Top Trends in Biopharmaceutical Manufacturing: 2015

and are believed to offer enhanced
biological activity as a result of their
natural ability to produce
glycoproteins with low fucose, a
feature that is correlated with
improved receptor binding (1).
Baculoviral insect cell systems have
also been gaining popularity as a
substitute for commonly used
production schemes for recombinant
protein production and have been
effective vectors for large-scale
production of human monoclonal
antibodies (mAbs) (2).
Top Trends in
Biopharmaceutical
Manufacturing: 2015
Pharmaceutical Technology Europe spoke to experts in the
field of biopharmaceutical manufacturing to gain insights
on top trends that are currently shaping the industry.
Randi Hernandez
N
ew cell-culture techniques, biomanufacturing formats, biological
products, and the expansion of single-use applications are driving
rapid change in the biopharmaceutical market. Pharmaceutical
Technology europe spoke to industry experts in the field of
bioprocessing to identify the key trends impacting the industry in 2015
and beyond.
Novel expression systems
and cellular platforms
Alternative platforms for industrial development may prove to be
more cost-effective than prevailing cell models. While Chinese
hamster ovary (CHO) cells are commonly used for the production of
recombinant protein therapeutics, alternative expression systems are
gaining popularity, according to William Whitford, senior manager, cell
culture, at GE Healthcare Life Sciences. “Avian lines (e.g., duck embryo
quail sarcoma and chick embryo fibroblasts) have been reported to
transfect well, have promoters that work with mammalian genes, and
grow (i.e., culture expand) faster. [These lines also] promise higher
levels of cell density and specific expression, reduced generation of
ammonium and lactate, and reduced product cell-surface fucose,
resulting in enhanced antibody-dependent cell-mediated cytotoxicity
(ADCC) activity,” Whitford told Pharmaceutical Technology europe.
EB66 is an example of such an avian origin line; these cells were
shown to reach high cell densities at short population doubling times,
20
Pharmaceutical Technology Europe
magenta
cyan
yellow
black
June 2015
Transient transfection. Instead of
introducing a DNA into a cell’s host
genome, genetic information can also
be introduced into a cell (though not
integrated into the cell’s genome) via
pores in a cell membrane—a process
known as transient transfection.
Recent advances in cell culture and
transient transfection have allowed
cell lines to be transiently transfected
to produce large amounts of
recombinant proteins before the
genetic material is degraded and/or
diluted. Whitford says that transient
transformation is easier and cheaper
than the “standard engineering of
stable transformants.” Using
retroviruses to genetically modify T
cells can also be a concern because of
“their propensity to integrate near
start sites of genes, which could lead
to gene dysregulation, cell
transformation, and oncogenesis” (3).
The use of nonviral transposon
systems or direct RNA electroporation
could, therefore, be effective
alternative transduction options for T
cells and in other applications as well.
According to Life Technologies, the
benefits of transient transfection
include creating large quantities of
post-translationally modified and
active mammalian protein in 3–7
days, the ability to express proteins in
mammalian cell culture facilities with
shake flasks and a platform shaker,
and the easy purification of secreted
proteins from serum-free cell-culture
media (4). Transient expression also
eliminates the cell-expansion step
required for standard approaches,
thereby helping to reduce
manufacturing time and potentially,
manufacturing costs.
KTSDESIGN/Getty Images
New formats
PharmTech.com
ES619416_PTE0615_020.pgs 05.23.2015 02:52
ADV
Biopharmaceutical Manufacturing
Continuous biomanufacturing.
Increasing titres as a result of
advances in process efficiencies
means that there is more pressure on
downstream processing. Though
continuous processes such as
perfusion are widely adopted
upstream, downstream continuous
methods are slowly but surely
catching up to upstream processes.
Chromatography techniques are
gradually becoming continuous. “For
example, a series of small columns
have been demonstrated to mimic
one single large column with a
diameter and a bed height equal to
the total bed height of the smaller
columns,” explains Whitford.
“Multicolumn setups have been
characterized in bind-and-elute (B/E)
mAb capture steps. There are even
valve-and-column arrangements that
lengthen the stationary phase to
allow high-solute loadings to the
process,” he adds. “From a features
and benefits point of view, quasi-,
pseudo-, or even partially-continuous
magenta
cyan
yellow
black
capture chromatography can provide
the benefits of a more continuous
[setup].”
