20 20

EXTRACTABLES/LEACHABLES
CONSIDERATIONS FOR
CELL & GENE THERAPY
DRUG PRODUCT DEVELOPMENT
EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Published by:
Bio-Process Systems Alliance (BPSA)
1400 Crystal Drive
Arlington, VA 22202

www.bpsalliance.org

AUTHORS
Manjula Aysola, MilliporeSigma
Dominic Clarke, PhD, Charter Medical
Ray Colton, VR Analytical
Paul Cummings, Smithers MDT
Jayanthi Grebin, Colder Products Company
Armin Hauk, Sartorius-Stedim Biotech
Eva Heintz, Solvay
Todd Kapp, Entegris
Brendan Lucey, Gemini Bio
Ruth McDermott, Sartorius-Stedim
Hernán Parma, American RENOLIT
Joseph P. St. Laurent, Chemic Laboratories Inc.

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Table of Contents
Topic Page
Part 1: Introduction 3
Part 2: Contrast Traditional E&L with Unique Requirements of CGT 4
Part 3: Risk Assessment 7
Part 4: Regulatory Considerations 10
Part 5: Unique Challenges of CGT 11
Part 6: Responsibilities for E&L Testing of CGT Delivery Systems 11
Part 7: Terms and Definitions 13
Part 8: Additional Information 15
Part 9: References 16

Figures Page
Figure 1: Typical Components Utilized in Cell & Gene Therapy (CGT) Manufacturing 5
Figure 2: Cell & Gene Therapy (CGT) Process Map 6
Figure 3: Leachability Risk Tolerance 10

Tables Page
Table 1: Examples of Process-Related Components and Their Uses in CGT 7

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Extractables/Leachables Considerations for Cell & Gene

Therapy Drug Product Development
Part 1: Introduction
Cell and gene therapy (CGT) research is both dynamic and promising. Cell therapies have the potential to treat various
disease states, including cancer, liver diseases, primary immunodeficiency diseases, etc. This paper focuses on cell
therapies and those differences associated with CGT versus classical biopharma production as it pertains to the topic
of extractables and leachables (E&L). It should be noted that, although viral-vector manufacturing (VVM) can be
considered part of CGT, this will not be discussed largely as VVM requires several downstream steps, which makes it
more similar to classical biopharma production.

According to the Alliance for Regenerative Medicine1, as of disorders, and (potentially) cancer. In this paper, per
September 2019, 1069 CGT clinical trials will be ongoing applicable FDA guidance, we will include gene-modified
worldwide (358 in Phase 1, 617 in Phase 2, and 94 in Phase cells like CART-T as a gene therapy process and/or consider
3.) The U.S. Food and Drug Administration released its first as a gene therapy component.
Guidance for Industry related to CGT in March 1998. This
Leachable chemical entities found in the CGT
Guidance defined “somatic-cell therapy” as “…the
administration to humans of autologous, allogeneic, or manufacturing automated or manual processes can
xenogeneic living cells which have been manipulated ex originate from single-use systems. These leachable
chemical entities can adversely affect the manufacturing
vivo.” The Guidance gives examples including
“implantation of cells as an in vivo source of a molecular process performance, safety and quality of CGT products.
species such as an enzyme, cytokine or coagulation factor; Certain leachable compounds can have adverse safety
infusion of activated lymphoid cells such as lymphokine implications when delivered directly to patients, especially
patients with compromised immune systems and organ
activated killer cells and tumor-infiltrating lymphocytes;
and implantation of manipulated cell populations, such as function. In addition, leachables can have compatibility
hepatocytes, myoblasts, or pancreatic islet cells, intended issues with CGT products through chemical reactivity, cell
toxicity, and potentially initiating cancerous changes in
to perform a complex biological function.”2
certain cells through genotoxic and mutagenic effects.
Gene therapy is defined in FDA Guidance as “medical Therefore, potential leachables (i.e., extractables) and
intervention based on modification of the genetic material actual drug product leachables in CGT products must be
of living cells.” 3 Gene therapy is therefore “an approach to assessed and controlled.
treat, cure, or ultimately prevent
disease by changing the pattern of There are various FDA Guidance
PART 1: HIGHLIGHT
gene expression.” The goal of gene Documents that discuss the issue of
therapy is to deliver specific Leachable chemical entities can adversely affect leachables related to drug product
sequences of DNA to target cells
the manufacturing process: performance, safety packaging systems,4 particularly for
and quality of CGT products. Understanding these
and, via incorporation of these DNA variables is vital for your success.
so-called “high-risk” dosage forms
sequences into the cells’ overall (e.g., inhalation and infusion).5 The
genetic structure, treat various Product Quality Research Institute
genetic-based disorders including cystic fibrosis, (PQRI) has created and published best practice guidelines
arteriosclerosis, amino acid and fatty acid catabolism for extractables and leachables in inhalation drug
products,6 and is currently in the final stages of releasing
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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

