Qualification, Validation and Maintenance of the Validated State of a Water for Injection System in the Biopharmaceutical Industry

Water is the most critical raw material in the biopharmaceutical industry, serving as a solvent, excipient, and processing aid in the formulation and manufacture of active pharmaceutical ingredients, drug intermediates, and finished products, as well as in analytical procedures. It is also indispensable for the cleaning of equipment and containers. Depending on its intended application and route of administration, specific quality grades and regulatory requirements are defined. Water for Injection (WFI) is produced in this facility by condensation of pure steam through a system comprising a condenser, a 1 m³ storage tank, a sanitary heat exchanger (cooler) with double tubing at the distribution outlet, a pump, and a closed-loop distribution. The system supplies five points of use located in the production area at the Center for Genetic Engineering and Biotechnology. At the return of the loop, a sanitary heat exchanger (heater) maintains the storage tank at ≥80 °C. During daytime operation, the cooler ensures a temperature of 35–40 °C at the points of use, while at night the loop remains hot, between 80 and 85 °C. Validation of pharmaceutical water systems has become increasingly critical for regulatory authorities, requiring detailed documentation of system design, equipment, and manufacturing processes [1]. The purpose of validation is to establish documented evidence that a process or system operates correctly and consistently delivers the required quality attributes. Performance Qualification (PQ) testing—including conductivity, nitrate determination, total organic carbon (TOC), endotoxin analysis, and microbiological monitoring—was conducted to demonstrate compliance of the WFI system with pharmacopeia specifications. In addition, continuous process verification ensures ongoing control, in alignment with the FDA’s 2011 Process Validation Guidance [2]. This work reports the qualification, validation, and maintenance of the validated state of a WFI system installed in the production plant, demonstrating sustained compliance with defined quality specifications throughout its operational lifecycle.

MATERIALS AND METHODS

The qualification, the three validation stages, and the maintenance of the validated state of the WFI system were carried out with the objective of evaluating the quality of the water produced and distributed, as established in the validation protocol and in accordance with national and international regulations.

Data analysis was performed through the preparation of trend charts in Microsoft Excel, where the values of the different measurements were compared against the alert and action limits established during Stage 1 of Performance Qualification.

Samples were collected for testing at all points of the Water for Injection system. The tests performed included: conductivity, presence of nitrates, total organic carbon (TOC) content, endotoxin determination by LAL assay, total aerobic mesophilic count, and detection of pathogenic microorganisms.

The assays were conducted by the laboratories of the Quality Control Directorate (total aerobic mesophilic count, presence of pathogens, conductivity, and presence of nitrates) and the Process Control Department (TOC and LAL). The alert, action, and specification limits for each of these WFI quality attributes are shown in Table 1.

The sampling points are described in Table 2.

Table 1. Tests and specification, alert, and action limits for each quality attribute evaluated in Water for injection

Table 2. Code and location of the sampling points of the system during Performance Qualification

Maintenance System

The SGestman program [3], a computer-assisted maintenance management system, was used to plan preventive maintenance and inspection of different components of the system.

RESULTS AND DISCUSSION

Qualification of the WFI System The qualification stages of the system were carried out, beginning with Design Qualification (DQ), Installation Qualification (IQ), followed by Operational Qualification (OQ), and finally Performance Qualification (PQ). The first step verified that all components and materials conformed to the specifications. During OQ, all equipment functions were tested in a single operation with satisfactory results, applying the manufacturer’s protocol, which covered the entire system, as described by Thiesset et. al., 2013 [4]. Table 3 shows the qualification results for each system element.

Table 3. Qualification process for each system element

Conductivity and Presence of Nitrates

Accurate conductivity measurements are essential to maintaining the high purity of Water for Injection (WFI), a critical factor for ensuring product safety and efficacy. As illustrated in Fig. 1, none of the 240 samples analyzed during Phases 1 and 2 of Performance Qualification exceeded the specification limit, based on the correlation between temperature and conductivity measured without temperature compensation, in accordance with USP <645> requirements [5]. Furthermore, nitrate concentrations were consistently below the specification limit (<0.2 mg/L), confirming the absence of nitrates in all system samples and compliance with the European Pharmacopoeia (EP) criteria for this test [6].

Figure 1. Results of the conductivity assay during Phases 1 and 2. The continuous line represents the specification for this parameter, based on the relationship between temperature and conductivity measured without temperature compensation, according to the current USP. The points correspond to the determinations obtained during the established period. Values are expressed in µS/cm.

Total Organic Carbon

Total Organic Carbon (TOC) analysis is a validated approach for monitoring water quality [7] including the evaluation of Water for Injection (WFI) in pharmaceutical applications. Fig. 2 summarizes TOC measurements obtained at each sampling location during Phases 1 and 2 of Performance Qualification. Across 240 samples collected over a four-week period, all results remained below the specification limit (≤ 500 ppb), thereby confirming the stability of TOC levels within the WFI system during both generation and distribution. Measured concentrations consistently ranged from 5.34 to 17.9 ppb, further demonstrating the robustness of the system in maintaining low organic carbon content.

Figure 2. Results of TOC determination assays at all sampling points during Phases 1 and 2 of Performance Qualification. Values are expressed in ppb.

