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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5886_Библиотеки_им_академика_М_И_Перельмана.pdf
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processing portion, typically clarication and concentration of the virus to standards that meet compliant safety, purity, and efcacy regulations.
The upstream process begins at vial thaw and continues on through a series of
cell expansion steps to generate a sufcient amount of cell inoculum to inoculate the
production vessel (N), the vessel the LVVs will be produced within (Fig.9.2). Cell
expansion can be carried out in shake ask vessels that are incubated in incubators
that support cell aeration and temperature requirements for growth. Alternatively, in
an effort to increase biomanufacturing capacity other alternatives such as the disposable wave bioreactor can prove promising in providing ease of operation by
eliminating the need to manipulate and manage multiple shake asks, no crosscontamination, while still ensuring good mixing and oxygen transfer without cell
damage [14]. In simple wave bioreactors allow for cell cultures to be inoculated into
a disposable sterile plastic bag that sits on a rocking platform that allows for efcient mixing and increased oxygen transfer due to the available high surface area
conditions.
Once the appropriate cell culture inoculum is generated, cells are then inoculated
into the production vessels where LVV production will occur. Bioreactors are
intended to provide suitable environments to maintain culture homogeneity while
ensuring the production of a product with desired quality attributes. Vector production is highly dependent on the producer cell and its` health during culturing.
Decline in cell health can have an effect on productivity and production of LVVs
thus resulting in low, if any, vector titers. When scaling up a bioprocess using bioreactors it is important to keep in mind that optimization of key bioengineering principles is critical to maintaining a desirable culture environment for cells to thrive.
Some of these parameters are the power input per volume ratio (P/V), impeller tip
speed, and the volumetric mass-transfer coefcient (kLa) as these all can have an
effect on culture conditions. In brief, the P/V is the rate of impeller energy transferred into the cell culture; this parameter can be indicative of shear stress to the
cells and can inuence mixing and oxygen transfer capabilities. Impeller tip speed
also inuences mixing time and if not optimized can lead to concentration gradients
within the culture, and kLa is the aeration capability of the bioreactor system, the
ability to transfer oxygen to the cell culture. In practice, it is recommended to use
bioreactors of similar design or geometry as this will facilitate the scaling up
process.
Once culture conditions or cell expansion are optimized, then mode of production is the next step in the upstream LVV bioprocessing. Whether the mode of production is performed via the transient transfection method or the use of a packaging
Fig. 9.2 Flow diagram of upstream bioprocessing steps for lentiviral vector production
9 CAR-T Bioprocessing
231
or stable cell line is dependent on the process and dosage requirements. There are
both pros and cons to the using either method. In one aspect transient transfection is
an attractive method due to its shorter process development times, though for largescale production this method is associated with higher running costs due to the
costly cGMP-grade plasmid DNA.On the other hand, stable producer cell lines are
associated with longer process development timelines and higher upfront costs with
lower exibility, but their use has the potential to generate more consistent harvest
titers and higher quality material while reducing running costs due to the elimination of the plasmid DNA requirement [15].
A decisional tool, such as one developed by Comisel etal., could be used to
determine the most cost-effective cell culture technology for the manufacturing of
LVVs. The group describes a cost of goods (COG) breakdown and rankings of all
the currently available cGMP-grade LVV manufacturing cell culture technologies
across a wide range of LVV products, clinical and commercial demand, and harvest
titer. Their analysis has suggested that single-use bioreactors (SUBs) are predicted
to be the most cost-effective cell culture technology with cost benets increasing
with increased demand, mostly attributed to the broad scalability of SUBs with the
largest scale at 2000L making SUBs superior for the purpose of commercialization
and large-scale production compared to other cell culture technologies [15].
