted with 1 ml saline, incubated for 15 min at room temperature without stirring and then for 60 min at 37 °C with agitation (250 rpm). Then, the vial was cooled in air to room temperature for 3–5 min. Two test tubes were supplied with 20 μl of apyrase solution and with 100 μl of the vaccine
2.5. Statistical analysis All measurements were performed in triplicate. Mean (X) and standard deviation (SD) for the bioluminescent signal and the ATP content were calculated using Excel software (Microsoft℗ Office Excel 2003 SPI). The ATP content was calculated and expressed as pmol ATP per vial or pmol ATP per 1 mg of BCG cells. 3. Results and discussion 3.1. Optimization of the intracellular ATP bioluminescence assay The conditions for the reconstitution and reactivation of a freezedried cell culture play a significant role in the correct determination of
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Luminescent signal, 103 RLU/s
50
Table 1 Comparison of different methods for intracellular ATP assays in BCG vaccine.
160
Literature data
Our method
120 y = 0.4401x R² = 0.9986
80 40 0 0
100
200
300
400
[ATP], pmol/ml Fig. 1. Linear correlation between the ATP concentration and bioluminescent signal for the ATP solutions in DMSO.
the intracellular ATP content. The viability of a freeze-dried bacterial preparation greatly depends on the reconstitution conditions (Morgan et al., 2006): the reconstitution solution, incubation time, temperature, etc. Two types of standard solutions are generally used to reconstitute BCG vaccines prior to the injection: diluted Sauton SSI and saline. According to protocols described in the literature (Jensen et al., 2008; Kolibab et al., 2012), the diluted Sauton SSI is used for reconstitution of a freeze-dried BCG vaccine. In our studies, we adjusted the reconstitution procedure to the saline-based protocol for the preparation of BCG vaccine suspension. Our preliminary experiments demonstrated that fluctuations of the incubation temperature cause discrepancies in the experimental results. Therefore, we chose the incubation temperature of 37 °C: determination of cell viability by plate counting is conducted under the same conditions and this temperature is easy to maintain. By measuring the intracellular ATP content during the incubation of reconstituted BCG vaccines at 37 °C, we found that the steady-state concentration of intracellular ATP is maintained within 30 to 120 min of incubation after which the ATP content starts to fluctuate. We selected the incubation period of 60 min as the standard condition for our experimental procedure. During freeze-drying, some bacterial cells get disrupted: consequently, their intracellular ATP enters the extracellular space. As the result, the total ATP content in the analyzed sample of the reconstituted cells can exceed the intracellular ATP content, the value of which characterizes the viability of the vaccine (Morgan et al., 2006). To solve this problem, the reconstituted samples are pretreated with apyrase, an ATP-hydrolyzing enzyme, before the extraction of intracellular ATP. In the protocol developed in (Kolibab et al., 2012), a BCG vaccine was reconstituted in diluted Sauton SSI medium and simultaneously treated with 0.5 U/ml apyrase with a subsequent 30-min incubation. To prevent the inactivation of apyrase and optimize the duration of the ATP depletion stage, we separated the reconstitution step and the step of the extracellular ATP depletion: the reconstituted BCG samples were treated with 1 U/ml apyrase at room temperature. We showed that the extracellular ATP content decreased by 98% in 10 min; extending the period of incubation of the apyrase–sample mixture provided no further decrease in the extracellular ATP content. The next step is the extraction of the intracellular ATP. Generally, ATP is extracted from the BCG cells by boiling them in a preheated EDTA-containing Tris-acetate buffer for 6 min (Prioli et al., 1985; Hoffner et al., 1999; Jensen et al., 2008; Kolibab et al., 2012). We simplified the ATP extraction procedure by using sterile DMSO as described in our previous studies (Ugarova et al., 1987; Ugarova, 1993; Romanova et al., 1997). We found that a one-minute incubation of BCG samples in 80% DMSO (room temperature) is sufficient to complete the extraction. We also demonstrated that the apyrase used at the previous step is irreversibly inactivated under these conditions.
Reconstitution of lyophilized BCG vaccine 1*): A freeze-dried A freeze-dried vaccine is vaccine is reconstituted reconstituted with 1 ml with 1 ml Dubos saline, incubated for 15 medium and incubated min at room temperature for 24 h at 37 °C. without stirring and then 2**): A freeze-dried for 60 min at 37 °C with vaccine is reconstituted agitation (250 rpm). The with 1 ml of diluted vial is then cooled in air to Sauton SSI media room temperature for containing 0.5 units of 3–5 min. apyrase and incubated for 30 min.
Degradation of extracellular ATP 1: The protocol for the In a test tube, 100 μl removal of extracellular vaccine suspension is ATP after the incubation gently mixed with 20 μl of with Dubos medium is apyrase solution and not provided. incubated at room 2: See above. temperature for 10 min without stirring. Extraction of intracellular ATP 1 and 2: BCG suspension The apyrase-treated is mixed with vaccine suspension is Tris-acetate buffer supplied with 1 ml DMSO, containing EDTA mixed, and incubated for (98–99 °C), boiled for 6 1 min at room temperature. min, and allowed to cool to room temperature. Measurement of ATP 1: 100 μl ATP reagent is added to 600 μl of cooled ATP extract; the bioluminescence signal is recorded (Ivial, RLU). 2: 100 μl ATP reagent is added to the sample; the bioluminescence signal is recorded (Ivial, RLU).
Advantages of our method This stage is the most important for obtaining correct and reproducible results. The use of saline solution for the reconstruction of a freeze-dried BCG vaccine and the precise control of the incubation duration and temperature standardizes the conditions and simplifies the preparation of the working samples.
A ready-to-use apyrase preparation provided in the kit reduces the labor intensity and duration of the analysis.
Using a highly efficient organic extractant, we extract ATP at room temperature. This reduces the duration and labor intensity of the ATP extraction and improves the reproducibility of the method.
100 μl ATP-reagent is added to 20 μl of the ATP extract in DMSO; the bioluminescence signal is recorded (Ivial, RLU). High operational stability of the ATP-reagent provides good reproducibility of the measurements.
Determination of the ATP content in BCG samples and the use of ATP standard 1 and 2: The ATP standard Freeze-dried ATP-control The linear relationship between the curve is obtained using is reconstituted with bioluminescence signal several dilutions of ATP saline and then treated (I) and the concentration Standard in Tris-acetate with DMSO as the BCG of ATP (Fig.1) eliminates buffer with EDTA. The vaccine. The amount of ATP bioluminescence signal is the need to obtain a calibration curve. extracted from BCG measured (Icontrol, RLU) as described above for the The calculation of samples is calculated vaccine. intracellular ATP content from the ATP standard The amount of in dry BCG vaccine is curve. intracellular ATP content simplified. in dry BCG vaccine is calculated by Formula (ATP), pmol = (ATP)control ∙ Ivial/Icontrol, pmol (ATP)control is the content of the ATP-control in the initial saline solution, pmol. Calculation of the specific activity (106 CFU/mg) from the measured values of the intracellular ATP content Calculation of the specific Using different BCG 1: Calculation of the activity: 106 CFU/mg = K vaccine batches, we specific activity: demonstrated that the 106 CFU/vial = 0.037 (ng · ATP, pmol/mg ATP/vial)1.398 K is the slope of the linear linear dependence exists
N.N. Ugarova et al. / Journal of Microbiological Methods 130 (2016) 48–53 Table 1 (continued) Literature data
Our method
Advantages of our method
2: No information
correlation between the intracellular ATP content and the CFU.
between the CFU values and the intracellular ATP content (Fig.2). This simplifies the calculation and increases its accuracy.
