Special Considerations for Extractables and Leachables Testing in Biological Products

February 2, 2015
Randi Hernandez

Randi Hernandez was science editor at Pharmaceutical Technology from September 2014 to May 2017.

Pharmaceutical Technology, Pharmaceutical Technology-02-02-2015, Volume 39, Issue 2

New guidelines focused on the materials of construction in biologic therapy packaging will help vendors prepare comprehensive extractable and leachable testing strategies.


Leachables from plastics in production equipment and packaging can be dangerous. Leachables can interact with active substances in biopharmaceuticals, and even change their physicochemical properties, often resulting in immunogenic reactions.

Pharmaceutical Technology spoke to Andrea Straka, senior specialist, technical customer support at West Pharmaceutical Services; Dr. Thomas Lehman, director of extractables and leachables and method development at Eurofins Lancaster Laboratories; Dr. Wayland Rushing, senior scientific advisor at ABC Laboratories; Tina S. Morris, vice-president of biologics and biotechnology, and Desmond Hunt, senior scientific liason for general chapters, both from the United States Pharmacopeial Convention (USP), about current trends in the extractables and leachables space and the specific considerations for leachables when dealing with biologic products.

Leachables in biologicsPharmTech: What are the special considerations for leachables when dealing with biologics?

Hunt and Morris (USP): With biologics, which are very large proteins with both hydrophilic and hydrophobic areas, there are many potential sites where leachables could bind to the proteins, leading to unfolding, truncation, aggregation, and precipitation. Also, there are other factors than just biologics that should be considered, such as patient safety issues around the leachable (which could be toxic or carcinogenic), impact of the leachable on efficacy of the drug product, and impact of the leachable on the packaging system itself.

Lehman (Eurofins Lancaster Laboratories): Leachables for biologics may come from a variety of materials, including final packaging or delivery devices and single-use systems utilized during manufacturing. When evaluating leachables, one must assess contact time and temperature with each material, as well as evaluate the potential impact the product matrix may have on solubilizing leachable compounds. Matrix components such as surfactants, pH extremes, or high ionic strengths may influence the product’s ability to promote leaching.

Straka (West Pharmaceutical Services): Biologics are large molecules with high surface areas and many sites of potential interaction. Structural and chemical modifications can occur as a result of a substance leaching from contact materials. Proteins are especially sensitive to metals that can bind or cause aggregation. It is important to characterize and understand the chemistry of biologic contact materials to enable the selection of best choices and mitigate potential interactions and issues related to stability.

Rushing (ABC Laboratories): When dealing with small molecules, the main concern for leachables is the specific toxicity/genotoxicity profiles. However, with biologics, there is the added concern of the ability of the leachable to interact with the biologic product itself. The first main concern involves leachable metals. Many biologics are very sensitive to metal content at levels below the currently accepted toxicological levels associated with the metals themselves.  As a result, depending on the specific biologic, additional monitoring of the metal content may be required. The second is the possibility of the leachable interacting directly with the biologic and causing post-translational modifications, thereby altering the nature of the biologic product. These alterations could result in the product losing its potency or other deleterious effects.

PharmTech: Which protein attributes decrease the potential impact of leachables?

Hunt and Morris (USP): It’s not a matter of the protein attributes, but a function of the formulations, which may have a lesser or greater propensity to extract leachables.

Lehman (Eurofins Lancaster Laboratories): One key attribute would be limiting the reactivity on the surface of the protein. Pegylation can be utilized to decrease the reactivity of the protein and improve its stability. In addition, the formulation of the biologic/drug product will have an effect on its ability to leach compounds from contact materials. The presence of surfactants, pH extremes, or ionic strength of the formulation can impact the ability of the product to solubilize leachable compounds from contact materials.

Timing of leachable analysesPharmTech: In what stage of manufacturing are leachable analyses most likely to occur? When is it most important to conduct a leachables analysis?

Hunt and Morris (USP): A leachable assessment is going to occur in two areas within the manufacturing suite:

  • With the manufacturing equipment itself, to determine what leachables may be coming from that equipment, and in what quantities. Further analysis would examine the levels and safety risks of various leachables identified.

  • With the packaging system, to examine what leachables are present, and in what quantities.