guidelines for parenteral drug products.7 The United States modify or manipulate the expression of a gene or to alter
Pharmacopeia (USP) has incorporated the essence of the the biological properties of living cells for therapeutic use.
PQRI guidelines into three general guidance chapters:
The principal difference is that the cells are the product,
(<1663>, <1664>, <1664.1, and (DRAFT) 1665>).8, 9 With
the exception of USP <1665>, none of the aforementioned and apart from a few washing steps, there is no opportunity
Guidance focuses on single-use to separate the impurities from the product. The patient
samples typically taken in a
systems.
hospital are transported in
PART 2: HIGHLIGHT
Standardized protocols, such as frozen bags to the centralized
USP <665> draft and/or the In cell and gene therapy, cells are the product
manufacturing facility. There are
BioPhorum Operations Group and there is little opportunity to separate several complex, manual steps
(BPOG) extractables protocol, impurities from the product. Therefore, this needed to isolate the target cells
completely changes how this needs to be
are available and may be applied and modify them to express the
approached.
for CGT products as well as all antigen before they can be
biological and biotechnology- expanded to produce sufficient
10, 11
based pharmaceutical products. cells to treat the patient. CGT therefore can be classified as
a small-scale production process requiring cell-sampling,
Part 2: Contrast Traditional E&L with manipulation and cultivation. Storage consumables and
Unique Requirements of CGT single-use devices are widely used in each of the process
Although classical biopharmaceutical manufacturing and steps.
CGT both involve cultivation of living cells, several The chemicals and devices used are frequently summarized
significant differences are obvious. In classical as ancillary materials 12, 13 and commonly used in various
biopharmaceutical manufacturing, the cells are utilized in CGT processes. Ancillary materials can include, but are not
the production of the drug substance (DS) (e.g., a protein), limited to, bioreactors and cell culturing systems,
an antibody or an enzyme. The cells, cell debris, host cell- components of culture media, drug- or biologic-like
related and process-related impurities are separated and components used to activate or otherwise change the
removed from the DS in a downstream purification process. biological characteristics of the cells, certain antisense
The downstream processing and formulation yields a drug polynucleotides and agents used to purge, select or
product (DP) with a high purity with very low levels of stimulate specific cell populations. These products are
impurities, including process equipment-related leachables largely intended to act on the cells, rather than have an
(PERLs). independent effect on the patient. The intended action of
Gene therapy can be used to modify cells inside or outside these products is not dependent upon incorporation into
the body. In gene therapy that is used to modify cells the somatic cell with maintenance of the product’s
outside of the body, blood, bone marrow or another tissue structural or functional integrity. Additionally, syringes,
can be taken from a patient, and specific types of cells can tubing, plastic pipette tips, plastic vials and blood collection
be separated out in the lab. The vector containing the bags, along with typical single-use technology (SUT) similar
desired gene is introduced into these cells. The cells are left or identical to those used in bio-manufacturing may be
to multiply in the laboratory and are then injected back into used. In some instances, these products can meet the
the patient, where they continue to multiply and eventually definition of medical devices and may be regulated as such,
produce the desired effect. Human gene therapy seeks to according to codified procedures.

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Figure 1: Typical Components Utilized in Cell & Gene Therapy (CGT) Manufacturing

From Charter Medical presentation

at 2017 BPSA International Single-Use Summit

Apart from cellular manipulation, there may be starting
materials like viral vectors whose activity can be impacted
by any impurities introduced by the manufacturing
systems. The upstream process is extensive and uses many
specialized reagents such as antibody-coated beads, while
the downstream process consists of cell harvest and
concentration/buffer exchange before formulation and
final fill. There may be certain “washing steps” to remove
select undesired process chemicals and impurities, but
there is no classical downstream processing to purify the
cells. The cells could be harvested by size exclusion or
centrifugation. As cells are the product, the process
streams tend to be close to physiological conditions with no
use of extremes of pH and/or organic solvents. The final
formulation typically includes dimethyl sulfoxide (DMSO) to
aid in the freeze down of cells to support cryogenic
transportation to the site of administration.
Given the extensive use of SUT for CGT manufacturing, the
cell-based product and the variety of possible contact
surfaces and contact time, it is reasonable to believe that
CGT products could be impacted by leachable compounds.
Regulatory requirements demand that drug products are
not exposed to contact items such that they would be
reactive, additive or absorptive, thus ensuring drug product
quality and safety.14 Organizations including the BPSA were
formed to support, encourage adoption and provide
guidance for the use of SUT for biopharmaceutical
production. These organizations also highly recommend
that E&L testing programs be implemented early in the
development process in order to reduce the risk for
possible late-stage manufacturing changes. Similarly, a
thorough understanding of the CGT process is necessary to
develop and conduct an effective E&L program.
Unintended process-related contaminants can enter
throughout the entire CGT manufacturing process and
accumulate. An impurity or contaminant (leachable)
entering a CGT production stream could affect cell growth
and/or expansion in process or ultimately impact a specific
critical quality attribute of the final cell-based product.

