Sterilization By Filtration Vs. Depyrogenation By Filtration For Parenteral Products
What is sterilization, and why is it essential for parenteral products?
Sterilization keeps patients safe from toxins and microbial illnesses when therapies or devices are consumed or used. Sterilization is any process that removes, kills, or deactivates all forms of life. Under the strictest definition of sterility, an item or product is sterile when there is the complete absence of viable microorganisms (bacteria, yeasts, viruses, and molds). For regulatory purposes, sterility is defined by acceptance criteria based on calculated contamination probability. An acceptable level of contamination risk for most items is the probability of a single contaminated product out of a million manufactured products. However, sterility criteria may be more stringent or lax depending upon the intended use of the medical device or product. Sterile products that undergo sterilization are often chemical, heat, or filter sterilized. Sterilization kills any microorganisms products collect during manufacturing. For chemical and heat sterilization, sterilization occurs after the product is placed in its final packaging. The product is often filtered and then aseptically filled into a sterile container for sterilization by filtration. This article covers filtration techniques for parenteral products including filtration sterilization vs. depyrogenation by filtration (endotoxin removal by filtration).
What is depyrogenation, and why is depyrogenation needed?
Parenteral products must be sterile and pyrogen-free. Even if a product is sterile, it can still contain pyrogens. Depyrogenation is a process for endotoxin removal. The most prevalent and problematic pyrogens are the bacterial endotoxins found in the outer cell walls of gram-negative bacteria. Thus, depyrogenation is a process that will either destroy or remove endotoxins. Products can accumulate pyrogens from raw materials or other parts of the manufacturing process. The best pyrogen removal or destruction processes are product-dependent. Standard depyrogenation methods are dry heat, rinsing, and filtration.
What are pyrogens, endotoxins, and lipopolysaccharide (LPS)?
Pyrogens are molecules or substances that cause a feverous reaction when they enter the human body. Endotoxins are the most common type of pyrogen. Endotoxins come from the cell walls of gram-negative bacteria. The endotoxins themselves are molecules with both fat components and complex sugar components. The presence of fat and sugar components is why endotoxins are also known in scientific literature as lipopolysaccharides (LPS). LPS is the biologically active portion of an endotoxin. In other words, LPS is the part of the endotoxin that triggers the innate immune system and causes illness in humans. Parenteral products and devices are contaminated with endotoxin through Gram-negative bacterial cells or cell wall fragments containing LPS. Lipopolysaccharide’s structure allows it to stick to hydrophobic (water-repellant) and hydrophilic (water-loving) surfaces. Thus, LPS components easily attach to molecules and proteins in solutions or material surfaces, causing endotoxin contamination. LPS also sticks to itself to form LPS chains known as aggregates.
What is sterilization by filtration?
Sterilization by filtration is a “cold” method of sterilization that removes microbes instead of killing them. Since sterilization by filtration works by removing microbes, sterilization by filtration (also called sterile filtration) is the only sterilization method that doesn’t rely on an elevated temperature, toxic chemicals, or another form of energy (such as gamma radiation) to destroy microorganisms. Sterile filtration is excellent for products that cannot be sterilized with heat or products containing a biological agent, such as an antibody or enzyme.
What is depyrogenation by filtration?
Depyrogenation by filtration is a filtration process that removes endotoxins from solutions. Endotoxin removal occurs primarily through adsorption and size exclusion for filtration processes. Bacterial endotoxins are extremely small. Thus, endotoxin removal through size exclusion requires an ultrafiltration membrane.
How is sterilization by filtration performed?
Simply speaking, sterilization by filtration is performed by flowing a non-sterile product through a sterile filter. The sterile filter then removes particulate matter and microorganisms from the non-sterile liquid formulation. In eliminating particulate matter and microorganisms, the filter sterilizes the product. Filters sterilize through a combination of sieving, screening, entrapment, impaction, and electrostatic attraction of particles (including microbes). Particles are collected on the surface of the filter during sieving and screening. In contrast, entrapment occurs when particles smaller than filter pores lodge themselves within the filter’s passageway. Electrostatic attraction absorbs particles of opposite charge to the filter surface. Note that entrapped particles, or particles held with an electrostatic charge, can be dislodged and end up in the product filtrate when flow rates or pressure is increased or varied. Keep this in mind for viscous products.
Membrane filters are used for sterilizing solutions because of their ability to be nonreactive with most products, retain particles, and not shed debris. Products with peptides should use polysulfone and polyvinylidene difluoride filters to prevent accidental protein adsorption to the filter membrane. Often filter membranes are composed of cellulose esters, nylon, polysulfone, polycarbonate, PVDF, or polytetrafluoroethylene. Structurally, filters are designed to increase surface area and thus increase the flow rate. Standard filter designs are flat membranes, pleated cylinders, and cartridge structures. During use, the product enters the outside of the filter cartridge with applied positive pressure forcing the fluid inward through the filter. The sterile filtrate then exits from the center of the cartridge. The filter and housing are steam sterilized before product filtration, typically by steam-in-place (SIP) systems. Pressurization during SIP sterilization must be gradual to maintain filter integrity. Often filters are dried with compressed gas after sterilization and before use.
