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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5335_Библиотеки_им_академика_М_И_Перельмана.pdf
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
of genetic material which produces therapeutic proteins [4]. Typically, this circumvents the restrictions related to the therapeutic use
of recombinant peptides, including low bioavailability, clearance
rates, and exorbitant production cost [5]. Tissue therapies aim to
mend and restore injury to organs and tissues through the engineering of components involving cells and tissue architectures [6],
combining cells from a patient with scaold biomaterials [7], which
can potentially plug the severe shortage in donated organs. Currently, there are over a hundred thousand potential recipients on
the waiting list in the United States alone [8].
CTGTPs have been defined and classified dierently by various
regulatory bodies, where a product will go through the regulatory
pathway according to the definition that they fall under. In some
countries, CTGTPs are also known as advanced therapy medicinal
products (ATMPs) or regenerative medicines (RM). In other countries, they may be further classified under categories such as cell
therapy medicines, gene therapy medicines, tissue-engineered medicines, or combined products, with dierent variations in naming
the respective product categories. These definitions take into consideration the degree of processing from the starting materials as well
as the purpose of the product, such as the restoration of function or
prevention of disease, which determines the extent of regulation a
product is subject to. However, there are also some countries where
little or no regulations for CTGTPs exist.
With rapid progress and strong interest in CTGTPs, as evident from
the increase in investments in these novel products over the last decade [7], many challenges have also arisen. These come from a lack
of knowledge regarding these novel products, where conventional
modes of manufacturing and regulation have not been adequately
adapted to ensure the safety and ecacy of CTGTPs. Considering
Cells, Tissues, and Gene Therapy Products
the lack of studies on CTGTPs, this article aims to juxtapose the
characteristics of CTGTPs with conventional biologicals, where
existing manufacturing trends from the latter will serve as a basis
for proposing solutions to solve the challenges faced in manufacturing and regulation. This article also aims to present the manufacturing processes and regulatory frameworks that CTGTPs are
subjected to in dierent countries.
CTGTPs and their principles of action
Cell therapy products use cells to repair or replace injured tissue or
cells in the body. Cells used may include mesenchymal stem cells
(MSCs), T cells, and pancreatic islet cells [9]. Cell therapy products
may contain only cells alone or exist as a part of gene therapy or
tissue therapy.
353
These products can be further categorized according to autologous
therapies or allogeneic therapies. Autologous therapies obtain cells
directly from the patient, thus circumventing immune reactions.
However, this also means that these cells are not suitable for use in
mass manufacturing as ‘o-the-shelf’ products. In contrast, allogeneic therapies utilize donated cells to treat multiple patients. These
cells are collected from healthy donors, rather than directly from the
patient to create a master cell bank (MCB). Consequently, the risk
of immunogenic reactions is higher [16]. Products for gene therapies
are manufactured via ex vivo or in vivo processes. An in vivo process
is one where a viral vector carrying the gene of interest is directly
transferred into the body via infusions, whereas an ex vivo process
involves removing a patient’s cells to be altered and then re-infusing the genetically modified cells back into the patient’s body [17].
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
For tissue engineering, current strategies include recreating organ
and tissue structure via scaold fabrication, 3D bioprinting and
self-assembly, integration of grafts to host via vascularization and
changing the host environment to create therapeutic responses [6].
Regulators often decide on the stringency of manufacturing requirements by considering if the therapy product is intended for homologous use, where the therapy product is administered at an identical
anatomical site and fulfils the same function in the recipient as in
the donor [1]. In addition, the extent of manipulation the cells or
tissues have undergone is also considered, where minimal manipulation implies that biological traits or functions of the cell or tissue
are unchanged [1].
Dierences between CTGTPs and conventional
biologicals
The type of therapy has an immense impact on the type of manufacturing style and challenges encountered. Conventional biologicals are biotherapeutic protein products made with recombinant
DNA technology, where cells are reprogrammed genetically to produce proteins that are insuciently produced in the body [10]. An
example includes synthetic insulin for the treatment of diabetes
and monoclonal antibodies for cancer treatment. The key dierence
between conventional biologicals and CTGTPs is that while proteins
are the final products in conventional biologicals, cells are the final
products in CTGTPs. Cells can produce specific proteins continuously as compared to the fixed number of specific proteins, which
may be degraded and thus depleted in the body. Hence, the cells
can potentially allow for longer lasting or permanent prophylaxis.
Cells, Tissues, and Gene Therapy Products
In general, the manufacturing process for chimeric antigen receptor
T (CAR-T) cell therapy products, one of the most common CTGTPs,
involves steps to preserve the cells and focus on purifying specific
target cells, while biologicals manufacturing involves an additional
step of isolation and purification of the protein. Since cells are relatively more sensitive to their environment, a more stringent process
in terms of the manufacturing environment must be in place to
maintain the quality and safety of the CTGTP. Overall, cell therapy products are generally not as well characterized as compared to
conventional biologicals, thus having dierent biomarkers in testing for ecacy.
Table 1 shows examples of the dierent types of products and how
they work. Figure 1 focuses on the manufacturing process of CAR-T
cell therapy product and conventional biologicals. Table 2 gives further details regarding the manufacturing, quality control storage
and transport between CTGTPs and conventional biologicals.