Lehman (Eurofins Lancaster Laboratories): It is important for materials and container closure systems to be evaluated for leachables throughout all phases of the manufacturing process; however, contact materials used in the final manufacturing steps, after filtration and dilution have been performed, pose the greatest risk for leachables. Therefore bioprocessing components in contact with the bulk drug substance or final drug product should be scrutinized more closely than components further upstream, as migrants from upstream components may be cleared through filtration or their levels reduced through dilution steps.

Straka (West Pharmaceutical Services): In my experience as a packaging expert working with drug manufacturers on extractables and leachables strategies, the best time to begin evaluations for leachables is as soon as the final package is chosen and the final manufacturing process is established. Since leachables are found in the final drug product, the actual analysis cannot truly begin until the contact materials are fully understood. The final packaging materials remain in contact with the finished drug for the longest period of time, so once they are chosen, the extractables evaluations should begin.

Rushing (ABC Laboratories): Historically, leachable analyses have been carried out as a requirement for product registration. As a result, many companies take the approach of performing E&L programs in conjunction with their Phase III stability studies. However, recently there seems to have been a shift of when E&L data evaluations are requested. Many sponsors have found themselves being asked for these data earlier in the development process. For example, several development programs over the last year were placed on clinical hold during Phase I trials until sponsors were able to provide E&L evaluations to FDA. I believe we will continue to see more requests during earlier phase development for E&L evaluations. These evaluations may not require a full leachable analysis, but rather a justification from the sponsor on why the contact materials were chosen and what assessments were performed to evaluate the potential risks from leachables. As a result, companies should implement E&L evaluation programs as early as possible to avoid potential delays.

PharmTech: Should leaching studies ever be conducted just prior to drug administration?

Hunt and Morris (USP): Leachable studies are very large, complex undertakings, and are not easily or rapidly conducted in a clinical setting. The goal of proper leachable control is to build quality into the process, which would be reflected in the quality of the end product.

Lehman (Eurofins Lancaster Laboratories): Any materials the product comes into contact with prior to administration should be evaluated from a risk perspective to determine if leachables could affect the drug product quality or surpass the limit of toxicological concern. Those components with higher risk should be fully evaluated for leachable compounds (e.g., an IV bag, tubing set, and infusion pump).

Leachables from the final container closure system should be evaluated prior to drug administration so appropriate evaluation of the data can occur. Leachable compounds should be monitored through established stability and assessed to ensure compatibility of the product with the final container closure system.

Straka (West Pharmaceutical Services): Leachables analysis should be performed on the final drug form as it would be administered to a patient. For example, if a lyophilized drug is being evaluated, it should be analyzed in its reconstituted form with the appropriate diluent. The packaging for the diluent should be profiled for extractables as well.

Detection challengesPharmTech: How can one be sure that all potential leachables in a product are being detected?

Hunt and Morris (USP): Most potential leachables are found in the extractable study-conducted under experimental conditions-during which potential chemical entities that could become a leachable are identified. Robust analytical techniques-those with the appropriate sensitivity to detect and identify such chemical entities-are crucial to the identification of potential leachables.

Lehman (Eurofins Lancaster Laboratories): Typical studies utilize a multitiered approach to fully characterize contact materials, beginning with a controlled extraction study, where a variety of extraction solvents and analytical techniques are utilized to characterize the material of interest. Next, a simulation study is conducted using drug product, placebo, or a model solvent. These data are then used to perform the leachable study where targeted compounds can be monitored. However, even with the multitiered approach, there is no way to ensure that all leachable compounds are going to be detected. Reactions can occur between the drug product and the container closure or delivery system that cannot be predicted in a laboratory setting. Environmental factors such as warehouse storage or transport can also generate unforeseen leachables.

PharmTech: Can ingredient material truly be used to forecast extractables? Are there some extractables that may not be measured by material testing?

Hunt and Morris (USP): An extractable study searches for all chemical entities that could come out of a material. In that regard, what can come out of a material is based on what goes into that material.
Today’s analytical technology is capable of detecting chemical entities in extremely small quantities, and that capability continues to improve. While it might be theoretically possible that some minute levels are not being detected during robust testing, it is extremely unlikely.

