Identifying, managing and controlling leachables and extractables is one of the foremost challenges facing biomanufacturers who want to use single-use systems to increase product throughput. Those seeking to fully realize the potential of single-use solutions must ensure that materials used in single-use systems, as well as disposable process containers, do not end up in the process stream and contaminate products being manufactured.

Scientific, quality control and regulatory approaches to control the risk of foreign substances inadvertently added to products for human consumption are evolving. They include toxicological studies that establish safe doses of leachables (either directly or indirectly), use of materials cleared after rigorous study, careful attention to proper manufacturing practices and quality design, and regulatory guidance.

Currently, biopharmaceutical manufacturers are working with toxicology approaches derived in large part on existing food and beverage regulations. New risk assessment approaches, more appropriate to the rapidly expanding use of single-use solutions in biopharmaceutical processing, will need to be developed. These should adhere to common sense solutions that neither overstate nor understate leachate concerns.

What are Extractables and Leachables?

Extractables are substances that might leach from a material’s surface into a solution because the substance has some potential to migrate under some conditions. Leachables are substances that actually migrate from the material surface into the solution under actual use conditions.

The addition of leachables to a pharmaceutical product stream has several potential negative effects, but the one most relevant to the use of single-use solutions is when the leachable is itself toxic and thereby poses a health risk to anyone exposed to it. This is the issue primarily discussed here. Other negative effects include the possibility that the leachable may interact with the drug product being produced and alter its stability and potency, or that the leachable could interfere with an assay that is critical to measuring an important property of the product.

Toxicity: the Dose Makes the Poison

When analyzing the potential toxicological risk of leachables, it is fitting to begin with the nearly 500-year-old notion, succinctly put by Paracelsus, who said, “All substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy.” Or, as we commonly put it, “The dose makes the poison.”

So, a recurring goal in toxicology is to establish this relationship between dose and response. One of the key tasks is to determine the substance’s threshold value, the dose below which no response occurs or can be measured.

For some substances, particularly residual solvents, the process used to establish a permitted daily exposure (PDE) to pharmaceutical impurity levels is done as part of very straightforward toxicological evaluations. Here, the impurity chemical is known, chronic animal studies of the toxicity of the chemical are available, and there is no indication that the chemical is genotoxic. Sometimes however, even these simpler toxicological studies can result in different conclusions when performed by different individuals or organizations. For example, Table 1 shows the conclusions of two toxicological analyses by the ICH Q3C committee and one by the EPA (drinking water standards) on common chemicals.

Table 1: Comparison of PDE and MCL in three toxicological studies

A different toxicological risk analysis is required for potentially genotoxic (mutagenic) compounds, because there is no lower threshold concentration for genotoxic compounds for which the risk is considered zero. Risk assessments for genotoxic materials attempt to calculate the probability of increased levels of adverse events, most typically carcinogenic events, from a particular (lifetime) exposure.

An acceptable risk is often 1 in 10⁶ additional events over a lifetime. A less stringent level, 1 in 10⁵, is often proposed for pharmaceuticals because there is a positive benefit to the user that can compensate for the negative risk, whereas the more stringent level is generally proposed for food and beverages that have no such benefit.

The Threshold of Toxicological Concern

When the identity of the chemical impurity is either wholly or partially unknown, scientists have adopted the concept of the threshold of toxicological concern (TTC) to define a generic human exposure threshold value for broad groups of chemicals below which there would be no appreciable risk to human health.

The TTC approach yields safe daily intake information if the chemical can be placed into a specific class of chemicals with known toxicology. A few classes of compounds are excluded from the TTC based on their molecular structure, including high potency carcinogens, compounds with bioaccumulation concerns, potential type 1 allergens, long half-life toxins like metals and metal containing compounds, and steroids. Table 2 shows permitted daily exposures for various chemical classes using the TTC approach.

Table 2: Permitted daily exposures for chemical classes

The TTC approach is now being widely used for a variety of biopharmaceutical applications. As more and more structures and toxicological information are entered into toxicology databases, TTC will offer even more value to biopharmaceutical manufacturers.

Materials Cleared for Usein the Food & Beverages

It is quite common for disposable devices used in the pharmaceutical industry to be made with materials that have already received 21CFR clearance for use in the food and beverage industry. Referring to Title 21 of the Code of Federal Regulations, which regulates food additives in the U.S., the clearance is obtained after a rigorous toxicological examination under worst-case use conditions that identifies substances that may migrate into food from use of a food contact substance (FCS).

Achieving 21CFR clearance means that the FCS has undergone examination under conditions similar to those used in pharmaceutical applications. After obtaining the clearance, no changes can be made in the manufacturing process that could affect safety.

Under the “mixture doctrine,” a manufacturer is permitted to physically blend two different polymers or other combinations of substances if each has 21CFR clearance for its intended use. For those using single-use solutions for biopharmaceutical manufacturing, the mixture doctrine means that manufacturers who assemble multiple materials that each have 21CFR clearance and without using any other chemicals than the assembled device, are assumed to also have 21CFR clearance. The doctrine would therefore apply to filtration devices (molded parts, spun bonded supports, and membranes) as well as disposable process containers (multilayer films, tubing, and valves).

One caveat: if combining the substances results in a chemical reaction that forms a new substance, then the new combination does not have clearance. Where all parts of a mixture are cleared in some way but one is only cleared for a limited purpose, that limit applies to the combination.

