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Volume 30, Issue 7
While the measurement of the toxicity of leachables is not always a required parameter, the information collected during these studies could inform future bioprocessing runs.
There has been a recent push by industry organizations and high-profile journals to encourage researchers to analyze their reference and raw materials thoroughly, and provide these data to journals as a condition of publication. This initiative was prompted by a fundamental interest in good science, with an overarching goal of promoting well-designed experiments that have reproducible findings.
Details about vendors and lots of materials used in experiments are not the only parameters that could be important to the overall success of an experiment or manufacturing run. Now that it has become standard practice to include single-use systems (SUS) in bioprocessing trains, researchers and manufacturers must consider if chemical entities from materials used are affecting cell health and/or growth.
At the very least, engineers should recognize the common concerns of SUS that could have negative effects on process variables, and ultimately, final drug product.
Despite the guidance documents that exist that attempt to provide recommendations for extractables and leachables (E/L) testing, there is no formal set of educational guidelines to inform how to best design test plans, how to conduct analyses, or how to interpret results from E/L studies, according to the BioPhorum Operations Group (BPOG). This was one of the reasons the group was compelled to release its own guide in 2017, titled Best Practices Guide for Evaluating Leachables Risk in Biopharmaceutical Single-Use Systems (1). The main learning objective of the guide is to “efficiently design studies that support a full range of manufacturing process conditions and that will provide a thorough understanding of leachables that may be present within products and in-process streams.” It also describes analytical techniques that effectively identify leachables.
Hopefully, the guide’s release will also stimulate others to incorporate its recommendations into purity and stability studies, including studies that are conducted in-house to inform manufacturing excellence, as well as those that are performed in academia. In the past, leachables tests have uncovered issues that ultimately impacted yield, patient safety, product release, and sample analysis (2).
As both old and new processes move to a more disposable environment, and new SUS suppliers enter the market, leachable studies may become increasingly important to protect the value of what is flowing through in-process streams. The studies will also help inform future bioprocessing runs, and perhaps, allow them to be more reproducible.
“It may be advantageous to the industry to expect more transparency in reporting results,” agrees Eric Isberg, director of life sciences at Entegris, a single-use materials supplier. “What I am hearing is that extractable protocols should be tied to experiments involving cell growth and product stability in single-use systems. For example, in single-use systems, there is a possible relationship between additives typically utilized in these system components and the inhibition of cell growth. If extractables are inhibiting cell growth, these conditions need to be characterized in order to have a proper experiment.”
Isberg says an extractables study typically precedes any leachables testing, and usually is done as a “worst-case scenario” of the potential compounds that could crop up during a leachables study. As the BPOG guide points out, E/L testing only assesses the risk of a leachable migrating into the drug product and does not consider the toxicity of the identified leachables. The assessment also does not consider the potential effect of leachables on the overall manufacturing process (1). After initial risk assessment and extractables testing, some companies subject extractables compounds to further toxicological assessment, while other companies only conduct additional extractables testing with model solutions. If extractables are below a certain toxicological threshold, sometimes leachable studies are not even conducted (2). As the BPOG guide points out, current risk-based approaches do not “necessarily require that leachables studies be conducted for all SUS” (1).
In the process planning scheme, testing is usually conducted at least six to eight months before the commencement of stability studies, but after the material list has been finalized, according to Eva Heintz, PhD, global market manager at Solvay Specialty Polymers, a company that is a raw materials supplier of specialty polymer used in many markets (including single use). Heintz consulted with NAMSA, a medical research organization, to come up with this estimate. “This allows time to perform the extractable studies, perform a risk assessment to determine compounds of potential concern, develop and validate methods, and then use the same pull points for [a] leachable analysis [that would have been done] for the stability study.”
The BPOG guide suggests that all E/L studies be done to scale, as the exposure surface area will influence the risk of leachables. It says that using process samples at scale will help prevent the need to conduct an additional leachables study when the project is ready for commercial manufacturing.