References. 1*) Jensen et al., 2008. 2**) Kolibab et al., 2012.
Table 2 Determination of the intracellular ATP content (pmol/mg) in different batches of the freeze-dried BCG vaccine. Batch
I II III IV V a b
The ATP content in the resulting extract was then determined by bioluminescence ATP assay. Fig. 1 shows that, for the ATP solutions in 80% DMSO, a strict linear dependence of the bioluminescence signal on the ATP concentration is maintained over a wide range of the ATP concentrations. The resulting procedure is simple and cost-effective. The BCG vaccine is reconstituted with 1 ml saline, incubated for 15 min at room temperature without stirring, and then incubated for 1 h under aerating conditions at 37 °C while stirring. The resulting solution is incubated with 1 U/ml apyrase for 10 min at room temperature after which the apyrase is inactivated and the extracellular ATP is extracted by treating the sample with 100% DMSO at room temperature. Finally, the intracellular ATP content is determined by the bioluminescence ATP assay. The differences between the known methods for intracellular ATP assays in BCG vaccine and the simplified method proposed by us are demonstrated in Table 1.
3.2. Reagent kit for the rapid viability control of BCG vaccines
106 CFU/mg
To simplify the preparation for the analysis and to standardize the procedure, we developed a reagent kit for the viability control of BCG vaccines (see Section 2.2). The kit makes it possible to reduce all the complex preparation procedures to the simple step of reconstituting the freeze-dried reagents in ready-to-use solutions. The use of the freeze-dried ATP-control, which contains the known amount of ATP, simplified the determination of the ATP content in BCG vaccines. The ATP content in the reconstituted ATP control is within the same range of values as the ATP content in reconstituted BCG vaccines. The ATP-control and the vaccine extracts are prepared similarly and contain 80% DMSO. Thereby, we account for the DMSO effect on the ATP-reagent activity.
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Assay 1a
Assay 2
Assay 3
Vial 1b
Vial 2
Vial 3
Vial 4
Vial 5
Vial 6
39.8 ± 1.5 36.9 ± 1.3 38.3 ± 1.0 39.9 ± 1.1 39.7 ± 0.4
36.8 ± 1.3 41.0 ± 0.6 38.6 ± 1.0 42.8 ± 0.2 40.3 ± 2.1
38.0 ± 1.4 41.3 ± 0.6 33.6 ± 2.3 53.7 ± 0.2 40.2 ± 2.2
37.2 ± 1.5 51.7 ± 0.1 37.6 ± 2.1 35.9 ± 0.6 36.2 ± 1.9
– 51.6 ± 0.1 34.1 ± 2.3 44.1 ± 0.6 42.3 ± 1.9
– 32.5 ± 1.8 37.0 ± 2.2 43.6 ± 0.2 42.4 ± 2.9
Assay run on separate days. Means of three measurements for each vial ± SD: standard deviation.
The kit was designed to meet the requirements of control laboratories. In these laboratories, one operator analyzes one batch of a freezedried vaccine per working day. The kit is optimized for this procedure: it is designed for 40 measurements, which is sufficient to measure bioluminescent signals of ATP-control and to characterize 5–6 vials of the analyzed vaccine batch. Thus, all the components of the kit are used in one working day. The use of the kit eliminates the need for time-consuming preparation of the buffer solutions and dilutions of the ATP standard, prevents contamination of the studied samples, and standardizes and simplifies the entire procedure. 3.3. Correlation between the ATP content and CFU level for freeze-dried BCG vaccine preparations To confirm that the simple ATP method for controlling the specific activity of a freeze-dried BCG vaccine, proposed by us, can be used to estimate CFU level in live freeze-dried BCG vaccines, we studied 48 vaccine samples with low and normal specific activity. As standardactivity samples, we used several different production batches of the freeze-dried BCG vaccine manufactured at the Microgen factory; the specific activity of these vaccines was in accordance with the technical specifications for this preparation. As low-activity samples, we used vaccine samples that were stored for different time periods after their expiration date. In all the samples, we determined the intracellular ATP content using the developed ATP assay and measured the CFU values by plate counting. Specific activity measured by plate counting is generally determined with ±15% deviation from the mean value. Fig. 2 shows that there is a strong linear dependence (R = 0.95) between the values of the intracellular ATP and CFU. The slope of the curve (K) is 0.36 ± 0.02. Therefore, we can use simple Formula (3) to calculate the specific activity (106 CFU/mg) from the measured values of the intracellular ATP content:
25
106 CFU=mg ¼ K ATP; pmol=mg
20
Five batches of standard-activity vaccines were used to examine repeatability and intermediate precision of the proposed intracellular ATP assay (Table 2). We analyzed from four to six vials from each batch to determine the intracellular ATP content. The measurements were performed on different working days. The ATP content (pmol/mg) value
Experimental data 15 Fitted Curve 10
95% Confidence Limit
y = 0.36x R² = 0.95
5
95% Prediction Limit
0 0
20 40 ATP, pmol/mg
60
Fig. 2. Linear correlation between the intracellular ATP content (pmol/mg) and the CFU value (106 CFU/mg) with 95% confidence limit and 95% prediction limit. Each point of the experimental data corresponds to a unique vaccine sample and represents an average of 2–3 independent measurements. The total number of samples was 48.
ð3Þ
Table 3 Correlation between the experimental and calculated CFU values in different samples of the freeze-dried BCG vaccine. Batch 106 CFU/mg, experimental
ATP, pmol/mg
ATP/cell, ×10−18 mol
106 CFU/mg calculateda
I II III IV V
38.0 ± 0.9 42.5 ± 6.1 37.1 ± 2.1 43.3 ± 3.8 40.2 ± 2.0
2.6 ± 0.4 2.7 ± 0.4 2.6 ± 0.4 3.4 ± 0.5 2.9 ± 0.4
13.7 ± 1.9 15.3 ± 2.1 13.4 ± 1.9 15.6 ± 2.2 14.5 ± 2.0
14.5 ± 2.2 15.5 ± 2.3 14.2 ± 2.1 12.8 ± 1.9 14.0 ± 2.1
a The calculated CFU values were found from the intracellular ATP content using formula (3): 106 CFU/mg = К × ATP, pmol/mg, where К = 0.36 ± 0.02.
