On Developing a Process for Conducting Extractable–Leachable Assessment of Components Used for Storage of Biopharmaceuticals

MINI-REVIEW On Developing a Process for Conducting Extractable–Leachable Assessment of Components Used for Storage of Biopharmaceuticals ADITYA A. WAKANKAR,1 Y. JOHN WANG,1 ELEANOR CANOVA-DAVIS,2 STACEY MA,3 DIETER SCHMALZING,4 JOSH GRIECO,5 TERRY MILBY,6 THERESA REYNOLDS,7 KELLEN MAZZARELLA,8 ED HOFF,2 STEPHEN GOMEZ,7 SHERRY MARTIN-MOE1 1

Department of Late Stage Pharmaceutical and Processing Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080 2

Department of Protein and Analytical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080

3

Department of Early Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080

4

Department of Corporate Quality, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080

5

Department of Clinical Quality, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080

6

Department of Regulatory Chemistry, Manufacturing and Control Systems, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080 7

Department of Safety Assessment, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080

8

Department of Pharmaceutical and Packaging Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080

Received 5 August 2009; revised 29 September 2009; accepted 30 September 2009 Published online 28 December 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22012 ABSTRACT: Extractables and leachables are product-related impurities that result from product contact with components such as gaskets, stoppers, storage bags, cartridges, and prefilled syringes that are used for processing, storage, and/or delivery of biopharmaceuticals. These impurities are a concern for patients due to potential effects on product quality and safety. It is possible that such an impurity could directly impact the patient or indirectly impact the patient by interacting with the protein therapeutics and forming protein adducts. Adducts and leachables may or may not be detected as product-related impurities in routine stability indicating assays depending on the rigor of the analytical program. The need for the development of a thorough and holistic extractable and leachable program based on risk assessment, review of existing literature, and consolidation of industry best practices is discussed. Standardizing component use within an organization enables streamlining of the extractable–leachable program. Our strategy for an extractable–leachable program is divided into different stages, each stage detailing the activities and the department within the organization that is responsible for execution of these activities. The roles and responsibilities of the key stakeholders are identified. The integration of analytical activities with health-based risk-assessment information into the design of an extractable–leachable program is highlighted. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2209–2218, 2010

Keywords: extractables; leachables; pharmaceutical development; health-based risk assessment; protein adducts; drug compatibility

INTRODUCTION Correspondence to: Aditya A. Wakankar (Telephone: 650-2256739; Fax: 650-224-2764; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 2209–2218 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Extractables are a class of compounds released from a component under aggressive treatment conditions; those that exceed what a component may endure during normal use (e.g., extended time, increased

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temperature and ionic strength, pH extremes). Leachables are a class of compounds that emanate from a component into the drug substance or drug product under normal conditions and are typically a subset of extractables. Wang and Chien1 extensively reviewed the kinetics models and factors that affect leaching phenomena in 1984. Leaching typically occurs upon migration of dissolved solute through the polymer matrix. The rate of leaching is diffusion controlled, and the amount of leachable is in proportion to the square root of time. The diffusion of organic substances in polymer matrix is also governed by activation energy that is in the range of 10–13 kcal/mol, thus one can expect the rate to double for every 108C increase in temperature.2,3 On the other hand, leaching of plasticizer from polyvinyl chloride (PVC) bags follows a linear relationship with time because the plasticizer is present in large concentration and encounters a nontortuous path by which the leaching substance can diffuse. Early efforts to detect extractables or leachables have employed UV spectroscopy or other nonspecific methodologies. A pioneering work on identifying extractables in a biotechnology product was conducted using LC in series with electrospray MS. In this study, butylated hydroxy toluene (BHT) and a polymer species were identified as extractables when acetonitrile was used as the extraction medium. However, these substances were not found in the drug product when extracted with the protein formulation or buffer formulation.4 In recent years the Eprex1 case has had a major impact on the regulatory scrutiny related to extractables and leachables. There were 175 cases of epoetin-associated pure red-cell aplasia (PRCA) reported for Eprex1 between 1998 and 2004. Most of these cases involved patients with chronic kidney disease who had received subcutaneous injections of epoetin.5 The phenolic derivatives that leached from the rubber stopper, used in prefilled syringes, into the formulation were postulated to be a causative agent for the immunogenic response observed.6–8 It was proposed that the presence of polysorbate 80, the stabilizer employed to replace human serum albumin in the second-generation drug product formulation, induced the leaching of these phenolic derivatives.8,9 Although a clear association between the presence of these leachables and the incidence of PRCA could not be demonstrated, the decrease in instances of PRCA did coincide with the change in the stopper configuration from the original uncoated stopper to the current Teflon-coated stopper.5,7 The incidences of Eprex1-related PRCA dropped significantly after additional changes were applied to storage and handling and the route of administration was changed from subcutaneous (SC) to intravenous (IV) route. Another factor that was identified as a JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010