Typically, the mode of production for fed batch cultures span a timeframe of 48h
post-transfection (hpt) or induction (hpi), with viral vector production peaking at 48
hpt and declining from 72h on, limiting the timeframe of when harvesting of the
LVVs can occur [8]. However, another area of interest in the production of LVVs is
process intensication. Process intensication or perfusion is another process that
can be used for the production of virus. The perfusion process extends over a period
of time without compromising cell health and LVV stability. It has enabled a scalable production process for the production of LVVs while increasing total vector
titer. Advantages of perfusion cultures are the ability to remove cell waste products
as they develop while maintaining nutrient and metabolite levels [8] required to support high cell density growth over extended periods of time. In addition, by retaining cells within the bioreactor this mode can facilitate the harvest step and subsequent
downstream processing. Some groups have demonstrated that LVVs are able to be
produced in 3L bioreactors operated under perfusion mode using novel approaches
that are scalable and are able to achieve increased viral titers of 30-fold compared to
a fed batch process [16]. Process intensication involves the use of a cell retention
device that retains cells inside the bioreactor while allowing for continuous media
exchange. As fresh media is continuously added, spent media containing the LVV is
removed and collected at the same rate.
The upstream process for the production of virus can vary dependent on the type
of cell line used, virus produced, and demand. Once the intended product, the virus,
is produced, next begins the downstream processing portion.
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9.2.6 Downstream Bioprocessing
The timepoint at which the upstream process ends is the timepoint when downstream processing steps commence. Once produced, LVVs within the cell culture
must then be harvested and processed through a series of downstream processing
steps to concentrate and generate a nal bulk vector that is of high purity and titer.
As high titers are important to meet the demand of doses required to treat patients,
purity of the product is also critically important to prevent potential inammatory
reactions both invitro and in vivo [3] as a result of common process and product
related impurities such as host cell proteins, residual DNA, transfection reagents,
media components, viral aggregates, or free vector components [8].
The harvest step begins by using a process that will aim to remove large host cell
proteins and cellular debris clarifying the vector (Fig.9.3). Two known methods can
be employed for the harvest clarication step; microltration and/or centrifugation.
Centrifugation can act as a pre-ltration step to help prevent lter fouling during
subsequent ltration steps, but not all processes involve a centrifugation step and is
dependent on the process. Cells and debris are separated from LVV based on the
mass properties of these components. Typically, as cells tend to weigh more than
LVV, at certain centrifugal speeds the cells and larger debris will sediment toward
the bottom, while the LVV will remain in the supernatant of the culture broth.
Whether a centrifuge step is carried out or not the harvest material will still be
passed through a membrane or depth lter for clarication of the virus. Filter choice
can impact the efciency of the clarication step, [8] therefore careful considerations should be applied in selecting a lter uniquely appropriate for the process at
hand as not all lter sizes and properties are compatible with all virus types.
Furthermore, seamless execution of the clarication step can be dependent on optimizing the upstream production. Take for instance culture viability at the time of
harvest, a lower cell viability will complicate the clarication step, due to the
increased amounts of cellular debris as a result of cell fragmentation during cell
death. This can potentially lead to lter clogging, increased pressures, and ultimately pre-mature lter fouling. These types of considerations should be kept in
mind not only for efcient processing of the vector but also to drive down costs of
purchasing additional lters. With proper optimization and choice of lters for the
process, this can be avoided.
Once the vector has been claried, the next step is to treat the harvest material
with a nuclease enzyme to digest residual DNA/RNA contaminants. Once treated,
the viral vector bulk can then be further puried through a series of sequential chromatography, or in some cases nonchromatographic steps that can either bind or
Fig. 9.3 Flow diagram of downstream bioprocessing steps for lentiviral vector production
9 CAR-T Bioprocessing
elute the vector, depending on the conductivity of the buffer solution used, separating the vector from impurities. Chromatography, in the case of ion exchange (IEX)
chromatography, separates the vector from impurities based on the charge characteristics of the components, while size exclusion chromatography (SEC) separates
based on size. Again, the choice of chromatography methods will be dependent on
the chosen process and virus produced. For LVV purication multiple types of chromatography have been applied resulting in varying recoveries [8]. Once puried the
vector is concentrated and exchanged into a nal ll formulated buffer of choice.
This step can be carried out using tangential ow ltration in an ultraltration/dialtration (UF/DF) mode that enables buffer exchange while removing low molecular
weight impurities [8]. The nal step is to sterile lter the nal vector product and
freeze for long-term storage until quality testing is carried for these products to be
dispositioned for use in CAR-T manufacturing. The next few sections will discuss
how the viral vectors are used to produce the therapeutic product once produced.