355
Ocial definitions of CTGTPs
In general, most countries adopt a risk-based approach in deciding
if a product is a CTGTP before subjecting it to licensing requirements. In countries such as the United States (US), the European
Union (EU), and South Korea, minimally manipulated and homologous use products are not subject to marketing authorization.
For CTGTPs which require marketing authorization, the extent of
regulatory requirements depends on the type of products manufactured. Countries such as Singapore include minimally manipulated
and homologous products under its definition of CTGTPs, and specifically exclude other products of the same type, e.g. bone marrow,
Product
Product name
category How it works Ref.
Spherox Autologous
cell therapy
product
Kymr ia h Autologous
cell-based
gene therapy product
Zolgensma In vivo gene
therapy
product
Vergenix™FG Tissue engi-
neered therapy product
Table 1: Examples of products and their mechanism of action
Spherox (Co.Don AG) is a product used to fix defects in the knee cartilage.
It contains spheroids of chondrocytes, derived from MSCs, and found in
healthy cartilage. A sample is extracted from the patient’s own tissues via
arthroscopy, cultivated in the laboratory to form a suspension of chondrocyte spheroids and then reintroduced into the patient’s cartilage. The
chondrocytes then attach to the cartilage to fill the defect
Kymriah (tisagenlecleucel) is indicated to treat of refractory B cell precursor
acute lymphoblastic leukemia (ALL). The patient’s T cells are genetically
modified by introducing deoxyribonucleic acids. These modified T cells
express chimeric antigen receptors (CARs), which facilitate the targeted
killing of CD19+ B cells by binding to them while stimulating the proliferation of the CAR-T cells. These CAR-T cells are dubbed a living drug as they
continue to exist in the body to fight cancer long after their infusion
Zolgensma (onasemnogene abeparvovec) is a one-time intravenous infusion
indicated for the treatment of spinal muscular atrophy (SMA) due to genetic
alterations in the SMN1 gene. The therapy replaces the missing/defective
SMN1 gene with a new copy of the SMN gene using the AAV9 virus carrier
Vergenix™FG is indicated for the management of acute and chronic wounds,
such as diabetic and pressure ulcers, and surgical wounds Vergenix™FG
provides a scaold for cellular and capillary growth and is supplied as a
lyophilized material contained in a syringe
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
[11]
[12],
[13]
[14]
[15]
Cells, Tissues, and Gene Therapy Products
Comparison of Manufacturing Processes for CTGTP and Biological Product
357
CTGTP (CAR-T-Cell)
Apheresis collection
Apheresis product wash/fractionation
T-cell selection
T-cell activation
Viral vectors, transposons, mRNA electroporation
Gene transfer
CAR-T cell expansion
CAR-T cell formulation
CAR-T cell cryopreservation
Infusion into patient
Biological (Therapeutic Protein)
Isolation of gene of interest
Introduction of gene to expression vector
Transformation into host cells
Cell banking system master cell bank,
working cell bank
Selection of the required sequence & propagation of cells
Extraction (lysis), clean-up, enrichment/isolation of protein
Downstream Upstream
Isolation & purification of protein
Formulation, packaging of protein product and QC
Sterilization, viral decontamination, pyrogen removal
Figure 1: Comparison of manufacturing process between CTGTP (CAR-T cell)
and biological (therapeutic protein) [18, 19]
CAR-T: chimeric antigen receptor T; CTGTPs: cell, tissue and gene therapy products; mRNA:
messenger ribonucleic acid; QC: quality control.
peripheral blood, cord blood and vaccines, without further classifying them as CTGTPs. Other countries such as China and India have
no formal definitions for CTGTPs.
Table 3 states the definitions and classifications by the US, EU, Australia, Japan, China, India, Malaysia, Thailand, Singapore, South
Korea, the World Health Organization (WHO) and the International Council for Harmonization of Technical Requirements for
Pharmaceuticals for Human Use (ICH). These countries have been
chosen to represent a heterogeneous regulatory environment with
diering levels of capacity and maturity.
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
Manufacturing of CTGTPs
In the manufacture and commercialization of CTGTP, two main
modes of manufacturing styles exist. Decentralized manufacturing
spreads production over dierent locations, allowing for a more
amenable response to demands [41]. An example of a decentralized
manufacturing process is the integration of good manufacturing
practice (GMP) facilities into a hospital setting to produce autologous therapies for patients. However, certain risks are associated
with this model, including diculty in quality control due to a lack
of central oversight and contamination risk [42]. On the other hand,
centralized manufacturing has been the prevalent way of manufacturing as it enables an economy of scale, due to well-established
processes and machinery to produce large, standardized batches of
the same product at a single location [41]. Some examples include
chemical drugs, allogeneic products and biologicals. However, this
characteristic makes it dicult for manufacturers to customize
products for specific patients. Currently, centralized manufacturing is utilized for licensed CTGTPs such as Kymriah (Novartis) and
Yescarta (Gilead), with a substantial interest in moving towards
decentralized manufacturing for autologous products at the point
of care [8]. The advantages and disadvantages of the manufacturing
methods will be further discussed in the subsequent sections.