potential cause for the immunogenicity was modification of epoetin when associated with polysorbate micelles.10 In a recent study, Mueller et al.11 investigated the ‘‘immunogenicity potential’’ of extractables and leachables from three drug product stoppers using dendritic cell models. Activation of dendritic cells was monitored by analyzing the expression levels of the costimulatory molecule CD86. The stoppers evaluated in this investigation included the PH 4106 stopper reported to be a component of the Eprex1 prefilled syringe. This investigation suggested that it is not the extractables and leachables from these different stoppers that lead to an increase in the dendritic readout. Instead, the hydrolytic breakdown product of polysorbate 80, oleic acid, as well as other follow-up products were identified as plausible factors responsible for the increase in the dendritic activation. Other factors such as leached silicone oil, a lubricant used in the prefilled syringe, have also been considered as potential causes for increase in immunogencity.9 Novel drug-delivery technologies such as prefilled syringes and pen-injector systems have also necessitated scrutiny of leachables as new materials are introduced to product contact. These devices consist of components such as plungers, stoppers, lined seals, and needles, each having the potential to leach chemicals into the drug product formulation.12–14 Additionally, these components utilize lubricants such as a silicone oil to coat the internal surface of the syringe barrel to ease motion of the plunger. Silicone oil is also applied to the exteriors of the hypodermic needle to ease movement through the epidermis during subcutaneous injections. Thirumangalathu et al.15 have demonstrated that the biophysical stability of monoclonal antibodies (MAbs) is adversely impacted by the presence of silicone oil in formulations. The presence of silicone oil at levels above 0.5% (w/v) was also shown to increase aggregation under accelerated conditions for four model proteins: ribonuclease A, lysozyme, bovine serum albumin, and concanavalin A.16 Studies with MAbs incubated in the presence of silicone oil have shown that the protein adsorbs as a monolayer to the surface of the silicone oil particle. The combination of silicone oil and agitation stress was also shown to lead to protein aggregation.15 Contamination of drug product caused by leaching of silicone oil has also been implicated as a factor responsible for aggregation of insulin in disposable plastic syringes.17–19 Prefilled syringe manufacture requires use of a heated tungsten rod to obtain a needle bore on the syringe. The tungsten wires are known to vaporize and erode during use, depositing a layer of tungsten along the needle bore that comes in contact with the product.20 Bee et al.13 demonstrated that the formation of soluble tungsten polyanions in formulations at DOI 10.1002/jps