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9.3 Cell Product Bioprocessing
9.3.1 End-to-End Systems
Generally speaking, the steps in cell product processing can be performed on different process unit operations, discussed in the following sections. However, there is a
growing interest in end-to-end production that requires minimal manipulation and
reduces the number of instruments needed per production. To this end, the
CliniMACS Prodigy from Miltenyi Biotec and the Cocoon from Lonza are two
available closed end-to-end systems that can accommodate selection, activation,
transduction, expansion, and harvest of the CAR-T product. The Prodigy has been
used for clinical trials at University College London [17] and is a popular option in
product development departments. The Prodigy can accommodate the isolation of
up to 3E9 CD3+ cells. While not yet as widely adopted, the Cocoon can accommodate real-time monitoring of cell culture characteristics like pH and dissolved oxygen, which is a key advantage over the monitoring available with the Prodigy system.
While it is possible to complete a fully closed CAR-T manufacturing process,
manufacturers need to ensure that all inputs, such as media, buffers and apheresis,
are available in weldable bags to maintain a functionally closed system. If necessary, open manipulations and transfer of material to a weldable bag can be performed in a BSC (biological safety cabinet), and then welded on to the closed
system. Weld compatibility of different materials should be taken into consideration
when designing a closed process. For example, closed reagents are available from
Miltenyi Biotec to ensure compatibility with the CliniMACS Prodigy system.
These end-to-end integrated systems will be discussed among the instruments
available for the different process units, but their integration of different process
units is worth highlighting to the reader (Table9.2).
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A. Adeniran et al.
9.3.2 Apheresis Collection andPreservation
To begin CAR-T manufacturing, a patient’s T cells must rst be isolated using leukapheresis, which separates leukocytes from a patient’s blood and returns the
remaining blood components to the patient.
Leukapheresis is typically performed with machines such as the Spectra Optia,
COBE Spectra (Terumo), or Amicus (Fresnius-Kabi) by highly trained nursing staff
at hospitals and clinical collection centers. Geothe University, one of the rst
German centers to be authorized for pediatric CAR-T treatment, used the Spectra
Optia for apheresis collection for Kymriah production [18].
In an apheresis machine such as the Spectra Optia, blood is rst mixed with an
anticoagulate like citrate before being separated by continuous-ow centrifugation.
The blood is separated into three layers: a red blood cell layer on the bottom, a buffy
Table 9.2 Example technologies for CAR-T bioprocessing
Cell processing step Example commercial technologies
Apheresis collection Spectra Optia (Terumo)
COBE Spectra (Terumo)
Amicus (Fresenius-Kabi)
Lymphocyte purication BCT Elutra (Terumo)
X-Lab (Corning)
Leukapheresis thawing Plasmatherm (Barkey)
Varitherm (Barkey)
ZipThaw (FreMon)
VIAThaw (Cytiva)
T-cell isolation Dynabead (ThermoFisher)
CD4, CD8 immunomagnetic reagents (Miltenyi Biotec)
RoboSep-C (Stemcell)
Sepax C-Pro (Cytiva)
WOLF cell sorter (NanoCellect)
MACSQuant Tyto (Miltenyi Biotec)
T-cell activation Dynabead (ThermoFisher)
TransACT (Miltenyi Biotec)
T-cell transduction LentiBOOST (Sirion Biotech)
Vectofusin-1 (Miltenyi Biotec)
T-cell transfection 4D Nucleofector (Lonza)
Cytokines for expansion GMP ProDots (Biotechne)
Lyophilized in vials (Miltenyi Biotec, CellGenix)
Closed processing systems CliniMACS Prodigy (Miltenyi Biotec)
Quantum (Terumo)
Cocoon (Lonza)
G-Rex (Wilson Wolf)
Xuri Wave (Cytiva)
1L+ scale cell expansion Hyperforma (ThermoFisher)
Biostat RM (Satorius)
Scale up systems G-Rex (Wilson Wolf)
Ambr (Sartorius)
9 CAR-T Bioprocessing
235
coat layer in the middle, and a plasma layer on top. The quality of layer separation
is determined by the packing factor, a numerical relationship between the inlet ow
speed and the centrifuge speed. The buffy coat contains the lymphocytes needed as
input for CAR-T manufacturing, as well as other contaminating cell types such as
monocytes and granulocytes. The nontarget layers, and replacement uids such as
plasma or saline, are returned to the patient to prevent electrolyte imbalances and
hypotension [19]. Plasma is often added to the isolated cells before further processing to promote cell healthy and viability.
