Considerations for E&L Analysis in Single-Use Systems

Extractables and leachables (E&L) must be analyzed for any product-contact material to determine the risk of leachable compounds contaminating a drug product. Container-closure systems, which contact the drug product during long-term storage, are often higher risk than process equipment, which has a short product-contact time. Processing equipment is not exempt, however. As polymer single-use systems (SUS) are increasingly being used in
biopharmaceutical manufacturing, there is a growing need to analyze E&L of this equipment. Industry organizations and consortiums have worked
to standardize analysis protocols that can be used throughout the supply chain. Standard methods facilitate comparison between suppliers.

BioPhorum, for example, first published a standardized extractables protocol in 2014, which was updated in 2020 (1). Because the organization was previously known as BioPhorum Operations Group (BPOG), the protocol is often called the BPOG protocol. SUS suppliers can use the protocol to measure the extractables in representative components. The purpose of the extractables testing information package, says BioPhorum, is “to allow the SUS end-user to rigorously estimate the types and amounts of leachables that could be generated by the SUS component during its intended use” (1).

Another set of standard methods was recently published in the United States Pharmacopeia (USP). USP General Chapter <665> Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products was published in 2022 (2), but the date for it to come into effect was postponed to 2026. USP’s accompanying procedural chapter <1665> became official on May 1, 2022 (3).

A key aspect of the BioPhorum and new USP documents is the use of risk assessment for end-users to determine the risk level of a component in a particular process. The analysis method depends on this risk level.

The International Council for Harmonisation (ICH) is working on a document related to E&L testing. A final concept paper for ICH Q3E was published in 2020 (4). Industry experts say that questions about how this upcoming ICH guidance will align with the USP and BioPhorum documents are yet to be answered.

The Extractables and Leachables Safety Information Exchange (ELSIE) Consortium, made up of 33 biopharma/medical device companies, is also active in this area and aims to provide expert input to efforts such as USP’s new chapter and the in-progress ICH Q3E, through working groups and formal commenting processes as well as white papers, workshops, and other public meetings. “ELSIE provides a collaborative environment where member companies can discuss and benchmark the approaches that companies are taking,” explains Lee Nagao, senior director of science, regulation, and policy at the ELSIE Secretariat. Harmonized guidelines or compendial chapters are crucial, because without them, “regulatory expectations can vary depending on the health authority and the experience of the specific [biologics license application] reviewer,” says Nagao.

Standardized protocols help in setting baselines and comparing differences. For end-users of SUS, standard protocols facilitate “like-for-like assessments between each supplier’s single-use technology (SUT)
products and components,” says Dan Rosen, vice-president and general manager of bioproduction at Thermo Fisher Scientific.

Extractables testing of the materials or components is used to identify which leachables might come from the material under standard conditions and in a simulation study. A challenge, however, is that different leachables could be formed during sterilization, storage, or use.

“Key challenges in characterizing E&Ls include identifying unknown molecules that arise during primary sterilization of the SUT products,” says Rosen.

The response of materials to commonly used gamma irradiation is generally well established, but due to limitations in capacity for gamma-radiation sterilization, X-ray sterilization is being considered as an alternative (5). Current theories suggest that the E&L profile should be similar after X-ray or gamma sterilization, but it is important to perform extractables testing, says the Bio-Process Systems Alliance (BPSA).

Cell and gene therapies

BPSA published a guide for E&L in cell and gene therapies (CGT) in July 2020 (6) to support companies developing manufacturing lines for scale up or scale out. “Since CGT was a new modality, the drug developers were almost exclusively using single-use components for economic, safety, and technical performance. This [situation] led to a need for BPSA to help coordinate a guide to assist developers on appropriate choice of materials and components for their process development,” says Brendan Lucey, chair of BPSA’s CGT Committee. CGT processes differ from other biopharma manufacturing using SUS because in classical biopharma, process-related impurities, including process-equipment related leachables, are removed in the downstream purification process. In CGT processes, however, there may be washing steps, but not the same kind of downstream purification. Because of this process set-up and the variety of contact surfaces and contact times, E&L testing to assess and then control leachables is crucial, concluded BPSA (6). The type of polymers used in SUS for CGT manufacturing also differs from classical biopharmaceutical manufacturing. In particular, the report noted that polyvinyl chloride (PVC) polymer may be used in transfer bags and tubing sets in CGT collection, processing, and storage. Because flexible PVC typically contains plasticizers and other additives that are not present in other polymers used in SUS, leachables from these additives should be considered (6).