EXTRACTABLES–LEACHABLES ASSESSMENT

or below pH 6.0 could lead to protein precipitation. Their research also demonstrates that only a very small number of tungsten particles are required to induce protein aggregation.13 The FDA industry guidance for container-closure systems for packaging human drugs and biologics states that the likelihood of component-dosage form interaction is highest for inhalation and injection products.21 Since a majority of biotechnology drugs are administered parenterally, it is understandable that in recent years there has been increased scrutiny of extractables and leachables associated with biotechnology drug products.22 Evaluation of extractables and leachables in biotechnology products is governed by regulatory documents such as CFR Title 21, part 600.11; CFR Title 21, part 211.94; ICH Q7 Section 7.4, and the FDA Guidance for Industry, Container Closure Systems for Packaging, Human Drugs and Biologics—Chemistry, Manufacturing and Controls for Packaging Documentation published in 1999. Each of these regulatory documents emphasize the importance of ensuring that the components used in the storage of drug products shall be noninteractive with the drug product formulation. Assessment of extractables and leachables is also an integral component of the FDA’s Quality by Design (QbD) initiative in the area of drug product design.23 Leachables are product-related impurities that could directly affect product quality and safety. In addition to these direct effects, they have the potential to react with protein molecules to form protein adducts. The routine stability-indicating assays employed to determine product quality may not be adequate for analysis of leachables and protein adducts. A program dedicated to assessment of these product-related impurities is desirable. The focus of this article is to provide an approach for the assessment of extractables and leachables emanating from components used for the long-term storage of drug substances and drug products with an emphasis on biotechnology products. This minireview provides a framework for developing an extractable–leachable program that encompasses different functional areas within a biopharmaceutical organization. A process for conducting an extractable–leachable program based on the integration of activities among different functional areas such as raw material selection, pharmaceutical development, analytical development, safety assessment, and quality control and assurance is presented. The activities associated with each individual stage of the extractable–leachables program, from selection of components through life-cycle management, are discussed using examples. Not included within the scope of this article are the components used for storage and delivery of orally inhaled nasal drug DOI 10.1002/jps

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products (OINDPs) which has been addressed by Norwood et al.24

DISCUSSION The different stages of the extractable–leachable program are shown in Figure 1. The development of an extractable–leachable program involves coordination of activities between component vendors and functional areas such as raw material selection, pharmaceutical development, analytical development, safety assessment, and quality within a biopharmaceutical organization. This cooperation should be initiated at the stage of selection of a component.

Figure 1. Stages of an extractable–leachable program. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010

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Selection of Components The primary packaging components are those elements of the container-closure systems that are in direct contact with the active pharmaceutical ingredient (API)/drug substance or drug product formulation. For parenterally administered formulations this includes bioprocess bags for API’s or drug substances, and for drug products this includes vials, stoppers, syringe barrels, lined seals, plungers, septa, and needles. Stoppers have been less of a concern in recent years due to the advent of coating technology that renders chemical inertness to their surfaces.8 However, components such as prefilled syringes and cartridges contain silicone oil used for lubrication that may leach into the drug product formulation thereby resulting in protein denaturation or aggregation.8,16,25,26 The ingredients of a formulation such as surfactants (e.g., polysorbate) also have the potential to interact with components and enhance leaching of materials.8,27 Such interactions have the propensity to alter the stability of the drug product formulation as well as the integrity of the container-closure system. As shown in Figure 1, the component selection process becomes a critical aspect to the development of an extractable and leachable program. During the component selection process, the drug product manufacturers must liaise with the component vendors to obtain information concerning materials of construction of components. A complete list of chemicals and additives that are present in the component, if available, should be obtained from the vendor. To enable such information sharing, the roles and responsibilities of the vendor and drug product manufacturer shall be preestablished. Such information sharing should be conducted within the scope of the confidentiality agreements that exist between the vendor and the drug product manufacturer. Jenke28 has emphasized this strategy for developing an extractable/leachable program in collaboration with component vendors. When possible, the drug product

manufacturer should select components that do not contain chemical species that could either leach or interact with the drug substance or drug product or pose a toxicological concern. In some instances, the component vendor may be able to provide information pertaining to extractables testing of their products. The information could then be reviewed based on the following criteria: list of extractables, information concerning extraction conditions, and details concerning assay methods for characterization of extractables that includes information such as limits of detection (LOD). The drug product manufacturer and the vendor should work closely in assessing the health-based risks associated with these extractables. This assessment is performed prior to qualification of a new component. The impact of processing conditions, during manufacturing, on the extractables profile of the components also needs to be evaluated and considered as a part of the assessment. If information cannot be obtained from the vendor or is deemed inadequate for the intended use of a component, the drug product manufacturer should assess the need for in-house extractable and leachable testing of a new component based on a risk-assessment approach. As shown in Table 1, this component risk assessment includes, but is not limited to: aspects such as drug product compatibility, surface area of exposure, duration of exposure, temperature during storage in the component, and information on USP testing of the new component.29–31 In Table 1, the risk-assessment score for components such as stoppers, bioprocess bags, and syringe barrels is shown to be high. This is mainly due to aspects of storage such as large contact surface area and the long residence time in the component. Additionally, the temperature of storage as well as the physical form of the formulation during storage (e.g., frozen, lyophilized) may significantly alter the risk associated with leachables. All these factors must be considered during the risk-assessment phase. As this riskassessment requires a thorough understanding of component use, product configurations, and product