Elutriation, the separation of cell types based on density and size, can be used if
further purication of the lymphocytes is desired before downstream processing.
The method is particularly good for separating lymphocytes from monocytes [20],
which can be benecial as monocytes can decrease nal product quality [21, 22].
Terumo’s BCT Elutra is a fully closed device available for performing elutriation.
The X-Lab from thermogenesis is available for a sedimentation-based approach to
cell separation.
To allow exibility in the supply chain and maintain health of the collected cells,
the apheresis is typically cryopreserved at the clinical collection site or a manufacturing facility. Both cryopreservation methods and supply chain challenges are further discussed in detail later in this chapter. Cryopreservation has been demonstrated
to eliminate myeloid-derived suppressor cells which have been shown to inhibit
T-cell proliferation in CAR-T manufacturing [23]. While cryopreservation is common, use of fresh apheresis material as an input material may help decrease overall
manufacturing time if logistical barriers to accommodate the required “just-in-time”
supply chain can be overcome. Kite pharmaceuticals has applied to the EMA
(European Medicines Agency) requesting use of fresh material to start manufacturing [24].
Apheresis collections can be highly variable from patient to patient and this variability can complicate manufacturing success. Factors contributing to variability
can include patient age, clinical indication, prior treatments, tolerance for anticoagulants, method of venous access, and apheresis center practices. Collecting apheresis before treatment with frontline chemotherapy or radiation regimens has been
suggested to combat challenges due to clinical indication and prior treatments,
though it remains to be seen if this would result in higher quality CAR-T drug products [20].
9.3.3 T-Cell Thawing andIsolation
Cryopreserved apheresis must be thawed before T-cell isolation can begin.
Though cryopreserved leukapheresis can be thawed in a water bath, dry thawing
reduces the likelihood of contamination and should be used in GMP facilities. The
Barkey Plasmatherm, used in by Novartis for Kymriah production [25], passes
warmed water through two cushions and the cryopreserved material is placed
between the two cushions until thawed. The ZipThaw from Fremon Scientic and
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the VIA Thaw and Smart-Max, both from Cytiva, are alternative dry thawing
devices. The ZipThaw differentiates itself for its light weight, the VIA Thaw for its
dry conduction technology, and the Smart-Max for its pneumatic mixing technology.
The T-cells must be separated from contaminating cells such as monocytes and
granulocytes in the apheresis as contaminating material can inhibit activation and
proliferation [20]. Additionally, there is emerging evidence that suggests dened
ratios of CD4 to CD8 cells yields uniform potency of CAR-T products [26].
Breyanzi is dosed as a 1:1 ratio of CD4 to CD8 cells [27]. However, Yescarta,
Kymriah, Tecartus, Abecma, and Carvykti do not specify a CD4:CD8 ratio for dosing [28–32]. T-cells can be specically isolated from the thawed apheresis using
immunomagnetic separation, uorescence-activated cell sorting (FACS), or
acoustics.
With immunomagnetic technology, positive and/or negative selection can be
used to isolate T-cells using beads. The beads can simultaneously display an antibody or ligand specic to the desired cell type and a magnetic particle, or the beads
themselves can be magnetic. In positive immunomagnetic separation, the beads
enable the desired population to be cross-linked to magnetic particle. In negative
immunomagnetic separation, the undesired cell types are targeted. Separation of the
bead-bound population can occur over a magnetic column or can be column-free.
ThermoFisher’s Dynabead technology is a column-free option for cell separation. With the Dynabead technology, the bead itself is magnetic, rather than being
linked to magnetic particle. Anti-CD3 and anti-CD4 Dynabeads are commercially
available, as well as the DynaMag CTS magnet, which can accommodate 50–330mL
for static separations, and >10L for continuous ow separations.
Miltenyi Biotec offers anti-CD4 and anti-CD8 immunomagnetic reagents, which
are compatible for use in the CliniMACS Prodigy. The reagent vial is spiked onto
the Prodigy tubing set and ltered before making contact with the cells. The cells
are ltered on a pre-separation column to reduce any clumped material from entering the magnetic column. The selected cells are then transferred to another bag
where they can be readily dispensed to a chamber to begin cell culture.