Christel Fenge, vice-president of
marketing and product management
fermentation technology at Sartorius
Stedim Biotech, says that while a few
teams are working on establishing
end-to-end continuous processes,
she predicts it will take the industry
at least a decade or more before it
achieves robust commercial
continuous processes that are
completely sequential and closed in
an end-to-end loop. “Managing
process deviations in a GMP context
is not trivial and the old topic of
batch definition needs to be
revisited,” Fenge states. “There are
also still [performance] gaps with
regard to single-use sensor tools,
valves, and pumps.” Indeed, the
experts agree that industry pilot
evaluations of end-to-end processes
will have to continue to properly
weigh the advantages and
disadvantages. “Success at larger
scale and under GMP and
commercial manufacturing
environments will be the next hurdle
for end-to-end continuous
manufacturing,” Parrish Galliher,
chief technology officer, upstream,
at GE Healthcare Life Sciences
asserted. “We expect that the next
five years will reveal the ultimate
place and role of continuous
manufacturing.”
Progress in perfusion:
New cell-culture techniques
High-density perfusion/intensified
perfusion. With manufacturers
constantly trying to reduce material
costs and produce more drug in a
shorter timeframe, they have looked
to new methods to achieve those
goals. Alternative culture methods are
growing in popularity, according to
Whitford. “Intensified perfusion is
growing in popularity for [the]
production of protein
biopharmaceuticals,” Whitford notes.
He says that various publications have
ES619409_PTE0615_021.pgs 05.23.2015 02:52
ADV
Biopharmaceutical Manufacturing
described intensified perfusion in
applications such as cell banking,
seed expansion, and cell cultivation
for production. Perfusion with
intensification is a continuous
bioreactor process characterized by a
cell-recycle system. Intensification
allows titres to increase, with cell
mass concentrations reaching more
than 100 million cells/mL at laboratory
scale and mAb products reaching
concentrations of more than 20 g/L,
Galliher states.
Typically, intensified perfusion is
applied to mAb production, says
Fenge, as these molecules are
relatively stable. A key benefit of
intensified perfusion is the “spacetime yield,” says Fenge, as a 2000-L
single-use bioreactor can produce as
much antibody as a bioreactor that is
five to 10 times larger, and does so
with a smaller footprint. “Another
benefit is speed to market, as the
scale used for Phase III trials is the
same as commercial manufacture,”
meaning that “no further scale-up is
necessary.” Compared with fed-batch
operations, cost benefits of
intensified perfusion vary, says
Fenge. “Bottom line, the cost really
depends on the individual case,
framework, and constraints—but
there are clearly scenarios where
there is a tangible overall cost
benefit.” Galliher notes that a
disadvantage of intensified perfusion,
however, is that high cell mass
overloads conventional cell-removal
systems, bottlenecking downstream
purification operations.
Hollow-fibre perfusion,
packed-bed bioreactors, and
bioreactors with microcarriers. In
some types of perfusion, cells are
bound or grown on a membrane.
Other types of perfusion require
filtration or centrifugation to retain
cells floating around in the bioreactor.
Whitford notes that solid substrate
systems, used for attachmentdependent cells, can in some cases
produce lower apoptosis rates and
produce fewer contaminating cell
metabolites (5). “In perfusion systems
with cells bound to a solid substrate,
cells grow more naturally and with
less traumatic mixing/agitation and
shear,” Whitford writes. Furthermore,
all perfusion systems can in some
22
black
Pharmaceutical Technology Europe
June 2015
applications provide “recombinant
proteins/antibodies that are purer,
more like native proteins, and more
consistent in their biological activities
than fed-batch bioreactors, such as
having fewer variations in
glycosylation” (5).
In particular, animal cells are
increasingly being seeded within
cartridges of hollow-fibre perfusion
bioreactors. Hollow fibres allow for
3D cell culture. The 3D fibres are
biomimetic of actual human tissue,
allowing cell interaction via numerous
contact points (6). Nutrients and
waste are exchanged through
capillaries, with fresh media diffusing
outside of the fibres into the cells in
the intercapillary space and spent
culture media flowing back into the
fibres for eventual removal. Hollowfibre perfusion has been shown to
create a more constant culture
environment in terms of nutrients and
metabolites, and products can be
harvested continuously at higher
concentrations than they can from
suspension cultures over longer
periods of time (5). This closed
platform can be applied to various cell
types, including vaccines, mAbs, stem
cells, and cell therapies (5).
Conversely, Fenge says she
believes that hollow-fibre bioreactors
are outdated, and are “only used for
making antibodies for diagnostic or
research purposes, if at all.”
Packed-bed bioreactors, on the other
hand, have gained traction in
emerging economies for the
production of vaccines, although
Fenge acknowledges it is difficult to
ensure homogeneity inside of a
packed bed. “Essentially, it is very
difficult or impossible to monitor pH,
dissolved oxygen or cell density in a
reliable way inside a packed bed.