Table 1. Use of Risk-Assessment Tools to Determine if a New Component Needs to Be Assessed for Extractables and Leachables

Contact surface area between the component and the stored solution Presence of organic solvents and/or surfactants in stored solutions Residence time of storage in the component Temperature of storage and/or processing pH of stored solutions USP testing 87,88,661 Total score

Stoppers

Bioprocess Bags

Syringe Barrels

8 4 8 8 4 1
33

8 4 8 4 4 1
29

8 4 8 8 4 1
33

1 ¼ low risk, that is, low probability of component, based on knowledge of component use and testing, to potentially result in leachables in API or drug product; 4 ¼ medium risk; and 8 ¼ high risk, that is, high probability of component, based on knowledge of component use and testing, to potentially result in leachables in API or drug product. The table lists the different storage and processing parameters based on which the risk score for a component was determined. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010

DOI 10.1002/jps

EXTRACTABLES–LEACHABLES ASSESSMENT

stability, a pharmaceutical development scientist should spearhead this stage of the extractable– leachable program. The extent of an extractable–leachable program can be substantially reduced and simplified by developing a ‘‘component-harmonization’’ program. In this program, a database of components that are evaluated and approved with respect to extractables and leachables is maintained. This database should be developed through coordination of functional areas such as pharmaceutical development, analytical development, quality, safety assessment, and raw material selection within the organization. Once a database is established, the drug product manufacturer should restrict component use to what is included in the scope of the component harmonization program. As the conditions of testing for extractables are usually standardized for a component and are not specific to the drug product formulation stored in the component, maintaining a database significantly lessens the extent of extractable characterization work and eliminates redundancies. Based on leachable data generated for different drug product formulations stored in a harmonized component, a bracketing strategy that precludes leachable testing for future drug products (having similar formulations and product configurations) may be possible. This database should be maintained by the quality function within the organization. Testing of Extractables As illustrated in Figure 1, following the risk-assessment procedure, an extractable program should be initiated during the component selection and qualification process. The extractable program should include component treatments that represent extremes of GMP processes and long-term storage conditions. The effect of processing steps, such as

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sterilization and washing on the extractable profile, is examined while designing such an extractable program. As mentioned in the FDA Guidance for Industry, Container Closure Systems for Packaging, Human Drugs and Biologics—Chemistry, Manufacturing and Controls for Packaging Documentation (1999), ‘‘the solvents that are to be used for extraction purposes should have similar propensity to extract chemicals from the components as that of the drug product. A stronger extracting solvent than the drug product would be used to obtain a qualitative extraction profile that would be used to establish quality control criteria.’’21 It is our recommendation that the extractable study be performed with one aqueous and one organic solvent (e.g., water and isopropanol mixtures, isopropanol) at a minimum. The use of extreme solvent conditions (e.g., hexane or methylene chloride) is recommended only for assay development purposes. Use of such extreme solvent conditions may assist with identification and characterization of those chemical species that are observed at low concentrations in water or isopropanol extracts. The choice of extraction conditions such as temperature of extraction and surface area of components should simulate worst-case exposure.29 For example, refluxing with extraction solvents for 8 h was reported for extraction studies on rubber stoppers.32,33 As demonstrated by different research groups, due to the variety of chemical species that can be extracted from a component, different analytical methods are required to obtain a complete extractable profile.24,32,34 Typically, analytical methods such as GC–MS, Headspace (HS) GC–MS, LC–MS, NMR, and ICP–MS may be utilized to characterize leachables. An example of extractable assessment of bioprocess bags is shown in Table 2 where different solvents ranging from water to 10% (w/v) polysorbate were employed in the extraction study. HS GC–MS, GC– MS, and LC–MS were used to analyze volatile,

