The Essential Evaluation of Pure Water in Pharmaceutical Applications

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BY JOSEPH PLURAD AND STÉPHANE MABIC, PHD

Uniquely found in every laboratory, water is sometimes used as a tool, and more often as a reagent, in laboratory experiments. Due to water’s ubiquitous nature, users frequently do not consider its quality or use. Selecting the right water purity grade can differentiate between success and challenges of given laboratory applications. Some causes for experimental difficulties include contaminated samples, tainted buffers or eluents, interference in chemical reactions or interference in biological processes (i.e., cell culture or DNA amplification). Additionally, increasingly sensitive laboratory instruments and scientific techniques compel researchers to use reagents of ever-increasing purity. Therefore, water purification processes and purity levels should not be taken for granted. This article will cover the quality guidelines and some pharmaceutical applications, the two-step purification process and a total water system concept.

The Impact of Water Quality on Pharmaceutical Applications Typically, R&D scientists — who are concerned with developing new compounds and biologicals — use water for buffer, media, eluent and reagent preparation to support technical applications, as well as in purification steps and purity analyses. Additionally, water is used to test a drug’s effectiveness in processes that require working with live cells or DNA/RNA interactions:

chromatography: HPLC, LC-MS, IC
spectrophotometry
PCR, blotting, electrophoresis
cell culture
sample preparation
other wet chemistry tests (pH measurements, titrations)
Conversely, the QA/QC laboratories support the manufacturing of these new and existing pharmaceutical agents. While the mission differs, the applications employed are similar to those used in the R&D laboratory:
chromatographic methods
bacteria sampling and testing
general wet chemistry uses
metal and contamination analyses
In these applications, water is normally linked to the preparation of the samples, buffers, reagents or eluents used to support the analytical test equipment.
A Two-step Purification Process
Most laboratory water needs can be reduced to two general classes: pretreated-grade water, produced using advanced purification technologies from a tap water source, and high-purity water, produced using a polishing unit. Both exceed the United States Pharmacopeia (USP) Purified Water recommendations. In this two-step process, the quality of the pretreated water will have a direct impact on the quality of the polished water. For example, an inefficient pretreatment will decrease the effectiveness of the polishing process.

Pretreatment: Taking Tap Water to USP Purified Water
Water purified by reverse osmosis (RO) passes through a semi-permeable membrane under pressure, which exceeds the natural osmotic pressure exerted against the membrane and opposes the natural osmotic flow. This results in purified water moving across the semi-permeable membrane and leaving behind a solution more concentrated in contaminants. The outcome is a relatively pure solution; well-maintained reverse-osmosis systems remove in excess of 95% of the dissolved ions, organic molecules and particles from a water supply.

The best pretreatment processes take the water processed by reverse osmosis and optimize it with downstream purification steps, such as deionization or electrodeionization. Millipore’s Elix® technology incorporates ion-exchange resins, ion-exchange membranes and a small electrical current to remove ionic species that have passed the RO process. Product water from this process can range from five to 15 megohms per centimetre purity and typically has less than 30 parts per billion of total organic carbon (TOC) contaminants, a grade above RO water but not yet ultrapure. An integrated 254-nanometre ultraviolet (UV) lamp further reduces bacterial counts in the water. The outcome is a consistent USP Purified Water supply that is less subject to fluctuations occurring as a result of the tap water condition changes.

Water of this quality has a broad range of general, laboratory-grade water applications. It is ideal for basic wet chemistry techniques, particularly with higher detection limits (per cent to high parts-per-million range) such as: pH analysis, electrochemical titrations and some UV spectrophotometric analyses. Additionally, it is well-suited for use anywhere distilled or deionized water is used: glassware washing or rinsing, as well as basic buffer or reagent use (reconstitution, dilution, dissolution, etc.). Electrodeionization water also is the best feedwater source for ultrapure water systems like Millipore’s Milli-Q® family of polishing systems.

Polishing — Ultrapure Water For Critical Applications
Polishers (or ultrapure water systems) take the pretreated water and further remove any remaining ions, organics, particles and biologics from the water stream. Better polishing systems do not use tap water directly as the contaminant levels often overwhelm the purification technologies in a polisher, resulting in poor water quality and increased cartridge-exchange frequency. Purification modes, such as ion-exchange resins, activated carbon, UV lamps and ultrafilters, can remove specific classes of contaminants, and should be combined to address the complete laboratory application needs.

All polishers use ion-exchange cartridges as the major purification step after pretreatment. The flow rates and the resin types used are optimized to remove all remaining ions from the Elix water, resulting in 18.2 megohms per centimetre ultrapure water. Ion-exchange resins attract positively and negatively charged ions from the water and substitute them with hydrogen and hydroxide ions. As these ion-exchange sites are consumed, cartridges containing the resin are simply exchanged. The product water in this process is considered to be free of ionic contamination, which would otherwise contribute electrical conductivity and decrease the resistivity. The Milli-Q Academic water system is suitable for any application where low levels of ions (parts-per-billion, parts-per-trillion levels) are necessary to the analytical or quality analysis. Typical applications can include ion analysis via ion-selective electrodes or conductivity analysis, ion-exchange chromatography and spectrophotometric techniques with very low detection limits or analysis levels.

