An Introduction to Extractables and Leachables Testing

Extractables Compounds that can be extracted from the container closure system when in the
presence of a solvent

• Leachables Compounds that leach into the drug product formulation from the container
closure as a result of direct contact with the formulation

Extractables studies are an assessment performed on a material. Extractables studies are

performed to identify substances that a patient may be exposed to. Leachables studies are an
assessment performed on the drug product. Leachables studies are performed to identify
substances that patients are exposed to.

The examination of Extractable and Leachable substances is extremely important for the
protection of patients and for regulatory documentation destined for authorities such as the FDA
and EMA.

The examination of Extractable and Leachable substances is extremely important for the
protection of patients and for regulatory documentation destined for authorities such as the FDA
and EMA.

In recent years, the pharmaceutical industry has developed a better understanding of the impact
of extractables and leachables on patient safety and drug product interaction, leading to increased
scrutiny by the regulators. When there is a change in a drug product’s immediate packaging
materials, there is a regulatory requirement to assess the impact of the change.

Smithers Rapra is a leading authority for E&L assessments. Our experts have a proven track
record of developing and delivering comprehensive E&L assessments tailored to your specific
needs in our FDA registered laboratories.

This expertise derives from Smithers Rapra's 90 year history of working with rubber and plastic
materials, our medical and pharmaceutical industry experience and our excellent understanding
of analytical chemistry.

You can benefit from one of our expert's extensive expertise in the E&L USA pre-conference
workshop, bringing you up to speed with all of the basics for E&L testing, but a brief overview
of the basics is included for your information below and more information can be found
at https://www.smithersrapra.com/testing-services/by-sector/medical-and-
pharmaceutical/extractables-and-leachables
What is E&L testing?

Regulators are concerned with substances migrating from different materials (polymers, metals,
glass etc.) which patients may be exposed to through many different routes of administration,
such as:

 Orally Inhaled Nasal Drug Products

 Parenteral
 Production equipment - Single Use Systems
 Medical Devices

Extractables studies - definition

Extractables studies are an assessment performed on a material. Extractables studies are

performed to identify substances that a patient may be exposed to.

Leachables studies - definition

Leachables studies are an assessment performed on the drug product. Leachables studies are
performed to identify substances that patients are exposed to. Leachables are typically a subset of
extractables, but not always.
Analytical Techniques for Extractables and Leachables Studies

Numerous analytical techniques are used for E&L studies because no single analytical technique
can detect them all.

The techniques used typically include:

 Gas Chromatography Mass Spectrometry (GC-MS)

 3D Gas Chromatography Mass Spectrometry (GCxGC-ToF-MS)
 Liquid Chromatography Mass Spectrometry (LC-MS)
 pH
 Conductivity
 Non-Volatile Residue (NVR) and Fourier Transform Infrared (FT-IR) spectroscopy
 Total Organic Carbon (TOC)
 Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES)
 Ion Chromatography (IC)

E&Ls are assessed for a number of different materials and medical devices, and a couple of the
main areas of testing are described below:

Single Use Systems

The use of Single Use Systems (SUS) can be hugely beneficial in biopharmaceutical
manufacturing processes. An assessment of Extractables and Leachables is important for patient
safety and to assess any potential impact on the process and product.

SUS are increasingly being used at every stage of biopharmaceutical manufacturing processes.
SUS components include:

 Filters
 Process containers (bags)
 Tubing
 Connectors
 Gaskets
 Valves

All product contact surfaces have the potential to release extractable material into a process. In a
biopharmaceutical process, a risk assessment for E&L should be made. Factors that affect that
assessment include:

 Nature of the extractable species

 Process fluid
 Contact time
 Contact temperature
  Downstream processes, (holding tanks, diafiltration, chromatography etc.)

Container / Closures

In order to obtain successful regulatory approval for new drug applications, packaging
interactions with the drug product should be evaluated to ensure product safety.

The degree of interaction between the drug product and the primary packaging is dependent on
the product type, as is the risk to the patient. The FDA summarises this in a table which is can be
viewed here:https://www.smithersrapra.com/testing-services/by-sector/medical-and-
pharmaceutical/extractables-and-leachables/container-closures

Smithers Rapra follow best practice guidelines, such as those provided by the PQRI for OINDP
and USP draft chapters <1663> and <1664>.