Table 2. Extractable Data Generated for a Drug Substance Storage Component Under Various Extraction Conditions Extractables Characterized

Methods Extraction solvents Component:

Bioprocess bag

DOI 10.1002/jps

GC–MS

LC–MS

Water, 0.1 M H3PO4, 0.1 M NaOH, IPA, 50% IPA/water, 10% polysorbate 20, 10% polysorbate 80, and hexane (if appropriate) 2,4-bis(1,1-dimethylethyl)-phenol, (Z)-13-docosenamide (Erucamide), tris phosphate, Irganox 1076, 3,5-di-ter-butyl-4-hydroxybenzaldehy de, 7,9-di-tert-butyl-1-oxaspiro(4,5) deca-6,9-diene-2,8-dione, n-hexadecanoic acid (palmitic acid), octadecanoic acid, hydrocarbon envelope, siloxane related, 1,3-bis(1,1-dimethylethyl)-benzene

Polyethylene glycol (PEG)related, docosenamide (Erucamide), Irganox 1076, oxidized Irgafos 168, hexadecenamide, Irgafos P-EPQ related, BHT, Irganox 1010, octadecenamide

HS GC–MS Water

Isopropanol, trimethylsilanol, octamethylcyclotet rasiloxane, siloxane related, decamethylcyclop entasiloxane (DMCPS), dodecamethylcycl ohexasiloxane, hydrocarbon related

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semivolatile, and nonvolatile species to obtain a complete extraction profile. A detailed discussion on the choice of analytical methodologies for conducting extractable studies is available in a publication by Wang.35 A qualitative assessment that primarily includes identification and characterization of the different chemical compounds observed during this stage of the extractable–leachable program is recommended. This qualitative assessment focuses on the chemical entities that are observed at high levels during the extraction study that, based on their physico-chemical properties, are likely to be leachables. In certain cases, such as components used in early clinical development, a controlled-extraction study similar to the one mentioned in Product Quality Research Institute’s (PQRI) recommendation for OINDPs may be performed.24 In a controlled-extraction study, the different extractables are identified, quantified, and assessed for potential impacts to patient health. Lack of health-risk concerns with these extractables may prevent the need to conduct a full-scale leachable assessment during the early clinical development phases. The design of an extraction study requires understanding of the manufacturing process, molecular properties of extractables and expertise in terms of choice of appropriate analytical tools. As shown in Figure 2, it is recommended that the analytical development groups within the organization lead this stage of the extractable–leachable assessment. Following the qualitative assessment, the list of characterized extractables is submitted to the toxicologist for the purpose of a health-based risk assessment (Fig. 1).

Selection of Target Leachables The list of characterized extractables is examined for the presence of chemicals that could be of potential toxicological concern. This is typically based on a review of the existing toxicological databases including the National Library of Medicine’s TOXNET (includes TOXLINE), PubMed, and RTECS. Based on available data, those chemical species that do not pose toxicological concerns and are generally regarded as safe (GRAS) are excluded from the list of extractables. Those chemical species that are not excluded are selected as target leachables. A representative compound may be selected, based on its relative abundance, as a surrogate for a group of chemical species that are structurally similar. Another consideration in the selection of surrogate compounds is solubility in the product formulation. In our experience, examples of surrogate molecules have included palmitic acid used as a surrogate for myristic acid, dodecanoic acid, and stearic acid and cyclohexane used as a surrogate for hexane, pentane, and cyclopentane. In such instances, the health-based risk assessment is performed for the surrogate compound. The selection of surrogate compounds as target leachables will be based on information obtained in the health-based risk assessment. As shown in Figure 2, the process for selecting target leachables requires cooperation between analytical development and safety assessment functional areas within the organization. An understanding of the analytical detection limits, chemical properties such as structure–activity relationships, and related toxicological concerns is desired during this stage of the