As mentioned, the unique nature of CTGTPs as compared to conventional biologicals makes the manufacturing of safe and ecacious CTGTPs a challenge. So far, there has been a clear distinction
between manufacturing for product development and conducting
clinical trials for academic research, with the former taking place in
pharmaceutical companies and the latter in hospitals. However, in
recent developments, personalized CTGTPs have demanded a higher
Cells, Tissues, and Gene Therapy Products
involvement of hospitals in the development of CTGTPs due to their
manufacturing process [20], which must be near to the patient due
to the sensitivity of the product, thus necessitating hospital premises to be of GMP standard. The problems of quality control in cell
therapies produced in the academic setting and industry, coupled
with strict regulations and diculty in harmonizing a standardized manufacturing process, hinder the availability of treatments to
patients [43] through manufacturing process challenges.
The manufacturing process of CAR-T cells is shown in Figure 2.
Peripheral blood mononuclear cells are first collected from the
patient via leukapheresis. This is followed by T-cell selection, removing adulterants such as gross red blood cells and platelets, while
simultaneously enriching T cells [18]. The T cells are then activated
using technologies involving antibody-coated nanobeads before
being transduced with a viral vector, which contains the anti-CD19
CAR transgene. The T cells are then expanded in bioreactors to produce doses for sucient therapeutic eect. Finally, the T cells are
removed from the beads, washed, cryopreserved in infusion bags,
and tested for the critical quality attributes of the product before it
is released and thawed for infusion into the patient [44].
359
In the case of tissue engineering and regenerative medicine (TERM)
therapies, multiple types of materials are required to come together.
As such, the manufacturing workflow is dierent for each component, including cells, scaolds or bio-printed therapies. The manufacturing workflow for cells is as shown in Figure 2, whilst the
manufacturing workflow for scaolds and bio-printed materials are shown in Figure 3. Bioprinting involves the fabrication of
three- dimensional anatomical structures to be used in therapies
[45] and in in vitro models since they can be engineered to mimic
Table 2: Comparison of manufacturing, quality control, storage and transport between CTGTPs and conventional
biological
Biologicals Autologous CTGTP Allogeneic CTGTP Comments Ref.
Sterility Sterility assurance
through aseptic
processing involving
protein products
Closed/open
system
Batch Scale; Batch
number
Stability in
processing
(between batch/
final product)
Stability of
product
(expiration
time)
QC test methods Relatively well established Less well established Both biologicals and CTGTPs may require
Closed Closed There is a move towards automated, closed sys-
Large batch scale;
Multiple
Relatively stable and
uniform
A few days to a month
(after reconstitution)
Sterility assurance through aseptic pro-
cessing involving live cells. All starting
materials and processes must be sterile
Individual patient
basis; Single
Cells are non-robust and sensitive to
environment
A few hours — [21]
Small batch scale;
Multiple
Live cells are unstable under heat, radiation, or
chemicals. They also need to be manufactured
aseptically with sterile filtration. Inadequate
time to complete sterility tests due to short
shelf life/ urgent medical need
tems to reduce variability of both biolog icals
and CTGTPs
Ty pic al ly, allogeneic therapies utilize scale-up
approaches while autologous therapies utilize
scale-out approaches
For biologicals, substitution and chemical
modification of protein or cha nging solvent
properties may improve stability of product
without compromising activity
new and specific quality control testing
methods
[20]
[20]
[20] ,
[21]
[20]
[20]
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Manufacture and Supply, Science and Reg ulation Towards High-Qua lity Medicinal Products
QC sampling
strategy
Purification Protein structure may
No minimum sample
volume
be altered during
elution steps Rapid
purification necessary
to prevent cleavage by
protease released by
lysed bacteria.
Elimination of residual
DNA (from hybridoma
of mammalian cell) is
required
Minimum sample volume The test results from minimum sample volume
for quality control may not accurately reflect
the properties of the batch. Reference samples
are not required to be retained due to the
small quantities of final CTGTP manufactured
for use by patient(s)
Vec tor s:
Dicult to separate vectors that have
taken up genetic material from those
that have not, or f rom empty capsids,
due to similar measurable biophysical
properties
Due to larger size, vectors also diuse
more slowly than proteins and hence
bind to bead surfaces of solid-phase base
chromatog raphy rather than diuse
into pore
Technology for the separation of cells:
Magnetic-Activated Cell Sorting
(MACS): separates cells via a single
surface antigen Fluorescence-activated
cell sorting (FACS): separates cells via
multiple sur face markers
Purification methods may aect transduction
ability, infectivity, and structural integrity.
Impurities can potentially cause immunogenic
reactions. The presence of empty capsids can
also reduce the therapeutic dose
Target cells may share common cell surface mark-
ers, necessitating multiple separations, leading
to decreased throughput and increased cost
The throughput of FACS is too low to achieve
manufacturing-scale quantities and is dicult
to integ rate into aseptic, closed systems
[20]
[18],
[19],
[22],
[23]
Cells, Tissues, and Gene Therapy Products
(Continued )
361