The extractables study:

 Design and execution of the extractables study on critical components of the primary
packaging, covering inorganic and organic extractables.
 Determination of the Analytical Evaluation Threshold (AET) from the Safety Concern
Threshold (SCT)
 Use of a number of extraction methods, employing multiple analytical techniques, as no
one analytical technique is capable of detecting all extractable substances
 Provision of a detailed technical report, containing full results and chromatograms
 Option for undertaking a literature based toxicological assessment of the detected
extractables to identify target leachables

The leachable study:

 Development and validation of methods for the assessment of leachables in the drug
product according to ICH Q2(R1)
 Analysis of stability time point samples for leachables

 Extractables and Leachables Studies


 Extractables and leachables (E/L) expertise to quantify and assess risk associated with
potential leachable impurities that originate from pharmaceutical container closures,
process equipment and medical device packaging
 Extractables and leachables (E/L) studies are critical to the identification and
quantification of harmful leachable impurities which could migrate from pharmaceutical
container closure systems, process equipment and packaging to contaminate
pharmaceutical products.
 A profile of extractable components must be obtained, via controlled extractables studies
(CES), in order to identify potential sources of leachables such as antioxidants,
plasticizers, dyes and metal catalysts. As pharmaceutical packaging, drug delivery
systems and implantable medical devices can be extremely complex, with mixtures of
plastic, polymer, rubber or glass materials, printed surfaces and coatings all utilized, it is
critical that E/L studies are designed specifically for your drug product and the container
materials so that the risks associated with leachable impurities can be assessed.
 Our scientists conduct extractables and leachables studies in accordance with regional
guidance, Good Manufacturing Practice (GMP) PQRI recommendation, United States
Pharmacopeia (USP) requirements (including USP chapters <1663>, <1664>, and
<1664.1>), and awareness of FDA guidelines. As an initial step, we aim to gather all
information about your packaging system in order to obtain a strong impression of
potential sources of extractables or leachables. The combination of our experience in
method development for controlled extractables studies, coupled with our vast knowledge
of leachable compounds, mean that we can anticipate and identify potential sources of
risk through strategic screening studies.
 We provide leachables method validation compliant to Good Manufacturing Practice
(GMP) for use in storage and stability programs and can support a wide range of closure
or drug delivery systems such as pre-filled syringes, large volume parenteral products,
orally inhaled and nasal drug products (OINDP), single use and disposable medical
equipment, printed packaging and bioprocessing equipment. Leachables associated with
drug delivery systems, implantable medical devices and other devices such as e-cigarettes
are also assessed.
 Our analytical teams can determine both organic and inorganic contaminants through the
application of a range of instrumentation including liquid chromatography (HPLC)
liquid chromatography-mass spectrometry (LC-MS(MS)), gas chromatography (GC), gas
chromatography-mass spectrometry (GC-MS(MS)), and inductively coupled plasma
spectroscopy (ICP, ICP-MS), dependent on the analysis.
 With specialists in glass delamination studies, we provide a range of solutions that help
mitigate the risks associated glass delamination and leaching of metal ions /elemental
impurities from glass into drug products for molded bottles, containers, vials or
prefillable syringes.
 Comprehensive evaluation and safety assessment review of laboratory E/L study data is
performed by our in-house toxicologist consultants in order to identify and address
potential risks and to assist with comprehensive impurity testing, whilst identifying any
gaps in your existing data packages. We also provide recommendations and support for
preparation of very low-risk data packages related to E/L testing.
 Extractable Leachables Specialists
Our experts have over 25 years’ experience in specialised analytical and toxicology
 assessment for extractables and leachables, and this is reinforced by our knowledge of
polymer, plastic and packaging materials, allowing us to identify components from the
most complex polymer formulation including adhesives, additives and stabilizer
ingredients and their degradation products. Our consultants provide a first-class
evaluation and assessment of existing E/L data gaps, and through strategic screening or
quantitative studies, we offer the right study for your product. Intertek also provides
recommendations and support for preparation of very low risk data packages related to
E/L testing.
 Global Expertise
Intertek offers extractable and leachable testing services through GMP-compliant
laboratories located in Whitehouse, NJ (USA) and Basel (Switzerland) with research
support from our Mumbai (India) facility, as part of our commitment to Total Quality
Assurance. Bringing quality and safety to life, we offer Total Quality Assurance expertise
to help you to meet and exceed quality, safety and regulatory standards.

