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 pro-
duct 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 leach-
ables 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 respon-
sible 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 informa-
tion 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 assess-
ment; protein adducts; drug compatibility
INTRODUCTION
Extractables are a class of compounds released from a
Correspondence to: Aditya A. Wakankar (Telephone: 650-225-
component under aggressive treatment conditions;
6739; Fax: [phone]; E-mail: [e-mail])
those that exceed what a component may endure
Journal of Pharmaceutical Sciences, Vol. 99, 2209 2218 (2010)
during normal use (e.g., extended time, increased
ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 2209
2210 WAKANKAR ET AL.
temperature and ionic strength, pH extremes). potential cause for the immunogenicity was modifica-
Leachables are a class of compounds that emanate tion of epoetin when associated with polysorbate
from a component into the drug substance or drug micelles.10 In a recent study, Mueller et al.11 inves-
product under normal conditions and are typically a tigated the   immunogenicity potential  of extracta-
subset of extractables. Wang and Chien1 extensively bles and leachables from three drug product stoppers
reviewed the kinetics models and factors that affect using dendritic cell models. Activation of dendritic
leaching phenomena in 1984. Leaching typically cells was monitored by analyzing the expression
occurs upon migration of dissolved solute through levels of the costimulatory molecule CD86. The
the polymer matrix. The rate of leaching is diffusion stoppers evaluated in this investigation included
controlled, and the amount of leachable is in the PH 4106 stopper reported to be a component of the
proportion to the square root of time. The diffusion Eprex1 prefilled syringe. This investigation sug-
of organic substances in polymer matrix is also gested that it is not the extractables and leachables
governed by activation energy that is in the range from these different stoppers that lead to an increase
of 10 13 kcal/mol, thus one can expect the rate to in the dendritic readout. Instead, the hydrolytic
double for every 108C increase in temperature.2,3 On breakdown product of polysorbate 80, oleic acid, as
the other hand, leaching of plasticizer from polyvinyl well as other follow-up products were identified as
chloride (PVC) bags follows a linear relationship with plausible factors responsible for the increase in the
time because the plasticizer is present in large dendritic activation. Other factors such as leached
concentration and encounters a nontortuous path silicone oil, a lubricant used in the prefilled syringe,
by which the leaching substance can diffuse. Early have also been considered as potential causes for
efforts to detect extractables or leachables have increase in immunogencity.9
employed UV spectroscopy or other nonspecific Novel drug-delivery technologies such as prefilled
methodologies. A pioneering work on identifying syringes and pen-injector systems have also necessi-
extractables in a biotechnology product was con- tated scrutiny of leachables as new materials are
ducted using LC in series with electrospray MS. In introduced to product contact. These devices consist
this study, butylated hydroxy toluene (BHT) and a of components such as plungers, stoppers, lined seals,
polymer species were identified as extractables when and needles, each having the potential to leach
acetonitrile was used as the extraction medium. chemicals into the drug product formulation.12 14
However, these substances were not found in the drug Additionally, these components utilize lubricants
product when extracted with the protein formulation such as a silicone oil to coat the internal surface of
or buffer formulation.4 the syringe barrel to ease motion of the plunger.
In recent years the Eprex1 case has had a major Silicone oil is also applied to the exteriors of the
impact on the regulatory scrutiny related to extrac- hypodermic needle to ease movement through the
tables and leachables. There were 175 cases of epidermis during subcutaneous injections. Thiru-
epoetin-associated pure red-cell aplasia (PRCA) mangalathu et al.15 have demonstrated that the
reported for Eprex1 between 1998 and 2004. Most biophysical stability of monoclonal antibodies (MAbs)
of these cases involved patients with chronic kidney is adversely impacted by the presence of silicone oil in
disease who had received subcutaneous injections of formulations. The presence of silicone oil at levels
epoetin.5 The phenolic derivatives that leached from above 0.5% (w/v) was also shown to increase
the rubber stopper, used in prefilled syringes, into the aggregation under accelerated conditions for four
formulation were postulated to be a causative agent model proteins: ribonuclease A, lysozyme, bovine
for the immunogenic response observed.6 8 It was serum albumin, and concanavalin A.16 Studies with
proposed that the presence of polysorbate 80, the MAbs incubated in the presence of silicone oil have
stabilizer employed to replace human serum albumin shown that the protein adsorbs as a monolayer to the