For sterile filtration, the product must not adversely affect the particle retention of the filter. Also, the product must not cause the filter to leach any materials into the product. Filter manufacturers provide information on the liquid volume a filter can flush before oxidizable substances are released. Further, filter manufacturers provide data on extractables obtained with exposure to various solvents. Common filter extractables include oligomers, mold release agents, antioxidants, wetting agents, manufacturing debris, plasticizers, and 0-ring material. Sometimes protein biologics will bind to the filter material. In this case, a pre-flush step may be used before filtration to occupy any available binding sites for the proteins and remove any potential extractables.
Factors affecting sterile filtration efficiency & particle retention:
- Type of particle—Particle source (metal, microbe, etc.), shape, charge, and size.
- Filter material—Filter composition affects the charge-related attraction of particles, including microorganisms. Van der Waals forces, hydrogen bonding, and hydrophobic attraction are filter properties affecting charge-related particle attraction.
- Filter membrane thickness—Filter thickness slows the fluid flow and affects particle adsorption. However, a rough and thick membrane can be as efficient as a fine, thin one depending on the filtered product.
- Filter porosity—The smaller the porosity, the greater the retention of microorganisms and the slower the flow rate. Product-filter incompatibilities increase as filter porosity decreases.
- Temperature—Higher temperatures increase microbial proliferation and viability, thus increasing the possibility of microbial adsorption with the filter’s pore walls.
- Type of fluid being filtered—Highly viscous products will require applied pressure to move the product through the filter. Thus, microbes or therapeutic biologics in viscous products will experience higher shear forces during filtration. Viscous products will disrupt particle adsorption to the filter membrane but will not affect size exclusion. Surface-active agents in products will bind to solid surfaces and can reduce or prevent bacterial adsorption to the filter. However, surface-active agents will not affect filter membrane structure or microbe size.
- Applied pressure, flow rate, and usage time—Pressure, flow rate, or usage time increases can harm filter integrity and affect microbe sizes. Higher pressures, flow rates, and usage times decrease filter integrity, especially when outside the manufacturer’s recommendations. Generally, commercially used membrane sterilizing filters can be used for up to one week without changes in functionality.
How is depyrogenation by filtration performed?
Depyrogenation filtration processes are often performed on solutions containing proteins and peptides. Factors to consider when selecting your filters for endotoxin removal are the protein types in solution, protein concentration, electrolyte concentration, pH buffer system, molecular weight and isoelectric point (pI) of the protein, filtration flow rate, and protein aggregation potential. Endotoxin removal within liquids may be accomplished employing filtration through more than one filter type. The primary filter types used for depyrogenation are described below. These filters are specific to depyrogenation and not intended for microorganism removal. However, some depyrogenation filters can retain certain microorganisms.
The following six filtration types are used for sterile product depyrogenation:
#1: Microporous Membrane Filters
Microporous membranes with pore size or retention ratings between 1.0 and 0.1 microns (μm) effectively remove intact bacteria via size exclusion. Filtering freshly prepared solutions with microporous membranes can remove microbes and prevent endotoxin formation. Endotoxins themselves are much smaller than microporous membrane filter pores. However, endotoxins are negatively charged and can be removed through adsorption with positively charged membranes.
#2: Reverse Osmosis Membranes
Reverse osmosis (RO) membranes are the tightest size separation filters and can remove dissolved salts and sugars from water. Essentially everything, including pyrogens, is extracted via size exclusion. RO membranes filter best at high pressure (200–1000 psi), which allows the filter to overcome osmotic pressure. RO membranes can be ultrafiltration system components (see #3 below) or single polymeric membranes.
#3: Ultrafiltration Membranes
Ultrafiltration (UF) membranes have pore sizes ranging from one to one hundred nanometers. These filters are traditionally rated by molecular weight cut-off (MWCO) and are made of polymeric porous structures. Since the endotoxin subunit, LPS, is about 10–20 kilo Daltons (kDa), membranes of 6–10 kDa MWCO are used for size exclusion depyrogenation. However, LPS is commonly in aggregated forms weighing 300 to 1000 kDa so that endotoxin can be successfully removed by MWCO membranes of 30–100 kDa. UF depyrogenation capability can be boosted using the adsorptive capabilities of hydrophobic membranes. Overall, UF is not recommended for depyrogenation of solutions containing large proteins but is highly effective at removing endotoxins for small molecule drugs, buffers, electrolytes, antibiotics, and antifungal agents.
#4: Charge-Modified Depth Filters
Depth filters function through size exclusion, via sieving or entrapment, and adsorption, via electrokinetic or hydrophobic interactions. Charge-modified depth filters are more advantageous in some situations than positively charged membrane filters because they allow less endotoxin to pass through the filtered product when the filter reaches its endotoxin filtration limit. Further, charge-modified depth filters are highly effective with a 4-log or 5-log reduction in endotoxin removal from products. In tricky filtrations, cost savings can be gained by using an appropriate depth filter.