Start components pre approved? Select DP/DS components

No

Evaluate need for Extractable/ Leachable testing

Compile Extractable Study Report

Evaluate need for additional extractable study

Compile Leachable Study Report

Design Leachable Study

Yes

Safety Assessment

Compile Extractable/ Leachable data for IND & BLA

Assess protein stability

Obtain relevant data from vendor Select target leachables

Evaluate for in vs outsource

Qualify assays for target leach

Define extractable study

Review and approve study report

In-House or Out-Sourced Testing

Characterize Extractables

Develop assay to monitor protein adducts

Compile Extractable Characterization Report

Characterize protein adducts Report Leachables

Clinical Product Quality

Protein Analytical Chemistry (PAC)

Pharmaceutical & Device Development (PDD)

Extractable & Leachable Test Development Flow

Review and approve study report

Perform Extractable Health Based Risk Assessment

Maintain Extractable/ Leachable Reports

Perform Leachable Health Based Risk Assessment

Figure 2. A business process model depicting the extractable–leachable process flow among different departments within an organization. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010

DOI 10.1002/jps

EXTRACTABLES–LEACHABLES ASSESSMENT

extractable–leachable program. Upon completion of the health-based risk assessment, analytical methods should be qualified to identify and quantify those extractables that are selected as target leachables. Performing a Leachable Study The leachables in drug substances and drug products should be monitored under real-time and accelerated conditions. These studies could be conducted as a part of the drug substance and the drug product stability program that is designed in accordance with the ICH Q1A (R2) guidelines.36 An example of a study program for a prefilled syringe component to assess organic leachables is shown in Table 3. The table shows the different target leachables selected for monitoring prior to initiating a leachable study (e.g., naphthalene, BHT, dodecanoic acid, 3,5-HBA, etc.) and the different temperature conditions that were used to incubate samples of the drug product as per the ICH Q1A (R2) guidelines. Most of the leachables were observed to be below the limit of quantitation. Accumulation of 9.9 mg/mL dodecanoic acid was observed following incubation at 258C and 60% relative humidity (RH) for up to 6 months. The leachable program also included evaluation of metal impurities using ICP–MS and analysis for the presence of silicone (data not shown). The conditions employed for incubating samples in a leachable study are required to simulate worst-case storage conditions during actual use. For example, for a liquid product filled into a vial/stopper assembly, vials are

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maintained in an inverted position during the leachable assessment. This procedure maximizes exposure to the rubber stopper, a potential source of leachables. For cartridges or prefilled syringes, ensure that the drug product is in contact with its components such as the lined seal, plunger, and surfaces that are typically lubricated with silicone oil. Jenke37 established the importance of demonstrating a qualitative and quantitative correlation between leachables and extractables that are observed. A qualitative correlation can be established by demonstrating that the leachables observed are the same chemical species as those present in the extractable study. A quantitative correlation is based on demonstrating that the concentrations of chemical compounds observed in a leachable study are below those observed during extractable characterization. Establishing such a correlation between extractables and leachables is important in validating the design of the extractable–leachable program. Establishing a correlation between the extractables and leachables may enable one to perform the health-based risk assessment based on levels of extractables. If the levels of extractables are determined to not have a potentially adverse impact on patient health, then future leachable testing may be precluded.37,38 A justification for lack of leachable data may need to be provided in cases wherein data from health-based risk assessment of extractables are used to qualify components. In certain cases a leachable species observed might not be a subset of either the target leachables selected

Table 3. Leachable Data Generated During Storage of a Drug Product Formulation in a Prefilled Syringe Target LOQ (mg/mL) LOD (mg/mL) Condition Time (8C/%RH) (month) 0 1 58C/ambient RH 3 6 12 18 24 258C/60% RH 1 3.5 6 308C/65% RH 0.25 1