Endotoxin Determination by LAL Assay

The LAL assay is an in vitro method employed to detect and quantify bacterial endotoxins. It represents the most critical quality control test mandated by the FDA for all parenteral pharmaceuticals and water for injection. As shown in Fig. 3, none of the 240 assays conducted during the four-week period of Phases 1 and 2 of Performance Qualification yielded results exceeding the specification limit (≤ 0.25 EU/mL). Endotoxin concentrations reported by the Process Control Laboratory using the LAL method were consistently < 0.063 EU/mL, corresponding to the assay’s quantification limit. For data analysis and graphical representation, the absolute value of 0.063 EU/mL was applied to all measurement.

Figure 3. Results of endotoxin determination assays at all sampling points during Phases 1 and 2 of Performance Qualification. Values are expressed in EU/mL

Microbiological Analysis

The control of water quality, with particular emphasis on microbiological integrity, is recognized as the most critical attribute of quality assurance in the production of Water for Injection (WFI). Microbial contamination represents a significant risk to patient safety and therapeutic efficacy, thereby necessitating stringent monitoring and validation of purification systems. Regulatory frameworks such as the United States Pharmacopeia (USP) and the European Pharmacopoeia (EP) mandate rigorous standards for microbiological quality, underscoring its central role in ensuring compliance and safeguarding pharmaceutical products. In Fig. 4, from a total of 240 samples analyzed during the two weeks of intensive sampling in Phases 1 and 2 of Performance Qualification, no values above the specification limit (≤10 CFU/100 mL) were obtained. The values ranged between 0 and 2 CFU/100 mL. No pathogenic microorganisms were detected in any of the samples analyzed.

Figure 4. Results of microbiological assays at all sampling points during Phases 1 and 2 of Performance Qualification. Values are expressed in CFU/100 mL.

Results of the Qualification performance Phase

The quality of pharmaceutical water is critical in manufacturing facilities. Sampling and testing are vital to validation and ongoing monitoring. Phase 3 of the WFI system PQ was conducted over one year of operation following Phase 2. The 149 values obtained for conductivity and nitrates complied with the specifications according to the current USP. In the case of TOC assays, values ranged between 9.17 and 242 ppb, with 4 values above the alert limit, representing 97.3% of the total assays remaining under control (Table 4). These findings did not compromise the integrity of the measured water quality parameter. For endotoxin determination by the LAL assay, all results were below 0.063 EU/mL. Phase 3 monitoring over one year confirmed microbial counts consistently below 2 CFU/100 mL. Continuous circulation under turbulent flow and elevated temperatures (80–85?°C) maintained system homogeneity, effectively suppressing microbial growth. These parameters ensured reliable compliance with United States Pharmacopeia (USP) water quality standards [7].

Table 4. Analytical Results of Phase 3 Performance Qualification

Maintenance of the Validated State of the System

Continuous process verification ensures that manufacturing processes remain under constant control, minimizing the risk of deviations and guaranteeing product quality. An important aspect in critical systems is compliance with the maintenance program and calibration of measuring instruments. During the analyzed period, 100% of the work orders (WO) planned by the SGestMan maintenance management system [3] were executed. Of a total of 20 WOs, 19 corresponded to General and Review activities, while 1 were corrective and additional interventions that did not result in significant changes to the system or affect the physicochemical parameters evaluated by Matsuda et. al., 1987 [8]. Preventive maintenance interventions included filter replacements, gasket changes, valve function checks, and cleaning of system components, which contributed to reducing unexpected failures and additional costs. Fig. 5 illustrates the relationship between unplanned and planned interventions, with 95% compliance of the planned WOs, an indicator that demonstrates system stability and adherence to the scheduled maintenance cycle. In addition, calibration of the main measuring instruments—such as pressure gauges, online TOC analyzers, and temperature and conductivity sensors—was verified. In all cases, compliance with the calibration plan was confirmed. This demonstrates that all information collected during the analyzed period is accurate and reliable, thereby ensuring the integrity of the data obtained throughout the monitoring and operation of the system, in accordance with Annex 5 of the WHO Technical Report Series No. 996 [9]. The control of water quality—particularly microbiological quality, conductivity, endotoxins, and TOC is the main reason why the pharmaceutical industry dedicates considerable resources to the development and maintenance of Water for Injection systems. Table 5 summarizes the total number of samples analyzed during one year of operation after completion of the validation stages, as well as the minimum, median, and maximum values obtained for each WFI quality attribute during continuous monitoring, in order to demonstrate the maintenance of the validated state of the system. In all cases of conductivity, the values obtained were below the specification limit, meeting the requirements established in USP <645> [10], when samples were measured using conductivity readings without temperature compensation. Similarly, nitrate presence was determined; in all cases, the values obtained were below the specification (≤ 0.2 mg/L). During the monitoring period, TOC measurements remained below the action levels, with a maximum value of 245 ppb. This result demonstrates compliance with the USP <643> criteria for this test [11]. The total aerobic mesophilic count remained below the alert and action levels for the evaluation of the microbiological quality of the system, with a maximum value of 2 cfu/100 mL. This demonstrates that this attribute is maintained in accordance with a consistent sampling program and standard analytical methods. No pathogenic microorganisms were detected. Endotoxin determination for quantifying water samples was performed according to USP37–NF32 specifications [5]. All values obtained were <0.063 EU/mL, below the quantification limit of the assay, which is consistent with the USP specification for the chromogenic endpoint LAL test. This demonstrates the highest quality of Water for Injection produced by the system. These results confirm the absence of contamination at the points of use and sampling with Gram-negative bacteria, as previously reported by Martínez et. al., 2004 [12].

Figure 5. Work orders of the WFI system during the evaluated period. R (review WO), G (general intervention WO), and C (corrective maintenance WO – unplanned).

Table 5. Results of Water for Injection quality attributes during maintenance of the validated state