Endotoxin is measured in Endotoxin Units (EU). An EU is defined as the activity of 0.2 nanograms of Reference Endotoxin Standard. Due to the nature of most tests requiring a liquid medium, endotoxin specifications and results are often written in EU/mL.

The presence of endotoxin in the bloodstream is called endotoxemia. If endotoxin-contaminated blood circulates through the liver, many immune cells can release too rapidly, leading to overexpression. Overexpression of an immune response within the body can, in serious cases, lead to shock, organ failure, and death.

Ingestible medications cannot cause endotoxemia because of the acid within the digestive tract. Endotoxin can be dissolved and avoid absorption through the intestinal walls. Contaminated injectable and ophthalmic medications can cause endotoxemia because of their interaction with the bloodstream

Sterile processes of pharmaceutical formulation cannot prevent endotoxin contamination entirely. Sterile processes often focus on killing bacteria, if present, which leads to the expression of endotoxin. Endotoxin can be introduced at any point and by anything in the process of formulating a pharmaceutical product, from the solvent water to the raw materials of the packaging.

The three Bacterial Endotoxin Tests (BET) that use Limulus Amoebocyte Lysate (LAL) are gel-clot assay, chromogenic, and kinetic turbidimetric testing. Infinity Laboratories offers all these methods of testing.

Limulus amoebocytes form coagulin when in the presence of endotoxin. During the gel-clot assay test, LAL reagent is introduced to a sample tube, and endotoxin is present if the sample coagulates into a clot or gel-like mass. Gel-clot assay is a qualitative test; it does not specify the amount of endotoxin present, only that it is or is not. This quick pass/fail test is available at the Crown Point testing facility.

Kinetic turbidimetric testing is the quantitative collection of coagulation data. The process is sensitive enough to detect down to 0.005EU/mL. The samples are tested in duplicate to collect quantitative coagulation data and compared to a standard curve. Each sample is tested unchanged and with a known amount of endotoxin in a 96-well microplate. The microplate is dosed with lysate, and the wells with endotoxin will coagulate. The microplate reader will measure the degree of coagulation. Kinetic turbidimetric testing is offered at the Infinity sites in San Diego, St Louis, and Crown Point.

The reaction that creates coagulin also breaks bonds between proteins and a specific dye. Once the dye is free of the protein, it can express its yellow color. This color is used in chromogenic testing to determine if endotoxin is present. Chromogenic testing can be qualitative and quantitative, depending on the number of data points taken.

Chromogenic endpoint testing is qualitative, with only one data point being recorded. Kinetic chromogenic testing takes multiple data points during the test, allowing for a quantitative result. Kinetic chromogenic testing is sensitive enough to detect down to 0.005EU/mL when done via cartridge or microplate testing. Kinetic chromogenic testing is offered at the Crown Point, Petaluma, and San Diego Infinity Laboratory locations.

1. Su W, Ding X. Methods of Endotoxin Detection. Journal of Laboratory Automation. 2015;20(4):354-364. doi:10.1177/2211068215572136. https://journals.sagepub.com/doi/10.1177/2211068215572136?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed

2. Dept. of Health, Education, and Welfare Public Health Service Food and Drug Administration *ORA/ORO/DEIO/IB*. Bacterial Endotoxins/Pyrogens. Inspection Technical Guides. 2014.

3. Charles River. Gel-Clot Endotoxin Test. https://www.criver.com/products-services/qc-microbial-solutions/endotoxin-testing/lal-reagents-accessories/gel-clot-lal?region=3611

4. Fujifilm Pyrostar. Use of Colorimetric Method in LAL Test. 2018. https://www.wakopyrostar.com/blog/kit-lal/post/use-of-colorimetric-method-in-lal-test/ – :~:text=The colorimetric analysis methods allow,Limulus amebocyte lysate) for endotoxins

Mycoplasma is the smallest prokaryotic organism that can live in cell-free culture media. Because they lack a cell wall, mycoplasma is resistant to the most common antibiotics, and they may pass through the conventional 0.2 μm filters used in cell culture, thus raising the potential for high contamination rates. Contamination can alter virtually every chemical and physical property of cells (depending on the contaminating species and the host type), causing them to yield unreliable results and perhaps unsafe biologicals, biopharmaceutical drugs, or viral vaccines. In addition, mycoplasma contamination can be present with no obvious change in the host culture, making it very challenging to detect without dedicated testing. Whether your product is derived from cell substrates or is used by your customers in cell culture, nobody wants mycoplasma in their facility.

Over 200 mycoplasma species are identified, originating from many sources ranging from soil to mammals, cells, and insects.

Laboratory Personnel: Did you know that most mycoplasma contamination comes from people? Many mycoplasma species are found on human skin and saliva (M. hominis, M. orale, and M. fermentans to name a few). A simple cough, sneeze, or even talking without a mask can inadvertently introduce mycoplasma to surfaces you do not want it to be. Due to their extremely small size, mycoplasma cannot be detected in routine Viable Environmental Monitoring of your cleanroom space or Biosafety Cabinet.

Raw Materials: Contaminated animal-derived supplements like fetal bovine serum and trypsin can lead to a full-scale contamination. Other reagents, such as plant-based serums, media, and feeder cells, are also susceptible to contamination. Ensure you have a robust material receiving system that includes receipt of C of As, qualified vendors, and testing available to ensure all your raw materials are mycoplasma-free.