Another closed system for cell separation is the RoboSep-C from Stemcell
Technologies. The RoboSep-C can process up to 2x10E7 cells but unlike the
CliniMACS Prodigy, the RoboSep-C is column-free. The cells of interest are isolated in a bag with PVC tubing that can be welded on to other downstream equipment. A unique feature of the RoboSep-C is its cell washing cartridge, which washes
and concentrates cells without pelleting or centrifugation [33].
Closed cell separation is also possible on Sepax C-Pro from Cytiva Life Sciences.
The Sepax C-Pro is multifunctional like the CliniMACS Prodigy, but uses a separate, closed kit for each of its isolation, transduction, harvest, and formulation functions. One kit is available for immunomagnetic separation and can accommodate up
to 880mL of input volume.
Fluorescence-activated cell sorting (FACS) can also be used to separate cell
types. For this ow cytometry-based separate method to be clinically feasible, the
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237
ow cytometer must be in a closed, aseptic system. Sorting cytometers such as the
WOLF Cell sorter or the cartridge-based MACSQuant Tyto can be used to aseptically separate cells.
Finally, buoyancy-activated cell separation (BACS) uses functionalized microbubbles to bind cells of interests, and after a gentle agitation, the microbubbles and
bound cells oat to top of the solution. Cesca Therapeutics and Akadeum Life
Sciences offer BACS kits. However, these kits are currently only available at the mL
scale and would need further scale-up development to accommodate the volumes
required for clinical manufacturing.
9.3.4 Activation
Activation of the selected T-cells is necessary for cells to be able to expand and
accept the vector encoding the CAR-T receptor. The TCR and CD28 receptors are
often targeted for cellular activation as this mimics the manner by which antigenpresenting cells naturally activate T-cells [34], and activation of the TCR alone by
anti-CD3 antibodies does not induce full activation [35]. There are a variety of activation technologies available that utilize components such as matrices, beads,
and cells.
Matrix- and bead-based activation reagents are advantageous for commercial
manufacturing as they allow for a consistent activation reagent to be used across
multiple manufacturing batches.
Novartis reported the use of Dynabeads for Kymriah manufacturing. Available
from ThermoFisher, Dynabeads are magnetic beads covalently linked to anti-CD3,
anti-CD28 and, if desired, anti-CD137 antibodies. Dynabeads can be removed in a
magnetic eld after activation is complete [36]. These beads can also be used for
cell isolation, streamlining the isolation and activation steps and reducing the number of reagents needed.
TransACT technology from Miltenyi Biotec is a polymeric matrix conjugated to
CD3 and CD28 agonists compatible with Miltenyi’s closed system Prodigy unit.
Following activation, TransACT is removed from the culture by exchanging culture medium.
Expamer technology has been introduced to temporally ne-tune T-cell activation by utilizing the reversible Twin-Strep tag interaction with Strep-Tactin.
Anti-CD3 and anti-CD28 antibody fragments are functionalized with Twin-Strep
tag, which binds a soluble Strep-Tactin multimer. This allows for muti-valent binding by one Strep-Tactin multimer. Adding D-biotin to the cell culture disrupts the
interaction between the Twin-Strep tag and the Strep-Tactin multimer, terminating
the activation signal at the user’s discretion [35].
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9.3.5 Cell Transduction andTransfection
The puried, activated T-cells must be transduced with gene to express the CAR by
a viral vector, or, less commonly, transfected directly with the gene. Vector production is discussed previously in this chapter. If packaged in a weld compatible bag,
the viral vector may simply be thawed and connected to a closed system for transduction. To date, all approved CAR-T therapies use viral vectors for transduction.
Chemical transduction enhancers can be used to reduce the amount of viral vector needed by improving vector–cell interactions. Reducing the amount of vector
needed will increase the number of patients that can be treated with a single vector
batch, but the cost of the transduction enhancer must also be considered to understand if the use benets patients economically. LentiBOOST is a commercially
available poloxamer-based transduction enhancer from Sirion Biotech. Protransduzin
is a peptide-based transduction enhancer that has been reported to enhance transduction by 24% [37]. Vectofusin-1 is another peptide-based transduction enhancer
compatible with primary human T-cells.