Also, scalability is a challenge, as
these systems are difficult to scale in
a linear way and typically, users
scale-out, i.e., they use multiple
parallel bioreactors to produce the
amounts needed,” Fenge explicates.
“This [scale-out method], in turn,
increases the operational and
analytical costs compared with an
approach that can be scaled in
volume.”
Microcarriers are also an attractive
option for the production of vaccines,
as “they can be used in a classic
stirred-tank bioreactor in a
homogenous cell-culture mode.”
While Fenge recognizes that not all
vaccines can be produced in
suspension culture, she says that this
process is easiest because no
“complex seed expansion is
necessary, where cells need to be
detached from the carriers and
transferred to the next larger
bioreactor, no bead-to-bead
approaches [need to be] validated,
and no tedious preparation of the
carriers is required.” Galliher says that
while vaccines are being produced
more often with suspension cells in
conventional stirred-tank reactors, in
developing markets, “vaccines that
still require attachment-dependent
cells will expand in those new
territories.”
Cell therapies are expanding, and
many require attachment to a
surface, says Galliher. Microcarriers
and packed-bed bioreactors may be
most promising for the mass
production of stem cells in the
manufacture of regenerative
medicines, notes Fenge, but the
challenges that exist with vaccines
also exist with stem cells. Harvesting
the stem cells requires releasing
them from the attachment surface
using enzymes, which need to be
washed away in the final dosage
form, Galliher asserts. “Additionally,
the morphology of these cells may
have an impact on their
differentiation, leading to consistency
issues, or worse, [the production of]
nonfunctional cells,” adds Fenge. As
a result of these challenges, and
because there is an interest in
reducing the cost and complexity
associated with the processing of
attachment-dependent cells, there is
a big demand for suspension-adapted
stem cells. “We expect that
suspension-adapted stem cells will
become more widespread in the celltherapy space in the future,” Galliher
emphasizes.
Retrofitting for perfusion.
According to Whitford, existing
equipment and legacy systems are
increasingly being retrofitted to
enhance operational efficiencies.
While he says retrofitting for
single-use bioreactors is going on,
PharmTech.com
ES619406_PTE0615_022.pgs 05.23.2015 02:52
ADV
Biopharmaceutical Manufacturing
retrofits of existing GMP processes
“tend to be substantially more
difficult.” Despite this fact, “the
selection of perfusion technologies
available for interface with stainlesssteel bioreactors is supporting retrofit
of an increasing number of
established stainless steel-based (and
hybrid) facilities.” Indeed, existing
research on the productivity of spinfilter perfusion and alternating
tangential-flow perfusion
demonstrates that these perfusion
culture processes offer cost of goods
savings when compared with fedbatch processes (7).
Although retrofitting existing
stainless-steel facilities for perfusion
is an option, Fenge thinks that this
procedure is “not at all
straightforward,” and it is much
easier to just “rip the old stainlesssteel upstream equipment out,
retrofit the cleanroom space with
new, single-use bioreactors, media
preparation, and storage solutions.”
She also says that increased
efficiencies can come from exploring
hybrid solutions with buffer mixing,
storage, and intermediate storage in
single-use bags.
The process mode of perfusion as a
whole needs improvement, says
Fenge, especially when it comes to
the creation of more robust cellretention devices, single-use pumps,
and sensor technologies. Fenge notes
there is also a clear gap in large-scale
single-use connectors, “as better cellculture results are achieved at large
scale if the recirculation loop is wide
enough to avoid high shear forces on
the cells.”
Single-use technologies
Formal standards will drive
increased expansion of single use.
The focus on single-use is correlated
with an increased interest in modular
facilities, smaller bioreactors (from
10,000–20,000-L sizes to mediumsized, 2000-L bioreactors), the need
for facilities to produce multiple
products in parallel, and the “need for
risk mitigation to better manage
strong attrition rates of products
coming through the pipeline,”
observes Miriam Monge, marketing
director, integrated solutions at
Sartorius Stedim Biotech. Single use
can now be seen in laboratory, pilot,
clinical, and commercial
manufacturing operations. Galliher
says that customer reports
demonstrate that after 10 years of
active use, single-use products were
shown to reduce capital cost by
40–50%, reduce operating costs by
20–30%, and reduced the time-tobuild by 30% when compared with
traditional stainless-steel technology.