Extractables and leachables in

pharmaceutical packaging
The potential for harmful chemicals to be extracted from packaging materials that pose a risk to
patients has spurred regulatory authorities to establish guidelines for extractable and leachable
(E&L) testing for primary drug packaging materials.1–3 Drug licence holders must establish
programmes that effectively assess and test these contamination risks. In 1999, following the
introduction of the asthma drug salmeterol in the UK, three cases of respiratory arrest occurred
shortly after patients started taking the drug; and, at about the same time, the United Kingdom
Committee on the Safety of Medicines reported 26 cases of deterioration in asthma soon after the
introduction of long-acting beta-agonists. In 2005, a paper published in the medical journal
Kidney International reported on the increased incidence of pure red cell aplasia with an Eprex
formulation in uncoated rubber stopper syringes.

Recently, other issues have been highlighted in the media, such as concerns regarding the safety
of phthalates, and there were reports of a postponement of the MMR vaccination of babies in
some parts of the Netherlands because of fears about glue leaking from needles. The regulatory
authorities have not been slow to react to these concerns and the very real risks of product
contamination from E&Ls, the US having enacted E&L legislation under the Federal, Food,
Drug & Cosmetic Act (FFDCA), and the EU having recently introduced decentralised and
mutual recognition procedures for the evaluation of E&L safety risks.

The legal position

FFDCA, 21 CFR Part 211.94, states the following:

 drug product containers and closures shall not be reactive, additive or absorptive so as to alter
the safety, identity, strength, quality or purity of the drug beyond the official or established
requirements
  container closure systems shall provide adequate protection against foreseeable external
factors in storage and use that can cause deterioration or contamination of the drug product
 drug product containers and closures shall be clean and, when indicated by the nature of the
drug, sterilised and processed to remove pyrogenic properties to ensure that they are suitable
for their intended use; such depyrogenation processes shall be validated
 standards or specifications, methods of testing and, when indicated, methods of cleaning,
sterilising and processing to remove pyrogenic properties shall be written and followed for drug
product containers and closures.

Compendial standards are now the law, with 21 USC 351 stating that “the Secretary shall
promulgate regulations prescribing appropriate tests or methods of assay in accordance with
which such determination as to strength, quality or purity shall be made.”

There is no shortage of guidance for industry regarding the selection of appropriate materials for
pharmaceutical packaging or for testing the stability of new drug substances, and products and a
range of E&L test procedures have been developed to ensure patient (and public) safety.
However, it is essential that both pharmaceutical/medical product and pharmaceutical/medical
packaging companies know which compendial standards and guidances apply to their particular
products and, if they do not know or are unsure, that they ask the relevant regulatory authority
for advice.

Assays: best practice

What makes a good assay? The guiding principles are that testing procedures must be
scientifically rigorous, be based on best practice by incorporating risk assessment and risk
management principles, and that biological and chemical assessments complement and inform
each other. Test regulations, procedures and standards need to be in alignment with other
international standards and best practices, while due consideration must be given regarding the
use of laboratory animals when selecting appropriate procedures.

According to ISO 10993-18, chemical characterisation is “the identification of a material and the
identification and quantification of the chemicals present in materials or finished medical
devices.” USP <1663> presents a framework for the design, justification and execution of an
extractables assessment for pharmaceutical packaging and delivery systems, while also
establishing the critical dimensions of an extractables assessment and discussing the practical
and technical aspects of each dimension. However, the standard does not establish specific
extraction conditions, analytical procedures or mandatory extractables specifications, nor
acceptance criteria for particular packaging and delivery systems or drug products. Neither does
it delineate every situation in which an extractables assessment is required.

