Evaluation of the polarographic technique for assay of the viability of freeze-dried BCG vaccine: I. The polarographic technique

Vaccine, Vol. 13, No. 3, pp. X9-272, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 026441ow95 $10.00 + 0.00

Evaluation of the polarographic technique for assay of the viability of freeze-dried BCG vaccine: I. The polarographic technique Maria Ivette Carboni Malucelli”, Rinaldo Niero, Pedro Hklio Lucchiari and Metry Bacila In this paper, a new assay based on oxygen uptake assessed by polarography was evaluated with the aim of establishing the viability of freeze-dried BCG vaccine. An oxygen electrode possessing a temperature sensor was designed for this purpose. The polarographic method used had several advantages, particularly its rapidity and use of small amounts of biological material. These advantages are ideal for quality control qf BCG vaccine. Keywords: BCG viability; polarographic methodology; oxygen electrode

Various techniques have been used for the in vitro quality control of BCG vaccine (see Refs 1 and 2 for review). These include assays which estimate: (1) the capacity for germination; (2) the number of culturable particles; (3) the cell ATP content; or (4) the rate of oxygen consumption. Such techniques are currently employed in order to establish the viability of BCG vaccine and as control methods during release of the vaccine for public health use. This paper describes a very sensitive oxygen electrode incorporating a temperature sensor3m5, which has been employed for assaying BCG vaccine viability. The design of this instrument and the electronic circuit were based on models described previously6-“. This methodology was shown to be reliable and much less time- and material-consuming than the classical Warburg respirometric technique. The latter is officially recommended by the World Health Organization (WHO) for the quality control of BCG vaccine. MATERIALS

AND METHODS

Batches of freeze-dried intradermic BCG vaccine produced by the Instituto Butantan, Sao Paulo, Brazil, according to the methods described by WH0’3,‘4, were used in this research. For reconstitution of the vaccine, the following solutions were tried as diluents: (i) Sauton’s solution (0.5 g MgSO,; 0.5 g K,HPO,; 0.05 g ferric ammonium citrate; 2.0 g citric acid; 4.0 g asparagine; 60.0 ml pure *To whom correspondence should be addressed at: lnstituto Butantan, Divisgo de Desenvolvimento Tecnol6gico e Produ$io, Avenida Vital Brazil 1500, CEP-O5503-900 SBo Paulo, Brazil. (Received 26 January 1994; revised 20 May 1994; accepted 23 May 1994) This article forms part of the PhD thesis of M.I.C. Malucelli

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glycerin; up to 1000 ml distilled water; pH 7.25); (ii) 2% sodium glutamate solution; and (iii) buffered saline solution containing 6.8 g NaCl, 1.48 g Na,HPO,, 0.43 g KH,PO, in distilled water up to lOOOm1,pH 7.2. All buffered saline solutions contained Tween-80 at concentrations of 0.1,0.2 or 0.3%. All solutions were previously sterilized by autoclaving for 15 min at 120°C. They were also tested for their possible effects on BCG respiratory activity. Solution (iii) was selected for the experiments. Polarographic assays of oxygen uptake by BCG vaccine preparations were carried out by means of a specially designed instrument (Figures 1-3). The polarographic circuit used was that described by Lucchiari et al.“.

Two main features of the polarograph used in this experiment were the oxygen electrode combined with a temperature sensor (Figure I) and the reaction chamber (Figure 2). Both the outlets of the digital thermometer and the polarographic circuit were connected to a two-channel model ECB-RB 202 potentiometric recorder (Equipamentos Cientificos do Brasil), thus allowing simultaneous recording of the oxygen concentration and the temperature in the reaction chamber. The polarographic circuit together with the digital thermometer and the respective power supplies were assembled on an aluminium chassis (21 cm wide x 11 cm high) connected to a magnetic stirrer driver by means of a 9 V Oxford microengine at 2000 rev min- ‘. The motor pulley (6.2 mm diameter) sets in motion another pulley (44mm diameter) through a rubber belt. A 24 x 14 x 6 mm magnet is fixed on the large pulley, allowing a speed for the magnetic bar of 280 rev min- i. The technical procedure for determination of oxygen uptake by BCG vaccine was carried out according to the following steps. The reaction chamber was cleaned with a neutral detergent (3% Extran) under stirring and then

