Single-Use-Systems: Leachable Study Design for Single-Use Components

Biopharmaceutical manufacturers can realize substantial gains in process flexibility, speed, and efficiency through implementation of polymeric single-use systems (SUS) for use in their manufacturing processes. The use of polymeric single-use-systems (SUS) and components has increased dramatically in biopharmaceutical manufacturing processes. SUS are comprised of polymers, and the manufacturing process for the SUS can utilize a variety of chemical processing aids (i.e. wetting agents, slip agents, plasticizers, antioxidants, blocking agents). Any polymeric processing component that has product or process fluid contact can contribute chemical entities to the fluid. Generally, the entities that can be contributed to the fluid from components include oligomers, plasticizers, antioxidants, slip agents, or degradation products. The potential to introduce these additives to the product or process fluid is an inherent risk when implementing SUS into biopharmaceutical manufacturing processes.
Potential additives may be in the form of extractables or leachables. Extractables are organic and inorganic chemical entities that can migrate from a material under aggressive conditions such as elevated temperature, extreme surface exposure, and/or aggressive solvent systems. Leachables are organic and inorganic chemical entities that can migrate from a material under normal processing conditions. Leachables are often, but not always, a subset of extractables.
As understanding of the potential risks related to extractables and leachables grows, the biopharmaceutical industry and health authorities are taking steps to ensure that appropriate and robust evaluations are performed that adequately protect patient safety. There are no specific regulations for extractables testing for polymeric SUS and components. Unlike extractables, there are regulatory guidelines and regulations from FDA (21 CFR 211.65), and EMEA (EMEA/CVMP/205/04) for leachables in drug product. 21 CFR 211.65 (a) specifically states that “Equipment used in the manufacture of drug product shall be constructed so that surfaces that contact components, in-process materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, strength, quality, or purity of the drug product beyond the official or other established requirements.”
Such regulatory guidelines do not provide a framework regarding how to assess risks, design a leachables study, perform associated analyses, or interpret the data sets that result from extractables and leachables testing.
In 2014, the Biophorum Operation Group (BPOG) Extractables Working Group published Standardized Extractables Testing Protocol for Single-Use Systems in Biomanufacturing¹, to provide a framework for generating comprehensive extractables profile. Following publication of the extractables protocol, biopharmaceutical manufacturers recognized the need for leachable standards. In 2015, the BPOG Leachables Working Group, comprised of subject matter experts from 22 biopharmaceutical manufacturers, was formed. The BPOG Leachables Working Group set out as a community of collaboration with the goal of developing a scientifically robust approach for leachables evaluation using the principles of quality risk management to identify, evaluate, communicate and mitigate leachables risks to product quality and patient safety. In March 2016, the BPOG Leachables Working Group published Biophorum Operations Group (BPOG) Best Practices Guide for Evaluating Leachables Risk From Single-Use Systems Used in Biopharmaceutical Manufacturing². The best practices guide provides a framework for industry to ensure a robust and streamlined approach to understanding the SUS materials used in the process, their chemical additives, and eventual product impact. The best practices guide is applicable to both drug substance and drug product manufacturing processes.
The Best Practice Guide is comprised of three sections: The Risk Assessment Model, Leachables Study Design and Analytical Methods. These sections are described in the subsequent sections.

The Risk Assessment Model
The Risk Assessment Model provided within the Best Practices Guide aligns with the ICH Q9 guidance on Quality Risk management. ICH Q9 states that the evaluation of risk to quality should be based on scientific principles and ultimately link to the protection of the patient. Using the BPOG Risk Assessment Model, biopharmaceutical manufacturers can leverage comprehensive process knowledge to perform science and risk based assessments for leachables from SUS used in their biopharmaceutical manufacturing processes.
The key considerations included in the risk assessment model are presented in Table 1.

Through the risk assessment model, a biopharmaceutical manufacturer is able to use methodology to systematically rank each SUS or component in a given manufacturing process into High, Medium, and Low Risk categories. Based on the ranking, the manufacturer is able to decide the level of rigor necessary to qualify a particular SUS or component for use in the process. In some cases, extractables data is detemined to be sufficient to support a given SUS application and further evaluation through leachables testing is not required.

Leachables Study Design
Based on the output from the risk assessment model, some SUS applications within biopharmaceutical manufacturing process will require a leachables study to support qualification of the SUS or component. In these cases, the Leachable Study Design section of the Best Practices Guide outlines the key parameters to consider when developing a test protocol, to ensure a robust and efficient study.
The key parameters that should be considered while designing a leachables study include, but are not limited to those presented in Table 2.

