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WATER PURIFIED SYSTEMS VALIDATION IN A PHARMACEUTICAL INDUSTRIAL PROCESS

Ravagnani. M. A. S. S.1*, Silva, N. L. 1 and Gazolza, Sebastio2
1

Departamento de Engenharia Qumica - Universidade Estadual de Maring 2 Departamento de Estatstica - Universidade Estadual de Maring

Abstract. Work methodology and professionals involved in pharmaceutical industry in Brazil has been changed in
the later years. At long Current Good Manufacturing Practices (cGMP) have been used by industrial processes, especially in the multinational industries ones that use the main branch procedures. On the other hand, smaller companies use own cGMPs demanding great effort from their employees. Anvisas RDC 134, reedit by RDC 210, is the document that formalizes the so called cGMPs of World Health Organization (WHO) approved in 1994, as well as obeying Mercosul standards. Since RDC was implemented, professionals of pharmaceutical industries have been working to answer all the requirements, especially process validation. Pharmaceutical industry, as well as the cosmetic, veterinarian and food ones answer with the qualification policies that are used to validate processes to the sources of pressure and stimulus which are represents by the market and govern health agencies. Validation is a typical procedure that refers mostly to Good Manufacturing Practices, whose main objective is to prevent medicines to offer risks to the consumer, to the environment and to the operator. Water is essential to the production of medicine and health products. In most cases it is added to the product during the manufacturing process. Since it is suitable to the human consume, being drinkable doesnt assure that it also can be used in machines, industrial facilities, medicine, production, food products, cosmetics or any kind of chemical or pharmaceutical products. In the present paper, a water purification system was studied to guarantee the requirements of United States Pharmacopoea XXIII and XXIV. Filtration system, deionizer unit and an ultraviolet (UV) light equipment, are part of the system. The monitorated parameters were Conductivity, Oxidizable Substances, pH, Carbon Dioxide and Bacteriological Purity. The system was under control and it was validated based on the results obtained.

Keywords: Process Validation, Water System and Purified Water.

1. Introduction
In the last years, mainly at the end of 90s, the validation approach in pharmaceutical industries has been discussed due to its real importance within a productive process in relation to the products quality attributes, like purity, safety and effectiveness, that constitutes the base of the thought and working of professionals involved to the process validation (Santos, 2001). The U.S. Food and Drug Administration (FDA) in 1987 in his most recently proposed named Guidelines on General Principles of Process Validation, has offered a definition to process validation, after a series of incidents involved cross-contamination problems by some pharmaceutical manufacturing establishments: Process validation is establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality characteristics.

To whom all correspondence should be addressed. Address: Departamento de Engenharia Qumica, UEM - Centro de Tecnologia, Bl.D90, 87020-900 Maring Brazil E-mail: ravag@deq.uem.br

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Therefore, validation is legible through the documentation establishment, the guarantee of that manufacturing process assures the product quality with homogeneity. The validation concept applies to the end product, and all the previous stages are called qualification. So, the validation consists of a series of qualifications that involves tests, systems verifications and also critical process parameters, that are its regulating keys, and can vary within an acceptance limit. The different qualifications form than, the stages of validation: Design Qualification, Installation Qualification, Operation Qualification and Performance Qualification (Jnck, 2002a). Each one must follow its own schedule and protocol. The water, in a pharmaceutical industry, is the most important raw material for the accomplishment of any process. Therefore, it is essential to not only assure that water, but also installations and procedures are validated. (Veneranda, 2004). So, it is necessary to choose a well-designed water system, by using a combination of methods that allows reaching the quality levels of water to a determinate application, by optimizing its particular capacity of remove contaminants. The objective of this work consists in to validate the Steviafarma Industrial S/A company water purification system, according to the specified requisites in United States Pharmacopeia (USP) XXIII and XXIV, adopting the concurrent validation and the Statistical Control Process. The software Statistica 6.0 was the tool work used for the data statistical treatment.