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Sample number
Characteristics of 106 CFU/mg, experimental the vaccine
(ATP), pmol/mg
106 CFU/mg calculated using К =
I
Liquid, before freeze-drying
~60–80
156 ±
56 ± 2.0
The data in Table 4 show that the intracellular ATP content and the specific activity of the liquid vaccine are about two times higher than those of the freeze-dried vaccine. These data demonstrate that, before freeze-drying, the specific activity of the BCG vaccine can be controlled by the intracellular ATP content, which makes it possible to adjust the vaccine composition if necessary.
After freeze-drying Liquid, before freeze-drying
N/A
3.0 73 ± 2.0
26 ± 1.0
4. Conclusion
~60–80
132 ±
48 ± 2.0
After freeze-drying Liquid, before freeze-drying
N/A
3.0 60 ± 1.0
21 ± 1.0
68.5
150 ±
54 ± 2.0
Liquid, before freeze-drying
59
3.0 126 ±
46 ± 1.0
Table 4 Specific activity of the BCG vaccine samples before and after freeze-drying.
0.36
II
III IV
2.0
determined for each vial is the mean value from four to five measurements. This working arrangement was used to estimate if the measured intracellular ATP content depended on the measurement conditions. The results indicate that the proposed ATP method is reproducible and resistant to the conditions of the analysis at different days of operation. Evidently, the mean values of the ATP content for the samples of the same batch are determined with an error of no more than ± 10%. It was only for the samples of the batch II that the measurement deviation was of ±14.4%. Mean values of the ATP content (pmol/mg) in different batches of the freeze-dried BCG vaccine are shown in Table 2. Using these data and the experimental values of CFU, we calculated the average ATP content in one cell of the studied BCG vaccine (Table 3). The resulting values ranged from 2.6 × 10−18 to 3.4 × 10−18 mol ATP per cell for all the studied samples. The mean value was of (2.8 ± 0.2) × 10−18 mol ATP per cell. This value agrees with the literature data on the intracellular ATP content in BCG vaccine cells (Janaszek et al., 1987; Kolibab et al., 2012). According to Kolibab et al. (2012), the ATP content per cell varies in a rather wide range for different strains of BCG vaccine. The high intracellular ATP content in the studied BCG samples indicates that the method that we proposed for the reconstitution and reactivation of the freeze-dried BCG vaccine proved to be very effective. The comparison of the measured and calculated values of the specific activity for different samples of the freeze-dried BCG vaccine shows that these values are similar; their differences fall within the allowable range of deviations for the specific activity values of the BCG vaccine.
3.4. Comparison of specific activities for liquid and freeze-dried vaccine preparations The ATP method developed for the determination of the specific activity of freeze-dried BCG vaccines can be used to determine the activity of a liquid vaccine prepared for the subsequent freeze-drying. We showed this for several vaccine samples. The data are presented in Table 4. In contrast with the freeze-dried vaccine, the liquid vaccine sample required no pretreatment by incubation at 37 °C. The volume of the liquid vaccine in the vials was of 0.2 ml; each vial contained 0.5 mg biomass. Before determining the ATP content, the suspension volume in the vial was adjusted to 1 ml by adding the required volume of saline. This step facilitated the comparison of the ATP content in the vials of liquid and freeze-dried vaccine because the suspension volumes for these vaccines became equal. We measured the intracellular ATP content (ATP, pmol/mg) in the liquid vaccine (Table 4). The specific activity (106 CFU/mg) of the liquid vaccine was determined with standard methods or calculated by Formula (3). The values of the respective parameters for the same vaccine batches were also determined after the freeze-drying.
We proposed a simple and cost-effective ATP method for controlling the specific activity of the freeze-dried BCG vaccine and the liquid BCG vaccine before its freeze-drying. We optimized the conditions for rapid rehydration and reactivation of the freeze-dried BCG cells and modified the conditions for their lysis and for the bioluminescence measurement. We also designed a reagent kit that does not require timeconsuming preparation before the assay. All of this reduced the duration of the analysis from 36 (Jensen et al., 2008) to 2 h and significantly reduced the complexity of the individual stages of the analysis. We showed that the measured intracellular ATP content (АТP, pmol/mg) is directly proportional to the specific activity (106 CFU/mg) (with the proportionality coefficient K) in the range of the manufactured BCG vaccine preparation. The coefficient K was determined and used to calculate the specific activity (106 CFU/mg) of the produced BCG vaccine based on the measured intracellular ATP content (ATP, pmol/mg). Our method has the following advantages: (1) the duration of the entire analysis (including all the preparation steps) was reduced to 2–2.5 h; (2) the use of commercially available and affordable ready-to-use reagents (ATP-reagent, ATP-control, apyrase-reagent, DMSO, saline, and water) simplifies the analytical procedures and reduces their duration thereby decreasing the cost of the analysis; and (3) standardization of the conditions for each stage increases the accuracy and reproducibility of the analysis. The method is characterized by good reproducibility, stability, and the ease of processing the measurement results, which is especially important when the method is implemented in the production environment. Conflicts of interest There are no conflicts of interest. Acknowledgements We thank Federal State Unitary Company “Microgen Scientific Industrial Company for Immunobiological Medicines” of the Ministry of Health of the Russian Federation (1360/15) for financial support of this research. References Askgaard, D.S., Gottschau, A., Knudsen, K., Bennedsen, J., 1995. Firefly luciferase assay of adenosine triphosphate as a tool of quantitation of the viability of BCG vaccines. Biologicals 23 (1), 55–60. Beckers, B., Lang, H.R., Schimke, D., Lammers, A., 1985. Evaluation of a bioluminescence assay for rapid antimicrobial susceptibility testing of mycobacteria. Eur. J. Clin. Microbiol. 4 (6), 556–561. Brandau, S., Suttmann, H., 2007. Thirty years of BCG immunotherapy for non-muscle invasive bladder cancer: a success story with room for improvement. Biomed. Pharmacother. 61 (6), 299–305. Chen, Z.R., Cheng, S.G., An, Y.Q., Cao, X.H., 1989. Experimental research on utilization of bioluminescent technique to substitute the current viability count for BCG vaccine. Chin. Med. J. 102, 906–910. Crispen, R.G., 1971. Rapid testing of freeze dried BCG vaccine for stability and viability. Symp. Ser. Immunobiol. Stand. 17, 205–210. Gheorghiu, M., Lagranderie, M., 1979. Mesure rapide de la viabilité du BCG par dosage de l'ATP. Ann. Microbiol. (Paris) 130B (2), 147–156. Ho, M.M., Markey, K., Rigsby, P., Jensen, S.E., Gairola, S., Seki, M., Castello-Branco, L.R., López-Vidal, Y., Knezevic, I., Corbel, M.J., 2008. Report of an international collaborative study to establish the suitability of using modified ATP assay for viable count of BCG vaccine. Vaccine 26 (36), 4754–4757. Hoffner, S., Jimenez-Misas, C., Lundin, A., 1999. Improved extraction and assay of mycobacterial ATP for rapid drug susceptibility testing. Luminescence 14 (5), 255–261.