in the second-generation drug product formulation, surface of the silicone oil particle. The combination of
induced the leaching of these phenolic derivatives.8,9 silicone oil and agitation stress was also shown to lead
Although a clear association between the presence of to protein aggregation.15 Contamination of drug
these leachables and the incidence of PRCA could not product caused by leaching of silicone oil has also
be demonstrated, the decrease in instances of PRCA been implicated as a factor responsible for aggrega-
did coincide with the change in the stopper config- tion of insulin in disposable plastic syringes.17 19
uration from the original uncoated stopper to the Prefilled syringe manufacture requires use of a
current Teflon-coated stopper.5,7 The incidences of heated tungsten rod to obtain a needle bore on the
Eprex1-related PRCA dropped significantly after syringe. The tungsten wires are known to vaporize
additional changes were applied to storage and and erode during use, depositing a layer of tungsten
handling and the route of administration was along the needle bore that comes in contact with the
changed from subcutaneous (SC) to intravenous product.20 Bee et al.13 demonstrated that the forma-
(IV) route. Another factor that was identified as a tion of soluble tungsten polyanions in formulations at
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 DOI 10.1002/jps
EXTRACTABLES LEACHABLES ASSESSMENT 2211
or below pH 6.0 could lead to protein precipitation. products (OINDPs) which has been addressed by
Their research also demonstrates that only a very Norwood et al.24
small number of tungsten particles are required to
induce protein aggregation.13
The FDA industry guidance for container-closure
DISCUSSION
systems for packaging human drugs and biologics
states that the likelihood of component-dosage form
The different stages of the extractable leachable
interaction is highest for inhalation and injection
program are shown in Figure 1. The development of
products.21 Since a majority of biotechnology drugs
an extractable leachable program involves coordina-
are administered parenterally, it is understandable
tion of activities between component vendors and
that in recent years there has been increased scrutiny
functional areas such as raw material selection,
of extractables and leachables associated with bio- pharmaceutical development, analytical develop-
technology drug products.22 Evaluation of extracta- ment, safety assessment, and quality within a bio-
bles and leachables in biotechnology products is
pharmaceutical organization. This cooperation
governed by regulatory documents such as CFR Title
should be initiated at the stage of selection of a
21, part 600.11; CFR Title 21, part 211.94; ICH Q7
component.
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 empha-
size 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 ema-
nating from components used for the long-term
storage of drug substances and drug products with
an emphasis on biotechnology products. This mini-
review provides a framework for developing an
extractable leachable program that encompasses
different functional areas within a biopharmaceutical
organization. A process for conducting an extracta-
ble 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 Figure 1. Stages of an extractable leachable program.
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
2212 WAKANKAR ET AL.
Selection of Components manufacturer should select components that do not
contain chemical species that could either leach or
The primary packaging components are those ele- interact with the drug substance or drug product or
ments of the container-closure systems that are in pose a toxicological concern. In some instances, the
direct contact with the active pharmaceutical ingre- component vendor may be able to provide information
dient (API)/drug substance or drug product formula- pertaining to extractables testing of their products.
tion. For parenterally administered formulations this The information could then be reviewed based on the
includes bioprocess bags for API s or drug substances, following criteria: list of extractables, information
and for drug products this includes vials, stoppers, concerning extraction conditions, and details con-
syringe barrels, lined seals, plungers, septa, and cerning assay methods for characterization of extrac-
needles. Stoppers have been less of a concern in recent tables that includes information such as limits of
years due to the advent of coating technology that detection (LOD). The drug product manufacturer and
renders chemical inertness to their surfaces.8 How- the vendor should work closely in assessing the
ever, components such as prefilled syringes and health-based risks associated with these extractables.
cartridges contain silicone oil used for lubrication This assessment is performed prior to qualification of
that may leach into the drug product formulation a new component. The impact of processing condi-
thereby resulting in protein denaturation or aggrega- tions, during manufacturing, on the extractables
tion.8,16,25,26 The ingredients of a formulation such as profile of the components also needs to be evaluated
surfactants (e.g., polysorbate) also have the potential and considered as a part of the assessment.