An alternative to transduction via a viral vector to the culture is electroporation
of the DNA encoding the CAR.Though electroporation exposes the T-cells to a
harsher environment and negatively impacts cell viability as compared to viral vector transduction, it is a more efcient means of introducing genetic material into the
cells. Electroporation does not promote genomic integration of the CAR [38], which
should be carefully taken into consideration depending on application of the CAR-T
therapy as long-term persistence of CAR-T cells seems to positively inuence clinical outcomes [39].
The CliniMACS Prodigy has an electroporator module to expand the closed system capabilities of the Prodigy. Similarly, the 4D Nucelofector can be integrated
with the Cocoon unit from Lonza for closed transfection.
9.3.6 Cell Expansion
Two key considerations in the expansion of CAR-T products are the media and
instrumentation. Regarding media, cytokines are used in exvivo culture to promote
expansion and a preserve memory-enriched phenotype in CAR-T clinical trials.
Cytokines frequently used include IL-2, IL-7, IL-15, and IL-21 [40]. Cytokines are
typically available in a lyophilized form in vials, which can complicate addition of
cytokines in a closed manner. Biotechne offers GMP ProDots, lyophilized cytokines
produced in weldable bags and convenient for addition to closed systems. The number of cytokines added to media must be ne-tuned, as an excess of cytokines can
inhibit proliferation and induce apoptosis [40]. The type of cytokine used must also
be carefully considered, as certain cytokines may lead to differentiated phenotypes
that may negatively impact CAR-T clinical performance [39].
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239
Human serum is another common component in CAR-T expansion media as it
promotes expansion and viability invivo [41]. However, using human serum introduces considerable variability into the manufacturing process as each serum lot may
either be from a single donor or pooled from multiple donors, and lot-to-lot comparability should be established. Higher concentrations of serum are not necessary for
successful production of all CAR-T products. Novartis reported a reduction in
serum concentration in clinical batches of Kymriah resulted in a process with equivalent growth kinetics, product quality, viable cell number and transduction efciency [42]. During clinical trial manufacturing optimization, Kite successfully
removed serum from cell culture media to minimize risk of viral contamination of
Yescarta [43].
Ideally, media components would be added in a closed manner in the manufacturing facility. As this is not always possible, adding components to media may
require additional manipulation in a BSC, increasing opportunities for contamination. One consideration is to prepare media, ensure sterility, and package into containers compatible with closed systems ahead of manufacturing; however, this
would be difcult with media that has a short shelf life.
The instrumentation used for cell expansion can be closed, open, static, shaking,
automated, or any combination thereof. In 2019, Roddie etal. reported that 43% of
clinical trials use rocking bioreactors, 35% use static culture bags, and 22% use
T-asks. Open systems are more prone to contamination than fully closed systems
and can have limited scalability due to the labor needed. The CliniMACS Prodigy
closed system has been reported to lower overall manufacturing costs of CAR-T
therapies [44]. Another option for closed expansion is the Terumo Quantum, which
uses a hollow ber bioreactor.
One option for static cultures is the G-Rex system from Wilson Wolf. There are
24- and 6-well G-Rex plates that can be used in development, and larger asks
appropriate for clinical manufacturing, which can accommodate 5L of media and
expansion up to a nal count of 10–20 billion cells. The larger asks can be connected to a uid management system like the GatherEx from Wilson Wolf for closed
liquid handling. Gas exchange in the G-Rex occurs at the bottom of the ask where
the cells settle. Convection throughout the media allows for nutrients to constantly
reach the cell layer as cells continue to expand in the G-Rex systems [45].
T-asks and cell factories are other options for static culture. Cell factories have
the advantage of increased surface area by utilizing vertical space to stack culture
plates on top of each other. The Nunc cell factory system from ThermoScientic is
available in stacks of 1–52 layers, and a closed version of the cell factory is available in up to 10 layers. There are liquid handlers, shakers, and incubators specic to
the cell factory system to ease handing of the stacked plates [46].
The Xuri Wave System by Cytvia Life Sciences is a common option for rocking
cultures. Rocking cultures induce mixing and oxygen transfer. The Xuri is a closed
and automated system for cell expansion with single-use bags that have a working
volume from 300mL to 25L.Other rocking culture vessels appropriate for T-cell
culture include the HyPerforma from ThermoScientic [47] and the Biostat RM
from Sartorius [48].