N.N. Ugarova et al. / Journal of Microbiological Methods 130 (2016) 48–53 Janaszek, W., Aleksandrowicz, J., Sitkiewicz, D., 1987. The use of the firefly bioluminescent reaction for the rapid detection and counting of mycobacterium BCG. J. Biol. Stand. 15 (1), 11–16. Jensen, S.E., Hubrechts, P., Klein, B.M., Hasløv, K.R., 2008. Development and validation of an ATP method for rapid estimation of viable units in lyophilized BCG Danish 1331 vaccine. Biologicals 36 (5), 308–314. Knezevic, I., Corbel, M.J., 2006. WHO discussion on the improvement of the quality control of BCG vaccines. Paster Institute, Paris, France, 7 June 2005. Vaccine 24 (18), 3874–3877. Koksharov, M.I., Ugarova, N.N., 2011. Thermostabilization of firefly luciferase by in vivo directed evolution. Prot. Eng. Des. Sel. 24 (11), 835–844. Kolibab, K., Derrick, S.C., Jacobs, W.R., Morris, S.L., 2012. Characterization of an intracellular ATP assay for evaluating the viability of live attenuated mycobacterial vaccine preparations. J. Microbiol. Methods 90 (3), 245–249. Lomakina, G.Y., Modestova, Y.A., Ugarova, N.N., 2015. Bioluminescence assay for cell viability. Biochem. Mosc. 80 (6), 701–713. Lundin, A., 2000. Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites. Methods Enzymol. 305, 346–370. Morgan, C.A., Herman, N., White, P.A., Vesey, G., 2006. Preservation of micro-organisms by drying; a review. J. Microbiol. Methods 66 (2), 183–193.
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Prioli, R.P., Tanna, A., Brown, I.N., 1985. Rapid methods for counting mycobacterial adenosine triphosphate (ATP) determined by firefly luciferase assay. Tubercle 66 (2), 99–108. Romanova, N.A., Brovko, L.Y., Ugarova, N.N., 1997. Comparative assessment of methods of intracellular ATP extraction from different types of microorganisms for bioluminescent determination of microbial cells. Appl. Biochem. Microbiol. (Moscow) 33, 306–311. Shi, M.F., Klegerman, M.E., Groves, M.J., 1989. Viability of freeze-dried tice-substrain BCG by bioluminescent measurement of adenosine triphosphate. Microbios 59, 145–155. Ugarova, N.N., 1993. Bioanalytical applications of firefly luciferase. Appl. Biochem. Microbiol. (Moscow) 29, 135–144. Ugarova, N.N., Brovko, L.Y., Trdatian, I.Y., Rainina, E.I., 1987. Bioluminescent methods of analysis in microbiology. Appl. Biochem. Microbiol. (Moscow) 23, 11–20. Ugarova, N.N., Koksharov, M.I., Lomakina, G.Y., 2009. Reagent for determination of adenosine-5′-triphosphate. RF Patent 2420594. WHO, 2004. BCG vaccine. WHO position paper. Wkly, Epidemiol. Rec. 79 (4), 27–38. WHO/TB/Technical guide/77/9, 1977. In Vitro Assay of BCG Products.
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
A simplified ATP method for the rapid control of cell viability in a freeze-dried BCG vaccine Natalia N. Ugarova a,b,⁎, Galina Yu. Lomakina a,b, Yulia Modestova a,b, Sergey V. Chernikov c, Natalia V. Vinokurova c, Elena V. Оtrashevskaya c, Vyacheslav Y. Gorbachev c a b c
Faculty of Chemistry, Lomonosоv Moscow State University, Moscow 119991, Russia LLC Lumtek, Vorobievy Gory 1/77, Moscow 119992, Russia FSUC SIC Microgen, MOH RF, 10, Vtoroi Volkonsky per., Moscow 127473, Russia
a r t i c l e
i n f o
Article history: Received 12 July 2016 Received in revised form 24 August 2016 Accepted 28 August 2016 Available online 29 August 2016 Keywords: ATP assay BCG viability Colony forming units Freeze-dried BCG vaccine Tuberculosis
a b s t r a c t We propose a simple and cost-effective ATP method for controlling the specific activity of a freeze-dried BCG vaccine. A freeze-dried BCG vaccine is reconstituted with 1 ml saline and incubated for 15 min at room temperature and then for 1 h at 37 °C. The vaccine is then treated with apyrase to remove extracellular ATP. After that, the cells are lysed with DMSO and the ATP content in the lysate is measured by the bioluminescence method. To implement the method, we developed a kit that requires no time-consuming preparation before the analysis. We demonstrated the linear relationship between the experimental values of the specific activity (106 CFU/mg) and intracellular ATP content (ATP, pmol/mg) for different batches of the studied BCG vaccines; the proportionality coefficient was К = 0.36 ± 0.02. We proposed a formula for calculating the specific activity from the measured content of intracellular ATP (ATP, pmol/mg). The comparison of the measured and calculated values of the specific activity (106 CFU/mg) shows that these values are similar; their differences fall within the allowable range of deviations for the specific activity values of the BCG vaccine. © 2016 Published by Elsevier B.V.
1. Introduction The vaccine based on an attenuated strain of freeze-dried viable bacteria Mycobacterium bovis (BCG vaccine) is widely used for vaccination against tuberculosis (Ho et al., 2008; Jensen et al., 2008; Knezevic and Corbel, 2006); it is also used as an immuno-therapeutic agent against bladder cancer (Brandau and Suttmann, 2007). Hundreds of millions of the vaccine doses are produced annually in different countries all over the world (WHO, 2004). One of the key characteristics of the BCG vaccine is the specific activity of freeze-dried М. bovis BCG bacteria. The cell viability of the BCG vaccine is traditionally characterized by a number of colony-forming units per 1 ml of the vaccine suspension (CFU/ml) or per 1 mg of biomass (CFU/mg). Cell viability of the vaccine is generally determined by plate counting. The mycobacterial cells grow very slowly, so three to five weeks are usually required to complete the testing of the vaccine samples. Moreover, the CFU assay is variable and often not reproducible because of the difficulties associated with the cultivation of mycobacteria: these organisms are prone to adhesion and aggregation and the composition of their cultivation medium is very complex.
⁎ Corresponding author at: Faculty of Chemistry, Lomonosоv Moscow State University, Leninskie Gory 1/3, Moscow 119991, Russia. E-mail address: [email protected] (N.N. Ugarova).
http://dx.doi.org/10.1016/j.mimet.2016.08.027 0167-7012/© 2016 Published by Elsevier B.V.