to interact with components and enhance leaching of If information cannot be obtained from the vendor
materials.8,27 Such interactions have the propensity or is deemed inadequate for the intended use of a
to alter the stability of the drug product formulation component, the drug product manufacturer should
as well as the integrity of the container-closure assess the need for in-house extractable and leachable
system. As shown in Figure 1, the component testing of a new component based on a risk-assess-
selection process becomes a critical aspect to the ment approach. As shown in Table 1, this component
development of an extractable and leachable pro- risk assessment includes, but is not limited to: aspects
gram. such as drug product compatibility, surface area of
During the component selection process, the drug exposure, duration of exposure, temperature during
product manufacturers must liaise with the compo- storage in the component, and information on USP
nent vendors to obtain information concerning testing of the new component.29 31 In Table 1, the
materials of construction of components. A complete risk-assessment score for components such as stop-
list of chemicals and additives that are present in the pers, bioprocess bags, and syringe barrels is shown to
component, if available, should be obtained from the be high. This is mainly due to aspects of storage such
vendor. To enable such information sharing, the roles as large contact surface area and the long residence
and responsibilities of the vendor and drug product time in the component. Additionally, the temperature
manufacturer shall be preestablished. Such informa- of storage as well as the physical form of the
tion sharing should be conducted within the scope of formulation during storage (e.g., frozen, lyophilized)
the confidentiality agreements that exist between the may significantly alter the risk associated with
vendor and the drug product manufacturer. Jenke28 leachables. All these factors must be considered
has emphasized this strategy for developing an during the risk-assessment phase. As this risk-
extractable/leachable program in collaboration with assessment requires a thorough understanding of
component vendors. When possible, the drug product 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
Stoppers Bioprocess Bags Syringe Barrels
Contact surface area between the component and the stored solution 8 8 8
Presence of organic solvents and/or surfactants in stored solutions 4 4 4
Residence time of storage in the component 8 8 8
Temperature of storage and/or processing 8 4 8
pH of stored solutions 4 4 4
USP testing 87,88,661 1
1 1
Total score
33 29 33
1 ź 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 ź medium risk; and 8 ź 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 2213
stability, a pharmaceutical development scientist sterilization and washing on the extractable profile, is
should spearhead this stage of the extractable examined while designing such an extractable
leachable program. program. As mentioned in the FDA Guidance for
The extent of an extractable leachable program Industry, Container Closure Systems for Packaging,
can be substantially reduced and simplified by Human Drugs and Biologics Chemistry, Manufac-
developing a   component-harmonization  program. turing and Controls for Packaging Documentation
In this program, a database of components that are (1999),   the solvents that are to be used for extraction
evaluated and approved with respect to extractables purposes should have similar propensity to extract
and leachables is maintained. This database should chemicals from the components as that of the drug
be developed through coordination of functional areas product. A stronger extracting solvent than the drug
such as pharmaceutical development, analytical product would be used to obtain a qualitative
development, quality, safety assessment, and raw extraction profile that would be used to establish
material selection within the organization. Once a quality control criteria.  21 It is our recommendation
database is established, the drug product manufac- that the extractable study be performed with one
turer should restrict component use to what is aqueous and one organic solvent (e.g., water and
included in the scope of the component harmonization isopropanol mixtures, isopropanol) at a minimum.
program. As the conditions of testing for extractables The use of extreme solvent conditions (e.g., hexane or
are usually standardized for a component and are not methylene chloride) is recommended only for assay
specific to the drug product formulation stored in the development purposes. Use of such extreme solvent
component, maintaining a database significantly conditions may assist with identification and char-
lessens the extent of extractable characterization acterization of those chemical species that are
work and eliminates redundancies. Based on leach- observed at low concentrations in water or isopropa-
able data generated for different drug product nol extracts. The choice of extraction conditions such
formulations stored in a harmonized component, a as temperature of extraction and surface area of
bracketing strategy that precludes leachable testing components should simulate worst-case exposure.29
for future drug products (having similar formulations For example, refluxing with extraction solvents for
and product configurations) may be possible. This 8 h was reported for extraction studies on rubber
database should be maintained by the quality stoppers.32,33 As demonstrated by different research
function within the organization. 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
Testing of Extractables
profile.24,32,34 Typically, analytical methods such as
As illustrated in Figure 1, following the risk-assess- GC MS, Headspace (HS) GC MS, LC MS, NMR, and
ment procedure, an extractable program should be ICP MS may be utilized to characterize leachables.
initiated during the component selection and quali- An example of extractable assessment of bioprocess
fication process. The extractable program should bags is shown in Table 2 where different solvents
ranging from water to 10% (w/v) polysorbate were
include component treatments that represent
employed in the extraction study. HS GC MS, GC
extremes of GMP processes and long-term storage
conditions. The effect of processing steps, such as 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 GC MS LC MS HS GC MS
Extraction Water, 0.1 M H3PO4, 0.1 M NaOH, IPA, 50% IPA/water, 10% polysorbate 20, Water
solvents 10% polysorbate 80, and hexane (if appropriate)
Component:
2,4-bis(1,1-dimethylethyl)-phenol, Polyethylene glycol (PEG)- Isopropanol, trimethylsilanol,
