Microbial Analysis of Water- Why an important protocol in the Pharma industry?

Posted On:24 March 2021

Water is the lifeline for humans. We cannot imagine our life without water as our cells will dry off and lose the activity of all the survival guaranteeing proteins inside the cytosolic and nucleus environments. But so is true for bacterial cells, although they don’t comprise a well-defined nucleus, they are still 70 percent water constituted. The proteins ensuring the viability of these cells can only function at a particular range of water concentrations inside the cell. This fact consolidates the need and importance of water in pharma industries where antibiotics and vaccines are produced in animal and bacterial cell cultures.

Research wings of many pharmaceutical companies engage in experimentation with these cultures. Hence the importance of water as a standard medium for nutrient and growth media preparation becomes irrefutable to small-scale laboratories and research facilities.

The Pharma Water

Pharma Industries use various grades of water. Broadly, pharma water can be classified as either bulk water or packaged water. Bulk water is typically extracted and treated at the site of usage whereas packaged water is extracted at a different place and subsequently packed and sterilized to maintain quality as per microbial load limitations in the various pharmacopeia.

Since pharma plants and research laboratories use sterile tools and chemical agents in order to maintain sterility, bulk water is expected to be treated in multiple units before use at pharma facilities. Finally, after various treatment processes, purified water and water for injection are used.

Knowing about the two main types-

  • Purified Water-

It is used in the production of non-parenteral products and agents. They are mostly used to clean different tools, equipment and other material which might come in contact with non-parenteral products.

For producing purified water, source water undergoes ion exchange, reverse osmosis (RO), ultrafiltration or electro-deionization processes, and ultraviolet/chemical disinfection as purification procedures. Purified water is highly sensitive to microbial biofilms and therefore it can become a source of tenacious biofilms that produce undesirable levels of pathogens and endotoxins. This is one of the reasons that purified water is not used to prepare media for the production of non-parenteral products as they come in direct contact with the human body.

  • Water For Injection (WFI)

The other grade of water used in pharma is water for injection (WFI) where microbial contamination, biofilms, and endotoxins must be eradicated from the sample before use. Either drinking water or purified water should be considered as a feed for the WFI production unit. No lesser grade of water could be used to produce WFI.

Distillation is one of the strongest available techniques used to form WFI from purified water. The apparatus used for distillation is carefully designed and fully covered to stop any external agents or contaminants from entering the unit. Storage containers are essentially validated and rechecked in order to minimize the levels of endotoxins and pathogenic growth.

Compliance test for WFI includes conductivity, total organic carbon & bacterial endotoxin test (levels of endotoxin not more than 0.25 IU per ml) along with tests done for purified water. Since WFI is highly sterile and void of all the endotoxins, it is used to prepare various formulations and aqueous solutions for production and research purposes.

Sources of contamination

The biggest source of pathogenic contamination at water sources like lakes, groundwater, etc. is leakage in the inefficient drainage system which carries the wastewater from thousands of households living in an area. Scientifically, pathogens in fecal waste of ill and infected people can come in contact with clean water pipelines and groundwater if there is leakage of any sort in wastewater drains. Pathogenic bacteria like Staphylococcus aureus live on the skin of an infected person. There is a high risk of contamination from the bodies of microbiologists and researchers if they do not follow the laboratory/industrial protocols properly. With a plethora of opportunities for the pathogen to enter purified water and WFI containers, it becomes essential for pharma companies to regularly conduct contamination tests to ensure quality standards and risk of microbial contamination.

Microbial Analysis-

  • Traditional Methods

There are many methods included in this section and each one is equally important for checking the microbial quality of water. These tests include Microbial Enumeration test, Tests for the pathogen, Bacterial Endotoxin Test (BET), and Sterility Test that are performed using harmonized solutions.

  • More Advanced Rapid techniques

The traditional tests have been used in laboratories and pharma facilities for years. They are the classic approach of determining contamination in water and many other samples. But they face a very big challenge to overcome, i.e. time constraints for production. These tests take 48 to 72 hours to cultivate results for the microbiologist to analyze. But within this time frame, the pharma production line must have produced tens and hundreds of product batches with contaminated water. The time comes at a very great cost to million dollar-making organizations like pharmaceutical companies. To overcome the loss which might incur in these cases, there has been a high demand for rapid contamination/coliform testing.

By using chromogenic (e.g. chromogenic coliform agar) or fluorogenic culture media, classic testing methods evolve into rapid detection strategies. On a medium containing fluorogenic and/or chromogenic substrate, the target organism is identified by the activity of an enzyme specific to that organism. In fluorogenic/chromogenic culture media the enzyme characterizing the target organism splits a fluorogenic substrate into two separate components, a sugar or amino acid and a fluorogenic which converts UV-light to visible light. The fluorescence produced is read in the dark under exposure to a UV light at 366 nm. The chromogen colors the broth and the colony. Since the color does not diffuse into the agar, therefore, only the target colonies are colored.

The differential system eliminates the need of sub-culturing and further for biochemical tests by offering highly specific identification. Most importantly, the results are visible after 24 hours of incubation as greater incubation period might lead to the migration of fluorogenic substrate into the agar, thereby making it difficult for a single target colony to be detected.

Similarly, when Rapid kits for coliform detection like P.A E.coli kit is used for coliform detection, the results are available in 24 hours and the indication is in terms of a color change of media to blue-green. In this way, the time taken for visualization of colonies is significantly reduced by one third or half the time taken by classic detection tests.

Water contamination in Pharma- The hazardous effects on the population

Since water is an essential element for survival of bacterial cells, one cannot ignore the possibility of attraction of pathogenic microorganisms towards water sources. This is backed by a November 2019 incident of cholera outbreak in Nashik village in Pune district of Maharashtra. Due to the mixing up of a leaking drinking water pipeline and contaminated muddy water, the pathogen caused a mass outbreak that has been suspected to have affected 130 people in the village. Last year in August, a renowned Pharmaceutical company was also asked to recall dozens of OTC cough syrups, nasal sprays and rectal suppositories because of a poorly designed water system that tested positive for Burkholderia cepacia.

These outcomes might be even more horrendous if pathogens make it to pharmaceutical finished products and infect thousands of consumers.

As the water is directly involved in formulations of parental drugs, its contamination can lead to some serious health issues like septicemia that ultimately leads to death. Therefore, it becomes very important on the drug manufacturer’s part to monitor such contamination before the pathogen find its ways into the raw materials or finished product.