Polarographic

thoroughly rinsed with distilled water. The reaction chamber was then filled with 70” GL ethanol for 15 min followed by a rinsing step with sterile distilled water. After the BCG vaccine assays, the reaction chamber was thoroughly cleaned with a 10% sodium hypochloride

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solution and rinsed with distilled water. Before any assay, the reaction chamber was always thoroughly rinsed with sterile buffered saline solution. After this step, 1.5 ml of the same solution was transferred to the reaction chamber allowing the whole system to reach a steady state; adjustments to 0 and 100% oxygen saturation were carried out. For vaccine reconstitution, care was taken in opening each vial in order to avoid aerosols and foam formation. By means of a sterile syringe, 0.5 ml of diluent was transferred to each vial containing 5 mg freeze-dried BCG, and gently shaken. The suspension was transferred to another vial containing another batch of 5 mg of freeze-dried vaccine. Complete suspensions of 10 mg of freeze-dried vaccine were always used for assaying the oxygen uptake, and were transferred to the reaction chamber through the access hole (FQurr 2) by means of a syringe. Protein assays were carried out by the Lowry method’ 5. RESULTS

Silver

Ring<

\ 1

) Platinum

Wire

Of the different solutions used for BCG vaccine reconstitution, the best results were obtained with buffered saline solution containing 0.3% Tween-80. In contrast, Sauton’s medium presented several disadvantages, producing large particles and being liable to contamination. Standardization

Figure 1 Schematic view of the oxygen electrode combined with a sensor for temperature. A 0.5 mm diameter platinum wire was sealed into soft glass tubing (4 mm diameter). A 99.95% pure silver ring was sealed around the tip of the tubing in which the platinum wire had been sealed. Immediately above the silver ring, a diode 1N 914 was fixed and used as a temperature sensor. Copper wires were soldered to the platinum wire and the silver ring and used to establish outside connections. The whole system was sealed inside acrylic tubing with epoxy resin (Dexter Corporation). After hardening of the resin, the electrode was carefully polished with water sandpaper (No. 600) and then with a flannel. Finally, a dialysis membrane (Sigma’s Dialysis ‘Sacks’) was used to cover the electrode and tied with a nylon thread

of the assay procedure

Figure 4 shows the results of a polarographic determination of oxygen uptake by BCG vaccine. It can be seen that the fall in the polarization current due to oxygen uptake from the reaction medium occurs immediately after transfer of the BCG suspension to the reaction chamber. Thus, knowing that the oxygen concentration in an air-saturated aqueous solution corresponds to 209 PM O2 l-r, one can estimate the oxygen uptake rate in nmol0, per second (nmol0, s- ‘). The correlation between the protein concentration in the BCG vaccine and the rate of oxygen uptake (Table I) was shown to be directly proportional in the range of l-20 mg of vaccine (Figure 5). The linear regression for the correlation between the rate of oxygen uptake and

Figure 2 Schematic view of the reaction chamber used for the assay of oxygen uptake by BCG vaccine: (A, 8) inlet and outlet of circulating water; (C) magnetic bar; (D) outlet for cleaning the reaction chamber by means of a vacuum pump; (E) insertion hole for the polarographic oxygen and temperature sensor; (F) access hole for introduction of components of the reaction system; (G) main body; (H) cover; (I) rubber rings for sealing; (J) reaction chamber (1.5 cm diameter by 2.0cm height), built in acrylic and fixed into the main body. The whole instrument is made in acrylic. Outlets A and B are connected to a circulating water bath for which the temperature could be monitored with a sensitivity of O.l”C