Bioprocess
(Z)-13-docosenamide (Erucamide), related, docosenamide octamethylcyclotet
bag
tris phosphate, Irganox 1076, (Erucamide), rasiloxane,
3,5-di-ter-butyl-4-hydroxybenzaldehy Irganox 1076, oxidized siloxane related,
de, 7,9-di-tert-butyl-1-oxaspiro(4,5) Irgafos 168, decamethylcyclop
deca-6,9-diene-2,8-dione, hexadecenamide, entasiloxane (DMCPS),
n-hexadecanoic acid (palmitic acid), Irgafos P-EPQ related, dodecamethylcycl
octadecanoic acid, hydrocarbon BHT, Irganox 1010, ohexasiloxane, hydrocarbon
envelope, siloxane related, octadecenamide related
1,3-bis(1,1-dimethylethyl)-benzene
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
2214 WAKANKAR ET AL.
semivolatile, and nonvolatile species to obtain a Selection of Target Leachables
complete extraction profile. A detailed discussion on
the choice of analytical methodologies for conducting The list of characterized extractables is examined for
extractable studies is available in a publication by the presence of chemicals that could be of potential
Wang.35 toxicological concern. This is typically based on a
A qualitative assessment that primarily includes review of the existing toxicological databases includ-
identification and characterization of the different ing the National Library of Medicine s TOXNET
chemical compounds observed during this stage of the (includes TOXLINE), PubMed, and RTECS. Based on
extractable leachable program is recommended. This available data, those chemical species that do not pose
qualitative assessment focuses on the chemical toxicological concerns and are generally regarded as
entities that are observed at high levels during the safe (GRAS) are excluded from the list of extractables.
extraction study that, based on their physico-chemi- Those chemical species that are not excluded are
cal properties, are likely to be leachables. In certain selected as target leachables. A representative
cases, such as components used in early clinical compound may be selected, based on its relative
development, a controlled-extraction study similar to abundance, as a surrogate for a group of chemical
the one mentioned in Product Quality Research species that are structurally similar. Another con-
Institute s (PQRI) recommendation for OINDPs sideration in the selection of surrogate compounds is
may be performed.24 In a controlled-extraction study, solubility in the product formulation. In our experi-
the different extractables are identified, quantified, ence, examples of surrogate molecules have included
and assessed for potential impacts to patient health. palmitic acid used as a surrogate for myristic acid,
Lack of health-risk concerns with these extractables dodecanoic acid, and stearic acid and cyclohexane
may prevent the need to conduct a full-scale leachable used as a surrogate for hexane, pentane, and
assessment during the early clinical development cyclopentane. In such instances, the health-based
phases. The design of an extraction study requires risk assessment is performed for the surrogate
understanding of the manufacturing process, mole- compound. The selection of surrogate compounds as
cular properties of extractables and expertise in target leachables will be based on information
terms of choice of appropriate analytical tools. As obtained in the health-based risk assessment. As
shown in Figure 2, it is recommended that the shown in Figure 2, the process for selecting target
analytical development groups within the organiza- leachables requires cooperation between analytical
tion lead this stage of the extractable leachable development and safety assessment functional areas
assessment. Following the qualitative assessment, within the organization. An understanding of the
the list of characterized extractables is submitted to analytical detection limits, chemical properties such
the toxicologist for the purpose of a health-based risk as structure activity relationships, and related tox-
assessment (Fig. 1). icological concerns is desired during this stage of the
Extractable & Leachable Test Development Flow
Start
Evaluate need
Compile
No
components for Extractable/
Extractable Study
pre approved? Leachable
Report
Compile
testing
Select DP/DS Leachable Study
Compile
Yes Design
components Report
Extractable/
Leachable
Leachable data
Study
for IND & BLA
Assess
Evaluate need
Obtain
protein
for additional
relevant data
stability
extractable
from vendor
study
Select target
leachables
Qualify
Define
Evaluate for in assays for
extractable
vs outsource target leach
study
Review and
In-House or Out-Sourced Testing
approve study
report
Develop assay to Characterize
Compile
monitor protein protein
Extractable
adducts adducts
Characterization
Characterize
Report
Report
Extractables
Leachables
Maintain
Review and
Extractable/
approve study
Leachable
report
Reports
Perform Perform
Extractable Leachable
Health Based Health Based
Risk Risk
Assessment 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
Development (PDD)
(PAC)
Safety
Clinical Product
Protein Analytical Chemistry
Pharmaceutical & Device
Assessment
Quality
EXTRACTABLES LEACHABLES ASSESSMENT 2215
extractable leachable program. Upon completion of maintained in an inverted position during the
the health-based risk assessment, analytical methods leachable assessment. This procedure maximizes
should be qualified to identify and quantify those exposure to the rubber stopper, a potential source
extractables that are selected as target leachables. 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
Performing a Leachable Study
surfaces that are typically lubricated with silicone oil.
