The removal of endotoxin from any system or substance that will come in contact with patients during medical therapies, implantation of a device or injection of a substance is critical to patient safety.
Endotoxin and Its Sources
Though the terms endotoxin and pyrogen are often used interchangeably, endotoxins and pyrogens are not the same. The larger group of substances is pyrogens, which are any fever producing agents from an organic source. Endotoxins are one form of pyrogen, perhaps the most common form.
They are remnants of cell walls of gram-negative bacteria (see Figure 1 below). As the cell wall of gram negative bacteria breaks down it releases lipopolysaccharide (LPS) molecules that are the actual endotoxin. Examples of gram negative bacteria include E. coli, Pseudomonas aeruginosa and the bacteria that cause cholera and meningitis.
Model of the cell wall of Gram-negative bacteria. Left: The Murein (M) layer adjacent to the cytoplasma membrane (CM) is covalently bound to the hydrophilic ends of lipoproteins. Their lipophilic ends are attached to the outer membrane (OM) consisting of phospholipids and lipid A of LPSs. The hydrophilic, O-specific heteropolysaccharide side chains of the LPSs are located on the surface of the bacterial cell.
Right:Schematic representation of a LPS molecule consisting of lipid A, R-core and OAntigen. Symbols: Glc-NAc= N-acetylglucosamine; gal = galactose; Hep = heptose; KDO = 2-keto-3- deoxyoctonate; PS = periplasmic space. (From H.G. Schlegel: Allgemeine Mikrobiologie, Thieme. Stuttgart, New York (1985): p. 50.)
Pyrogen can be detected by the Pyrogen Test conducted with rabbits. It is a costly and time-consuming test that is not practical for monitoring potential contamination of any substance or process on a realtime basis. The Bacterial Endotoxin Test, also termed the Limulus Amebocyte Lysate (LAL) Test can detect, in a short period of time, the LPS molecules that make up endotoxin. For more on the history and science behind these two tests, consult the book “Endotoxins: Pyrogens, LAL Testing, and Depyrogenation” (Second Edition, 2001) by Kevin L.
Williams, published by Marcel Dekker. The most common source of endotoxin is water. Water borne bacteria, several of which are gram negative bacteria, are destroyed by chlorination and other treatment at the municipal level. However, the cell walls are not completely destroyed in the process, leaving endotoxin in the water. In fact, tap water can contain endotoxin levels of over 100EU/ml. Ingesting endotoxin is, generally, not harmful to humans. However, when endotoxin reaches the bloodstream of a human, the results can be, as described above, catastrophic.
Water for specific medical and biopharmaceutical uses has defined limits on the levels of endotoxin allowed. Water used in dialysis has an endotoxin limit of 2EU/ml. Water for Injection (WFI), with the highest level of water purity in the biopharmaceutical industry, has an endotoxin limit of 0.25EU/ml. These levels are set based on the obvious need to protect patients from the terrible effects of endotoxin exposure.
Critical Situations for Endotoxin Removal There are three major areas of concern for endotoxin contamination. First is in therapeutic processes, like dialysis. Second is in the manufacture and packaging of medical devices used for patient diagnosis, surgery or for implantation in a patient. Finally, and perhaps most important, is in the making of drugs and biological products for injection.
Therapeutic Processes Healthcare practitioners, from dialysis nurses to hospital administrators to family physicians and dentists, are concerned with bacterial and organic contamination of water in their systems. Dialysis centers use water directly in the treatment of patients, many of whom suffer from immune system deficiencies.
The water purification system in the dialysis center is expected to reduce or even remove endotoxins from the water used for patient treatment. Hospitals and clinics are concerned with water and with the potential for endotoxin contamination in any product that will contact patients. Water is used for rinsing instruments, hand washing, laboratory tests and even feeding sterilizing systems. Both bacteria and endotoxin could contaminate the water and the instruments or equipment that it touches. Some form of water treatment or filtration is being used to control bacteria and endotoxin in almost all hospital and clinic water use areas.
Dentists use water for mixing treatment products and rinsing patients’ mouths, especially after causing bleeding. The prevention of patient exposure to bacteria and endotoxins in that water is becoming more of a concern in dental offices.
As a result, water filtration for the specific control of bacteria and endotoxin is becoming more common in dental applications. Medical Devices The term “medical devices” covers a wide range of items. Any device used for the examination, diagnosis or treatment of patients is considered a medical device. Even the equipment used to clean or reprocess devices is considered a medical device.
Items included range from obvious things like pacemakers and artificial hips to the not-so-obvious like endoscope reprocessors and even the cartridge filters used in water systems feeding treatment or reprocessing devices. Bacterial or endotoxin contamination in many of the devices could be catastrophic to a patient. The most likely source of contamination, other than handling by production personnel, is the water system used to make the devices or for reprocessing the devices.
Rinse water is used in the making of many devices. The quality of the rinse water is, in many cases, Water for Injection. Water for Injection, or WFI, is made to a standard that DOES NOT require sterility. In fact, up to 10cfu/100ml of bacteria are allowed. Endotoxin levels up to 0.25 Eu/ml are also allowed. However, most systems are operated to produce water that is as close to sterile and endotoxin-free as possible. Bacterial retention filters and endotoxin removal filters are used safeguard the water quality.