In the last decade, an alternative method for the rapid quantitative determination of viable cells in BCG vaccine was proposed and approved by the World Health Organization (WHO). This method is based on the bioluminescence assay of intracellular adenosine-5′-triphosphate (ATP) (Ho et al., 2008; Jensen et al., 2008; Knezevic and Corbel, 2006). The bioluminescence method makes it possible to determine the amount of intracellular ATP rapidly. The intracellular ATP content is the main indicator of cell viability. When a cell dies, the production of ATP stops and the intracellular ATP content rapidly decreases by tens and hundreds of times. The intracellular ATP content is an indicator of the metabolic status of cells; it depends on the nature and size of cells, their energy state, and the external conditions (Lomakina et al., 2015). Bioluminescence ATP assay is based on the ability of the firefly luciferase enzyme to catalyze the oxidation of D-luciferin by oxygen in the presence of a magnesium salt and ATP. The formation of the reaction product, oxyluciferin, is accompanied by the emission of visible light. If the contents of luciferase, D-luciferin, and the magnesium salt in the reaction mixture are fixed, the light intensity is proportional to the ATP content in the analyzed sample. Thus, the viability of cells in the sample can be determined using the bioluminescence method (Ugarova et al., 1987; Ugarova, 1993, Lundin, 2000). Since 1970s, the bioluminescence method has been used by many authors to determine the activity of BCG cells and to study their properties in different systems (Crispen, 1971; Gheorghiu and Lagranderie,
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1979; Beckers et al., 1985; Chen et al., 1989). It was shown that there is a correlation between the intracellular ATP content and the number of viable cells in liquid and freeze-dried preparations of the BCG vaccine (Askgaard et al., 1995; Janaszek et al., 1987; Prioli et al., 1985; Shi et al., 1989). However, it was only in 2008 that the paper was published describing the validation of the ATP method for the rapid assessment of the viability of freeze-dried BCG vaccine preparations (Jensen et al., 2008). In this variant of the method, the BCG vaccine is reconstituted using a full mycobacterial growth medium (the Dubos medium with Tween and bovine albumin) and incubated for 24 h at 37 °C to restore the metabolic activity of the cells and, consequently, increase the intracellular ATP content. The intracellular ATP is then extracted by boiling the vaccine suspension in an EDTA-containing Tris-acetate buffer solution after which the intracellular ATP content is determined by the bioluminescence ATP assay. The cell viability is then estimated by the correlation between the ATP content and the CFU values. The developed method has reduced the total duration of analysis from 30 days to 36 h. The authors showed that the method is characterized by a good reproducibility, robustness, accuracy, linearity, and high sensitivity. On the initiative of the WHO, the developed method was tested in six laboratories in different countries. However, only three laboratories managed to obtain the same results because the conditions, the testing procedures, the reagent base, and the equipment varied somewhat in different laboratories (Ho et al., 2008). Kolibab et al. (2012) later modified the protocol proposed in (Jensen et al., 2008) by eliminating the timeconsuming incubation step: BCG vaccines were reconstituted in diluted Sauton's SSI media and incubated for 30 min in the presence of apyrase at room temperature. The aim of the present study was to develop a simplified ATP method suitable for use in the factory for the rapid control of the specific activity (viability) of the production batches of a freeze-dried BCG vaccine. 2. Materials and methods 2.1. BCG samples We used a BCG vaccine containing viable mycobacteria of a BCG Mycobacterium bovis production strain, substrain BCG-1 (Russia), freezedried in a 1.5% sodium glutamate solution and sealed in evacuated vials. Each vial contained 0.5 mg BCG cells and 3 mg sodium glutamate. In several experiments, a liquid BCG vaccine was assayed before its freeze-drying.
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suspension (sample no. 1 and no. 2). The samples were gently mixed and incubated at room temperature for 10 min without stirring. 2.3.2. ATP extraction and measurement of bioluminescence signal for the resulting extracts The first test tube (sample no. 1) containing the apyrase-treated vaccine suspension was supplied with 1 ml DMSO and mixed. After 1 min of incubation, 100 μl ATP-reagent was added to 20 μl of the ATP extract in DMSO and the bioluminescence signal was measured in a LUM-1 luminometer (Lumtek, Russia). Each measurement was repeated at least twice and the arithmetic mean of the two repeats was calculated. If the difference between the two repeats exceeded 20%, the measurement was performed for a third time. Similarly, the measurements were conducted for the sample no. 2 from the same vaccine vial. The arithmetic mean value of the bioluminescence signal was determined for the analyzed vial (Ivial). 2.3.3. Measurement of bioluminescence signal for the ATP-control and calculation the ATP content (ATP, pmol/mg) Two test tubes were supplied with the mixture of 100 μl of the reconstituted ATP-control solution, 20 μl saline, and 1 ml DMSO. The resulting solution was mixed and the bioluminescence signal for the ATP-control was measured as described above for the BCG vaccine. The arithmetic mean value of the bioluminescence signal for the ATPcontrol (Icontrol) was calculated; the value was then used to calculate the intracellular ATP content in a sample of the dry BCG vaccine using Formula (1). ðATP Þ; pmol ¼ ðATP Þcontrol
I vial ; pmol I control
ð1Þ
Here, (ATP)control is the content of the ATP-control in the initial saline solution, pmol; Icontrol is the mean value of the bioluminescence signal for the ATP-control; and Ivial is the mean value of the bioluminescence signal for the vial. The intracellular ATP content in 1 mg of the BCG vaccine was calculated by Formula (2) using the obtained value of the ATP content in the vial (pmol) and the mass of BCG cells in the vial (0.5 mg): ðATP Þ; pmol=mg ¼
ðATP Þ; pmol BCG mass; mg
ð2Þ
2.2. Analytical reagents
2.4. Determination of CFU
For this study, we developed a reagent kit based on the ATP-reagent (Lumtek, Russia). This kit contained freeze-dried ATP-reagent, ATP-control and apyrase; liquid sterile deionized water; saline (0.9% NaCl in water); and dimethylsulfoxide (DMSO). The ATP-reagent (Ugarova et al., 2009) comprised of the thermostable firefly luciferase Luciola mingrelica (Koksharov and Ugarova, 2011), luciferin, buffer components, and stabilizers was reconstituted with 4 ml of deionized water 1 h before the experiment. The ATP-control, 10 pmol, was reconstituted in 1 ml saline. The apyrase was also reconstituted in saline; the resulting solution contained 5 U/ml apyrase. The reconstituted solutions were stored at room temperature and used within one working day.
Six BCG vials were reconstituted in saline to a concentration of 0.5 mg BCG/ml. The BCG suspensions from the vials were pooled and diluted in saline. Three serial dilutions of the suspension were inoculated onto Löwenstein Jensen medium and incubated at 37 °C for four weeks before plate counting and calculating the number of CFU (106 CFU/mg) (WHO, 1977).