UV photo-oxidation is often employed between deionization steps to decrease two classes of contaminants: any remaining organic compounds in the Elix system water stream as well as bacteria. Organic compounds can foul ion-exchange resin by coating the active reaction sites on the resin. Most water purification systems employ a dual-wavelength UV lamp to address these organic contaminants. UV light at 185 nanometres generates OH radicals that initiate oxidation of carbon bonds, leading to the formation of bicarbonate that can be removed using ion-exchange resin. UV light at 254 nanometres can deactivate bacteria by impairing their DNA. Millipore’s Milli-Q Gradient A10® system not only employs this dual-wavelength lamp, but also incorporates an in-line, calibrated and validatable total organic carbon (TOC) monitor to capture the ultrapure water quality. Ultrapure water that has been purified using a UV lamp is essential for chromatography techniques, including HPLC and LC-MS, TOC analyses and other analyses that are sensitive to organic or bacterial contamination.

One final purification technique often combined with ion-exchange resins in the polishing process is ultrafiltration (UF). By taking a membrane filter with a nominal pore size at the molecular level, it is possible to remove large organic molecules, including bacterial byproducts like endotoxins (also called pyrogens). UF membranes are effective in removing nucleases like RNase and DNase from the water purification stream as well as removing endotoxins with a log reduction value (LRV) of six. Using UF-based purification techniques provides RNase-free water and eliminates the need to further treat the ultrapure water, either with diethyl pyrocarbonate (DEPC) or via autoclaving. The combination of UF technology with an integrated UV lamp in a single polisher, gives users an equivalent to bottled “molecular biology-grade” water.

UV/UF-treated water is especially suitable for most critical biological research: cell culture, DNA microarrays, PCR, RNA research, and protein research and analysis. Essentially, any application requiring DEPC-treated water is a viable candidate for UF-treated water.

Advantages of the Two-step Purification Process
Contrary to distillation or service deionization processes, a two-step purification method provides the scalability and flexibility to meet a variety of needs. A combination of purification and monitoring technologies provides multiple water qualities as well as better product water control (see Table 1).

The best systems provide an operating-cost benefit, compared with techniques such as distillation or bottled water. Similarly, quality offerings provide better control of the purification process, compared with building-wide, central water purification systems. As industry experts, water purification companies are accustomed to matching proper water qualities with the needs of laboratory technicians and scientists. This often varies based on analytical goals, detection limits and contamination constraints. For example, some users rely on USP Purified Water regardless of their application needs. However, USP Purified Water is defined broadly not only in the way it is produced, but also with regard to its acceptable ionic and organic levels. Therefore, Purified Water is often not pure enough for many of the analytical and research techniques employed in pharmaceutical laboratories. Millipore systems produce water that meets or exceeds the Purified Water standard and provides the optimal water quality that is suitable for purity levels demanded by the analytical test, chemical reaction or biological process in pharmaceutical laboratories.

Total Water System Considerations
In such a regulated environment, pharmaceutical laboratories should seek to have as much process control as allowable. This responsibility includes selecting water purification suppliers who know the pharmaceutical industry and applications, are intimately familiar with water purification systems and technologies, and are sensitive to the regulatory demands of the industry.

Suppliers, in general, should have multiple water purification technologies available. A combination of purification technologies better addresses the multiple applications in a single laboratory. With water, one size does not fit all. The two-step purification concept is adaptable to most, if not all, applications that may be found in a single lab. However, there are other parts of the water “package” to consider:

Knowledge of the laboratory applications: ensure that the proper selection and implementation of purification technologies matches the work done in the laboratory.
Cost of ownership: costly delays can be eliminated by relying on quality pure water that is always available.
Technical support: better water system suppliers have technical resources available by phone or on-site to install, maintain, and repair the systems they build.
Regulatory support: purification systems such as Millipore’s can be validated to meet the needs of the pharmaceutical industry. In addition to providing comprehensive documentation of the Installation Qualification, Operational Qualification and maintenance of the system, better manufacturers facilitate compliance with regulatory agencies like the U.S. Food and Drug Administration.
Good design: suppliers design their water purification systems to minimize purified water re-contamination. This includes not only the purification media and methods, but also the materials of construction and flexible distribution that minimizes “standing” time.
Conclusion
Water is clearly an essential laboratory tool. From cleaning or rinsing glassware to growing cells or developing the newest blockbuster drug, its purity can never be underestimated or taken for granted. Clearly, the key is to match the correct purity levels to the criticality of the application. This can often be achieved by pretreating tap water with the appropriate technology. The pretreated water should be suitable enough for basic work and serve as the feedwater source for an ultrapure water system. At the ultrapure level, the systems used should integrate the best combination of purification and monitoring technologies, and address all of the critical chemical or biological applications.

In determining the best means for purifying water in your pharmaceutical laboratory, it is necessary to consider all of the possible uses for water as well as any regulatory requirements. These water systems should meet all the laboratory application needs and be validated to published norms. The proper selection and maintenance of such a laboratory water system can guarantee not only consistently pure water, but also peace of mind.

Joseph Plurad is product marketing manager, Lab Water Division, Millipore Corp., 290 Concord Rd., Billerica, MA 01821. Phone: 978-715-2390; e-mail: joseph_plurad@millipore.com.
Stéphane Mabic, PhD is worldwide applications support manager, Lab Water Division, Millipore Corp.