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Figure 2: Cell & Gene Therapy (CGT) Process Map

Transfer
Bag

From Charter Medical presentation

at 2017 BPSA International Single-Use Summit

While manufacturing of CGT products is highly reliant upon
SUT, the conditions experienced during manufacturing
(e.g., length of contact material exposure time, solvent/
solutions) are typically less invasive in comparison to those
in the biopharma industry. Common SUT contact materials
for CGTs may include blood collection bags, processing
containers, cell expansion bags, tubing sets, filters,
connectors, syringes and final container vials or bags. SUT
used in mAb bioprocessing are typically made of the same
polymers used in CGT, with a notable exception such as PVC
(a plastic more commonly found in medical devices).
While there is widespread usage of PVC in medical
applications, its utilization in single-use bioprocessing
systems has been limited to tubing sets. In contrast,
multiple transfer bags may be employed in the CGT process
(as observed in Figure 2), which in combination with blood
collection containers and tubing sets, create significant
product contact to PVC. Although length of exposure to
these PVC components is limited in transfer bags and
tubing sets, the type of extractables and leachables that
may result from PVC components in the SUT is different
than what has been typically seen in container data
generated from extractables protocols designed for the
traditional single-use bioprocess industry. PVC contains
different types of additives such as plasticizers and heat
stabilizers that are not normally present in other
thermoplastic materials. Some of these additives,
plasticizers for example, are physically absorbed by the PVC
(no chemical bond is present), which facilitates their
leaching or extraction at high rates under appropriate
conditions. In addition, PVC is particularly sensitive to
degradation by heat, aging or radiation. Although additives
in the formulation may protect the polymer from oxidation,
a variety of molecules may be created during this
degradation/stabilization process. Special attention may
have to be given to PVC-related extractables and
leachables, as well as to their potential interactions with
the cell-based product and with other factors that play a
role in the CGT process.

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Table 1: Examples of Process-Related Components and Their Uses in CGT

Single-Use Intended Purpose Material (common)

Collection bags Starting materials PVC (polyvinyl chloride)

Transfer/processing bags Wash; manipulation PVC

Transfer (tubing) sets
Cell expansion
containers
Media containers
Cryopreservation
containers
Fluid transfer; sample removal;
reagent addition
Cell culture or expansion
Culture media; cryomedia; buffer
storage
Final fill; in-process frozen storage
PVC, ABS (acrylonitrile butadiene styrene), thermoplastic
elastomer, silicone
Polyolefin, EVA (ethylene vinyl acetate), FEP (fluorinated
ethylene propylene), PE (polyethylene)
PVC, polyolefin, EVA, FEP, PE, PETG (polyethylene
terephthalate glycol), LDPE (low density polyethylene)
Polyolefin, EVA, FEP, PP (polypropylene), COC (cyclic olefin
co-polymer)

Regardless, a risk analysis of each product contact material
used in the process should be conducted. Some of the
variables to consider include:
• Proximity to the final product
• Extraction capability of the solution
• Contact time
• Contact temperature
• Product contact surface area
• Pre-treatment of the material
• Material compatibility/resistance
• Supporting extractable testing provided by supplier
While leachable contaminants from SUT can affect a CGT
product throughout the entire process, the final product
and final product storage container are often deemed the
most critical due to proximity to the intended patient.
Furthermore, most of these products are cryopreserved,
which involves the inclusion of DMSO as a critical
cryoprotective agent. Although DMSO/water mixtures can
be tolerated, neat DMSO can be one of the stronger
extracting solvents associated with current CGT
manufacturing processes.
CGT-specific process steps exist that are not currently
supported by many of the routine extractable studies.
Some areas of consideration include the number and types
of solvents, temperatures and time. For example, the
inclusion of DMSO as a solvent should be considered.
Elevated (warmer) temperatures are typical, but as many
CGT final products are cryopreserved, perhaps E&L studies
need to consider the total exposure time (prior to
cryopreservation and during) for the plastic components to
DMSO.
Part 3: Risk Assessment
There are three major areas of concern to address in a risk
assessment of CGT applications:
1. The influence of leachables to the viability of the
cells, along with undesired stimulation effects are a
critical parameter, which must be considered with
regards to the process performance.
2. There are only very limited procedures to purify the
cells. That means process-related impurities
including leachables can—under certain
circumstances—remain in the product due to
absorption and/or adsorption to the cells. Such
process-related impurities can have a negative
impact on product quality or patient safety.
3. With stem-cell therapies, the manipulation of the
cells may result in degenerative cells. One can
envisage the possibility that degenerated cells (i.e.,
cancer cells) may be produced and are applied
together with the therapeutic cells to a patient, and
is of highest criticality regarding patient safety.
Fortunately, it is possible to consider these three points by
taking into account our already-available knowledge on
extractables for SUT and translating it into the dedicated
requirements and concerns regarding CGT.