Raw material suppliers to the SUS industry may conduct E/L studies in-house, but they often do not publish these results, says Heintz. She says publishing these studies could hurt a company’s intellectual property. “The results of E/L studies reveal proprietary information about the molecule a company has developed. Sharing this publicly poses a significant risk. How companies can manage this risk is to publish results to the FDA in a Master Access File or Drug Master File. By working with the FDA, one can evaluate the test result without compromising companies’ confidentiality.”
The BPOG guide recommends manufacturers “persist with reasonable requests for supplier information. Ensure suppliers understand the significance and importance of your requests for information. Generally, the more they understand, the more open they will be.” But, according to Heintz, requesting certain information on product formulation could constitute an excessive request. “From a specialty raw material plastic supplier’s perspective, biopharma is not the only market that we serve, and our innovation (i.e., formulation and know-how) is our bread and butter. Requesting formulation information in addition to or in lieu of going through the FDA, when a leachable and extractable profile has been completed per the BPOG guideline, is unreasonable. Similarly, requesting extraction studies with solvents and solutions not previously agreed upon can quickly become unreasonable.”
One of the study design parameters outlined in the BPOG guide includes the examination of the typical dose range in manufacturing and the measurement of time between manufacturing or incubation and gamma irradiation. The primary function of gamma sterilization, says Heintz, is “to generate a sterile environment by reducing or eliminating any initial bioburden without imparting any residual radiation.” She notes that although there are some microorganisms that can survive low-dosage gamma irradiation, “few are able to do so at 25kGy or higher.”
Documenting levels and the extent of gamma sterilization is increasingly important for biologics; cell health has been shown to suffer following exposure to gamma radiation (3). In the aforementioned study, gamma radiation broke down the polymers in single-use equipment and the breakdown of the antioxidant additive leached into cell culture fluid (3).
Polymers from different suppliers, however, do not always behave the same way after undergoing irradiation (4), as the method of manufacture and quality of each polymer resin can vary (1). Thus, the BPOG guide suggests researchers do not “blindly rely on extractables data” (1). Rather, manufacturers should independently evaluate extractables following the steps in the BPOG Extractables Protocol and communicate which compounds should be measured to any contract manufacturing organizations with which they work.
“Gamma radiation of some single-use materials can result in increased extractable and leachables, just like other sterilization methods, such as autoclaving,” notes Isberg. “It is important to select single-use systems made not only with gamma-stable materials, but also with materials that inherently have lower extractables and leachables in order to compensate for any negative effect seen from the sterilization method.”
Gamma sterilization not only has the capability to be detrimental to cell viability, it also can affect the function and circuitry of the sensors in disposable bioreactor bags that are inserted into the bags and into flow paths prior to gamma exposure, according to the copy included in a patent filing from Finesse Solutions, Inc., a company that was recently acquired by GE Healthcare Life Sciences. Finesse’s patent covering a new type of sensor, sensor carrier, and methods for installing a sterilized peripheral in a bioprocessing vessel or component, seeks to tackle the problems that gamma radiation present to disposable bioprocessing equipment. The invention permits packaged sensors and carriers to be sterilized together, rather than separately, which Finesse says reduces “the negative effects that sensors endure during gamma sterilization” (5).
1. BioPhorum Operations Group, BioPhorum Operations Group (BPOG) Best Practices Guide for Evaluating Leachables Risk from Polymeric Single-Use Systems Used in Biopharmaceutical Manufacturing (BPOG, November 2016).
2. BPOG Extractables and Leachables Team, “Survey of Industry Leachables Best Practices Completed,” PDA Letter (March 2016).
3. M. Hammond et al., PDA J. Pharm. Sci. Technol. 67 (2), 123-134 (March-April 2013).
4. D. Sinha, Adv. Appl. Sci. Res. 3 (3), 1365-1371 (2012).
5. Selker et al. (Finesse Solutions Inc.), “Aseptic Connectors for Bio-Processing Containers,” US patent 9,335,000, May 10, 2016.
Volume 30, Number 7
When referring to this article, please cite it as R. Hernandez, “Balancing Protocols for Leachables," BioPharm International 30 (7) 2017.