#5: Activated Carbon Depth Filters
Activated carbon is a filter adsorbent that binds colors, odors, bacterial endotoxins, and nucleic acids. As one of the best endotoxin removal agents, activated carbon can reduce endotoxin from liquids by a 4-log or 5-log reduction. However, active carbon provides nonspecific adsorption and can remove other important elements or molecules from a liquid during filtration.
#6: Membrane Adsorbers
When the therapeutic protein in solution has the same molecular weight range as endotoxins (10–20 kDa), ultrafiltration will not work to separate endotoxins from the therapeutic protein formulation. Charge-modified membrane adsorbers are used instead of ultrafiltration in this case. In membrane adsorbers, ligands attached to the filter membrane’s surface provide endotoxin removal. Two primary strategies are used for product pyrogen removal with these filters: 1) using a strongly basic anion exchanger (type Q) or 2) using a strongly acidic ion exchanger (type S). In type Q membrane adsorbers, the basic exchanger (quaternary amine) is in a buffer with lower pH than the isoelectric point of the therapeutic protein. Type Q adsorbers bind endotoxin to the charged membrane surface and allow the therapeutic protein to pass through the membrane. In contrast, type S adsorbers use an acidic ion exchanger (also in a buffer with a pH lower than the isoelectric point of the therapeutic protein) to bind the therapeutic protein and allow the endotoxin to pass through the membrane. After initial type S filtration, the bound protein can be eluted using special buffers. Type S filters are commonly used to process monoclonal antibodies and recombinant proteins. Type S and Type Q filters are highly effective and provide a 4-log or greater reduction in endotoxins from products. Mixed-mode membrane adsorbers can be used as an alternative to type Q and type S membrane adsorbers. These membrane adsorbers only provide endotoxin removal on 3-log or 4-log reduction scale. However, mixed-mode membranes are superior at depyrogenation of protein solutions with high salt concentrations (e.g., 100–500 mM) compared to Q-type and S-type filters. In mixed-mode membranes, anionic and hydrophobic chemistries are used to bind endotoxins to the membrane surface. At the same time, salt and pH balancing flushes harness the power of charge repulsion to allow therapeutic proteins to flow through the membrane.
What are the similarities and differences between filtration processes used to depyrogenate products vs. sterilize them?
Filtration for depyrogenation is more complex than filtration for sterility due to the small sizes of endotoxins. For many sterile filtration options, a simple 0.2 µm membrane filter will be sufficient. However, depyrogenation requires a charge to the membrane filter or a completely different filter (or filtration method) altogether. For most protein solutions, a charge depth filter, ultrafiltration, or membrane absorber will need to be used to depyrogenate liquid products. Depyrogenation and sterilization processes that utilize filtration come with similar filtration difficulties. For example, it is difficult to define an appropriate “worst-case” condition for filter validations, whether they are depyrogenation or sterilization validations. Further, the filter’s effects on the product are a primary consideration when selecting an appropriate filter for sterilization or depyrogenation. Overall, it is critical to consider filtration techniques for sterilization and depyrogenation separately, as filters that remove bacteria do not always remove pyrogens. Also, pyrogen removal techniques may not work well for bacteria removal.
Summary
Parenteral products must be sterile and pyrogen-free. Sterilization is any process that removes, kills, or deactivates microbes, whereas depyrogenation is a process that removes pyrogens. The most prevalent and problematic pyrogens are the bacterial endotoxins found in the outer cell walls of gram-negative bacteria. Filtration is a technique that can provide heat-free depyrogenation and sterilization for parenteral products. There are several factors affecting sterile filtration efficiency & particle retention. These factors include particle type, filter material, filter membrane thickness, filter porosity, filtration temperature, the type of fluid being filtered, filtration pressure, filtration rate, and filter usage time. Filtration for depyrogenation is more complex than filtration for sterility due to the small sizes of endotoxins. The most prominent types of filters used for depyrogenation are: 1) microporous membrane filters, 2) reverse osmosis membranes, 3) ultrafiltration membranes, 4) charge-modified depth filters, 5) activated carbon depth filters, and 6) membrane adsorbers. All in all, ensure you choose a contract manufacturing and testing organization that can provide appropriate sterility testing, bacterial endotoxin testing, and depyrogenation for your product needs.
MycoScience is a contract manufacturing organization specializing in sterile syringe and vial filling. MycoScience also offers Preservative Efficacy Testing, Sterilization Validations, Bioburden Testing, Cleaning Validations, Microbial Aerosol Challenge Testing, Accelerated Aging, Microbiology Testing, Cytotoxicity Testing, Bacterial Endotoxin Testing, EO Residual Testing, Package Integrity Testing & Environmental Monitoring services medical devices and allied industries. MycoScience is an ISO 13485 certified facility.
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