Lehman (Eurofins Lancaster Laboratories): Materials testing can be used as a starting point to provide a list of potential extractables, but this information alone is not sufficient. There could be extractables generated by the manufacturing process that would not be observed from the material testing. Extractable formation could be the result of reactions that occur during the manufacturing process, or from interactions with product matrix. In addition, certain pretreatments of components, such as sterilization, can affect the extractables profile even though the resin was otherwise well characterized.

Straka (West Pharmaceutical Services): Understanding the ingredients used in a complicated material, such as an elastomer, is extremely helpful when forecasting extractables. Of course, rubber formulations are mixtures designed to react, so extraction studies must be performed on the material in its final form, after all of the post processing (washing, lubrication, sterilization, etc.) has been completed. The ingredient information will make identifying unknown extractables easier in the long run.

Rushing (ABC Laboratories): Material ingredients can be used to predict some of the potential extractables, but should never be used exclusively for this purpose. Many polymer formulations are proprietary and only limited information about their formulations is provided to the sponsor. As a result, the sponsor may not have enough information to truly predict extractables. Another important note is that polymer manufacturing (of the source resin) is not performed to the same level of standards as drug product manufacturing and the purity of the compounds used can result in unexpected leachables. For example, if the polymer material contains an antioxidant, it is not uncommon to sometimes observe related antioxidants as extractables. This is the result of these related antioxidants being impurities in the antioxidant being used in the original formulation.

Common questions and regulatory updatesPharmTech: What common questions do manufacturers have about extractable and leachable testing?

Hunt and Morris (USP): There is no single common question about extractable and leachable testing. Rather, it is a matter of organizational knowledge. Some companies have been doing extractable and leachable testing for 20 to 30 years, providing a large base of experience within their organizations. Others, who may be newer or smaller, do not have that background, and must work to build their institutional knowledge.

Lehman (Eurofins Lancaster Laboratories): Manufacturers commonly question how to conduct an extractables and leachables study that would satisfy end users and the agencies worldwide. There are many different approaches one can take to assess extractables and leachables. When designing a study, one should ensure that the study is scientifically justified and meaningful for the intended use of any and all contact materials.

Straka (West Pharmaceutical Services): Drug manufacturers commonly inquire about the need to perform leachables testing. Some recurrent vendor questions include: Does using a barrier-faced stopper negate the need for leachables testing? If the same packaging components were evaluated for drug X, can we assume they are safe for drug Y? The answer to both questions is, no, leachables evaluations need to be performed for every drug product/packaging configuration. Drug manufacturers must understand the importance of evaluating the package-drug interaction, and allocate a sufficient budget required to do such testing in advance.

PharmTech: The new chapters in the United States PharmacopeiaNational Formulary (USPNF) focus on using well-characterized materials. What are the biggest changes these updates introduce?

Hunt and Morris (USP): The biggest change from the current chapters is a move away from focusing on individual packaging components. The new chapters take a revised approach, examining the materials of construction, including the importance of using well-characterized materials, and also include procedures and guidelines for defining the meaning of ‘well-characterized.’ Additionally, the new chapters set standards for the suitability and safety of a packaging system for a finished drug product.

Lehman (Eurofins Lancaster Laboratories): The new chapters in the USPNF take a quality-by-design approach, placing responsibility on the material manufacturer to test resins prior to manufacturing. In addition, the end user would be expected to evaluate the manufactured component for their own particular use, taking into consideration their process conditions.

Straka (West Pharmaceutical Services): The new chapters in the USP-NF are above <1000> and are, therefore, considered guidance, not regulation. They are good references for drug manufacturers preparing their strategy for extractables and leachables evaluations. They emphasize the need to gather the understanding for every drug product, and how to go about doing the task. In contrast, the existing chapter for plastics, <661>, is enforceable regulation and introduces major changes, which might have a greater impact on suppliers and drug manufacturers.

Article DetailsPharmaceutical Technology
Vol. 39, No. 2
Pages: 52-55
Citation: When referring to this article, please cite it as R. Hernandez, “Special Considerations for Extractables and Leachables Testing in Biological Products,” Pharmaceutical Technology39 (2) 2015.