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT
PERLs can be detrimental to cell viability and growth, and
fortunately to date, very few identified extractables (e.g.,
PERLs) from SUT effectively inhibit cells. The most
prominent case is bDtBPP, where the leachables bis (2,4-di-
tert-butylphenol) phosphate was found to be detrimental
to cell growth and cell viability of CHO cells, which are
commonly used as host cells.15-17 After identifying bDtBPP
as a degradant of a plastic additive (antioxidant), it was
directly possible to develop appropriate and safe
materials.17, 18 Secondly, nitro-BPA is involved, where a
reaction product of a monomer caused cell inhibition.19
And lastly, the effects of PVC plasticizers on HeLa cells have
been published elsewhere.20 “This observation is insofar
remarkable, as there are several structurally similar
compounds identified as extractables, for which no effect
on typical host cells were found. In this context, it is also
necessary to mention that the detrimental effects on cells
are often associated with serum-free medium, while
serum-containing medium have been documented to
mitigate these effects.
It is currently unclear whether the above statements can be
extended to the cell lines used in the CGT area and/or to
cells, which are manipulated under the dedicated
conditions of CGT.21 Currently, the best way to detect
materials which may be detrimental to cell growth and cell
viability is an appropriate bio-test with sensitive cells. For
that purpose, it would be highly desirable to develop
dedicated and sensitive assay for CGT applications similar
to those developed for SUT used in biomanufacturing.22, 23
In this context, it is worth considering that any material
which is used in approved medical devices applications
such as implants, was already subjected to extensive
cytotoxic investigations. If materials previously used to
construct medical devices are used, human safety data
could be extrapolated and present a low risk for their use
in cell therapies. These tests are commonly executed in
animal models and allow for an extrapolation to safety in
human cells and tissues. Such materials might be a low risk
option for use in cell therapy processes, whereas materials
that have not already been extensively evaluated may have
to be sufficiently qualified to demonstrate biocompatibility.
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Additionally, to ensure that extractables and leachables
always remain under control, it is critical to ensure that
proper change notifications24 are followed throughout the
supply chain. This may help prevent changes in the
components (processing, polymer and/or additives) that
are part of the SUT, which can represent a risk to the
patient or to the cell-based product.
The second point touches on the considerations on residual
of PERLs, which are released from SUT and remain in the
product, and ultimately are exposed to the patient with the
therapeutic product. In particular, it is necessary to ask
whether current extractables protocols used for SUT
qualification are appropriate to generate data for CGT
applications. Common extractables protocols for SUT
require extraction with organic solvents (e.g., EtOH),
mixtures of organic solvents and water, and water with low
and high pH values. Extraction temperatures typically do
exceed the physiological condition, and the surface-to-
volume ratio is high. Therefore, one can conclude that
these extraction conditions are fulfilling the “worst case”
criteria for CGT applications as well. The extracts are
subjected to a full analytical evaluation (GC/MS, UPLC/MS,
Headspace GC/MS, ICP-MS, IC, etc.). With these methods,
one can obtain a comprehensive extractables profile
suitable to conduct a risk assessment regarding product
quality and patient safety. Considering that most CGT
applications will be associated with low volume doses of
product, one could conclude the patient exposure to
leachable compounds would be diminished. In contrast to
a classical SUT application, the risk assessment for CGT
applications requires not only to consider dissolved
leachables in the DP formulation, but also leachables that
may be adsorbed on or incorporated into the cells. It has
been reported that CHO cells, E.coli and yeast-cells can
adsorb typical leachables;25 however, it is currently
unknown whether—and to what extent—typical CGT cells
can adsorb, take in or even metabolize leachables.
Furthermore, extractables data can be used to assess the
effect on adherent cells and provide a deeper knowledge of
the mechanism of a potential direct transfer of leachables
from a material to the cells.
8
 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

In opposition to the perception that PERLs may be a major possible to extend the analysis to known or suspected
risk in producing degenerative cells (point 3 above), one CMRs in a target analysis. In addition, it can be envisaged
may argue that this risk is eventually of low or minor to develop biological test systems to detect CMRs from the
relevance. Until recently, there were only very rare cases materials with special sensitive cell lines. Also, methods to
published where carcinogenic, mutagenic or genotoxic assess cells directly (e.g., with cell painting) shall be
compounds (CMRs) were identified in extractable studies considered in detecting CMRs from plastic extracts.
from contact materials used in container closure systems
As discussed above, it should be
(CCS) or SUT applications. The
most prominent case—going considered to use plastic
back into the 1990s—was the
PART 3: HIGHLIGHT materials for CGT application,
which are also used as implants,
identification of N-
In addition to the typical concerns, process- for which successful cytotoxic
nitrosamines and polycyclic
related impurities can have a negative impact investigations in the frame of a
aromatic hydrocarbons on product quality or patient safety, with the
(PAHs) in metered dose medical device submission were
potential to produce degenerated (i.e.,
inhalers, where incompatible cancer) cells. made. These tests allow an
elastomer components were interpretation, whether a certain
used for the construction of metering valves. In contrast, material can be used in contact with human tissue or cells,
there are publications which show that the wide majority without inducing undesired effects. Suggesting one would
of commonly found extractables do not show any alerts to use only materials for the manipulation in CGT applications,
be a CMR. Nevertheless, these results cannot be used to which are also used in approved medical devices (LOW
eliminate the possibility of cell degeneration when exposed RISK), would reduce the risk of generating degenerative
to leachable compounds. The reason is that our knowledge cells to a minimum.
on CMRs originates from animal studies and additionally This approach would be in line with the four-tier approach
from epidemiological data for human exposure (e.g., (Tier 1: “Low-risk, highly qualified materials that are well-
occupational hygiene and/or occupational health data). suited for use in manufacturing…currently used in
That means our knowledge on CMRs is based on data approved drug and/or medical device”; Tier 2: “Low-risk,
referring to systemic effects (i.e., the response of an entire well-characterized materials that are well-suited for use in
organism.) Therefore, it cannot be excluded that isolated manufacturing… with an intended use in a drug or medical
cells manipulated in CGT applications are more sensitive to device”; Tier 3: “Moderate risk materials that will require a
CMRs than complete organisms. higher level of qualification than previous tier materials”;
and Tier 4: “Highest risk level where extensive qualification
To assess whether CMR compounds can be released from a
material, the qualitative extractables analysis can be used is necessary prior to use in manufacturing”) as further
as a powerful tool. The techniques which are commonly defined in USP <1043> to classify ancillary material based
applied in extractables studies would detect most of the on risk.
known CMRs, which can originate from plastics. It is even