The leachables in drug substances and drug products Jenke37 established the importance of demonstrat-
should be monitored under real-time and accelerated ing a qualitative and quantitative correlation
conditions. These studies could be conducted as a part between leachables and extractables that are
of the drug substance and the drug product stability observed. A qualitative correlation can be established
program that is designed in accordance with the by demonstrating that the leachables observed are
ICH Q1A (R2) guidelines.36 An example of a study the same chemical species as those present in the
program for a prefilled syringe component to assess extractable study. A quantitative correlation is based
organic leachables is shown in Table 3. The table on demonstrating that the concentrations of chemical
shows the different target leachables selected for compounds observed in a leachable study are below
monitoring prior to initiating a leachable study (e.g., those observed during extractable characterization.
naphthalene, BHT, dodecanoic acid, 3,5-HBA, etc.) Establishing such a correlation between extractables
and the different temperature conditions that were and leachables is important in validating the design
used to incubate samples of the drug product as per of the extractable leachable program. Establishing a
the ICH Q1A (R2) guidelines. Most of the leachables correlation between the extractables and leachables
were observed to be below the limit of quantitation. may enable one to perform the health-based risk
Accumulation of 9.9 mg/mL dodecanoic acid was assessment based on levels of extractables. If the
observed following incubation at 258C and 60% levels of extractables are determined to not have a
relative humidity (RH) for up to 6 months. The potentially adverse impact on patient health, then
leachable program also included evaluation of metal future leachable testing may be precluded.37,38 A
impurities using ICP MS and analysis for the justification for lack of leachable data may need to be
presence of silicone (data not shown). The conditions provided in cases wherein data from health-based
employed for incubating samples in a leachable study risk assessment of extractables are used to qualify
are required to simulate worst-case storage condi- components.
tions during actual use. For example, for a liquid In certain cases a leachable species observed might
product filled into a vial/stopper assembly, vials are 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 Naphthalene BHT Dodecanoic Acid 3,5-HBA Palmitic Acid 2246 Irganox 1076
LOQ (mg/mL) 0.101 0.200 7.943 0.198 7.930 1.199 2.999
LOD (mg/mL) 0.031 0.061 2.407 0.060 2.403 0.363 0.909
Condition Time
(8C/%RH) (month)
0 ND BLOQ ND ND ND ND ND
1 ND BLOQ ND ND ND ND ND
58C/ambient RH
3 ND BLOQ ND ND ND ND ND
6 ND BLOQ ND ND ND ND ND
12 ND BLOQ ND ND ND ND ND
18 BLOQ BLOQ BLOQ BLOQ BLOQ ND ND
24 ND BLOQ BLOQ ND ND ND ND
258C/60% RH 1 ND BLOQ ND ND ND ND ND
3.5 ND BLOQ 8.9 ND ND ND ND
6 ND BLOQ 9.9 ND ND ND ND
308C/65% RH 0.25 ND BLOQ ND ND ND ND ND
1 ND BLOQ ND ND ND ND ND
BHT ź 2,6-di-tert-butyl-4-methylphenol
3,5-HBA ź 3,5-di-tert-butyl-4-hydroxybenzaldehyde
2246 ź 2,2-Methylenebis(6-tert-butyl-4-methylphenol)
Irganox 1076 ź Octadecyl 3-(3,5-de-tert-butyl-4-hydroxyphenyl)-propionate
ND ź Not Detected
BLOQ ź Below Limit of Quantitation
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
2216 WAKANKAR ET AL.
or even a subset of the extractables that are reported. Health-Based Risk Assessment of Leachables
An example of such a situation was reported for
Irganox 1076, an antioxidant used in rubber stoppers. Health-based risk assessment is conducted following
In this case, the leachables observed were the the completion of the leachable study stage (Fig. 1).
chemically modified degradants of the parent com- This risk assessment is the responsibility of the safety
pound Irganox 1076.37 Such chemically degraded assessment/toxicology group within the biopharma-
species that have structure activity relationships to ceutical organization (Fig. 2). Risk assessment is
the parent compound should be monitored. The typically performed using data on leachables obtained
identification and quantitation of such chemical under real-time storage conditions for drug sub-
species also need to be performed as a part of stances and drug products. In certain cases leach-
leachable study. Assay qualification for such leach- ables observed under accelerated storage conditions
ables should be performed if these species are may be included as a part of the risk assessment. A
observed in significant amounts and/or present a summary describing the observed leachables and
toxicological concern. The origin of such leachables their concentrations, the drug product indication,
should also be determined. route of administration, dosing regimen, frequency of
An aspect of leachable assessment that has not dosing, and patient population should be prepared.