2. Materials and Methods

2.1. Equipaments The water purified system is composed by a pre-filtration unit, with a 1 m cellulose filter (Cunolatina) followed by an activated carbon bed (Cunolatina). The first one is responsible to remove non-dissolved ions and the second one to remove chlorine compounds and low molecular weight organic compounds, present in the feed water, to the deionizer column protection. After the carbon bed, comes the deionizer unit, represented by a mixed bed ion exchange column (Permution), with the objective of to removal the dissolved ions. The purified water than passes through the ultraviolet equipment with a 254 nm wave length (Germetec), with germicide action for its sterilization. The layout of the system in this study can be visualized in Figure 1.
Feed Water - Sanepar

Deionizer Column Coluna Deionizadora

Celulose (1m)

FiltroFilter de Celulose Filtro de Carvo Bed

Carbon

Point 1

Ponto 2 UV Equipment Point 3 Point 4

Poin of Use

Point 5

Fig. 1. Water purified system layout. 2

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One of the most important variables in the process of purified water for pharmaceutical apllications is the quality and the credibility of the water supplying system. In the present case, the water supplying is obtained from SANEPAR Paran State Sanitation Company. The conductivity data were measured in a portable equipment (Alpax) and pH data were obtained by using a Quimis pHmeter.

2.2. Methodology The validation methodology applied to this system was the concurrent one, which was realized during the routines operations. The installation qualification was only applied to UV equipment, because it was obtained with the others equipment suppliers, documents certifying their own technical and functional specifications. To UV equipment, an installation verification were made. Also, the technical specifications were checked, in accordance with the design requirements, supplier instructions and supported documentation. The operational qualification, had the purpose of to verify that the equipment operates as intended in all expected operational range, according to their functional specifications. The project qualification was not carried through, because it is more specific to new equipments or systems. According to Jnck (2002b), this stage is an activity that, through critical process analysis, objectives to certify that the technical documentation incorporates the concepts of the User Requirements Specifications (URS). Therefore, as the case was an installed system, the validation effort started with the installation of the qualification. The performance qualification consisted in to evaluate the system performance during the water production in attempting to USP requirements. This stage was done in two phases. During the first phase, the system was kept under normal operating levels during extensive and frequent samplings for four months in order to profile the system. The second phase is the phase 1 continuation, but with lower frequency of sampling than phase 1, being also carried out in 4 months. Generally each phase of testing should take 4-6 months to complete or longer, if necessary, as proposed by Johnson (1993). The author also describes that a full validation of a system could require as long as a year because of the operating problems, equipment failures, and maintenance errors, which should be expected to occur during the validation period. The system sampling procedure sampling consisted, first, in opening the water register and, after that the feeding valve. Water was circulated through out the system during 15 minutes and, all the valves were sanified with a 70% alcohol solution. Table 1 presents the sampling points and the analyses that had been realized.

Table 1. Sampling points of water system. Sampling Points 1 Feed Water SANEPAR 2 After Pre Filters 3 After Deionizer Column 4 After UV Equipment 5 Point of Use Microbiological Analysis Total coliform, bacteria, yeasts Total coliform, bacteria, yeasts Bacteria, yeasts Bacteria, yeasts Total coliform, bacteria and yeasts Physical Chemical Analysis pH, conductivity, chlorine, hardness Chlorine, oxidizable substances pH, conductivity, carbon dioxide, oxidizable substances -----------------------pH, conductivity, carbon dioxide, oxidizable substances 3

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The physical chemistry analyses were done daily for points 1, 2, 3 and 5, as well as the yeasts and bacteria for points 3, 4 and 5. The microbiological analyses for points 1 and 2 were done once a week, in the same way, the total coliform for points 1,2 and 5. Tables 2 and 3, present the acceptance criteria for drinking and purified water, consulted in the literature. Table 2. Specified limits for drinking water. Potable Water Total coliform and Escherichia coli* Bacteria** Yeasts pH* Chlorine* Conductivity Specified Limits Absent in 100 mL 500 CFU/mL -------6,0 to 9,5 5 mg/mL -------Table 3. Specified limits for purified water. Purified Water Bacteria** Yeasts pH** Conductivity** Carbon dioxide* Oxidizable substances* Specified Limits 100 CFU/mL -------5,0 to 7,0 1,3 S/cm The mixture remains clean The pink color does not completely disappear

Hardness* 500 mg/mL * Portaria n 518 (2004) ** Groves, Anisfeld and Olson (1991). Chlorine Technique

* The Unites States Pharmacopeia XXIII (1990). ** The Unites States Pharmacopeia XXIV (1995).

For free chlorine evaluation, it was placed 1 mL of orto-toluidine in an erlenmeyer, and added 100 ml of the water sampling. After the mixture, the color was compared immediately with permanent chlorine standards (Pregnolatto e Pregnolatto, 1985).