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Figure 3: Leachability Risk Tolerance

Likelihood of Leaching

From www.usppf.com HIGH MODERATE LOW

HIGH Moderate Risk

Likelihood of Persisting

High Risk

MODERATE Moderate Risk

Low Risk
Moderate Risk
LOW

From USP 〈1665〉 Characterization of Plastic Materials, Components, and Systems Used in the Manufacturing of
Pharmaceutical Drug Products and Biopharmaceutical Drug Substances and Products, PF 43(3) [May–June 2017]

As depicted in Figure 3, each individual drug product require the acknowledgement that all polymeric and
sponsor must establish the manufacturing circumstances elastomeric materials are the source of extractable
that dictate the degree of risk, although how this is analytes, which may result in potential product
accomplished is not specified in USP <665>. When adulteration as leachables. Furthermore, it is this potential
assessing this risk, the sponsor must consider the multitude for adulteration that is quoted by the Code of Federal
of factors that impact the likelihood of leaching and the Regulations 21.CFR 211.65: “Equipment shall be
likelihood to persist. Although not prescriptive, USP <665> constructed so that surfaces that contact components, in-
describes the following as parameters that may be process materials, or drug products shall not be reactive,
considered: “1) The chemical and physical nature of the additive, or absorptive so as to alter the safety, identity,
contacted material/component, establishing the strength, quality, or purity of the drug product beyond the
material’s/component’s ‘propensity to be leached’; 2) the official or other established requirements.”14 It is the
chemical nature of the contacting process stream,
establishing the process stream’s ‘leaching power’; 3) the
conditions of contact, addressing the ‘driving force’ for
leaching; 4) the ability of upstream process operations to
eliminate the PERLs from the process stream or to dilute
the PERLs to such an extent that an adverse effect is
unlikely;’ and 5) the inherent risk associated with the
manufactured drug product, considering such factors as the
nature of the manufactured dosage form”.

responsibility of the industry to recognize that the presence

Part 4: Regulatory Considerations
and persistence of extractable analytes directly result in
Regulatory guidance supporting and governing CGT
relative risk to the product and ultimately the patient. It is
product manufacturing where single-use and multi-use
this relative risk that supports the basis in which industry
systems are used is absent. The regulatory implications
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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT
and regulatory experts agree a thorough yet realistic
approach to assess potential extractables must be
established. The study design should accommodate certain
specific parameters (e.g., varying organic and aqueous
extraction solvents, reasonable pH extremes and rational
extraction temperatures) which final results will support
comprehensive conclusions. It is clear that product risk
assessment is recommended by the U.S. FDA and other
global regulatory agencies, and general guidance to
conduct this activity has been described within the body of
the FDA’s “Final Report on ‘Pharmaceutical cGMPs for the
21st Century – A Risk-Based Approach’ provides clear
guidance on utilizing scientifically sound, risk-based
approaches to pharmaceutical cGMP requirements.” As of
the date of this writing, there are no published final
adopted regulations or established extractable
concentration limits specifically supporting CGT
production. Several regulatory and industry workgroups
(most notably USP and ICH) are moving towards this goal,
however.
The USP chapter <665> (Pharmacopeia Forum 43 (3), May
5, 2019) and by way of reference, chapter <1665>, are the
current benchmark documents providing regulatory
direction surrounding the conduct of a SUT-CGT extractable
investigation. It is noted that although USP chapter <665>
is currently not officially promulgated and <1663> is
considered an informational chapter and, as such, not
enforceable, they do provide a basis for the design,
justification, and execution of an extractables investigation
supporting CGT.
Now it is understood that CGT products are administered
via injection, intravenous, or infusion and, as such, one
should take into consideration those USP revision bulletins
and industrial guidance documents governing parenteral
products when contemplating the process and product’s
regulatory risk.
Part 5: Unique Challenges of CGT
Extractables studies seek to identify and quantify
compounds, which are released from the contact material
into an extraction fluid 8, 26, 27 and therefore are conducted
under forced or exaggerated laboratory conditions. Results
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from extractables studies require a subsequent assessment
of the detected compounds with regards to process
performance, product quality and patient safety.28
Consequently, a leachable study can provide information
about the presence and persistence of leachables as
process-related impurities.
E&L studies, as they are currently conducted for SUT, may
not provide all relevant extractables data required for CGT
processes. Currently, a significant issue associated with E&L
PART 5: HIGHLIGHT
Existing extractables and leachables studies
may provide part of the necessary
information relevant for SUT.
studies supporting CGT is that these studies are asking for
a qualitative and quantitative analysis in the contacting
liquid phase. Currently, a significant issue associated with
E&L studies supporting CGT is the study designs largely
investigate the contacting liquid phase (e.g. extractable[s]),
as this information can provide a direct correlation to the
degree of absorption of said analytes to the cells (e.g.,
leachables). However, one must consider the adherent cells
where a direct transfer of PERLs from the contact material
into the cells may occur. Although these correlations can be
assumed, one must consider that the semi-quantified
extractables data may not directly point to the influence on
cell viability. Also, the question of whether there are
leachables that can cause stimuli and/or cell degeneration
cannot be answered with analytical tools alone.
Part 6: Responsibilities for E&L Testing of
CGT Delivery Systems
As a part of the responsibility for producing a drug product
or device submission, the sponsor is required to prepare a
thorough and appropriate E&L study. As the responsibility
for the filing lies solely with the sponsor, the ultimate
responsibility for the completion of valid E&L studies also
belongs to the sponsor. The sponsor has many options as
to how that study is completed.
11
 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