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Figure 4 Polarographic determination of the rate of oxygen uptake by BCG vaccine. The system involved 1.5ml of a sterile buffered saline solution (containing 174pmol NaCI, 15pmol Na,HP04, 0.45pmol KPHPOb, pH 7.2) and 10 mg of freeze-dried BCG vaccine suspended in 0.5 ml of the same sterile buffered saline solution containing 0.3% Tween-80. The experiment was carried out at 37°C and the rate of oxygen uptake was calculated as being equal to 209 x 10W3 Vr,/180 A 1. The rate of oxygen uptake is l.lGlVrq/At expressed in nmol 02s’. V, is the volume of fluid; q is the oxygen consumed during the period of time considered; 209 nmol O2 ml-’ is the solubility of oxygen at 37°C; A is the amplitude in cm between 0 and 100% of oxygen; and t is the distance in cm projected by deflection of the curve

Table 1 Values of oxygen uptake (nmol O2 s’) of freeze-dried vaccine (batch l.B. 36L38) assayed by polarography

System*

Freeze-dried BCG vaccine (mg)

1 2 3 4 5 6 7 8

1 2 3 5 10 20 50 100

BCG

Protein content (mg ml-‘)

Oxygen uptake (nmol0, s’)

0.12

0.0194 0.0240 0.0333 0.0616 0.1054 0.2177 0.3762 0.5075

0.21 0.36 0.56 0.87

*I.5 ml sterile buffered saline and l-100 mg freeze-dried BCG vaccine suspended in 0.5 ml buffered saline containing 0.3 ml Tween 80 at 37°C

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vaccine concentration gave values of a =0.0048 and b = 0.0105, r = 0.9987. The correlation value for protein concentration and oxygen uptake gave r=0.9859, a = - 0.0239 and b = 0.2626. Table 2 shows the results of an experiment designed to study the reproducibility of the polarographic method used. According to variance analysis and Student’s t test, no significant statistical differences were found in assays of the same batch, at a level of x=0.05. DISCUSSION The development of a specific methodology based on an oxygen electrode for assay of oxygen uptake by freeze-dried BCG vaccine is described in this paper. This

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et al.

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Table 2 Reproducibility of the polarographic assay of oxygen uptake for different batches of BCG vaccine. The system involved 10 mg of freeze-dried BCG vaccine suspended in 0.5 ml of buffered saline solution containing 0.3% Tween-80 at 37°C

Batch number

Number of tests/batch

09/89

4 10 4 7 7 7 4 7 6 11 14 17

Ill89 12189 13189 15189 17189 24189 25189 29189 31189 32189 87107124

Oxygen uptake (nmol O2 s-’

per 10 mg)

0.0904 0.0995

0.2034 0.1425 0.0789 0.0944 0.0898 0.1034 0.1089 0.1152 0.1136 0.1047

Standard deviation

Variability coefficient (%)

0.0055 0.0099 0.0234 0.0106 0.0047 0.0083 0.0090 0.0163 0.0173 0.0173 0.0212 0.0151

6.1 9.9 11.5 7.0 5.9 8.8 10.0 15.7 15.8 15.0 18.7 14.4

performed with 12 batches of BCG vaccine produced at the Butantan Institute (Table 2). The variability coefficient indicated that the values were much more reliable than those obtained by the classical Warburg technique. Thus, it can be concluded that the polarographic method is very reliable and convenient for BCG quality control. It gives rapid results with the use of a minimum amount of biological material. The use of polarography could also be very important in standardization and quality control during production of the vaccine and control before any immunization programme. REFERENCES 1

2

0

50

20 VACCINE

100 (mgl CONCENTRATION

Figure 5 Polarographic determination of oxygen uptake by freeze-dried BCG vaccine with respect to its concentration

may improve the quality control of BCG vaccine production and of BCG batches. One of the main features of this instrument is the fact that it possesses a platinum electrode and that the reference electrode (Ag-AgCl) is embodied in a single unit combined with a temperature sensor which allows control of temperature variations in the reaction chamber. Furthermore, the electronic circuit is very easily assembled. The polarographic assay of the oxygen uptake rate, given in nmol O2 s- ‘, was shown to be sensitive to as little as 1 mg of BCG and was linear up to 20 mg (Table I), with correlation coefficients between the oxygen uptake rate and the concentrations of BCG or protein of 0.9987 and 0.9859, respectively. Buffered saline solution containing 0.3 % Tween-80 gave the best results in our experiments. One of its main features was that the Tween-80 contributed to production of a very homogeneous suspension with no pellet formation. This methodology gave reproducible results in assays