been specifically mentioned in earlier literature This information is used to compute the estimated
reports is the impact of leachables on protein quality daily intake (dose) of the leachable. A detailed
and product purity. Monitoring changes such as discussion on safety qualification of extractables
formation of aggregates, fragments, acidic and basic and leachables is available in a review by Northup.40
variants, and protein adducts should also be per- The PQRI group introduced the concept of safety
formed concurrently with the leachable assessment. thresholds for leachables that were observed in
In a recent study by Ji et al.,39 formation of high OINDPs.24 Based on their investigations a safety
molecular weight protein adducts due to protein concern threshold of 0.15 mg/day and a qualification
leachable interactions as well as the presence of threshold of 5 mg/day is recommended for leachables
polymeric leachables was detected in a topical and chemical impurities observed in OINDPs.41
methylcellulose gel formulation of a recombinant The health-based risk assessment is based on
humanized protein, rhVEGF, that was stored in knowledge of systemic toxicity, route specific toxicity,
contact with an Adaptic1 wound dressing material. and mutagenic potential of a compound. Information
Such interactions between proteins and leachables such as compound estimated daily intake (EDI) and/
may also occur during long-term storage of drug or acceptable daily intake (ADI) values and/or
product in contact with components such as rubber thresholds of toxicological concern (TTC) and/or other
stoppers, lined seals, plungers, polyethylene bottles, risk-assessment determinations are established.
etc. In such instances, in addition to monitoring drug ADIs are calculated for leachables for which adequate
product stability using routine stability-indicating toxicology data are available. The EDI is a risk-
assays, the analytical scientist may need to monitor assessment calculation used for pesticides, food
samples for the presence of protein adducts. A direct additives, etc., used to define the daily intake of a
method to monitor formation of protein adducts chemical that during an entire lifetime appears to be
uses mass spectrometry. An indirect method without appreciable health risk on the basis of all
employed to monitor formation of protein adducts is known facts. ADIs typically are calculated from No
to include a placebo formulation in the leachable Observed Adverse Effect Level (NOAEL) values by
assessment. Any difference in the level of leachables dividing by safety and/or modifying factors (e.g., 10-
between drug product formulation and the placebo fold). Dividing by these factors allows for animal-to-
formulation may suggest interaction of the leachable human and human-to-human variability, experimen-
with the protein. tal differences, and mechanistic or pharmacokinetic
The leachable study must be initiated using considerations. Each leachable-specific ADI is then
appropriate representative drug substance or drug compared to its corresponding EDI. In the absence of
product material prior to NDA or BLA filing. It is our sufficient toxicity data to calculate an ADI, a
recommendation that this study should be initiated qualitative structure activity relationship, QSAR,
using Phase III material or material from the assessment (DEREK, version 11.0) is conducted.
qualification lots. As shown in Figure 2, the pharma- The qualification threshold of 5 Mg/day is applied to
ceutical development and quality control scientists leachable structures for which the QSAR assessment
should be responsible for developing the design of this does not result in mutagenicity and/or carcinogenicity
leachable study as well as addressing any sample alerts. Leachable structures for which a QSAR
handling issues. The testing of leachables should be assessment results in mutagenicity and/or carcino-
performed in an analytical laboratory using qualified genicity alerts are assigned a safety concern thresh-
assay methods. old of 0.15 mg/day.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 DOI 10.1002/jps
EXTRACTABLES LEACHABLES ASSESSMENT 2217
Documentation and Life-Cycle Management extractable leachable assessment should also include
information on the effects of leachables on protein
Documentation of extractable and leachable reports
stability for, for example, formation of protein
(Fig. 2) is critical to every stage of the extractable
adducts. Harmonizing component use within a
leachable program. At the completion of the extrac-
biopharmaceutical organization is the cornerstone
table study an extractable characterization report is
of a successful extractable leachable program. To
drafted that includes information such as: (a)
conduct an effective extractable leachable program,
rationale for conducting the extractable study, (b)
the key stakeholders should be identified earlier in
choice of extraction conditions, (c) analytical assays
the process and their roles and responsibilities should
utilized for generating the extractable profile, (d) list
be well understood and documented. Integration of
of extractables, (e) health-based risk assessment of
analytical and health-based risk-assessment infor-
extractables, (f) list of target leachables selected with
mation throughout the different stages of the
justification, and (g) qualification of analytical assays
extractable leachable program is a key element of
for target leachables. The leachable study is typically
the program.