2.3. Determination of intracellular ATP 2.3.1. Reconstitution of freeze-dried BCG vaccine and degradation of extracellular ATP A vial containing freeze-dried vaccine was reconstituted with 1 ml saline, incubated for 15 min at room temperature without stirring and then for 60 min at 37 °C with agitation (250 rpm). Then, the vial was cooled in air to room temperature for 3–5 min. Two test tubes were supplied with 20 μl of apyrase solution and with 100 μl of the vaccine
2.5. Statistical analysis All measurements were performed in triplicate. Mean (X) and standard deviation (SD) for the bioluminescent signal and the ATP content were calculated using Excel software (Microsoft℗ Office Excel 2003 SPI). The ATP content was calculated and expressed as pmol ATP per vial or pmol ATP per 1 mg of BCG cells. 3. Results and discussion 3.1. Optimization of the intracellular ATP bioluminescence assay The conditions for the reconstitution and reactivation of a freezedried cell culture play a significant role in the correct determination of
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Luminescent signal, 103 RLU/s
50
Table 1 Comparison of different methods for intracellular ATP assays in BCG vaccine.
160
Literature data
Our method
120 y = 0.4401x R² = 0.9986
80 40 0 0
100
200
300
400
[ATP], pmol/ml Fig. 1. Linear correlation between the ATP concentration and bioluminescent signal for the ATP solutions in DMSO.
the intracellular ATP content. The viability of a freeze-dried bacterial preparation greatly depends on the reconstitution conditions (Morgan et al., 2006): the reconstitution solution, incubation time, temperature, etc. Two types of standard solutions are generally used to reconstitute BCG vaccines prior to the injection: diluted Sauton SSI and saline. According to protocols described in the literature (Jensen et al., 2008; Kolibab et al., 2012), the diluted Sauton SSI is used for reconstitution of a freeze-dried BCG vaccine. In our studies, we adjusted the reconstitution procedure to the saline-based protocol for the preparation of BCG vaccine suspension. Our preliminary experiments demonstrated that fluctuations of the incubation temperature cause discrepancies in the experimental results. Therefore, we chose the incubation temperature of 37 °C: determination of cell viability by plate counting is conducted under the same conditions and this temperature is easy to maintain. By measuring the intracellular ATP content during the incubation of reconstituted BCG vaccines at 37 °C, we found that the steady-state concentration of intracellular ATP is maintained within 30 to 120 min of incubation after which the ATP content starts to fluctuate. We selected the incubation period of 60 min as the standard condition for our experimental procedure. During freeze-drying, some bacterial cells get disrupted: consequently, their intracellular ATP enters the extracellular space. As the result, the total ATP content in the analyzed sample of the reconstituted cells can exceed the intracellular ATP content, the value of which characterizes the viability of the vaccine (Morgan et al., 2006). To solve this problem, the reconstituted samples are pretreated with apyrase, an ATP-hydrolyzing enzyme, before the extraction of intracellular ATP. In the protocol developed in (Kolibab et al., 2012), a BCG vaccine was reconstituted in diluted Sauton SSI medium and simultaneously treated with 0.5 U/ml apyrase with a subsequent 30-min incubation. To prevent the inactivation of apyrase and optimize the duration of the ATP depletion stage, we separated the reconstitution step and the step of the extracellular ATP depletion: the reconstituted BCG samples were treated with 1 U/ml apyrase at room temperature. We showed that the extracellular ATP content decreased by 98% in 10 min; extending the period of incubation of the apyrase–sample mixture provided no further decrease in the extracellular ATP content. The next step is the extraction of the intracellular ATP. Generally, ATP is extracted from the BCG cells by boiling them in a preheated EDTA-containing Tris-acetate buffer for 6 min (Prioli et al., 1985; Hoffner et al., 1999; Jensen et al., 2008; Kolibab et al., 2012). We simplified the ATP extraction procedure by using sterile DMSO as described in our previous studies (Ugarova et al., 1987; Ugarova, 1993; Romanova et al., 1997). We found that a one-minute incubation of BCG samples in 80% DMSO (room temperature) is sufficient to complete the extraction. We also demonstrated that the apyrase used at the previous step is irreversibly inactivated under these conditions.
Reconstitution of lyophilized BCG vaccine 1*): A freeze-dried A freeze-dried vaccine is vaccine is reconstituted reconstituted with 1 ml with 1 ml Dubos saline, incubated for 15 medium and incubated min at room temperature for 24 h at 37 °C. without stirring and then 2**): A freeze-dried for 60 min at 37 °C with vaccine is reconstituted agitation (250 rpm). The with 1 ml of diluted vial is then cooled in air to Sauton SSI media room temperature for containing 0.5 units of 3–5 min. apyrase and incubated for 30 min.
Degradation of extracellular ATP 1: The protocol for the In a test tube, 100 μl removal of extracellular vaccine suspension is ATP after the incubation gently mixed with 20 μl of with Dubos medium is apyrase solution and not provided. incubated at room 2: See above. temperature for 10 min without stirring. Extraction of intracellular ATP 1 and 2: BCG suspension The apyrase-treated is mixed with vaccine suspension is Tris-acetate buffer supplied with 1 ml DMSO, containing EDTA mixed, and incubated for (98–99 °C), boiled for 6 1 min at room temperature. min, and allowed to cool to room temperature. Measurement of ATP 1: 100 μl ATP reagent is added to 600 μl of cooled ATP extract; the bioluminescence signal is recorded (Ivial, RLU). 2: 100 μl ATP reagent is added to the sample; the bioluminescence signal is recorded (Ivial, RLU).
Advantages of our method This stage is the most important for obtaining correct and reproducible results. The use of saline solution for the reconstruction of a freeze-dried BCG vaccine and the precise control of the incubation duration and temperature standardizes the conditions and simplifies the preparation of the working samples.
A ready-to-use apyrase preparation provided in the kit reduces the labor intensity and duration of the analysis.
Using a highly efficient organic extractant, we extract ATP at room temperature. This reduces the duration and labor intensity of the ATP extraction and improves the reproducibility of the method.
100 μl ATP-reagent is added to 20 μl of the ATP extract in DMSO; the bioluminescence signal is recorded (Ivial, RLU). High operational stability of the ATP-reagent provides good reproducibility of the measurements.
Determination of the ATP content in BCG samples and the use of ATP standard 1 and 2: The ATP standard Freeze-dried ATP-control The linear relationship between the curve is obtained using is reconstituted with bioluminescence signal several dilutions of ATP saline and then treated (I) and the concentration Standard in Tris-acetate with DMSO as the BCG of ATP (Fig.1) eliminates buffer with EDTA. The vaccine. The amount of ATP bioluminescence signal is the need to obtain a calibration curve. extracted from BCG measured (Icontrol, RLU) as described above for the The calculation of samples is calculated vaccine. intracellular ATP content from the ATP standard The amount of in dry BCG vaccine is curve. intracellular ATP content simplified. in dry BCG vaccine is calculated by Formula (ATP), pmol = (ATP)control ∙ Ivial/Icontrol, pmol (ATP)control is the content of the ATP-control in the initial saline solution, pmol. Calculation of the specific activity (106 CFU/mg) from the measured values of the intracellular ATP content Calculation of the specific Using different BCG 1: Calculation of the activity: 106 CFU/mg = K vaccine batches, we specific activity: demonstrated that the 106 CFU/vial = 0.037 (ng · ATP, pmol/mg ATP/vial)1.398 K is the slope of the linear linear dependence exists
N.N. Ugarova et al. / Journal of Microbiological Methods 130 (2016) 48–53 Table 1 (continued) Literature data
Our method
Advantages of our method
2: No information
correlation between the intracellular ATP content and the CFU.
between the CFU values and the intracellular ATP content (Fig.2). This simplifies the calculation and increases its accuracy.