Chemical characterisation can, therefore, provide an understanding of materials, components or a

system, and assist with risk assessment. It also enables failure analysis to support a scientifically
sound rationale for a failed biocompatibility test while advancing the so called 3Rs initiative
(replacement, reduction and refinement) to reduce unnecessary in vivo testing. Prior to
biocompatibility testing, the judicious application of chemical characterisation may provide
information permitting the pre-emptive toxicological evaluation of chemical entities that may be
irritants or sensitisers, or that may elicit an acute toxicity response. However, such procedures
may not unequivocally declare the absence of such entities, and do not preclude a comprehensive
evaluation of all available data to permit a risk-based-testing approach.

Determination of risk factors

The risk factors associated with extractables and leachables include general quality
considerations associated with leachables, such as the pharmaceutical product’s stability and the
possible formation of aggregates, their interference with analytical methods or diagnostic tests,
and reduced process performance and associated medical effects. These include cell culture
growth, the rate of drug release, drug solubility and drug efficacy. There is also the possibility
that the leachable may be toxic and pose a health risk to the consumer.

The first requirement before doing any E&L testing procedure is to collect information from
packaging suppliers regarding the possible medical effects of their products. The construction
materials should be resistant against chemical attack, UV light, moisture, temperature and e-
beam or gamma irradiation (through a sterilisation procedure) to avoid the altering of a
material’s chemical profile or its physicochemical properties.

There are cases in which the contact materials have not shown compatibility whilst in contact
with a drug product. An example is a case when a non-coated rubber stopper was incompatible
with a drug formula, and calcium phosphate crystals were observed in a finished drug. The root
cause of this was found to be precipitation of the phosphate following the leaching of calcium
ions from the stopper. Calcium stearate is used by the rubber and plastic industries as an
effective elastomer, processing aid and release agent, which may have been the source of the
leaching calcium ions that then reacted.

Needles can pose a risk to patients if harmful chemicals are extracted from the packaging during
use. In the Netherlands, the fear of glue leaking from needles postponed the MMR vaccination of
babies.
Another potential cause of drug contamination is the photo- or gamma-induced degradation of
cyclo-organic copolymers, which are often used in primary packaging such as in blisters for solid
drugs, or in packaging for injectables. Possible mechanisms of degradation include oxidation,
cracking and structural rearrangement.

When performing extractables testing for finished packaging, it is important to select appropriate
extraction conditions and employ suitable analytical methods. Nevertheless, no specific
guidelines exist regarding the suitability of analytical screening methods for extractables in
conjunction with plastic components.

The methods cannot be properly validated owing to the fact that the target analytes are not
known. There is therefore a need to adopt a broad screening strategy to demonstrate the presence
or absence of residues for subsequent quantitative efforts. The analytes of interest must be
extracted from the materials and then subsequently identified, semi-quantified and then
characterised using multiple analytical techniques.

Standard extractable protocols do not take into account the different chemical nature of polymers
or laminated materials. The qualitative analysis of extractable analytes should be guided by an
analytical evaluation threshold that must be correlated with the sensitivity of the analytical
methods. Controlled extraction studies need to be done using different solvents to establish
“worst case” conditions that maximise extraction, or to bracket use conditions under exaggerated
conditions. The focus should be on the identification and calculation of the amount of
extractables in the packaging or closure device. This is necessary to ensure the quality and
comparability of the analytical results generated by laboratories for compliance purposes, and for
the creation of data for risk assessment purposes. This should be achieved by using quality
assurance systems and, specifically, by applying methods that have been qualified according to
common procedures, that meet defined performance criteria and by ensuring traceability to
common — or commonly agreed — standards.

Leachables are detected through the qualitative analysis of either freshly prepared product or
product taken from real-time or accelerated storage programmes, with quantitation determined
above an acceptable safety threshold. The determination of the limits of quantitation and the
resultant typical chromatograms obtained for known targets provide the means of validating the
test methods used.

These tests should also identify and quantify unforeseen leached substances above a safety limit
for unknown contaminants, and the root cause of the leaching, with the test report tabulating
levels of leachables seen from several shelf-life batches of the drug product or medical device.