With the increasing usage of single-use components, the appropriate expertise to perform an E&L study
specialty packaging devices, unique drug delivery platforms internally.
and novel container closure systems, the number and type
For the execution of the E&L testing, the sponsor may
of devices that require extractables and leachables testing
engage a partner laboratory for some or all of the study.
continues to grow at a rapid pace. As a result, E&L study
Furthermore, sponsors may engage directly with the
design has become increasingly
supplier’s technical team(s) as the
complex in order to address the
supplier(s) can be the ideal
unique requirements of diverse PART 6: HIGHLIGHT
partners when it comes to
products. As always, regulatory
As a part of the responsibility for producing extractables investigations. The
requirements will only increase
a drug product or device submission, the suppliers are typically well versed
and it will be incumbent upon the sponsor is required to prepare a thorough
in the composition and
sponsor to demonstrate that their and appropriate E&L assessment.
compatibility of their devices.
E&L study design will satisfy
Today, some suppliers can provide
regulatory expectations.
fully elucidated and identified extractables profiles for their
The sponsor may choose to perform their studies internally. SUT. Additionally, industry groups are encouraging the
The advantages of this approach are apparent: the sponsor placement of SUT testing responsibility back on the
retains complete control over the study, the drug product component manufacturer. While this may supply some of
expertise is close at hand with no confidentiality concerns, the required extractables data, the final responsibility lies
and potentially shorter timelines can be set if the resources with the sponsor.
are available. However, many organizations do not have

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Part 7: Terms and Definitions

Allogeneic Denoting, relating to or involving tissues or cells that are genetically dissimilar and hence
immunologically incompatible, although from individuals of the same species.
(www.lexico.com)
Antibody-coated beads A bead coupled with a specific antibody or set of antibodies which is often used to prompt
specific cell responses.
Antigen A toxin or other foreign substance which induces an immune response in the body,
especially the production of antibodies. (www.lexico.com)
Antisense polynucleotide A linear polymer made up of multiple nucleotide units that is complementary to distinct
functional regions of mRNA.
Autologous A situation in which the donor and recipient are the same person.
bDtBPP Bis (2,4-di-tert-butylphenyl)-phosphate.
Container Closure System (CCS) The sum of packaging components that together contain and protect the dosage form.
This includes primary packaging components and secondary packaging components, if the
latter are intended to provide additional protection to the drug product. (www.fda.gov)
Cryopreserved A method used to preserve the viability of tissue, organs and cells by storing them at
extremely low temperatures.
Cytotoxic A substance that has a toxic effect on certain cells.
DMSO Dimethyl sulfoxide.
Elastomeric material A component comprised of an elastic substance occurring naturally, as natural rubber, or
produced synthetically, as butyl rubber or neoprene. (www.dictionary.com)
Extractable Organic and inorganic chemical entities that are released from a pharmaceutical
packaging/delivery system, packaging component, or packaging material of construction
and into an extraction solvent under laboratory conditions. (www.usp.org)
IC Ion chromatography.
ICP-MS Inductively coupled plasma mass spectrometry.
Leachable Foreign organic and inorganic chemical entities that are present in a packaged drug
product because they have leached into the packaged drug product from a
packaging/delivery system, packaging component, or packaging material of construction
under normal conditions of storage and use or during accelerated drug product stability
studies. (www.usp.org)
GC-MS Gas chromatography-mass spectrometry.
Genotoxic A toxic agent that damages DNA molecules in genes, causing mutations, tumors, etc.
Monomer-caused cell inhibition A molecule which can be bonded to other identical molecules to form a larger polymer
chain, and which causes the cell to stop replicating.
Mutagenic A physical or chemical agent that changes the genetic material, usually DNA, of an
organism and thus increases the frequency of mutations above the natural background
level. (www.wikipedia.org)
Nitro-BPA Nitrogen substituted Bisphenol A.
Polymeric A large molecule, or macromolecule, composed of many repeated subunits.
(www.wikipedia.org)