There are limitations to the value of 21CFR-listed materials when performing safety risk assessments in pharmaceutical applications. First, most information submitted to the FDA

to obtain 21CFR clearance is not publicly available, and therefore any data contained in the submission for a food orbeverage cannot be reapplied towards a pharmaceutical application. Even the chemical identity of leachables from the evaluated FCS often remains unknown unless specified in the 21CFR listing.

While the FDA is currently in the process of consolidating 50 years of toxicity data into a publicly available database, the exact compositions of the submitted materials and their methods of manufacture are usually proprietary information and unlikely to be available. Also using 21CFR-cleared materials with solutions that have different solubility parameters than those used in the migration studies would mean that the original studies cannot be used.

Another limitation is that toxicity can vary depending on the route of administration, for not all drugs are taken orally, as food and beverages are. Nonetheless, even with these potential limitations, there are significant advantages of using 21CFR-cleared materials in biomanufacturing processes.

Gamma-Derived Leachables

Some leachables are chemically derived from extractables. For example, using gamma irradiation to sterilize polymeric materials causes the formation of free radicals, which can affect both the base polymer and any additives.

Even materials like polyethylene, considered to be gamma stable, can generate hundreds of gamma-derived leachables in ultra-trace levels. Because these leachables are structurally related to the original extractables present, typically either fragments or oxidative derivatives, the risk assessment of such leachables can use the TTC approach, based on the relationships to the original extractable structures.

The very low concentrations observed as gamma-derived leachables can be confirmed by specific measurements. In fact several polymeric films have been approved for direct contact with food during gamma sterilization (and afterward), after the FDA found that gamma-

derived leachables are generally well below the Thresholdof Regulation.

Regulatory Guidance inPharmaceutical Applications

Currently applicable FDA guidance documents do not directly address impurities from in-process leachables, but merely say extraneous contamination should be controlled by Good Manufacturing Practices. The most specific FDA guidance in the area of leachables relates to final container closure. Guidance on upstream, in-process leachables is less detailed, since the risk is lower, because of purification steps that occur downstream of the addition of the leachables.

One of the common difficulties in the use of polymeric materials is that the commercial lifetime of any polymeric material, or one of its components, is likely to be shorter than the commercial lifetime of a successful pharmaceutical drug. To address this issue, the EU formed the Polymer Forum to foster better communication and strategies between polymer and pharmaceutical manufacturers.

Quality by Design

One relatively recent approach that applies to extractables and leachables risk assessments is the use of the quality by design (QbD) approach to manufacturing. The goal of QbD is to design in the quality of the final product by having an understanding of all critical parameters and implementing robust manufacturing processes to control those parameters, as opposed to attempting to “test in” the quality from an unstable, poorly understood manufacturing process.

Figure 1 shows the preferred QbD process for achieving safety. Each level is sized to show the degree to which it lowers the risk that leachables will affect safety. The base of the pyramid is the responsibility of the tool manufacturer – this is where most of the safety is built in. The green levels represent application-specific steps that only the tool’s user can perform. The brown level represents steps that both the tool’s manufacturer and user can perform. The manufacturer of the tool would tend to perform generic analytical testing, whereas the end user is more likely to perform analytical testing more closely aligned with their application of the tool.

Figure 1: The preferred QbD process for achieving safety

Common SenseShould Play a Big Role

In the pharmaceutical arena there have been a few examples of leachables that potentially might affect patient health, virtually all from container closures. Examples in the past few decades have included polycyclic aromatic hydrocarbons from carbon black fillers in elastomers, N-nitrosoamines or mercaptothiazole in rubbers, and diethylhexylphthalates from plasticized PVC blood and intravenous bags and tubing. Even permeation of leachables from labels and their adhesives has been observed.

There are fewer examples of leachables in the biopharmaceutical arena, possibly because of the relatively short time that they have been manufactured. The issues in biopharmaceutical seem more centered on Active Pharmaceutical Ingredient (API) interactions with leachables, and less about potential direct toxicological issues, undoubtedly due to the greater inherent instability of biologicals relative to traditional small molecule pharmaceuticals. Nevertheless, there was one report in Europe of a rubber leachable after a formulation change that apparently caused an increased risk of red cell aplasia in patients receiving EPO therapy, but this was apparently a unique interaction between the leachable and the drug protein and not due to the leachable’s toxicity.

Because of their manufacturing process, cured elastomers have a much greater chance of having leachables with direct health risks than thermoplastics. Drug-leachable instability interactions are much more prevalent problems than direct leachable toxicity concerns. The higher risk of cured elastomer issues should be addressed by minimizing contact area and time, and/or selecting non-cured thermoplastic elastomers or over-molded elastomers. Drug stability studies should be performed early in the material evaluation process, and analytical leachables studies done to characterize the performance of acceptable materials.

In this era of spiraling healthcare costs, it is more important than ever that appropriate risk assessments are performed on leachables issues so that resources and costs can be aligned where science, knowledge and understanding tell us they should be focused. Common sense must be used to guide us towards best practices for leachables that do not rely on superficial, overly worst-case risk assessments. These will continue to evolve, based on improved scientific understanding of material science, solubility parameters, the impact of such sterilization procedures as gamma irradiation, application specific parameters, and relevant toxicology.

Thomas E. Stone, Ph.D., is a principal scientist at EMD Millipore in the Analytical Technologies Group and has been responsible for the technical leadership of EMD Millipore extractables and leachables strategy for more than 20 years. He can be reached at EMD Millipore at thomas.stone@merckgroup.com.