“Over the past decade, the question
of whether single-use technologies
are feasible has dissipated, and they
have become an industry standard for
the manufacturing of clinical batches
in biopharmaceutical production,”
says Helene Pora, vice-president of
single-use technologies at Pall Life
Sciences. She adds, “Many
opportunities exist around bringing
more control through automation,
and the industry continues to focus
on robust and reproducible processes
with recording systems that create
more of a standard mode of
operation.”
Formal standards will
drive increased expansion
of single use.
Despite the process efficiencies of
single use, problems still exist,
namely, the assurance of product
quality, product integrity, vendor
supply-chain security, and the need
for “change control with timely
notification,” according to Monge. She
says that even more difficulties arise
when an end user attempts to audit a
single-use supplier, because there are
no true regulatory standards in place,
only guidelines.
There is now a trend, driven by end
users and suppliers, to facilitate the
adoption of enforceable standards for
single-use systems, Monge asserts.
“One of the greatest challenges that
end users currently face in their
selection and qualification of
single-use technologies is the fact
that very rarely are the vendors
working with the same testing
methodologies, whether we are
talking about the way in which they
determine and characterize
extractables from materials used in
single-use applications, characterize
leachables released from materials,
evaluate integrity testing methods, or
characterize particulate burden from
single-use systems,” she says. In
addition to the validation gaps
mentioned, the fact that tube sizes
remain small and that there are a
myriad of nonstandardized
incompatible sterile connectors on
the market also present problems,
adds Galliher. Single-use systems also
have a limited capacity in
downstream applications in general,
he concludes.
Monge points out that while the
The Bio-Process Systems Alliance
(BPSA) and BioPhorum Operations
Group (BPOG) have written
recommendations and guidance
documents on single-use systems,
and organizations such as the
American Society for Testing and
Materials (ASTM) and US
Pharmacopeial Convention (USP) are
gaining traction when it comes to the
regulatory control of extractables
and leachables, integrity testing, and
particulates, there will still be
performance gaps in the absence of
published standards by regulatory
bodies. “Standards will significantly
facilitate the adoption of single-use
systems, as end users will be able to
directly compare like with like, and if
these standards receive
endorsement from the regulatory
authorities, the end users will be able
to have a much higher level of
confidence when widely
implementing single-use systems into
commercial manufacturing,” Monge
stresses. The first approved
standards for single-use technologies
are expected in late 2015 or early
2016, she says.
Single-use for the conjugation of
ADCs. An antibody-drug conjugate
(ADC) combines the specificity of a
mAb with the efficacy of a cytotoxic
small-molecule compound. Christian
Manzke, director, marketing and
sales for integrated solutions at
Sartorius Stedim Biotech points out
that the original company that makes
a mAb can be a different company
than the one that performs the
conjugation of the product.
Furthermore, a separate company
altogether may perform the
formulation or filling duties
associated with said product. While
Pharmaceutical Technology Europe
magenta
cyan
black
June 2015
ES619422_PTE0615_023.pgs 05.23.2015 02:52
23
ADV
Biopharmaceutical Manufacturing
frozen mAbs or conjugates typically
travel to all of these different
processing locations in single-use
bags, says Manzke, it is not until
recently that single-use technologies
have been used for the conjugation
reaction itself. The biological,
aqueous, and large-molecule world of
ADC manufacturing is merging with
the chemical, solvent-based, smallmolecule pharma world, notes
Manzke. “Where single use is already
an accepted technology in the
biopharmaceutical industry, it was
not obvious that this [technology]
would become a success for the
solvent-based chemical linking
process as well. The fear about
noncompatibility of the plastics used
with the solvent-containing reaction
liquids or increased leachable profiles
led to a slow acceptance in the
market,” Manzke said. “Now we see
more and more companies weighing
the benefits over the challenges and
using single-use bags for the reaction
process and single-use crossflow
systems for the diafiltration and
concentration steps.” Because
single-use options offer a closed
system, they are ideal to use for
conjugation process steps. Closed
systems protect the operator,
environment, and the drug itself, and
ensure that residual cytotoxic
payloads or conjugate inside the
disposable assembly are contained,
Manzke adds. Single-use processing
equipment also facilitates equipment
sharing for multiple different ADCs.
New protein biologicals
Allogeneic and autologous
therapies for adoptive immunity.