PERLs Process equipment-related leachables. (www.usp.org)

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Reagent A substance that, because of the reactions it causes, is used in analysis and synthesis.
Single-Use Systems (SUS)/ Fluid path components that replace reusable stainless steel components. The most typical
Single-Use Technologies (SUT) systems are made up of bag chambers, connectors, tubing, and filter capsules.
(www.bpsalliance.org)
Somatic cells Any cell of a living organism other than the reproductive cells.
UPLC-MS Ultraperformance liquid chromatography-tandem mass spectrometry.
Viral vectors Vehicles for gene delivery usually derived from parental wild type viruses whose viral
genes, which are necessary for replication and virulence, have been substituted with the
genes intended for cell manipulation. These can be used to create gene-modified cell
therapies such as chimeric antigen receptor cell therapy or directly used for in vivo gene
therapy.
Virus-Vector Manufacturing Large scale manufacture of viral vectors for therapeutic use.
(VVM)
Xenogeneic Cells belonging to individuals of different species.

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Part 8: Additional Information

Cell & Gene Therapy White Paper

https://bpsalliance.org/cell-and-gene-therapy-
resources/
This white paper, The Role of Single-Use Polymeric
Solutions in Enabling Cell and Gene Therapy Production:
 Defines and maps commonalities between
bioprocessing and CGT
 Provides high level documentation to identify
information gaps
 Shares best practices to scale up and scale out
 Identifies regulatory and technical requirements of
each stage
 Provides education around single-use technologies
and what is available
 Defines the language

Cell and Gene Therapy Process Map: A beginning…

https://bpsalliance.org/cell-and-gene-therapy-
resources/

Get an introduction to the white paper and learn

about Single-Use Technology and the role it may
play in the CGT marketplace.

 Haddock, R., S. Lin-Gibson, N. Lumelsky, R. McFarland, K. Roy, K. Saha, J. Zhang, and C. Zylberberg. 2017.
Manufacturing cell therapies: The paradigm shift in health care of this century. NAM Perspectives. Discussion
Paper. National Academy of Medicine, Washington, DC. https://nam.edu/manufacturing-cell-therapies-the-
paradigm-shift-in-healthcare-of-this-century.

 Molecular Therapy — Methods & Clinical Development (2016) 3, 16073; doi:10.1038/mtm.2016.73

Official Journal of the American Society of Gene & Cell Therapy.

 Heathman, Thomas & Nienow, Alvin & Mccall, Mark & Coopman, Karen & Kara, Bo & Hewitt, Christopher.
(2015). The translation of cell-based therapies: Clinical landscape and manufacturing challenges. Regenerative
Medicine. 101473. 49-64. 10.2217/RME.14.73.

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 EXTRACTABLES/LEACHABLES CONSIDERATIONS FOR CELL & GENE THERAPY DRUG PRODUCT DEVELOPMENT