Analytical sensitivity

It is essential to choose realistic extraction conditions that provide analytical sensitivity. Thus,
solvent volumes must be correlated with the analytical evaluation threshold (AET) and the limit
of quantitation (LOQ) of the analytical method using Equation 1 to provide the required
sensitivity.
Equation 1: AET = the allowable amount/substance to be released representing the actual
analytical evaluation threshold, ExVol = the volume of extraction solvent, factor = the
concentration factor to adjust method sensitivity and MS = the analytical sensitivity of the
proposed method, with MS > LOQ

However, if the AET of the extractable for the chosen method is inappropriate or impractical to
use, the sensitivity of the analytical method may be adjusted by using a sample concentration
factor. It is important to avoid the risk that any sample preparation of extract solution might
result in the loss of analytes, or the risk that high concentration factors could result in the
accumulation of environmental impurities. In addition, blank values cannot be validated in
screening methods for extractables with regard to a specific target substance.

The correct method is to analyse the extract both in its original state (without concentrating the
analyte by sample preparation) and by introducing a concentration factor. The concentration
factor should not exceed 5–10x, with the more concentrated extract possibly being produced by
increasing the extracted mass while employing a constant volume of solvent. It may be necessary
to find an alternative normative rationale to determine the extraction stoichiometry; this method
is highlighted, for example, in ISO 10993-12.

Completeness of an assessment

According to USP <1663>, the completeness of an extractables assessment can only be judged
against the overall goals of the assessment, and the generation of extracts should be
accomplished with multiple solvents or extracting media with varying extracting power based on
the known extracting power of the drug product vehicle. Multiple and complementary extraction
techniques need to be employed, including those with the capability for volatiles analysis, as do
extraction conditions that allow equilibrium to be achieved.

In the E&L test extraction procedure for finished containers, 3–4 various solvents should be used
to mimic real-use conditions. It is also important to choose the appropriate tools for separation
and detection — volatile organics being evaluated using gas chromatography (GC) and semi-
volatile organics being measured by liquid injection GC using a flame ionisation detector (FID)
and a mass spectrometer (MS) detector with accurate mass assignments for fingerprinting. Non-
volatile organics can be assessed by HPLC combined with diode-array detection (DAD) and a
high resolution MS, whereas typical methods for elemental analysis include inductively coupled
plasma mass spectrometry (ICP-MS) and inductively coupled plasma atomic emission
spectroscopy (ICP-OES).

Cationic and anionic targets are evaluated using ion chromatography techniques. Specialist
techniques may have to be employed for critical compounds, such as GC-thermal energy analysis
(GC-TEA) for nitrosamines from rubber, or LC-MS/MS for perfluorinated carboxylic acids,
amides and sulphonamides that may be present in perfluorinated polymeric materials.
Complementary or additional techniques that may be employed include NMR, infra-red and
ultraviolet spectrometry.

Leachables strategies

For leachables, target analysis is completed by validation combined with analytical screening.
Thus, all methods need to be validated and target analysis may be appropriate or reasonable to
monitor the expected leachables when extractables/simulated use profiles undoubtedly represent
an identified profile. However, when extractables/simulated use profiles show many “known
unknowns,” target analysis may be combined with multiple analytical methods to also discover
unexpected leachables and/or poorly identified leachables that are “subsets” of the extractables.
Storage conditions and suggested points for leachables analysis are presented in Table I.

The US Food and Drug Administration expects three production lots to be tested, and USP
<1663/1664> states: “Leachables studies may include accelerated storage conditions, but they
cannot be limited to accelerated conditions and must include real-time assessment.”
A leachables strategy must establish a quantitative and qualitative correlation between profiles
and demonstrate that compounds detected in the leachable studies were also present in the
controlled extractables studies. It is also important to demonstrate that the levels of leachables
obtained from studies are generally less than the levels of extractables obtained in the
quantitative controlled extraction studies. This requirement emphasises why it is necessary to use
multiple batches in extractable and leachable studies.

References

1. FDA Guidance for industry, Container Closure Systems for Packaging Human Drugs and Biologics:
www.fda.gov/downloads/drugs/ guidances/ucm070551.pdf (May 1999).
2. FDA Guidance for Industry: Nasal Spray and Inhalation Solution, Suspension, and Spray Drug
Products: www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/
guidances/ucm070575.pdf (July 2002).
3. EMEA Guideline on Plastic Immediate Packaging Materials (CPMP/QWP/4359/03):
www.ema.europa.eu/docs/
en_GB/document_library/Scientific_guideline/2009/09/WC500003448.pdf (May 2005).