Depyrogenation of pharma glass

When I decided a couple years ago to start writing more regularly about pharmaceutical glass packaging, I suppose my intended audience was more on the pharma/biotech side of things.?It’s been interesting to see that a significant fraction of my readers also appear to be suppliers of primary packaging components for sterile drug products, and so I’ve also tried over time to include some application-specific posts that might be more relevant to their interests.?For example, a colleague involved in the container manufacturing side of our business once asked me about why our pharma customers need to run vials through a depyrogenation process prior to filling the drug product.?It seems like the annealing process (see Footnote 1) used to relax stress in a freshly formed glass container was the source of confusion.?Why do we need to heat the vials a second time when they’re already experiencing temperatures in excess of 500°C during the initial manufacturing process?

A primary goal of an aseptic fill-finish operation is to manufacture drug products that are safe and effective.?Safe drug products should be free of contaminants such as microbes and particulates.?In this case, we’re focused on contamination that is intrinsic to bulk-packed glass vials at the time of receipt by the aseptic manufacturing site.?Vials are unloaded and initially washed to remove particulate and some level of bioburden.?Next, the vials are subjected to dry heat (see Footnote 2).?The first objective of this process is to sterilize any remaining microbial contamination.?The concept of sterilization is easily understood here – it’s simply the absence of any living microorganisms on the surfaces of the glass vial.?The heat is literally “cooking” the microbial contamination to death.?However, sterility does not automatically equate to a container that is safe for use.?Sub-cellular components known as “pyrogens” remaining on a sterilized surface can still trigger a biological response ranging from a mild fever (see Footnote 3) to deadly septic shock.

For simplicity, let’s assume that the major pyrogen of concern is a molecule called lipopolysaccharide (LPS).?LPS is also known as an endotoxin, a substance intrinsic to bacterial cell walls (see Footnote 4) that is capable of eliciting an inflammatory response.?Figure 1 is a simplified schematic of the cellular envelope for Gram-negative bacteria.?You can see LPS molecules anchored within the outer membrane, specifically the exterior-facing half of the membrane known as the “outer leaflet”.?LPS functions include but are not limited to providing structural stability and reducing permeability of the cell membrane to inhibitory molecules such as antibiotics.

In contrast, an exotoxin is a substance that is actively secreted by virulent bacteria, generally with the goal of facilitating the spread or ongoing maintenance of an infection.?For example, the cholera toxin secreted by Vibrio cholerae causes an increased efflux of chlorine ions and reduced influx of sodium ions in affected cells.?The elevated concentration of salt triggers the osmotic flow of large volumes of water into the intestine, thereby forcing the infected host to help spread the bacteria into the environment.?The exo versus endo distinction is important – why would an endotoxin (i.e., a substance that is not specifically used by bacteria against an infected host) still have the ability to make us ill?

Our immune system has evolved to constantly monitor for signs of infection.?For example, innate immunity relies on pathogen recognition receptors (PRRs) that are capable of detecting the presence of molecular patterns typically associated with microbial invaders.?Toll-like receptors (TLRs) are one class of PRRs in mammals that are capable of detecting signature molecules associated with viruses, bacteria, fungi, and other parasites.?In particular, TLR4 (with some help from other proteins) is capable of binding to the lipid component of the LPS molecule.?The activation of TLR4 by LPS triggers a complex series of biochemical reactions that can result in an inflammatory response and the marshalling of our immune system to combat the perceived infection.?The strength of this response is dependent on the amount of detected LPS.?An implantable medical device or a parenteral drug contaminated by a high level of endotoxin (and, let’s not forget, all while being initially sterile) can produce an intense, uncontrolled inflammatory response at a systemic level that causes tissue damage, loss of blood pressure, and ultimately death through a process called sepsis or septic shock.?Multiple factors must be considered to minimize the pyrogen load – the components of the drug formulation, the primary packaging components, and the syringe used for injection.?Fortunately, the process for achieving depyrogenation of glass vials is straightforward in principle.?Dry heat is capable of decomposing the LPS structure to an extent that prevents recognition by TLR4.

USP <1228.1> includes basic information on dry heat depyrogenation, including procedures for process validation and process control (see Footnote 5).?The general objective is to demonstrate that the depyrogenation process is capable of achieving a so-called “3-log reduction” in endotoxin load – i.e., a 1,000-fold reduction.?The introduction of <1228.1> states that typical depyrogenation processes are run at temperatures between approximately 170°C up to about 400°C.?This is a pretty extreme range, in my experience.?The time needed to achieve a 3-log reduction at the lowest temperature would be excessively long and limit the throughput of the fill-finish operation, while operating at the highest temperature would be quite energy intensive.?In practice, a majority of the customers that I have worked with generally run their depyrogenation tunnels somewhere between 300°C to 350°C.?I should emphasize that depyrogenation is not a “one size fits all” process.?At a minimum, validated processes must be developed that bracket the expected range of container formats (small to large), which in turn can impact the thermal load and heat transfer behavior that governs inactivation of endotoxin.?

The temperature range used for depyrogenation impacts more than just residual endotoxin.? Physically and chemically bound species (e.g., water molecules and hydroxyl groups, respectively)) can be removed from the surfaces of glass containers.? This increases the surface energy of the glass containers, leading to behavior such as increased friction.? Some contaminants that are not fully removed during the washing process can also get “baked on” during depyrogenation.? For example, I’ve seen cases in which small pieces of plastic (presumably residue from the plastic shrink wrap used as secondary packaging material for bulk vials) can char in the depyrogenation tunnel to form dark colored deposits that are firmly adhered to the glass container.

Finally, let’s circle back to the start of this post and revisit the question – why do we need to heat glass vials a second time when they have already been annealed?? Technically speaking, they don’t – the temperatures involved with annealing should be more than sufficient to sterilize and depyrogenate a glass vial.? However, that vial now needs to be handled and packaged in an environment that limits further contamination.? You might also be thinking about ready-to-use (RTU) components, which arrive to the fill-finish site without requiring washing and sterilization.? It turns out that RTU vials are often made today in a batch process that still requires a second depyrogenation step.? In other words, vials are made and then held in inventory in the “bulk” form.? Those vials are then re-processed by washing and depyrogenation before being placed into secondary packaging that undergoes a terminal sterilization step.? One could in theory design a continuous RTU process in which vials exit an annealer and go straight into secondary packaging.? Perhaps we’ll see this actually happen as RTU container adoption continues to grow over time.

Questions or comments??– please leave them below or feel free to directly contact me.

Footnotes

1. Refer to my posts on glass viscosity and residual stress in containers for more information.

2.?Use of the qualifier “dry” is intentional here.?It’s meant to distinguish from processes that use a combination of moisture and heat, such as terminal sterilization in an autoclave.

3.?The etymology of the word pyrogen is directly linked to the ability to induce a fever.

4.?LPS is specifically associated with what are called Gram-negative bacteria, which are distinguished from Gram-positive bacteria by the general structure of their cellular envelope.

5.?The entire USP <1228.x> series of chapters covers other depyrogenation-related topics, including depyrogenation by filtration, chemical depyrogenation, depyrogenation by rinsing, etc.