Equipment: Bioreactors, tubing, water baths, liquid nitrogen, even pipettes can harbor mycoplasma. These can all be considered potential culprits when testing and troubleshooting cultures. Ensure that all equipment is cleaned adequately and frequently. Adopt a robust and validated Cleaning and Disinfection Program for your cleanrooms and work surfaces.

Strict adherence to Aseptic Technique procedures: It is essential to have well-trained, accountable personnel working in aseptic conditions. Limit talking in controlled environments, never pipette by mouth, contain all work in a Biosafety Cabinet, and maintain strict adherence to gowning procedures. Working in closed systems is also recommended, but remember, there will always be some form of human exposure to the raw materials and intermediates, so stay vigilant and work carefully.

Trust your sources: Can you be sure that the companies that provide your primary components are doing their due diligence? Many media and supplement producers will test each batch of their products for mycoplasma. If this data is unavailable or your Quality department requires third-party testing, include routine testing for mycoplasma as a requirement for releasing or green-tagging raw materials such as media, serum, or enzymes for your process.

Test Frequently: You may never know if you have a mycoplasma contamination unless you test for it. Demonstrating that your product is free of adventitious agents (like mycoplasma) is not only a good checkpoint in your process, it’s also a regulatory requirement. Let’s take a look at the test points in a generic cell culture process where samples can be tested for mycoplasma. The further upstream you perform your testing, the earlier you know and can mitigate any contamination to preserve labor and material costs.

Several traditional test methods follow USP, EU, and FDA Standards. This series of testing, known as direct and indirect culture methods, can be cumbersome due to the long incubation times and reliance on human observance for the growth of the smallest free-living organism known to man. In the age of accelerated timelines to push product out the door, are you prepared to quarantine your raw materials or product for a month before resuming production? Time is money, right?

In 2007, the European Pharmacopeia accepted nucleic acid tests, such as real-time PCR, as an alternative method for traditional mycoplasma detection after validation. PCR-based detection methods are highly sensitive and can provide rapid results, allowing researchers to respond quickly to isolate and eliminate contamination once it is detected compared to the time required using traditional techniques.

At Infinity Laboratories, Inc., we employ the MycoSEQ® mycoplasma detection system from Thermo Fisher Scientific, which was designed to meet the guidance provided by the European Pharmacopoeia in 2007 of rapid-response tests. MycoSEQ, which utilizes quantitative PCR (qPCR) to detect more than 90 Mycoplasma species, can provide results in hours rather than 10 to 28 days. Using state-of-the-art automated equipment, mycoplasma DNA is extracted by magnetic beads (also known as Solid Phase DNA Extraction) and then amplified in a thermal cycling machine. With the aid of SYBR® Green fluorescence technology, amplified DNA can be detected in real-time to determine whether mycoplasma is present or absent in a sample.

Government regulations demand it, and consumer health depends on it. That’s why — for manufacturers and labs that are involved in developing products for the pharma and biotech sectors — water testing is such a critical part of the process.

But how can manufacturers and labs ensure that their methods for water testing produce results that are 100 percent accurate every time?

It’s a question worth considering, as any contamination could lead to serious business and regulatory repercussions. Standards for water purity are set by the U.S. Pharmacopeia (USP), a scientific nonprofit organization that sets the bar for the strength, quality, and purity of medicines, with the goal of improving human health.

USP’s drug standards are enforceable in the United States by the Food and Drug Administration, and these standards are used in more than 140 countries.

To comply with these standards, pharmaceutical manufacturers and labs need reliable and reproducible confirmation that their water is pure, and they need that information quickly in order to prevent delays in the manufacturing and sale of critical medicines.

In-house testing is one option, but not always a reliable one. The process is inevitably time-consuming and inefficient when done on a micro-scale. Depending on the method of in-house testing and the person who’s doing it, results can differ from test to test. In-house testing does give your scientists more control over the assay, but they still risk sample contamination through the containers they buy.

The other option, outsourcing, is an alternative with many benefits. It can allow lab workers to focus on their core competencies while also providing an added layer of assurance that the water is meeting quality control standards.

However, choosing an outsourcing partner isn’t a decision to take lightly. Here are five key elements to look for in a water-testing partner:

• A kit that’s easy to acquire and use. The Ulti-Pak test kit from Ultimate Labs is available on Amazon.com, and includes certified sampling containers for Total Organic Carbon (TOC), bioburden and conductivity testing. It’s an easy, three-step process that reduces the time your team needs to spend on quality control, while increasing the reliability and reproducibility of your results. Containers arrive pre-sterilized and ready for sampling.
• Quick-turnaround results. Ultimate Labs delivers quality assured results to your lab just three days after sample receipt. Reports are easy to read and interpret with an overall PASS or FAIL grading that eliminates confusion.
• Testing by an experienced lab. Whether testing USP Purified Water or Water for Injection, using a lab that understands the differences between the compendial water monographs is critical for the quality control of the water system and ultimately the quality and safety of your product.
• Knowledge of the industry and regulations. The control of the microbiological quality of water systems is an important consideration for most pharmaceutical and consumer product manufacturing sites. In addition, chemical purity (often represented by TOC or TOC and Conductivity) is another important constituent in establishing control of purified water systems. Ultimate Labs understands these things well and stays on top of evolving regulations because it’s the company’s specialty.
• An open dialog. By working closely with your testing partner, you will better understand compliance and assess the risks in your processes or environment. With Ultimate Labs, you’ll receive a tailored, problem-solving compliant solution, whether it is data for an FDA submission for a medical device or on-going monitoring for a pharmaceutical process or utility.