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
ACKNOWLEDGMENTS
characterization report (Fig. 2). The resulting extrac-
table leachable characterization report includes, in
The authors thank Suzanne Weck and Timothy Finan
addition to the information summarized in the
for their contributions towards developing the extrac-
extractable characterization report, information on
table leachable program and business process at
(a) leachable study design, (b) list of leachables and
Genentech, Inc.
their concentrations, (c) information on effects of
leachables on drug product stability and protein
adducts if applicable, and (d) health-based risk
REFERENCES
assessment of leachables. This report is then main-
tained as a document within the quality group of the
1. Wang YJ, Chien YW. 1984. Sterile pharmaceutical packag-
biopharmaceutical organization. The documentation
ing: Compatibility and Stability, Chapter IIIB, Leaching.
of the extractable leachable program is beneficial for
Parenteral Drug Association. Bethesda, MD. pp 87 106.
managing vendor-initiated changes (VICs) in compo- 2. Hung GW, Autian J. 1972. Use of thermal gravimetric analysis
in sorption studies. II. Evaluation of diffusivity and solubility of
nents during the product life cycle. In the case of
a series of aliphatic alcohols in polyurethane. J Pharm Sci
VICs, the vendor is responsible for notifying the drug
61:1094 1098.
manufacturer prior to initiating a change in the
3. Hung GW, Nunez LJ, Autian J. 1975. Correlation of kinetic
component ingredient and/or its manufacturing
parameters and thermal behavior of segmented polyurethane
process. A detailed description of the change in elastomers with biological responses. J Pharm Sci 64:1492
1497.
component ingredient and/or the manufacturing
4. Wu SL, Wang YJ, Hu J, Leung D. 1997. The detection of the
process must be provided. The drug product manu-
organic extractables in a biotech product by liquid chromato-
facturer assesses the impact of the VIC on product
graphy on-line with electrospray mass spectrometry. PDA J
quality and safety and evaluates the need for any
Pharm Sci Technol 51:229 237.
additional extractable leachable assessment. Details 5. Bennett CL, Luminari S, Nissenson AR, Tallman MS, Klinge
SA, McWilliams N, McKoy JM, Kim B, Lyons EA, Trifilio SM,
concerning how to manage VICs should be described
Raisch DW, Evens AM, Kuzel TM, Schumock GT, Belknap SM,
in contractual agreements between drug product
Locatelli F, Rossert J, Casadevall N. 2004. Pure red-cell aplasia
manufacturer and component vendor.
and epoetin therapy. N Engl J Med 351:1403 1408.
6. Schellekens H. 2002. Immunogenicity of therapeutic proteins:
Clinical implications and future prospects. Clin Ther 24:1720
CONCLUSION
1740, discussion 1719.
7. Boven K, Stryker S, Knight J, Thomas A, van Regenmortel M,
Leachables emanating from components used for Kemeny DM, Power D, Rossert J, Casadevall N. 2005. The
increased incidence of pure red cell aplasia with an Eprex
long-term storage of API/drug substance and drug
formulation in uncoated rubber stopper syringes. Kidney Int
product are a concern for biotechnology products due
67:2346 2353.
to potential effects on product quality and patient
8. Sharma B. 2007. Immunogenicity of therapeutic proteins. Part
safety. Protein formulations are susceptible to leach-
2: Impact of container closures. Biotechnol Adv 25:318 324.
able contamination due to surfactant activity of 9. Locatelli F, Aljama P, Barany P, Canaud B, Carrera F, Eckardt
KU, Macdougall IC, Macleod A, Horl WH, Wiecek A, Cameron
proteins themselves as well as the presence of
S. 2004. Erythropoiesis-stimulating agents and antibody-
surfactants that are present as excipients in a
mediated pure red-cell aplasia: Here are we now and where
majority of protein formulations (e.g., polysorbates).
do we go from here? Nephrol Dial Transplant 19:288 293.
Conducting extractable leachable studies is required
10. Hermeling S, Schellekens H, Crommelin DJ, Jiskoot W.
per regulatory guidance documents. A complete 2003. Micelle-associated protein in epoetin formulations:
DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
2218 WAKANKAR ET AL.
A risk factor for immunogenicity? Pharm Res 20:1903 25. Treuheit MJ, Kosky AA, Brems DN. 2002. Inverse relationship
1907. of protein concentration and aggregation. Pharm Res 19:511
11. Mueller R, Karle A, Vogt A, Kropshofer H, Ross A, Maeder K, 516.
Mahler HC. 2009. Evaluation of the immuno-stimulatory 26. Mizutani T. 1980. Decreased activity of proteins adsorbed onto
potential of stopper extractables and leachables by using den- glass surfaces with porous glass as a reference. J Pharm Sci
dritic cells as readout. J Pharm Sci 98:3548 3561. 69:279 282.
12. Swift R, Nashed-Sauel Y, Liu W, Narhi L, Davis J. 2007. 27. Hill SS, Shaw BR, Wu AH. 2003. Plasticizers, antioxidants,
Tungsten, prefilled syringes and protein aggregation. ACS and other contaminants found in air delivered by PVC tubing
Meeting (BIOT 15), Boston, MA. used in respiratory therapy. Biomed Chromatogr 17:250 262.