Analytical Methods
A robust leachables study requires use of appropriate analytical methods to detect any leachables present in the solution following incubation. The Analytical Methods section of the Best Practices Guide outlines the key analytical methods to consider when developing a leachables study. The key analytical methods for use in leachables studies are presented in Table 3.

Best Practice Case Studies
Three case studies are presented below to illustrate the use of the BPOG Best Practices Guides for Leachables Study Design.

Case Study 1: Bulk Drug Substance Filtration Initial Risk Assessment and Testing
A 0.2 µm polyethersulfone (PES) filter is used to filter bulk drug substance in a biopharmaceutical manufacturing process. The drug substance is a protein solution with a circumneutral pH. The filtration is performed at ambient temperature, and is limited to 12 hours duration through the in-process control strategy.
A risk assessment was performed for the filter/process stream interaction. Based on the process step and processing conditions, it was determined that the filtration of BDS using the 0.2 µm PES filter represented a high risk filtration application. A review of existing data for the 0.2 µm PES filter was performed, and it was determined that sufficient applicable extractable/leachable data to support the usage of this filter was not available. Therefore, a study was required to qualify the filter for use in the bulk drug substance filling application.
A leachables screening study was designed to support the filtration of BDS with the 0.2 µm PES filter. The test solution selected was the bulk drug substance, following filtration under normal manufacturing conditions. A sample of the bulk drug substance prior to final filtration was taken from the formulation vessel immediately prior to filtration to serve as a negative control. As the test sample was taken from manufacturing, a scale down model was not required. The filter in contact with the solution was the filter to be qualified, and had been flushed and gamma irradiated prior to use in the process. The exposure temperature was within the normal manufacturing process temperature. Based on the study design, the test sample was taken from the manufacturing line during normal manufacturing operations as opposed to using controlled contact conditions within a laboratory environment.
The control sample and the test sample after contact with the filter under normal manufacturing conditions were analyzed for leachable compounds. The target analyte classes and analytical methods used were:

• Headspace Gas Chromatography with Mass Spectrometric Detection (HS/GC/MS)
o Target analytes: Volatile Organic Compounds (i.e. residual solvents)
• Gas Chromatography with Mass Spectrometric Detection (GC/MS)
– Target analytes: Semi-Volatile Organic Compounds (i.e. plasticizers)
• Liquid Chromatography with Mass Spectrometric Detection (LC/MS)
– STarget analytes: Non-Volatile Organic Compounds (i.e. fillers, plasticizers, antioxidants, and slip agents)
• Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES)
o Target analytes: Trace elements and Heavy Metals (i.e. elemental impurities)

No organic compounds were detected in the direct injection GC/MS, headspace GC/MS, or LC/MS analyses. No trace or heavy metals were detected in the ICP-OES analysis. Based on the results, the 0.2 µm PES filter was deemed appropriate for use in the biopharmaceutical manufacturing process for bulk drug substance filtration.

Case Study 2: Bulk Drug Substance Filtration: Re-evaluation for Technical Transfer
During subsequent technical transfer of the biopharmaceutical manufacturing process to a new facility at a larger scale, the filtration application was reviewed and a new risk assessment was performed based on the process changes required for facility fit (i.e. changes to exposure duration and surface area to volume ratio).
The larger scale manufacturing process uses a 0.2 µm polyethersulfone (PES) filter to filter bulk drug substance. The drug substance is a protein solution with a circumneutral pH. The filtration is performed at ambient temperature, and is limited to 24 hours duration through the in-process control strategy.
The outcome of the risk assessment was the same as for the original process, and the filtration application was deemed a high risk filtration. A review of the qualifying documentation was required to determine whether sufficient data to mitigate the high risk was available. Due to the larger scale, additional time was required to complete the filtration application. While the study was representative of the original process, the data generated could not provide sufficient justification for the increased contact time required after scale up of the process. Additional testing was required to represent worst-case conditions that are possible in the larger scale manufacturing operation.
A leachables screening study was designed to support the filtration of BDS with the 0.2 µm PES filter under the updated conditions expected in the larger scale manufacturing process. The test solution selected was the formulated bulk drug substance prior to final filtration. A sample of the formulated bulk drug substance prior to final filtration was taken from the formulation vessel. This sample was split into a control and test sample. The scaled down test filter was gamma irradiated prior to use in the study. In the manufacturing process, there is a defined filter flush required prior to use. The flush was not performed according to the manufacturing requirements to provide a worst-case condition for testing. Determination of the worst-case processing conditions considered the longest potential exposure duration at the warmest potential exposure temperature based on the process description and allowable ranges. This assessment included a review of the in-process control strategy, time out of storage, and time out of refrigeration data. The test sample was placed in contact with a scale down version of the manufacturing scale filter for 24 hours at 25°C.
Following the 24 hour hold, both the control sample and the test sample after contact with the filter were analyzed for leachable compounds using the same target analyte classes and analytical methods used for the original study. Three organic compounds and one metal were detected in the test samples following contact with the filter for 24 hours at 25°C. The presence of these leachables required review for toxicological impact. A toxicologist performed an assessment based on the identity and concentration of the leachables, as well as the dosing regimen for the drug product. The toxicology assessment concluded that based on these considerations; there is no impact to patient safety based on the data provided.
Based on the results, the 0.2 µm PES filter was deemed appropriate for use in the biopharmaceutical manufacturing process for bulk drug substance filtration up to 24 hours in duration.