References. 1*) Jensen et al., 2008. 2**) Kolibab et al., 2012.
Table 2 Determination of the intracellular ATP content (pmol/mg) in different batches of the freeze-dried BCG vaccine. Batch
I II III IV V a b
The ATP content in the resulting extract was then determined by bioluminescence ATP assay. Fig. 1 shows that, for the ATP solutions in 80% DMSO, a strict linear dependence of the bioluminescence signal on the ATP concentration is maintained over a wide range of the ATP concentrations. The resulting procedure is simple and cost-effective. The BCG vaccine is reconstituted with 1 ml saline, incubated for 15 min at room temperature without stirring, and then incubated for 1 h under aerating conditions at 37 °C while stirring. The resulting solution is incubated with 1 U/ml apyrase for 10 min at room temperature after which the apyrase is inactivated and the extracellular ATP is extracted by treating the sample with 100% DMSO at room temperature. Finally, the intracellular ATP content is determined by the bioluminescence ATP assay. The differences between the known methods for intracellular ATP assays in BCG vaccine and the simplified method proposed by us are demonstrated in Table 1.
3.2. Reagent kit for the rapid viability control of BCG vaccines
106 CFU/mg
To simplify the preparation for the analysis and to standardize the procedure, we developed a reagent kit for the viability control of BCG vaccines (see Section 2.2). The kit makes it possible to reduce all the complex preparation procedures to the simple step of reconstituting the freeze-dried reagents in ready-to-use solutions. The use of the freeze-dried ATP-control, which contains the known amount of ATP, simplified the determination of the ATP content in BCG vaccines. The ATP content in the reconstituted ATP control is within the same range of values as the ATP content in reconstituted BCG vaccines. The ATP-control and the vaccine extracts are prepared similarly and contain 80% DMSO. Thereby, we account for the DMSO effect on the ATP-reagent activity.
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Assay 1a
Assay 2
Assay 3
Vial 1b
Vial 2
Vial 3
Vial 4
Vial 5
Vial 6
39.8 ± 1.5 36.9 ± 1.3 38.3 ± 1.0 39.9 ± 1.1 39.7 ± 0.4
36.8 ± 1.3 41.0 ± 0.6 38.6 ± 1.0 42.8 ± 0.2 40.3 ± 2.1
38.0 ± 1.4 41.3 ± 0.6 33.6 ± 2.3 53.7 ± 0.2 40.2 ± 2.2
37.2 ± 1.5 51.7 ± 0.1 37.6 ± 2.1 35.9 ± 0.6 36.2 ± 1.9
– 51.6 ± 0.1 34.1 ± 2.3 44.1 ± 0.6 42.3 ± 1.9
– 32.5 ± 1.8 37.0 ± 2.2 43.6 ± 0.2 42.4 ± 2.9
Assay run on separate days. Means of three measurements for each vial ± SD: standard deviation.
The kit was designed to meet the requirements of control laboratories. In these laboratories, one operator analyzes one batch of a freezedried vaccine per working day. The kit is optimized for this procedure: it is designed for 40 measurements, which is sufficient to measure bioluminescent signals of ATP-control and to characterize 5–6 vials of the analyzed vaccine batch. Thus, all the components of the kit are used in one working day. The use of the kit eliminates the need for time-consuming preparation of the buffer solutions and dilutions of the ATP standard, prevents contamination of the studied samples, and standardizes and simplifies the entire procedure. 3.3. Correlation between the ATP content and CFU level for freeze-dried BCG vaccine preparations To confirm that the simple ATP method for controlling the specific activity of a freeze-dried BCG vaccine, proposed by us, can be used to estimate CFU level in live freeze-dried BCG vaccines, we studied 48 vaccine samples with low and normal specific activity. As standardactivity samples, we used several different production batches of the freeze-dried BCG vaccine manufactured at the Microgen factory; the specific activity of these vaccines was in accordance with the technical specifications for this preparation. As low-activity samples, we used vaccine samples that were stored for different time periods after their expiration date. In all the samples, we determined the intracellular ATP content using the developed ATP assay and measured the CFU values by plate counting. Specific activity measured by plate counting is generally determined with ±15% deviation from the mean value. Fig. 2 shows that there is a strong linear dependence (R = 0.95) between the values of the intracellular ATP and CFU. The slope of the curve (K) is 0.36 ± 0.02. Therefore, we can use simple Formula (3) to calculate the specific activity (106 CFU/mg) from the measured values of the intracellular ATP content:
25
106 CFU=mg ¼ K ATP; pmol=mg
20
Five batches of standard-activity vaccines were used to examine repeatability and intermediate precision of the proposed intracellular ATP assay (Table 2). We analyzed from four to six vials from each batch to determine the intracellular ATP content. The measurements were performed on different working days. The ATP content (pmol/mg) value
Experimental data 15 Fitted Curve 10
95% Confidence Limit
y = 0.36x R² = 0.95
5
95% Prediction Limit
0 0
20 40 ATP, pmol/mg
60
Fig. 2. Linear correlation between the intracellular ATP content (pmol/mg) and the CFU value (106 CFU/mg) with 95% confidence limit and 95% prediction limit. Each point of the experimental data corresponds to a unique vaccine sample and represents an average of 2–3 independent measurements. The total number of samples was 48.
ð3Þ
Table 3 Correlation between the experimental and calculated CFU values in different samples of the freeze-dried BCG vaccine. Batch 106 CFU/mg, experimental
ATP, pmol/mg
ATP/cell, ×10−18 mol
106 CFU/mg calculateda
I II III IV V
38.0 ± 0.9 42.5 ± 6.1 37.1 ± 2.1 43.3 ± 3.8 40.2 ± 2.0
2.6 ± 0.4 2.7 ± 0.4 2.6 ± 0.4 3.4 ± 0.5 2.9 ± 0.4
13.7 ± 1.9 15.3 ± 2.1 13.4 ± 1.9 15.6 ± 2.2 14.5 ± 2.0
14.5 ± 2.2 15.5 ± 2.3 14.2 ± 2.1 12.8 ± 1.9 14.0 ± 2.1
a The calculated CFU values were found from the intracellular ATP content using formula (3): 106 CFU/mg = К × ATP, pmol/mg, where К = 0.36 ± 0.02.