Hardness Techinique 50 mL of water were tranfered to a 250 mL erlenmeyer and added 1 mL of ammonia buffer solution and a small quantity (0,05 g) of Eriocromo Black T indicator. After that, the solution were titulated with EDTA 0,01 M until purple color turn blue (Pregnolatto e Pregnolatto, 1985). The calculation presented in Equation (1) was done:

1000 . V . f = mg of calcium carbonate per liter . A

where: V = EDTA (mL) used volume in the titulation. - f = factor of EDTA solution -A = water volume (mL).

(1)

Viable Bacteria Count (Multiple Tube Fermentation Technique)

It were used 12 tubes series containing 10 mL of lauryl tryptose broth. The test was realized in triple. 1 mL of

water was added to 10 mL of lauryl tryptose in the first tube series and the dilutions 1:100 and 1:1000 were prepared from the dilution 1:10. The three tubes left were the controls.
After the dilution has finished, they were taken to stoven during 48 hours at 30-35 C. If in a tube gas were present, it were considered positive for grown of microorganisms. The results were
0

expressed in MPN/mL according to a specific table (Farmacopia Brazileira, 1988).

The positive tubes were submitted to a confirmative test, as follow:

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Durhan tubes and BGLBB (Brilliant Green Lactose Bile Broth) were introduced into a tube. After that, the tubes

were submitted to a sterilization process for 15 minutes at 121C.

A part of the culture was transferred to BGLBB broth and incubated the tubes for 48 +/- 3 horas at 35+/- 0,5C.

The gas formation in any tube consisted of a positive result for the confirmative test.

Yeasts Count (Pour Plate Method)

The plates were prepared in duplicata. 2 mL of sampling was separated, and in each one 1,0 mL were used. After the Sabouraud Dextrose Agar culture medium be cold between the temperature of 44 to 48C, 20 mL was

added to the plates.

The plates under were rested for 10 to 15 minutes until the solidification. After that, the plates were taken to

stoven to 22-25C for 48 - 72 hours, with a control (Farmacopia Brasileira, 1988).

Heterotrophic Bacteria Count (Pour Plate Method) The applied technique for yeasts determination was the same to bacteria. The only differences were in the culture medium used: Plate Count Agar (PCA) and in the time and temperature of incubation: 30-35C during 24 to 48 hours (Farmacopia Brasileira, 1988).

Oxidizable Substances Determination To 100 mL of purified water, 10 mL of 2 N H2SO4 was added and after that, the solution was heated until boiling. Then, 0,1 mL of potassium permanganate 0,1 N were mixed to the solution, marking 10 minutes of boil (USP XXIII, 1995). Carbon Dioxide Determination To 25 mL of purified water was added 25 mL of calcium hidroxide SR. The mixture had to remain clear (USP XXIII, 1995).

3. Results and Discussion

Tables 4 and 5 presented the results to coliform total technique. Table 4. Percentages of de samples according and not according to the Anvisa Regulations (Portaria n 518, 2004) for total coliform. According to N Samples % 33 94,3 34 97,1 35 100 Not According to N Samples % 2 5,7 1 2,9 -------

Point 1 Point 2 Point 5

Table 5 presents the results of tubes which gas formation was observed. Two samples carried for point 1, one in May and the other in October, had the same result (3 MPN/mL). However in the confirmative test nothing was confirmed. 5

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Table 5. Results for samples according and not according for total coliform. Month May October October Dilution 1 0 0 0 Dilution 2 0 1 0 Dilution 3 1 0 1 MPN/mL 3 3 3

Point 1 Point 2

The values obtained to point 2 in the same day, didnt present any positive result. So, the result of point 1, can be false, because the point 2 was located after the pre-filter unit, which means that the water could become contaminated after circulate through it. In this point, could be noted a positive answer at the third series of dilutions. However, in the confirmative test, the answer was negative. Therefore, one can say that total coliform tests for these two points attended the specified limits presented in the Anvisa Regulations (Portaria n 518, 2004), assuring phyto-sanity to the feed water, according to the microbiological standards. During the performance qualification, all samples collected at point 5 presented negative results for total coliform, as can be noted in Table 4. So, the results found at point 3, could be wrong, and gas formation could be delivering from a external contamination. The pH values to feed water presented an average 6,60, indicating that didnt occur any significant change in the water quality during this monitoring. All values were in according to the Anvisa Regulation (Portaria n 518, 2004), with a standard deviation of 0,1.Figures 2 and 3, present the control chart of the obtained values of pH to points 3 and 5. The calculated average for both points were 5,17 and the standard deviation was 0,1.
10 9 8 7 6 pH 5 4 3 2 1 20 40 60 Samples 80 100 LCL = 5,00
pH 10 9 8