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World Health Organization. In vitro assays of BCG products. WHO/TB Technical Guide 77.9, 1977, unpublished working document Milstien, J.B. and Gibson, J.J. Quality control of BCG vaccine by the World Health Organization: a review of factors that may influence vaccine effectiveness and safety. Bull. World Health Organ. 1990, 88(l), 93-108 Malucelli, M.I.C. 0 metodo polarografico na determinacao da viabilidade da vacina BCG liofilizada, PhD thesis, Faculdade de Satide Publica da Universidade de 6&o Paula, Brazil, 1991 Malucelli, M.I.C., Niero, R., Lucchiari, P.H. and Bacila, M. Posible utilizaci6n del metodo polarografico, en la determinacidn de la viabilidad de la vacuna BCG liofilizada. In: Proceedings of the 24th Congreso Panamericano de la Union Latinoamericana de Sociedades de Tfsiofogia (ULAST), Buenos Aires, Argentina, 7990, p. 20 Malucelli, M.I.C., Niero, R., Lucchiari, P.H., Bacila, M., Souza, M.D.C., Bruzzo, D. and Alves, R.C.B. Estudo comparative da viabilidade da vacina BCG pelos metodos polarografico, Warburg e contagem de colhias (UFC). In: Abstracts of the 4th Simpdsio Brasileiro em Micobacterias, Bauru, S&o Paula, Brazil, 1991, p. 12 Davies, P.W. and Brink, F. Direct measurement of brain oxygen concentrations with a platinum electrode. Fed. Proc. 1942, l(l), 19 Chance, B. and Williams, G.R. A simple and rapid assay of oxidative phosphorylation. Nature 1955, 175, 1120-1121 Voss, D.O., Cowles, J.C. and Bacila, M. A new oxygen electrode model for the polarographic assay of cellular and mitochondrial respiration. Anal. Biochem. 1963, 6, 211-222 Lucchiari, P.H. Circuitopolarografico optimizadopara determinaqlo de nfveis de oxigt%io intracerebraf de rato. Professor’s Thesis, lnstituto Basic0 de Biologia Medica e Agricola, SBo Paula, Brazil, 1978 Lucchiari, P.H. and Hoshino, K. Inovacao de circuit0 polarografico para a determinacao de variaees dos niveis de oxigenio em meios biol6gicos. Rev. Cienc. Biomed. 1980, 1, 51-62 Lucchiari, P.H., Feofiloff, E.F., Boscardim, A.T. and Bacila, M. A technique for the determination of the available oxygen in living carp (Cyprimus carpio) muscle. Comp. Biochem. Physio/l984,78A, 675-679

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Mickel, T.J., Quetin, L.B. and Childress, J.J. Construction of a polarographic oxygen sensor in the laboratory. In: Polarographic Oxygen Sensors. Aquatic and Physiological Applications (Eds Gnaiger, E. and Forstner, H.) Springer Verlag, Berlin, 1983, p. 81 World Health Organization: Cornit OMS d’experts de la standardisation biologique. Revised requirements for dried BCG vaccine. WHO Tech. Rep. Ser. 1979, 838, 118-147

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World Health Organization. Manual of tests on vaccines in the expanded programme on immonization, BLGIUNDPl82.1 Rev. 1, unpublished working document, 1982, pp. 31-52 Lowry, O.H., Rosebrough. A.J., Farr, L. and Randall, R. Protein measurement with the folin phenol reagent. J. Biof. Chem. 1951, 193, 265-275