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Sample number
Characteristics of 106 CFU/mg, experimental the vaccine
(ATP), pmol/mg
106 CFU/mg calculated using К =
I
Liquid, before freeze-drying
~60–80
156 ±
56 ± 2.0
The data in Table 4 show that the intracellular ATP content and the specific activity of the liquid vaccine are about two times higher than those of the freeze-dried vaccine. These data demonstrate that, before freeze-drying, the specific activity of the BCG vaccine can be controlled by the intracellular ATP content, which makes it possible to adjust the vaccine composition if necessary.
After freeze-drying Liquid, before freeze-drying
N/A
3.0 73 ± 2.0
26 ± 1.0
4. Conclusion
~60–80
132 ±
48 ± 2.0
After freeze-drying Liquid, before freeze-drying
N/A
3.0 60 ± 1.0
21 ± 1.0
68.5
150 ±
54 ± 2.0
Liquid, before freeze-drying
59
3.0 126 ±
46 ± 1.0
Table 4 Specific activity of the BCG vaccine samples before and after freeze-drying.
0.36
II
III IV
2.0
determined for each vial is the mean value from four to five measurements. This working arrangement was used to estimate if the measured intracellular ATP content depended on the measurement conditions. The results indicate that the proposed ATP method is reproducible and resistant to the conditions of the analysis at different days of operation. Evidently, the mean values of the ATP content for the samples of the same batch are determined with an error of no more than ± 10%. It was only for the samples of the batch II that the measurement deviation was of ±14.4%. Mean values of the ATP content (pmol/mg) in different batches of the freeze-dried BCG vaccine are shown in Table 2. Using these data and the experimental values of CFU, we calculated the average ATP content in one cell of the studied BCG vaccine (Table 3). The resulting values ranged from 2.6 × 10−18 to 3.4 × 10−18 mol ATP per cell for all the studied samples. The mean value was of (2.8 ± 0.2) × 10−18 mol ATP per cell. This value agrees with the literature data on the intracellular ATP content in BCG vaccine cells (Janaszek et al., 1987; Kolibab et al., 2012). According to Kolibab et al. (2012), the ATP content per cell varies in a rather wide range for different strains of BCG vaccine. The high intracellular ATP content in the studied BCG samples indicates that the method that we proposed for the reconstitution and reactivation of the freeze-dried BCG vaccine proved to be very effective. The comparison of the measured and calculated values of the specific activity for different samples of the freeze-dried BCG vaccine shows that these values are similar; their differences fall within the allowable range of deviations for the specific activity values of the BCG vaccine.
3.4. Comparison of specific activities for liquid and freeze-dried vaccine preparations The ATP method developed for the determination of the specific activity of freeze-dried BCG vaccines can be used to determine the activity of a liquid vaccine prepared for the subsequent freeze-drying. We showed this for several vaccine samples. The data are presented in Table 4. In contrast with the freeze-dried vaccine, the liquid vaccine sample required no pretreatment by incubation at 37 °C. The volume of the liquid vaccine in the vials was of 0.2 ml; each vial contained 0.5 mg biomass. Before determining the ATP content, the suspension volume in the vial was adjusted to 1 ml by adding the required volume of saline. This step facilitated the comparison of the ATP content in the vials of liquid and freeze-dried vaccine because the suspension volumes for these vaccines became equal. We measured the intracellular ATP content (ATP, pmol/mg) in the liquid vaccine (Table 4). The specific activity (106 CFU/mg) of the liquid vaccine was determined with standard methods or calculated by Formula (3). The values of the respective parameters for the same vaccine batches were also determined after the freeze-drying.
We proposed a simple and cost-effective ATP method for controlling the specific activity of the freeze-dried BCG vaccine and the liquid BCG vaccine before its freeze-drying. We optimized the conditions for rapid rehydration and reactivation of the freeze-dried BCG cells and modified the conditions for their lysis and for the bioluminescence measurement. We also designed a reagent kit that does not require timeconsuming preparation before the assay. All of this reduced the duration of the analysis from 36 (Jensen et al., 2008) to 2 h and significantly reduced the complexity of the individual stages of the analysis. We showed that the measured intracellular ATP content (АТP, pmol/mg) is directly proportional to the specific activity (106 CFU/mg) (with the proportionality coefficient K) in the range of the manufactured BCG vaccine preparation. The coefficient K was determined and used to calculate the specific activity (106 CFU/mg) of the produced BCG vaccine based on the measured intracellular ATP content (ATP, pmol/mg). Our method has the following advantages: (1) the duration of the entire analysis (including all the preparation steps) was reduced to 2–2.5 h; (2) the use of commercially available and affordable ready-to-use reagents (ATP-reagent, ATP-control, apyrase-reagent, DMSO, saline, and water) simplifies the analytical procedures and reduces their duration thereby decreasing the cost of the analysis; and (3) standardization of the conditions for each stage increases the accuracy and reproducibility of the analysis. The method is characterized by good reproducibility, stability, and the ease of processing the measurement results, which is especially important when the method is implemented in the production environment. Conflicts of interest There are no conflicts of interest. Acknowledgements We thank Federal State Unitary Company “Microgen Scientific Industrial Company for Immunobiological Medicines” of the Ministry of Health of the Russian Federation (1360/15) for financial support of this research. References Askgaard, D.S., Gottschau, A., Knudsen, K., Bennedsen, J., 1995. Firefly luciferase assay of adenosine triphosphate as a tool of quantitation of the viability of BCG vaccines. Biologicals 23 (1), 55–60. Beckers, B., Lang, H.R., Schimke, D., Lammers, A., 1985. Evaluation of a bioluminescence assay for rapid antimicrobial susceptibility testing of mycobacteria. Eur. J. Clin. Microbiol. 4 (6), 556–561. Brandau, S., Suttmann, H., 2007. Thirty years of BCG immunotherapy for non-muscle invasive bladder cancer: a success story with room for improvement. Biomed. Pharmacother. 61 (6), 299–305. Chen, Z.R., Cheng, S.G., An, Y.Q., Cao, X.H., 1989. Experimental research on utilization of bioluminescent technique to substitute the current viability count for BCG vaccine. Chin. Med. J. 102, 906–910. Crispen, R.G., 1971. Rapid testing of freeze dried BCG vaccine for stability and viability. Symp. Ser. Immunobiol. Stand. 17, 205–210. Gheorghiu, M., Lagranderie, M., 1979. Mesure rapide de la viabilité du BCG par dosage de l'ATP. Ann. Microbiol. (Paris) 130B (2), 147–156. Ho, M.M., Markey, K., Rigsby, P., Jensen, S.E., Gairola, S., Seki, M., Castello-Branco, L.R., López-Vidal, Y., Knezevic, I., Corbel, M.J., 2008. Report of an international collaborative study to establish the suitability of using modified ATP assay for viable count of BCG vaccine. Vaccine 26 (36), 4754–4757. Hoffner, S., Jimenez-Misas, C., Lundin, A., 1999. Improved extraction and assay of mycobacterial ATP for rapid drug susceptibility testing. Luminescence 14 (5), 255–261.
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