Case Study 3: Drug Product Formulation Bioprocess Bag
A 1000-L ultra low density polyethylene (ULDPE) bioprocess bag is used to formulate and then hold to formulated drug product in a biopharmaceutical manufacturing process. The drug product is a protein solution with a circumneutral pH. The formulation and hold are performed at ambient temperature, and the maximum contact duration for the drug product in the bioprocess bag is 9 days. Based on the lowest potential drug product volume, a worst case surface area to volume ratio is 0.09 m²/L.
A risk assessment was determined that the formulation and hold of the drug product in the ULDPE bioprocess bag represented a high risk application. A review of existing data for the ULDPE bioprocess bag was performed, and determined that sufficient applicable extractable/leachable data to support the usage of this bag was not available. Therefore, a study was required to qualify the bioprocess bag for it’s intended use in the bulk drug product formulation and hold application.
A leachables screening study was designed to support the formulation and hold of the drug product in the ULDPE bioprocess bag. Two test solutions selected were selected: bulk drug product, and placebo. The placebo was included to provide a very similar solution to the drug product, without the potential for analytical interference presented by the presence of the protein. A control sample of each solution was aliquoted out from the pool prior to contact with the ULDPE bioprocess bag. A scale down test bioprocess bag was selected to allow for an appropriate surface area to volume ratio for analysis. Prior to use in the study, the bioprocess bags were gamma irradiated. The irradiated test bioprocess bags were filled with test solution and held at 30°C for 14 days to ensure that the study supported the worst case manufacturing conditions.
Following the incubation, samples were analyzed for leachable compounds using the same methods and target analytes described above.
Four organic compounds and three metals were detected in the test samples following storage in the bioprocess container for 14 days at 30°C. The presence of these four organic and three trace metal leachables required review for toxicological impact. A toxicologist performed an assessment based on the identity and concentration of the leachables, as well as the dosing regimen for the drug product. The toxicology assessment concluded that based on these considerations; there is no impact to patient safety based on the data provided.
Based on the results, the ULDPE bioprocess bag is appropriate for use in the biopharmaceutical manufacturing process for bulk drug product formulation and hold.

Conclusion
The recommendations set forth in the BPOG Leachables Best Practices Guide provide a standardized framework for biopharmaceutical companies to develop a strategy for managing the risks posed by leachables. The risk assessment model enables efficiency through knowledge-based prioritization of studies. This allows biopharmaceutical manufacturers to focus attention on applications that present the most risk, therefore provides increased assurance of patient safety. The leachables study design recommendations enable a robust study design, which is tailored specifically for the SUS in question. The robust study design, coupled with the use of appropriate analytical methods, ensures that the data derived from the study is accurate, reliable, and applicable to the full range of possible manufacturing or storage conditions. Given the above mentioned risk assessment and study evaluation, the resulting data enables a thorough toxicological understanding of the potential impact of the additives profile and how it relates to patient safety, maintaining product quality overall; product quality and the protection of our patients is the foremost responsibility of any biopharmaceutical manufacturer and end-user.

References
1. Ding W, Madsen G, Mahajan E, O’Connor S, Wong K, Standardized Extractable Testing Protocol for Single-Use Systems in Biomanufacturing, Pharmaceutical Engineeringm 2014; 34(6), 1-11.
2. BPOG E/L Working Group (15 authors including: McGohan, K.) BPOG Best Practices Guide for Evaluating Leachables Risk from Polymeric Single-Use Systems Used in Biopharmaceutical Manufacturing. www.biophorum.com, March 2017

Author Biography
Kathryn McGohan is a Scientist I within the Materials Science group of the Global Manufacturing Sciences & Technology organization at Bristol-Myers Squibb. Kathryn is responsible for the selection, qualification, and validation of single-use products for use in late stage development and commercial biopharmaceutical manufacturing processes (U.S. and ex-U.S.). Kathryn also supports the authorship of CMC regulatory filing sections, new product launches, and continuous improvement efforts. Kathryn currently represents Bristol-Myers Squibb on the BioPhorum Operation Groups (BPOG) Disposables Workstream.

 

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