Naphthalene

BHT

Dodecanoic Acid

3,5-HBA

Palmitic Acid

2246

Irganox 1076

0.101 0.031

0.200 0.061

7.943 2.407

0.198 0.060

7.930 2.403

1.199 0.363

2.999 0.909

ND ND ND ND ND BLOQ ND ND ND ND ND ND

BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ BLOQ

ND ND ND ND ND BLOQ BLOQ ND 8.9 9.9 ND ND

ND ND ND ND ND BLOQ ND ND ND ND ND ND

ND ND ND ND ND BLOQ ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND ND

BHT ¼ 2,6-di-tert-butyl-4-methylphenol 3,5-HBA ¼ 3,5-di-tert-butyl-4-hydroxybenzaldehyde 2246 ¼ 2,2-Methylenebis(6-tert-butyl-4-methylphenol) Irganox 1076 ¼ Octadecyl 3-(3,5-de-tert-butyl-4-hydroxyphenyl)-propionate ND ¼ Not Detected BLOQ ¼ Below Limit of Quantitation DOI 10.1002/jps

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or even a subset of the extractables that are reported. An example of such a situation was reported for Irganox 1076, an antioxidant used in rubber stoppers. In this case, the leachables observed were the chemically modified degradants of the parent compound Irganox 1076.37 Such chemically degraded species that have structure–activity relationships to the parent compound should be monitored. The identification and quantitation of such chemical species also need to be performed as a part of leachable study. Assay qualification for such leachables should be performed if these species are observed in significant amounts and/or present a toxicological concern. The origin of such leachables should also be determined. An aspect of leachable assessment that has not been specifically mentioned in earlier literature reports is the impact of leachables on protein quality and product purity. Monitoring changes such as formation of aggregates, fragments, acidic and basic variants, and protein adducts should also be performed concurrently with the leachable assessment. In a recent study by Ji et al.,39 formation of high molecular weight protein adducts due to protein– leachable interactions as well as the presence of polymeric leachables was detected in a topical methylcellulose gel formulation of a recombinant humanized protein, rhVEGF, that was stored in contact with an Adaptic1 wound dressing material. Such interactions between proteins and leachables may also occur during long-term storage of drug product in contact with components such as rubber stoppers, lined seals, plungers, polyethylene bottles, etc. In such instances, in addition to monitoring drug product stability using routine stability-indicating assays, the analytical scientist may need to monitor samples for the presence of protein adducts. A direct method to monitor formation of protein adducts uses mass spectrometry. An indirect method employed to monitor formation of protein adducts is to include a placebo formulation in the leachable assessment. Any difference in the level of leachables between drug product formulation and the placebo formulation may suggest interaction of the leachable with the protein. The leachable study must be initiated using appropriate representative drug substance or drug product material prior to NDA or BLA filing. It is our recommendation that this study should be initiated using Phase III material or material from the qualification lots. As shown in Figure 2, the pharmaceutical development and quality control scientists should be responsible for developing the design of this leachable study as well as addressing any sample handling issues. The testing of leachables should be performed in an analytical laboratory using qualified assay methods. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010