Drugs and Biological Products
The industry that uses the most filtration products is the biopharmaceutical industry. The many makers of parenteral, or injectable, drugs need to make sure that their products are as pure as possible. For obvious reasons, any contamination by bacteria or endotoxins could lead to serious or even deadly consequences for patients. As with any system of manufacturing, the best of contamination control systems will still allow bacteria, including gram negative bacteria, into the system. Drug manufacturing processes, even with some of the most stringent contamination control systems in existence, are still subject to this contamination. There are multiple filtration functions in any biopharmaceutical operation.
Filtration functions include water treatment and purification as well as filtration of ingredients and final product. One purpose for all filtration applications in the biopharmaceutical industry is the protection of the process and product from bacterial, endotoxin or other contamination. However, most filtration that is specifically for endotoxin removal or control is done at the end of the process, just before final packaging of the drug product.
Endotoxin Removal Technologies
Moving from endotoxin levels of 100EU/ml or higher to levels of 2EU/ml of lower requires several activities. Monitoring the sources of raw materials and water used in processes to assure the highest quality and lowest potential contamination is an obvious step. We will not discuss those activities as part of this paper. Preventive activities can limit the amount of contamination in any system, but will not prevent the introduction of bacteria and endotoxin.
Once bacteria and the endotoxin from the bacteria invade any system, aggressive steps must be taken to either a) remove the bacteria and endotoxins from the system or b) prevent the passage of bacteria and/or endotoxin from the system to the final product and/or patient. Removal or destruction of bacteria and endotoxin is the goal, and there are only a few methods of accomplishing that goal. Those are heat, chemical treatment and filtration and separation.
Endotoxin and pyrogen removal/destruction can be accomplished through the use of very high temperatures. The process essentially burns the bacteria, endotoxin and pyrogen away. This is a common practice for objects made of glass, metal or high temperature plastics. However, using high temperatures for liquids is not possible. Chemical destruction of bacteria and endotoxin is also possible.
Strong oxidizing chemicals like Minncare Cold Sterilant (peracetic acid and hydrogen peroxide mix) can destroy bacteria and oxidize most organic contaminants. This is effective for the immediate removal and destruction of any contaminants in a system, but will not prevent further contamination unless continuously used. This is impractical in most water or process systems.
Filtration and Separation Systems
Filtration and separation systems provide a continuous barrier to the spread of bacteria or endotoxins from systems contaminated with either. There are two alternative filtration and separation technologies for the removal of endotoxin from liquids, size exclusion and adsorption. Size Exclusion Size exclusion requires the filtration media to have pore sizes smaller than the particles/molecules to be removed. Semi-permeable membranes, similar to those used for reverse osmosis, are required to remove endotoxin from a fluid stream. The very small size of endotoxin (Molecular Weight of 1,000 to 30,000) forces the use of ultrafiltration and even nanofiltration membranes to exclude endotoxins.
The advantage of size exclusion is the absolute removal of the endotoxin. Using a membrane that rejects all molecules with a molecular weight of 1,000 or higher will almost guarantee an endotoxin-free liquid. However, there are some significant disadvantages to using these types of membranes. First, the small molecular weight requires the use of tangential flow, or “crossflow” membrane systems.
The membranes in these systems divide the incoming flow into a purified permeate stream that passes through the membrane and a concentrate stream that flows across the membrane surface to clean it of particulates and other foulants. The concentrate stream is often recirculated back through the membrane system, but at least a portion of the stream is sent to drain with the excess particulates and potential foulants. Unless the fluid is water, the loss of the concentrate stream means the loss of product.
The second disadvantage of using size exclusion systems is the potential for the rejection of product along with endotoxin. Any membrane that will block the passage of small particles and 1,000 molecular weight molecules will also block the passage of any large molecule product. In the case of biological products, it is entirely possible that the therapeutic product will be blocked by the membrane, defeating the purpose of the system. The form of the membrane devices used in tangential flow systems can also have an effect on the performance of the systems. Spiral wound membrane elements use flat sheets of membrane that are wrapped around a core tube and sealed with adhesives.
The flow path of the fluid is through a tortuous path to reach the membrane surface, through the membrane after shearing and then along a permeate carrier at low pressure until collection in the core tube and transport out of the system. This complex flow path subjects the contents of the fluid to forces that may cause the substances of interest to the manufacturer, especially biological substances, to break down during the separation and purification process.
Hollow fiber membranes create fewer stresses on the fluids with a simple path along and through the membrane. Though the possibility of blocking the product from passing through the membrane still exists, in cases where the product can pass through the membrane, it will be less stressed with hollow fiber membrane systems. Regardless of the membrane type, both the initial cost and the operating the cost of a tangential flow system can be quite high.
