ABSTRACT Purified water and water for injection (WFI) must meet acceptance criteria set by the United States Pharmacopeia (USP), and be produced from water complying with the drinking water regulations set by the Environmental Protection Agency (EPA), European Union or Japan. This poster summarizes the water quality tests offered by Boston Analytical in conjunction with meeting hold times, and using the electronic based reporting systems Laboratory Information Management System (LIMS) and Boston Analytical Secure Information System (BASIS).

BACKGROUNDUSP in conjunction with the Food and Drug Administration (FDA) has strict guidelines for the water used in. manufacturing pharmaceutical products. The required testing per USP is outlined in the table below. The USP has specific methodology for each water quality test associated that outlines the testing to be performed, as well as assay validity acceptance criteria. Acceptance criteria for water quality results obtained are set by the specific monograph within USP for each water type, or in the associated USP method. Purified water cannot be used in the manufacturing of parenteral products, therefore WFI is used.

TOTAL ORGANIC CARBON (TOC)TOC measures the amount of organic carbon present in water samples. Carbon can be introduced into water systems in many ways, but the most common are from microbial contaminants, or instrumentation used within the system. At Boston Analytical, testing is performed using a GE Sievers M9 TOC analyzer. Samples are tested from 40mL glass, low TOC vials with a septa top. Prior to initiating a sample run, a “blank” of Milli-Q water is performed and standards of sucrose and 1,4-Benzoquinone at a concentration of approximately 500ppb of TOC are performed. The autosampler needle depresses into the septa top of the TOC vial and draws up a volume of liquid to be tested. The instrument then measures the amount of Total Carbon (TC) and Inorganic Carbon (IC) within samples. Carbon present in the samples is transformed into to carbon dioxide (CO2) through the use of an oxidizer (ammonium persulfate). The sample is acidified with phosphoric acid to keep the CO2 produced from ionization, which could bias results low. The IC result is then subtracted from the TC result, to produce the TOC result.

CONDUCTIVITY Conductivity is the process in which ions in water are measured to determine the propensity of ion-facilitated electron flow through it. Gases, such as carbon dioxide dissolve in water and will interact to form ions, increasing the conductivity of the water the more carbon dioxide is present. This is why USP requires both TOC and Conductivity to be performed on purified water and WFI, as they correlate with each other. The more carbon dioxide present, the higher the conductivity and TOC results will be. Extraneous ions such as chloride and ammonia will also affect the conductivity of water. It is these two ions in which the USP measures the impurity of water against. Chloride anions counter ammonium ions and sodium cations counter chloride ions to keep the water electroneutral. Any imbalance in the ions present will indicate an impurity in the water, making it unsuitable for use in pharmaceutical applications.In order to accurately determine the conductivity of a water sample, a conductivity instrument, such as the Mettler Toledo S230 Conductivity meter used by Boston Analytical, is utilized. A standard of a known conductivity (50µS/cm) is tested prior to initiating testing of samples. The measurement of conductivity is dependent on the temperature of the standard and sample. The standard temperature for testing water for conductivity is 25˚C, but can be tested anywhere between 0˚C and 100˚C, as long as the instrument used can correct conductivity for the temperature.

ENDOTOXIN The bacterial endotoxin test serves to measure the amount of endotoxins present in a sample. Endotoxin refers to the lipopolysaccharide complex in the outer cell membrane of gram negative bacteria such as E. coli, Salmonella and Pseudomonas. The presence of endotoxin in mammals will activate an inflammatory response. Endotoxins are hearty, and cannot be denatured with high temperatures. Testing for bacterial endotoxin in a laboratory environment utilizes limulus amoebocyte (white blood cell) lysate (LAL) from Limulus Polyphemus, also commonly known as the horseshoe crab. The LAL will cleave the chromogenic substrate and the sample will turn yellow, which the instrument will read the absorbance at 405nm. The rate at which the color change happens is inversely proportionate to the concentration of endotoxin present. Therefore, the more endotoxin present, the faster the solution turns yellow. Utilizing the standard curve, the concentration of endotoxin present in an unknown sample can then be calculated.At Boston Analytical, rapid based technology is performed utilizing the Charles River Endosafe® nexgen-PTS™ system.This cartridge based technology allows the user to inoculate the cartridge with a small volume of sample and place the cartridge within the system. The cartridge has a built in standard curve and controls, and all necessary reagents, allowing the user to obtain fast and accurate results.

BIOBURDENBioburden is a test used to measure the amount of microbial contamination in a finished product or in the materials used to manufacture a product. Bioburden testing does not discriminate the different microbial contaminants that may be present, but uses a general growth media that will allow a diverse number of microorganisms to grow. Boston Analytical uses membrane filtration methodology to test for bioburden in water samples. All samples are received and tested within 24 hours of sample collection. A predetermined volume of water (generally 100mL) is passed through a 0.45µm microcellulose ester (MCE) filter. The filter is then aseptically placed on Reasoner’s 2A Agar (R2A, developed in 1985 to study microorganisms that inhabit potable water) and incubated. After the appropriate incubation time has elapsed, the plates are enumerated.

RESULTS & REPORTINGResults for all water quality tests are documented in electronic laboratory notebooks (ELNs) within LIMS. Each ELN utilized within the Boston Analytical Microbiology laboratory is custom designed for each test performed. All documentation and instrumentation utilized is 21CFR compliant and adheres to strict GDPs. All results obtained are evaluated against the given specification. Specifications set by USP are summarized in the table below. Any results obtained that exceed the given specification are investigated by Boston Analytical. All results obtained are then reported on a certificate of analysis. All testing progress and released results may be monitored by our clients through BASIS. All results obtained are reported on a certificate of analysis (COA) and issued to our clients electronically.

BOSTON ANALYTICAL CGMP AND USP COMPLIANT RAPID WATER QUALITY TESTING AND REPORTING UTILIZING LIMS AND BASISKayla Lane - Group Leader Routine USP Testing, Microbiology. Boston Analytical, Salem, NH

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