UCL = 7,00

7 6 5 4 3 2 1

UCL = 7,00

LCL = 5,00

20

40

60 Samples

80

100

Fig. 2. Control chart for pH values for point 3.

Fig. 3. Control chart for pH values of point 5.

The results are in accordance with the acceptance criteria to purified water according to USP XXIV, between 5,0 to 7,0, being under statistical control. Figure 4 presents the conductivity results. There are no critical changes in the feed water quality, because the results were show around the calculated average (130,64S/cm). The standard deviation was 0,6.

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140

135 Condutivity (S/cm)

130

Average = 130,64

125

120 20 40 60 Samples 80 100

Fig. 4. Control chart for conductivity values for point 1.

Figures 5 and 6 shows conductivity for points 3 and 5. It can be observed a systematic increase of values and, also two points above the specified control limit, for both cases. The average, 0,33 S/cm, and standard deviation were the same for both points, as was expected. Through the trend observed in these conductivity graphs and the points that exceeded the upper control limit, it was determinated the exchange period for the deionizer column, which must be 22 days. The conductivity reduction from 130,64 S/cm, at point 1, to 0,33 S/cm at point 3, corresponds to a remove efficiency of 99,7% by the deionizer column.
1,3 1,2 1,1 1 Condutivity (S/cm) 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 20 40 60 Samples 80 100 Average = 0,33 UCL = 1,3

1,3 1,2 1,1 1 Condutivity (S/cm) 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 20 40 60 Samples 80 100

UCL = 1,30

Average = 0,33

Fig. 5. Control chart for conductivity values-point 3.

Fig. 6. Control chart for conductivity values-point 5.

Table 6 shows results for bacteria and yeasts analysis. The small deviations on the average are probably occasioned by contaminations during the sampling or during the analysis. This can be affirmed, because the results of others days were under control, without deviations that could be interpreted as a microbiological growth. 7

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Table 6. Bacteria and yeasts analysis - Averages and standard deviation. Bacteria Average (CFU/mL) 9,7 9,9 11 0,2 0,2 SD 0,8 0,7 0,3 0,5 0,6 Yeasts Average (CFU/mL) 3,5 3,6 3,8 0,2 0,3 SD 0,9 0,8 0,9 0,5 0,7

Point 1 Point 2 Point 3 Point 4 Point 5

The bacteria analysis of point 3 (11 CFU/mL), was a little bit higher than other points, probably because the deionizer column constitutes a promising way to microbiological growth. Therefore, the obtained results for points 4 and 5 indicated that the UV equipment had an excellent performance of 98,6%, when the points 1 and 4 were compared. So, the values obtained to points 4 and 5 were in accordance with the USP XXIV requirements. It is observed in the point 3 a small increase in the yeasts counting, when compared with other points. However, the microbiological reduction from point 3 to 4 was satisfactory achieving 94,2 %. The calculated average for hardness was 40,45 ppm and the standard deviation was 0,4. The obtained values are presented in Figure 7. This figure shows the continuous behavior and the distribution around the average line. All the points in the graph, corresponds to specified limit of 500 ppm from Portaria n 518 (2004).
500 450 400 Hardness (ppm) 350 300 250 200 150 100 50 20 40 60 Samples 80 100 Average = 40,45 UCL = 500,00

Fig. 7. Control chart for hardness values for point 1. The chlorine analysis in feed water is shown in Figure 8. It folows the specification in Anvisa Regulations (Portaria n 518, 2004), whose limit is 5 ppm. The average was 0,5 ppm and the standard deviation was 0,1. All the points in the graph were next to the average, showing that no problems were detected. Figure 9 shows point 2, where the main objective was to remove all of the chlorine. As a result, the carbon bed exchange period for the deionizer column was found, and it corresponds to 10 days. The calculated average was 0,01 ppm with a standard deviation of 0,03. It means that 98,3% of chlorine reduction has been observed, comparing averages for points 1 and 2. 8

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0,3

UCL = 5

4 Chlorine (ppm) Chlorine (ppm)

0,2

0,1

Aaaa
1
Average = 0,51 Average = 0,01

20

40

60 Samples

80

100

20

40

60 Samples

80

100

Fig. 8. Control Chart to chlorine values for point 1.