The flow control and pumping system required is complex and requires an expensive monitoring and control system. The tangential flow membrane devices also retain a significant amount of product, in addition to the product that must be sent to drain as part of the process. If the system is in a biopharmaceutical process system, then that product must be removed from the system and the system cleaned and sterilized before it can be used for another batch of product. The cleaning and sterilization process makes system use both time-consuming and expensive.
There are two methods of capturing endotoxin through adsorption, without using size exclusion. The first is by electrostatic charge and the other is by chemical affinity. Electrostatically charged membranes, particularly membranes and media with a positive charge(positive Zeta potential), are often used to remove organic contaminants.
Most organic molecules have a negative charge, and are attracted to the positively charged media or membrane. Examples of charged media and membranes include the Cuno Zetapor and Pall Posidyne charged nylon membrane cartridge and capsule filters and the Cuno Zeta Plus charged lenticular media filters. Charged media can be effective in removing endotoxin from a fluid stream. However, any other negatively charged molecules will also be removed from the fluid.
That means that the molecules of a biological product, which are likely to have a negative charge, will be removed along with the endotoxin, if a charged media is employed. Charged media also is given its charge through a modification of the surface of the media or membrane. That surface modification affects only a limited number of sites on the surface of the membrane or media. Not all of the surface will be charged, making it possible to exhaust the charge during a filtration process.
Affinity adsorption is different from charge adsorption in two very important ways. First, the effects of the affinity are limited to specific chemicals or molecular structures. This differs from charge-based adsorption in that only certain substances will be attracted to the surface of the membrane or media, not all substances with a certain charge.
Second, the affinity is the result of the basic molecular structure of the membrane or media, not the result of a modification of the material. That makes the entire surface of the membrane or media available for the attachment of the targeted substance, not just certain sites that have been successfully modified. An example of an affinity membrane is the Polyphen® Hollow Fiber membrane used in FiberFlo brand cartridge and capsule filters.
FiberFlo hollow fiber filters use adsorption to remove endotoxin from fluids. The polysulfone hollow fiber membrane has an affinity for the polysaccharide site on the endotoxin. Therefore, the result of endotoxin coming in contact with the Polyphen membrane is the permanent bonding of the endotoxin to the membrane. Because the adsorption is the result of the affinity of the membrane for endotoxin, the membrane still allows the passage of proteins and other large molecules. This is a significant advantage over charged membrane or media. Charged membrane or media can adsorb proteins and other large molecules, preventing their passage.
The FiberFlo filters have been validated to remove 100% of endotoxin at challenge rates up to 5EU/ml. With test sensitivity to 0.06EU/ml, Polyphen membrane is tested to remove different levels of endotoxin with each filter micron rating. The FiberFlo 50, 0.05 micron absolute rated filters are validated to remove 100% of endotoxin at levels of up to 5EU/ml. The FiberFlo 100, 0.1 micron absolute rated filters are validated to remove 100% of endotoxin at levels up to 1EU/ml. These levels of endotoxin are commonly found in water systems and process fluid streams, regardless of the diligence of the contamination control personnel.
As discussed above, the hollow fiber structure of the FiberFlo hollow fiber filters subjects the fluid being filtered to fewer stresses. The relatively simple and gentle flow path through the hollow fiber filter means that the solution contents are less likely to be damaged by the filtration process. Hollow fibers are also compact in size, allowing more surface area in the filter, which can reduce the flow rate per square area of membrane and increase the throughput life of the filter.
All of the technical advantages, the endotoxin adsorption capabilities of the FiberFlo filters, plus a robust contamination prevention system, can prevent the contamination of products with endotoxin while improving system life and product quality. Conclusion Preventing the introduction of endotoxin to patients is a high priority in all medical and biopharmaceutical operations. In order to accomplish this priority, several steps are needed, including:
1. Diligent monitoring of bacterial and endotoxin levels in raw materials used in all processes is critical for the limiting of system and product contamination.
2. Aggressive remediation action is required when bacteria or endotoxin contamination is detected, including system disinfection or sterilization.
3. A contamination control system design that utilizes a combination of raw material monitoring and built-in protection of the product and system is more likely to prevent the contamination of final products with either bacteria or endotoxin.
Raw material monitoring programs can be simple or complex, and are not discussed in detail here. System and final product protection, in the form of filtration technologies, provides a reliable barrier that will prevent the contamination of final product or contamination of a treatment technology. The filtration technologies available each have advantages and disadvantages. After an examination of the features of each technology, it is clear that the FiberFlo hollow fiber technology has the most advantages.
The FiberFlo filters use the hollow fiber technology that is less likely to stress the fluid and cause chemical breakdown in a process solution. FiberFlo filters are “normal” flow filters and avoid the issues inherent in tangential flow filtration devices. The FiberFlo filters have a higher effective filtration area per device than most flat sheet membrane filters, allowing higher flows and throughputs. The FiberFlo membrane also uses chemical affinity to adsorb endotoxin, with more capacity to hold endotoxin than filters using a charge mechanism. Endotoxin removal by FiberFlo filters is also supported by substantial documentation.
The membrane performance is validated. Using FiberFlo filters in conjunction with due diligence in contamination control can be very effective in preventing the contamination of products and/or systems with bacteria or endotoxin.