Tuesday, April 24, 2012

Leachables and Extractables Affect Single-Use and Disposable Systems

Nigel J Smart, PhD

Take steps to limit undesirable effects

Leachables and extractables have become a significant driver in the potential application of single-use and disposable systems. The increase in industry activity associated with these systems and products reflects their perceived relevance and importance for biopharmaceutical drug manufacturing.
Issues connected with the use of leachables and extractables with various plastics and polymers have been discussed for more than 25 years in the pharmaceutical industry, where products have been in widespread use and have been associated with a variety of applications. Many applications have included use in water-based systems such as syringes and various other sealing components, so there is a long history with regulatory authorities such as the FDA.
However, in the past 10 years there has been an explosion in the use of disposable bags, wave bags, disposable plastic bioreactor inserts, and a myriad of associated tubing parts, samplers, and other components.
Some of this growth has been catalyzed by the need for rapid, inexpensive mechanisms for production as well as for prompt deployment of countermeasure products in the case of pandemic disease situations or terrorist attacks in which biological agents are required in significant quantities at very short notice.
The most notable application of disposable systems is in the area of clinical trial material supply, in which relatively small quantities of active drug substance are often required. Producing these materials using disposable systems in openly designed standardized production facilities provides for significant flexibility in the supply of multiple drug agents without the high cost of dedicated-purpose “machine in place” facilities.
This increase in the application of disposable systems for the production of biological drug substances is driving the application of new standards and methodologies.
There is a regulatory requirement to assure that the drug substance and drug products are in no way contaminated or adulterated by materials that could be either leaching from the various polymers used in the construction and fabrication of these new bioreactor systems or, conversely, extracted from these same polymer materials as a result of some elution process occurring due to solvent action.
The relevance of this increased activity related to materials use translates into a potential increase in the probability of, for example, some elastomer eluting from the components or a breakdown product washing off the material into the product stream.
So why is this important?
It is now well recognized in the modern biopharmaceutical industry that disposables and single-use systems can potentially offer significant advantages over conventional production systems. One important advantage includes the probability of rapid product changeovers, reduced or eliminated validation, cleaning validation, and a higher degree of process flexibility. These all propel the possibility of driving lean manufacturing principles into bioprocessing and biopharmaceutical processes, which ultimately leads to more efficient use of resources. Because the potential for driving efficiency and the effective use of resources is high on the agenda of manufacturers, the use of these systems is highly desirable. That said, nothing in life comes without a cost.
The real issue, as previously mentioned, is that if the disposable processing adds something to the product stream, there might be a safety issue for the patient taking the drug. From a regulatory compliance perspective, any unwanted contaminants would render the product adulterated and unsafe for use.

Addressing the Issue

Although the potential for issues with leachables and extractables has been known for decades due to the pharmaceutical industry’s experience with various polymers, this involved their characterization using selected USP tests. Using these tests for extractables profiles, subsequent leachables profiles have been developed based upon these and additional tests.
These generally recognized as safe (GRAS) limits were developed with the assumption that there was no drug interaction; however, with biopharmaceutical products, there have been some examples where an interaction of the leachable with the products has given rise to an increase in immunogenicity. This occurred in the case of EPREX, as reported by Sharma and colleagues as well as Schellekens and Jiskoot.1,2
In cases like this, new pathways of degradation will likely result. If this occurs, existing USP tests may well be ineffective due to interference; new, more specific testing methodologies may need to be developed.
Due to the importance of this issue for the whole biopharmaceutical industry, companies and user groups have pooled their resources to address the issues. Both have a vested interest in making sure that these highly flexible and lean solutions don't become derailed through unaddressed regulatory questions.
At the forefront of these initiatives are several industry associations, including:
  • The Bio-Process Systems Alliance (BPSA), which generally reflects the input and opinions of component suppliers;3-5
  • The Extractables and Leachables Safety Information Exchange (ELSIE), which represents pharmaceutical manufacturers’ experiences with leachables and extractables; and
  • The Product Quality Research Initiative (PQRI), which publishes data connected with extractables and leachables for a variety of pharmaceutical product types.
In particular, the BPSA published a 2010 series of recommendations for testing and evaluating extractables from single-use process equipment, providing a very helpful starting point for biopharmaceutical companies that use this type of technology to manufacture their products.6
Another overview of this area, which opens an interesting window on what the FDA’s Center for Drug Evaluation and Research believes are important issues related to leachables and extractables for single-use systems, was also recently provided by the FDA. This is a must review.7
continues below...