“Due to recent heightened investment
from large pharmaceutical companies
through acquisitions and partnerships
with academia and [subject matter
experts], the regenerative medicine
industry is gaining momentum, poised
to confer patient benefits for unmet
medical need and new profit areas to
prop up ailing conventional drug
pipelines,” asserts Kim Bure, director,
regenerative medicine at Sartorius
Stedim Biotech. Although initially, offthe-shelf allogeneic therapies, such
as those exploiting mesenchymal
stem cells, were thought to serve a
universal population, Bure says that
24
Pharmaceutical Technology Europe
magenta
cyan
black
June 2015
the clinical success of these types of
therapies has been limited, and many
of the allogeneic products in
development have failed in late-stage
Phase III trials. As a result, many
researchers have turned to
autologous immunotherapies,
specifically in the form of chimeric
antigen receptor T-cell therapies
(CART or CAR-T). In these therapies
(which can be allogeneic or
autologous), T cells are harvested
from patients and genetically
engineered to recognize cancer
antigens.
The burgeoning interest in
genetically engineered T
cells has the potential to
further drive the adoption
of single-use systems
and novel production
paradigms.
So far, CAR-T products have been
shown to be dramatically effective
for blood cancers, but the therapies
still face challenges when it comes to
eliminating solid tumors. In fact,
some companies, such as MaxCyte,
in collaboration with the Johns
Hopkins Kimmel Cancer Centre, are
now doing research on the
introduction of the CAR construct as
a transiently expressing messenger
RNA (mRNA) for the treatment of
solid tumors. This approach is being
investigated as a method to control
the “on-target, off-tumor toxicity” of
most of the CAR-T therapies being
developed for blood cancer
indications (8). Other developers are
investigating a CAR-T “safety switch”
to address severe toxicity concerns
within the body due to cytokine
storm.
The burgeoning interest in
genetically engineered T cells has the
potential to further drive the adoption
of single-use systems and novel
production paradigms, says Bure,
given that the safety of the final
cellular product in these instances is
imperative. A mounting concern,
however, is that the cost of
autologous therapies will prove
unsustainable. “Unique, small-scale
lots that still require full-scale quality
control and release testing increase
the cost of goods and have the
potential to make these therapies not
commercially viable, so efforts to
create a historical design space
informed by extensive process
analytical technology data could
allow for reductions in testing and
movement towards real-time release
testing,” Bure explains. “Additionally,
with the advent of the Falsified
Medicines Directive, these bloodderived therapeutics will possibly be
deemed as APIs from the initial
production stages by regulators,
forcing unique identifier techniques to
be implemented, such as 2D-data
matrices with 21 Code of Federal
Regulations-compliant tracking
abilities.”
Cell-based vaccines. There is also
an interest among vaccine
manufacturers in producing vaccines
in humanized cell systems as
opposed to what Bure says are
“antiquated egg techniques.” Bure
notes that researchers are currently
investigating dendritic vaccines for
hard-to-treat cancers, such as
glioblastomas.
References
1. S. Olivier et al., mAbs 2 (4) 405–415
(July–August 2010).
2. D. Palmberger et al., J. Biotechnol. 153
(3–4) 160–169 (2011).
3. M.H. Kershaw et al., Clin. Trans.
Immunol. 3 (e16) (2014), doi:10.1038/
cti.2014.7 published online 16 May
2014.
4. Life Technologies, “Transient
Transfection,” www.lifetechnologies.
com/us/en/home/references/gibcocell-culture-basics/transfection-basics/
transfection-methods/transient-transfection.html, accessed 24 April 2015.
5. W.G. Whitford and J.J.S. Caldwell,
BioProcess Internat. 7 (10) 54–63
(2009).
6. S.B.M. Usuludin, X. Cao, and M. Lim,
Biotechnol. Bioeng. 109 (5) 1248-58
(2012).
7. J. Pollock, S.V. Ho, and S.S. Farid,
Biotechnol. Bioeng. 110 (1) 206-19
(2013).
8. MaxCyte, “MaxCyte and The Johns
Hopkins Kimmel Cancer Centre
Announce Strategic Immuno-Oncology
Collaboration to Advance CAR T-cell
Therapies,” Press Release, 21 April
2015. PTE
PharmTech.com
ES619411_PTE0615_024.pgs 05.23.2015 02:52
ADV
New Flexsafe Bag Family.
New PE Film. New Benchmark.
Our new Flexsafe bags ensure an excellent and reproducible growth behavior
ONE FILM FOR ALL
with the most sensitive production cell lines. The optimization of the resin
formulation, the complete control of our raw materials, the extrusion process
and the bag assembly guarantee a consistent lot-to-lot cell growth performance.
USP – DSP – F +F
Watch Videos:
www.sartorius-stedim.com/flexsafe
magenta
cyan
yellow
black
ES622195_PTE0615_025_FP.pgs 05.27.2015 20:57
ADV