Health-Based Risk Assessment of Leachables Health-based risk assessment is conducted following the completion of the leachable study stage (Fig. 1). This risk assessment is the responsibility of the safety assessment/toxicology group within the biopharmaceutical organization (Fig. 2). Risk assessment is typically performed using data on leachables obtained under real-time storage conditions for drug substances and drug products. In certain cases leachables observed under accelerated storage conditions may be included as a part of the risk assessment. A summary describing the observed leachables and their concentrations, the drug product indication, route of administration, dosing regimen, frequency of dosing, and patient population should be prepared. This information is used to compute the estimated daily intake (dose) of the leachable. A detailed discussion on safety qualification of extractables and leachables is available in a review by Northup.40 The PQRI group introduced the concept of safety thresholds for leachables that were observed in OINDPs.24 Based on their investigations a safety concern threshold of 0.15 mg/day and a qualification threshold of 5 mg/day is recommended for leachables and chemical impurities observed in OINDPs.41 The health-based risk assessment is based on knowledge of systemic toxicity, route specific toxicity, and mutagenic potential of a compound. Information such as compound estimated daily intake (EDI) and/ or acceptable daily intake (ADI) values and/or thresholds of toxicological concern (TTC) and/or other risk-assessment determinations are established. ADIs are calculated for leachables for which adequate toxicology data are available. The EDI is a riskassessment calculation used for pesticides, food additives, etc., used to define the daily intake of a chemical that during an entire lifetime appears to be without appreciable health risk on the basis of all known facts. ADIs typically are calculated from No Observed Adverse Effect Level (NOAEL) values by dividing by safety and/or modifying factors (e.g., 10fold). Dividing by these factors allows for animal-tohuman and human-to-human variability, experimental differences, and mechanistic or pharmacokinetic considerations. Each leachable-specific ADI is then compared to its corresponding EDI. In the absence of sufficient toxicity data to calculate an ADI, a qualitative structure–activity relationship, QSAR, assessment (DEREK, version 11.0) is conducted. The qualification threshold of 5 Mg/day is applied to leachable structures for which the QSAR assessment does not result in mutagenicity and/or carcinogenicity alerts. Leachable structures for which a QSAR assessment results in mutagenicity and/or carcinogenicity alerts are assigned a safety concern threshold of 0.15 mg/day. DOI 10.1002/jps

EXTRACTABLES–LEACHABLES ASSESSMENT

Documentation and Life-Cycle Management Documentation of extractable and leachable reports (Fig. 2) is critical to every stage of the extractable– leachable program. At the completion of the extractable study an extractable characterization report is drafted that includes information such as: (a) rationale for conducting the extractable study, (b) choice of extraction conditions, (c) analytical assays utilized for generating the extractable profile, (d) list of extractables, (e) health-based risk assessment of extractables, (f) list of target leachables selected with justification, and (g) qualification of analytical assays for target leachables. The leachable study is typically a long-term study spanning the intended shelf-life of a product. Upon completion of the leachable study, data related to leachables are appended to the extractable characterization report (Fig. 2). The resulting extractable–leachable characterization report includes, in addition to the information summarized in the extractable characterization report, information on (a) leachable study design, (b) list of leachables and their concentrations, (c) information on effects of leachables on drug product stability and protein adducts if applicable, and (d) health-based risk assessment of leachables. This report is then maintained as a document within the quality group of the biopharmaceutical organization. The documentation of the extractable–leachable program is beneficial for managing vendor-initiated changes (VICs) in components during the product life cycle. In the case of VICs, the vendor is responsible for notifying the drug manufacturer prior to initiating a change in the component ingredient and/or its manufacturing process. A detailed description of the change in component ingredient and/or the manufacturing process must be provided. The drug product manufacturer assesses the impact of the VIC on product quality and safety and evaluates the need for any additional extractable–leachable assessment. Details concerning how to manage VICs should be described in contractual agreements between drug product manufacturer and component vendor.

CONCLUSION Leachables emanating from components used for long-term storage of API/drug substance and drug product are a concern for biotechnology products due to potential effects on product quality and patient safety. Protein formulations are susceptible to leachable contamination due to surfactant activity of proteins themselves as well as the presence of surfactants that are present as excipients in a majority of protein formulations (e.g., polysorbates). Conducting extractable–leachable studies is required per regulatory guidance documents. A complete DOI 10.1002/jps

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extractable–leachable assessment should also include information on the effects of leachables on protein stability for, for example, formation of protein adducts. Harmonizing component use within a biopharmaceutical organization is the cornerstone of a successful extractable–leachable program. To conduct an effective extractable–leachable program, the key stakeholders should be identified earlier in the process and their roles and responsibilities should be well understood and documented. Integration of analytical and health-based risk-assessment information throughout the different stages of the extractable–leachable program is a key element of the program.

ACKNOWLEDGMENTS The authors thank Suzanne Weck and Timothy Finan for their contributions towards developing the extractable–leachable program and business process at Genentech, Inc.

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DOI 10.1002/jps