Categories of Leachables and Extractables

Although leachables and extractables often have differing mechanisms of interaction, they frequently have well-defined breakdown patterns that can be characteristic of the material degrading. This is very helpful for new materials that degrade by somewhat regular reactions, producing related subsets of breakdown products even when the endpoint is new. Listed here are some common leachables and extractables:
  • Polyethylene
  • Polypropylene
  • Polyvinyl chloride
  • Elastomeric materials
These include the following metal ions:
  • Tungsten
  • Iron
  • Aluminum
  • Calcium
  • Barium
  • Boron
  • Silicon
  • Nickel
  • Magnesium


As referenced earlier, a plethora of regulations and guidances provide instruction for the assessment of leachables and extractables in pharmaceutical systems. Some useful regulations include the following:
  • 21CFR600.11(b), which references guidance for equipment;
  • 21CFR600.11(h), which refers to containers and closures;
  • 21CFR211.65(a), which refers to equipment construction;
  • ICH Q9 5.1, covering equipment connected to the manufacture of APIs and intermediates; and
  • ICH Q5C, which refers to the stability of biotechnology and biopharmaceutical products.
Navigating these various regulations successfully enables the development of a useful strategy to address potential issues before they can have a detrimental effect on the product.

Useful Definitions

Several characteristics have been identified to help distinguish extractables from leachables. Some of these are listed below:
  • Extractables have been characterized as entities extracted from the component material;
  • Extractables have been characterized as entities occurring as a result of the exaggerated conditions of use;
  • Extractables are produced in organic, water/aqueous, or dried products vehicles; and
  • Extractables have been identified as being useful in predicting leachables.
The following lists some useful characteristics of leachables:
  • Leachables have been described as migrants from the component material;
  • Leachables often occur within the specifications connected with the recommended conditions of use and storage;
  • Leachables occur in the drug product’s vehicle; and
  • Leachables are often, but not always, a subset of extractables.
All this presents some challenges for biopharmaceutical products. Several issues are relevant:
  • Accurate processing and storage conditions must be derived to help reduce and manage the potential for leachable and extractable issues to arise;
  • Suitable limits and ranges for detection analysis and product stability must be defined;
  • Better standards are needed for toxicological characterization to quantify risks; and
  • Appropriate risk management, risk analysis, and risk mitigation strategies are needed to deal with any problems that are detected.
Important considerations that may influence these limits and ranges include those relative to the specific toxicity of the product in question and the intended therapeutic dose of the drug being evaluated. Information about the patient treatment population might also be important. For example, immunocompromised, elderly, or infant patients might be more susceptible to any potential contaminants than mainstream adult populations.

Are They a Problem?

For all new biopharmaceutical products, the FDA will require a comprehensive risk assessment for biologics license application submissions. This includes an analysis of the overall production process for the products, with a plan for data collection from early clinical trials material production through to the BLA submission and new drug application approvals. That plan should include a study that incorporates both accelerated and real-time data studies.
If there is a problem, suppliers and users have a role to play. Suppliers address this by being supportive and providing extractables information about their materials.
Typically, they develop a profile of extractables and identify individual chemical species. Often these are done using alcohol on water-based systems, because these are often the systems in use in biopharmaceutical manufacturing processes. The following points are particularly important to record during this process:
  • Quantitate all extractables using quantitative tests. These should have sufficient sensitivity and specificity to provide accurate results.
  • Provide this data to drug sponsors as an approved statement of the material’s suitability to be used in their processes to produce biopharmaceutical products.
Sponsor companies use this data to develop strategies to look at leachables, which are often a subset of chemical species seen during extraction processes performed by the suppliers.
Leaching processes may occur in a variety of situations, including the following:
  • In upstream systems, which includes media preparation and buffer preparation operations;
  • In midstream process operations, which includes cell culture and fermentation operations;
  • In downstream process operations, which includes concentration of the product and exchange and purification operations;
  • In bulk storage operations, which includes API and formulated bulk; and
  • In drug products storage, which includes final product containers such as vials and pre-filled syringes.

Assess, Mitigate Risk

From the user’s point of view, there is a need to be proactive and characterize the chemical species to assure safety. This involves developing appropriate solutions to potential problems before they arise. The risk assessment required by the FDA needs to be data-driven and will involve the construction of a dossier that will be a compilation of all the relevant information to support the use of the material connected with the manufacture of the product.
Typical components examined during the risk assessment process will include insert liners, filters, bags, containers closures, multiuse assembly components, and other product contacting component surfaces.
Now, using a decision tree-type chart to rank situations that may require action and mitigation, it becomes possible to create an action strategy to deal with these issues.
A series of compendial tests can be used as part of this risk analysis process, including USP 381, which deals with elastomer closures, USP 661 covering containers, USP 87 covering in vitro biological testing, and USP 88 covering in vivo biological testing. By using these, integrated with other information, it is possible to decide which situations probably require mitigation and which involve low risk.
However, it should be noted that compendial methods may not always be adequate for identifying extractables or leachables in the system under consideration, and in situations like this, other methods may be required to identify degradation species. Depending upon the nature of the species being produced during a leachables study, a variety of analysis techniques may be required to accurately identify them. Some techniques that may be used in the identification of these compounds are high performance liquid chromatography, gas chromatography HPLC/mass spectrometry, GC MS, ion chromatography, infrared, and inductively coupled plasma.