13. Bee JS, Nelson SA, Freund E, Carpenter JF, Randolph TW. 28. Jenke D. 2007. An extractables/leachables strategy facilitated
2009. Precipitation of a monoclonal antibody by soluble tung- by collaboration between drug product vendors and plastic
sten. J Pharm Sci 98:3290 3301. material/system suppliers. PDA J Pharm Sci Technol 61:17 23.
14. Jenke DR, Jene JM, Poss M, Story J, Tsilipetros T, Odufu A, 29. 2008. (661) Containers and plastics. In the United States
Terbush W. 2005. Accumulation of extractables in buffer solu- Pharmacopoeia, USP 31.
tions from a polyolefin plastic container. Int J Pharm 297:120 30. 2008. (88) Biological reactivity tests, In Vitro. In the United
133. States Pharmacopoeia, USP 31.
15. Thirumangalathu R, Krishnan S, Ricci MS, Brems DN, 31. 2008. (87) Biological reactivity tests, In Vitro. In the United
Randolph TW, Carpenter JF. 2009. Silicone oil- and agita- States Pharmacopoeia, USP 31.
tion-induced aggregation of a monoclonal antibody in aqueous 32. Zhang F, Chang A, Karaisz K, Feng R, Cai J. 2004. Structural
solution. J Pharm Sci 98:3167 3181. identification of extractables from rubber closures used for pre-
16. Jones LS, Kaufmann A, Middaugh CR. 2005. Silicone oil filled semisolid drug applicator by chromatography, mass spec-
induced aggregation of proteins. J Pharm Sci 94:918 trometry, and organic synthesis. J Pharm Biomed Anal 34:841
927. 849.
17. Bernstein RK. 1987. Clouding and deactivation of clear (reg- 33. Jenke DR. 2001. Evaluation of model solvent systems for
ular) human insulin: Association with silicone oil from dispo- assessing the accumulation of container extractables in drug
sable syringes? Diabetes Care 10:786 787. formulations. Int J Pharm 224:51 60.
18. Baldwin RN. 1988. Contamination of insulin by silicone oil: A 34. Pan C, Harmon F, Toscano K, Liu F, Vivilecchia R. 2008.
potential hazard of plastic insulin syringes. Diabetes Med Strategy for identification of leachables in packaged pharma-
5:789 790. ceutical liquid formulations. J Pharm Biomed Anal 46:520 527.
19. Chantelau E. 1989. Silicone oil contamination of insulin. Dia- 35. Wang Q. 2005. Selection of analytical techniques for pharma-
betes Med 6:278. ceutical leachable studies. Am Pharm Rev 40:42 44.
20. Rathore N, Rajan RS. 2008. Current perspectives on stability of 36. 2005. ICH Q1A (R2) Stability testing of new drug substances
protein drug products during formulation, fill and finish opera- and products.
tions. Biotechnol Prog 24:504 514. 37. Jenke DR. 2005. Linking extractables and leachables in con-
21. Guidance for Industry. 1999. Container closure systems for tainer/closure applications. PDA J Pharm Sci Technol 59:265
packaging human drugs and biologics. Rockville, MD: 281.
US Department of Health and Human Services, Food and Drug 38. Jenke D. 2007. Evaluation of the chemical compatibility of
Administration. plastic contact materials and pharmaceutical products; safety
22. Markovic I. 2007. Evaluation of safety and quality impact of considerations related to extractables and leachables. J Pharm
extractable and leachable substances in therapeutic biologic Sci 96:2566 2581.
protein products: A risk-based perspective. Expert Opin Drug 39. Ji JA, Borisov O, Ingham E, Ling V, Wang YJ. 2009. Compat-
Saf 6:487 491. ibility of a protein topical gel with wound dressings. J Pharm
23. Yu X, Wood D, Ding X. 2008. Extractables and leachables: Sci 98:595 605.
Study approach for disposable materials used in bioprocessing. 40. Northup S. 2005. Assessing the biological safety of extractable
Biopharm Int 21:42 51. and leachable chemicals in pharmaceutical and medical pro-
24. Norwood DL, Paskiet D, Ruberto M, Feinberg T, Schroeder ducts. Am Pharm Rev 20:38 42.
A, Poochikian G, Wang Q, Deng TJ, DeGrazio F, Munos 41. Ball D, Blanchard J, Jacobson-Kram D, McClellan RO, McGo-
MK, Nagao LM. 2008. Best practices for extractables and vern T, Norwood DL, Vogel W, Wolff R, Nagao L. 2007. Devel-
leachables in orally inhaled and nasal drug products: An opment of safety qualification thresholds and their use in orally
overview of the PQRI recommendations. Pharm Res 25:727 inhaled and nasal drug product evaluation. Toxicol Sci 97:226
739. 236.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 DOI 10.1002/jps