Fig 9. Control chart for chlorine values for point 2.

Table 8 express the analysis of oxidizable substances. For point 2, only 3 determinations presented positive results. However, by analyzing point 3 in the same day, nothing was found.

Table 8. Percentages of de samples according and not according to the USP XXIII regulations for oxidizable substances According to N Samples % 102 97,1 103 98,1 105 100 Not according to N Samples % 3 2,9 2 1,9 -------

Point 2 Point 3 Point 5

It were founded two positive results in point 3, but the microbiological results remained under control. For point 5, all the results were negatives, and the purified water attested its acceptance criteria. Carbon dioxide determination was done for points 3 and 5, as shown in Table 9.

Table 9. Percentages of samples according and not according to the USP XXIII regulations for Carbon Dioxide analysis . According to N Samples % 105 100 105 100 Not according to N Samples % -------------

Point 3 Point 5

4. Conclusions
All the equipments installed and analyzed presented satisfactory performance, attesting their functional and technical specifications. It means that the purification water production is qualified. The carbon bed in the deionizer column presented an efficiency of 98,3% in removing chlorine. One can conclude that in normal operations it needs to be substituted each 10 days. 9

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The deionizer column showed an efficiency of 99,7% in removing the dissolved ions. So, to maintain this level for the purified water, the column must be substituted each 22 days. It was shown that the UV equipment was the adequate alternative to reduce the contamination coming from bacteria and yeasts. It is assured by its efficiency of 98,6% and 94,2% respectively. Finally, it is important to say that any system of purified water can be validated, as shown in this paper. The industrial system of purified water of Steviafarma Industrial S/A is qualified to produced water, according to the USP XXIII and XXIV requirements, respectively, and once conclude that it is validated.

5. References
Agncia Nacional de Vigilncia Sanitria. Portaria n 518, de 25 de maro de 2004. Agncia Nacional de Vigilncia Sanitria. Resoluo RDC n 210, de 04 de julho de 2003. Farmacopia Brasileira (1988). 4 ed. Ateneu, So Paulo. Groves, M. J., Anisfeld, M. H., Olson, W. P., 1991, Sterile Pharmaceutical Manufacturing: Applications for 1990s. 1st ed. Interpharm Press. Guidelines on General Principles of Process Validation, Division of Manufacturing and Product Quality (HFN-320) Center for Drugs and Biologics (FDA), Rockville, Maryland (May 1987). . Johnson, W. M. (1993). Validation of Water Systems for Sterile and Nonsterile Products. In: Berry, I.R., Nash, R.A. (eds), Pharmaceutical Process Validation, 2 ed., chapter 9, New York, USA, Marcel Dekker, Inc. Jnck, R. H. (2002a). Qualificao do Projeto e da Instalao em Indstria Farmacutica. Controle de Contaminao, 39, 28-35. Jnck, R. H. (2002b). Purificao da gua para Fins Farmacuticos, Alimentcios e Cosmticos. Controle de Contaminao, 34, 12-25. Pregnolatto, W., Pregnolatto, N. P. (1985). Normas Analticas do Instituto Adolfo Lutz. 3ed. So Paulo. Santos, M. P. (2001). Validao de Processo na Indstria Farmacutica no Brasil. Controle de Contaminao, 25, 20-28. The United States Pharmacopeia XXIII. By authority of the United States Pharmacopeial Convention, Inc., meeting at Washington, D.C., March 8-10, 1990. Prepared by the Committee of Revision and published by the Board of Trustees. Official from January 1, 1995. The United States Pharmacopeia XXIV. By authority of the United States Pharmacopeial Convention, Inc., meeting at Washington, D.C., March 9-12, 1995. Prepared by the Committee of Revision and published by the Board of Trustees. Official from January 1, 2000. Veneranda, N. (2004). Qualificao Fundamental para a Qualidade do Sistema. Controle de Contaminao, 57, 16-20.

Acknowledgments

The authors are grateful to Steviafarma Industrial S/A. 10