Risk and the Process Stream

FIGURE 1: Example of a Risk Assessment Chart for Leachables
In shaping risk assessment practices that are helpful in the development of protein therapeutics, it is important to first quantify the extent of the potential risk and then subsequently ameliorate any possible negative outcomes that are identified.
Figure 1 illustrates a typical risk assessment tool that may be employed in the evaluation of potential issues associated with leachables.
In terms of the process stream, risk is increased when the disposable contact surface is in contact with the product process stream toward the end of the manufacturing process.
This higher risk is highlighted in red in the chart, where there is substantially higher risk associated with any function late in the manufacturing process.
In other circumstances, systems failing existing USP testing regimes will also fall into a higher risk category and will require additional mitigation measures to assure safety and meet prevailing regulatory requirements.
One factor of special note is that excipients and other materials that may be added to the final formulation can increase the potential for leaching and should be monitored carefully. Supplementary decision trees, which deliver a multifactorial analysis that requires individual ranking of factors in terms of their importance on the final outcome, can help with this consideration.
This type of approach should always produce a low-risk outcome. Any other result requires a comprehensive and detailed mitigation strategy that would support the components’ use. Inevitably, this would involve additional extraction of the components and further identification of any resulting chemical species that are produced, using reliable analytical methods.

A Check List

It is important to make sure you develop a complete and accurate dossier of the risk analysis and data package for each component unit or component type to be used in your single-use /disposable manufacturing system.
Remember that toxicity studies do not measure chronic response to potential long-term leachable exposure, so this should be factored into data collection programs. Product end-of-shelf life dating is rarely evaluated clinically, which could be an issue for some systems. As a result, studying the leachables profiles over the life of the product is an important consideration.
Because new supplier information may become available, it is imperative to update the product dossier to keep it current and relevant to your product system. Revisit your system periodically to make sure that nothing has changed over time. Remember that these dossiers are living documents and will require periodic attention to remain accurate, especially if parts of the process are changed or new equipment substituted. Make sure that the team conducting the risk assessment is multidisciplinary so that all aspects of the study are appropriately covered.
Finally, do not overthink the methodology used to analyze whether there is a significant risk. There are many existing methodologies available and documented for this purpose, and it is important that you choose one that fits well with your system needs. Experience teaches us that the best approaches are often the simplest.


  1. Sharma B, Ryan MH, Boven K. Reactions to Eprex’s adverse reactions. Nat Biotechnol. 2006;24:1199-1200.
  2. Schellekens H, Jiskoot W. Eprex-associated pure red cell aplasia and leachates. Nat Biotechnol. 2006;24(6):613-614.
  3. Ding W, Martin J. Implementation of single-use technology in biopharmaceutical manufacturing: an approach to extractables and leachables studies, part one–connectors and filters. BioProcess Int; 2008;6(9):34-42.
  4. Ding W, Martin J. Implementation of single-use technology in biopharmaceutical manufacturing: an approach to extractables and leachables studies, part two–tubing and biocontainers. BioProcess Int; 2009;7(5):46-51.
  5. Ding W, Martin J. Implementation of single-use technology in biopharmaceutical manufacturing: an approach to extractables and leachables studies, part three–single-use systems. BioProcess Int; 2010;8(10):52-61.
  6. Bio-Process Systems Alliance. BPSA guides to extractables and leachables from single-use systems. Available at: www.bpsalliance.org/guides.html. Accessed January 28, 2012.
  7. Markovic I. Considerations for extractables and leachables in single-use systems. A risk-based approach. Paper presented at: Parentarel Drug Association Single Use Systems Workshop; June 22-23, 2011; Bethesda, Md.

Editor’s Choice

  1. Sauerborn M, Brinks V, Jiskoot W, Schellekens H. Immunological mechanism underlying the immune response to recombinant human protein therapeutics. Trends Pharmacol Sci. 2010;31(2):53-59.
  2. Rathore N, Rajan RS. Current perspectives on stability of protein drug products during formulation, fill and finish operations. Biotechnol Prog. 2008;24(3):504-514.
  3. Mueller R, Karle A, Vogt A, et al. Evaluation of the immuno-stimulatory potential of stopper extractables and leachables by using dendritic cells as readout. J Pharm Sci. 2009;98(10):3548–3561.
  4. Yu X, Wood D, Ding X. Extractables and leachables study approach for disposable materials used in bioprocessing. BioPharm Int website. Feb. 1, 2008. Available at: www.biopharminternational.com/biopharm/article/articleDetail.jsp?id=490803. Accessed Jan. 23, 2012.
  5. MacDonald JS, Robertson RT. Toxicity testing in the 21st century: a view from the pharmaceutical industry. Toxicol Sci. 2009;110(1):40-46.

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