Part 9: References
1 “Manufacturing Challenges Facing Cell & Gene Therapy”, Alliance for Regenerative Medicine, September 19, 2019,
Webinar, https://alliancerm.org/wp-content/uploads/2019/09/Informa-ARM-Manufacturing-Challenges-
Webinar_FINAL.pdf
2 https://www.fda.gov/media/72402/download
3 Gene & Cell Therapy FAQs | ASGCT - American Society of Gene & Cell Therapy, https://www.asgct.org/education/gene-
and-cell-therapy-faqs
4 Peri P., ONDQA FDA. Regulatory Perspectives on Extractables and Leachables, PQRI Workshop on Thresholds and Best
Practices for Parenteral and Ophthalmic Drug Products (PODP) Bethesda, MD. Feb 2011.
5 U.S. Food and Drug Administration - Guidance for Industry: Container Closure Systems for Packaging Human Drugs and
Biologics. 41 (1999).
6 Norwood DL, Paskiet D, Ruberto M, et al. Best Practices for Extractables and Leachables in Orally Inhaled and Nasal Drug
Products: An Overview of the PQRI Recommendations. Pharm Res. 2008;25(4):727-739.
7 PQRI –PODP Working Group Recommendations: Parental Drug Products (PDP) Ophthalmic Drug Products (ODP) 3 rd
PQRI/FDA Conference on Advancing Product Quality Washington DC, 22 March 2017.
8 United State Pharmacopoeia <1663> Assessment of Extractables Associated with Pharmaceutical Packaging/Delivery
Systems. 41, 7910–7924 (2018).
9 United State Pharmacopoeia <1664> Assessment of Drug Product Leachables Associated with Pharmaceutical
Packaging/Delivery Systems. 41, 7924–7937 (2018).
10 United State Pharmacopoeia DRAFT <665> Polymeric Components and Systems Used in in the Manufacturing of
Pharmaceutical and Biopharmaceutical Drug Products. 2019. http://www.uspnf.com/notices/comment-period-extended-
packaging-general-chapters-appearing-pharmacopeial-forum-43-3
11 Ding W, Madsen G, Mahajan E, O’Connor S, Wong KM. Standardized Extractables Testing Protocol for Single-Use Systems in
Biomanufacturing. Pharm Eng. 2014;34(6):1-11.
12 Atouf, F., Provost, N. M. & Rosenthal, F. M. Standards for Ancillary Materials Used in Cell- and Tissue- Based Therapies.
Bioprocess Int. 11, 12–22 (2013).
13 Solomon, J. et al. Current perspectives on the use of ancillary materials for the manufacture of cellular therapies.
Cytotherapy 18, 1–12 (2016).
14 CFR - Code of Federal Regulations Title 21. (n.d.). Retrieved January 17, 2018, from
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=211.65
15 Hammond, M. et al. Identification of a leachable compound detrimental to cell growth in single-use bioprocess containers.
PDA J. Pharm. Sci. Technol. 67, 123–34 (2013).
16 Hammond, M. et al. A cytotoxic leachable compound from single-use bioprocess equipment that causes poor cell growth
performance. Biotechnol. Prog. 30, 332–337 (2014).
17 Jurkiewicz, E., Husemann, U., Greller, G., Barbaroux, M. & Fenge, C. Verification of a new biocompatible single-use film
formulation with optimized additive content for multiple bioprocess applications. Biotechnol. Prog. 30, 1171–1176 (2014).
18 Blaschczok, K. et al. Evaluating New Film for Single-Use Bags: Growth Performance Studies with Animal and Human Cells.
Bioprocess Int. 14, (2016).
19 Peng, J. et al. Chemical Identity and Mechanism of Action and Formation of a Cell Growth Inhibitory Compound from
Polycarbonate Flasks. Anal. Chem. 90, 4603–4610 (2018).
20 Ekwall B., Nordensten Ch., Albanus L.: Toxicity of 29 Plasticizers to HeLa Cells in the MIT-24 System; Toxicology, 24 (1982),
199-210
21 Brindley D. et al. Extractables and Leachables in Cellular Immunotherapy Biomanufacturing, BioProcess Int. 13(10) 2015:
S28-S37.
22 Eibl, R. et al. Recommendation for Leachables Studies Standardized cell culture test for the early identification of critical
films. Dechema (Dechema, 2014).
23 ASTM E3231-19 Standard Guide for Cell Culture Growth Assessment of Single-Use Material
24 Carter J., Restrepo S., Vogel J., Isberg E., BPOG An Industry Proposal for Change Notification Practices for Single-Use
Biomanufacturing Systems.
25 Paudel K, Hauk A, Maier TV, Menzel R. Quantitative characterization of leachables sinks in biopharmaceutical downstream
processing. Eur J Pharm Sci. 2020;143:105069. doi:https://doi.org/10.1016/j.ejps.2019.105069.
26 Ball, D. J., Norwood, D. L., Stults, C. L. M. & Nagao, L. M. Leachables and Extractables Handbook: Safety Evaluation,
Qualification, and Best Practices Applied to Inhalation Drug Products. (Wiley, 2012).
27 Jenke D. Compatibility of Pharmaceutical Products and Contact Materials - Materials Used in Pharmaceutical Constructs
and Their Associated Extractables. John Wiley & Sons, Inc.; 2009
28 Li, K. et al. Creating a Holistic Extractables and Leachables (E&L) Program for Biotechnology Products. PDA J. Pharm. Sci.
Technol. 69, 590–619 (2015).

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DISCLAIMER
The information in this document is intended to capture the current state of the Single-Use-Technology (SUT) Industry in regards to
Particulate Control, Testing and Evaluation. The material presented herein is intended to help characterize levels and types of particles,
as well as to provide methods to assure minimal levels of particulate in SUT. This information is offered in good faith and supported
by the expertise of its contributors. However, BPSA, its members and contributors do not assume any responsibility or obligation for
the reader’s compliance to the content of this document. This is not a standard, but a set of recommendations.
This document is not intended to, nor should it be used to support a cause of action, create a presumption of a breach of legal duty,
or form a basis for civil liability. Nothing expressed or implied in this informational document is intended, or shall be construed, to
confer upon or give any person or entity any rights or remedies under or by reason of this informational document.
Determination of whether and/or how to use all or any portion of this document is to be made in your sole and absolute discretion.
Use of this document is voluntary.
BPSA shall not be responsible or liable for any inaccuracies in the document or the information presented. All warranties express or
implied are disclaimed and waived.
Manufacturers, suppliers and end users should consult with their own legal and technical advisors relative to their SUT use and
participation. No part of this document constitutes legal advice.

About BPSA
The Bio-Process Systems Alliance (BPSA) was formed in 2005 as an industry-led corporate member trade association dedicated to
encouraging and accelerating the adoption of single-use manufacturing technologies used in the production of biopharmaceuticals
and vaccines. BPSA facilitates education, sharing of best practices, development of consensus guides and business-to-business
networking opportunities among its member company employees.
For more information about BPSA, visit www.bpsalliance.org.

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