Adapting to changes

The rapid pace of change in guidelines has required a quick response by contract labs to create experiment designs that are compliant, says Dujuan Lu, manager and global lead of extractable and leachable testing at SGS Health Science. SGS labs are following the USP <1665> procedures, because these have been official since May 2022, but the postponement of USP <665>’s official date is helpful for the industry, Lu says. “The transition period gives pharma/biopharma manufacturers time to requalify as needed. We are seeing lots of activity now because clients want to be ready for 2026,” she explains.

Some clients want to cover both the USP and BioPhorum protocols, says Lu, who explains that the two differ slightly in recommended solvents for acid and base conditions. Lu points out that USP considers the different solvents for acid conditions to be equivalent; for the base conditions, USP recommends choosing the alkaline solvent depending on the specific process pH value.

Designing extraction studies

Design of extraction studies for single-use components is complicated by multi-component assemblies and complex geometries. “The recommended ratio of surface area to volume is 6 cm2/mL. Calculating the surface area is straightforward for a tube or a bag, but can be challenging for a connector or valve, for example,” explains Lu. In designing an extraction study for single-use filters, both the housing and the membrane must be evaluated. “The procedure depends on the shape. For a capsule filter, for example, we can fill the filter with solvent and cap the ends. For a smaller filter, we might need to remove the membrane and submerge it in solvent,” explains Lu.

Because many different polymeric materials are used to construct single-use components, the extractable profile is complex and multiple analytical techniques are needed to identify and quantify the compounds, says Lu. She explains that volatile organic compounds can be measured with headspace gas chromatography (HS-GC) coupled with high-resolution mass spectrometry (HRMS) or flame ionization detection (FID). Semi-volatile organic compounds can be measured with direct sample injection in GC-HRMS/FID. Non-volatile organic compounds can be measured with ultra high-performance liquid chromatography (UHPLC) coupled with HRMS and a photodiode-array detector. “Unknown identifications are crucial for the extractables testing, so high-resolution mass spectrometers are recommended for both GC and LC testing,” says Lu. She adds that elemental impurities can be measured with inductively coupled plasma-mass spectrometry.

Calculating the analytical evaluation threshold (AET) for processing equipment is another challenge. “The AET is set up based on the toxicology concerns; above [the AET], analytes need to be identified and quantified for health risk assessment,” explains Lu. “The instrument detection limits need to be at, or lower than, the AET to make sure all compounds above the AET can be detected. The process equipment AET is typically calculated based on the final product AET by applying the dilution factor during the process. The difference between the batch size and the extraction stoichiometry during the extractable study will be considered during the calculation.”

Questions remain

To perform risk assessment, it is crucial to have a good understanding of the manufacturing process. “[Understanding] any clearance, concentration, or dilution steps [is] critical, particularly to help narrow down the number of materials requiring in-depth characterization,” says Nagao.

Questions remain, says Nagao, about what point in the process a risk assessment should begin, how to define component risk, and what the minimum data would be to qualify low-risk components. “For example, gaskets, seals, or connectors used upstream of a [filtration] step will be inherently low risk and unlikely to contribute significantly to the leachables profile. Is it necessary, then, to fully characterize the extractables profile? This [type of question] is where a harmonized guidance that is implemented across the globe, perhaps based on risk-management principles, would be helpful,” Nagao suggests.

References

1. BioPhorum, BioPhorum Best Practices Guide for Extractables Testing of Polymeric Single-Use Components Used in Biopharmaceutical Manufacturing (BioPhorum, 2020).

2. USP, General Chapter <665>, “Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products,” USP–NF, (Rockville, MD, 2022).

3. USP, General Chapter <1665>, “Characterization and Qualification of Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products,” USP–NF (Rockville, MD, 2022).

4. ICH, Final Concept Paper ICH Q3E: Guideline for Extractables and Leachables (ICH, June 30, 2020).

5. BPSA, X-Ray Sterilization of Single-Use Bioprocess Equipment, Part 1: Industry Need, Requirements, and Risk Evaluation (BPSA, 2021).

6. BPSA, Extractables/Leachables Considerations for Cell & Gene Therapy Drug Production Development (BPSA, 2020).

About the author

Jennifer Markarian is manufacturing reporter for Pharmaceutical Technology.