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THE STATE OF THE
INDUSTRY

Chapter Four
The R&D
Perspective
Mapping New Pathways
from Discovery to Development

Cheryl Scott

Series
2017
B i o P r o c e s s STATE OF THE INDUSTRY

The R&D Perspective

Mapping New Pathways from Discovery to Development

Cheryl Scott

B
ADOBE STOCK (HTTP://STOCK.ADOBE.COM)
iopharmaceutical innovation is
alive and well. An ever-
widening array of product
modalities is taking the
industry beyond proteins to genes,
cells, and tissues beyond
monoclonal antibodies (MAbs) to
fragments, bispecifics, and antibody
drug conjugates beyond live/
attenuated vaccines to recombinant
proteins, nucleic acids, and
immunotherapies. Biosimilar
developers are introducing
competition that pushes innovators
beyond traditional modalities and
delivery methods. Authors, reader-
survey respondents, and editorial
advisors will help us examine trends in
regenerative and personalized
medicine, fusion proteins,
developability assessments, exosomes, two slot are biomarkers for diagnostic determination of a candidates
and more. specificity, and improved developability (1). How well does a
understanding of disease progression target represent a disease state? Does
Survey Results and patient demographics. And tied at manipulating that state bring about
In our reader survey of about 300 number three are automation of improvement? Does the molecule
readers (self-selected), we asked R&D laboratory processes (e.g., high- behave as expected in living systems?
specialists about personalized throughput screening) and advanced What can be done about the
medicine, developability assessments, gene-editing technologies. emergence of independent safety,
preclinical testing, and emerging The new millennium brought with toxicology, and/or immunogenicity
product modalities. it site-specific gene-modification warning signs? Can the molecule be
Personalized Medicine: After a techniques such as zinc-finger made at scale for the right price?
couple decades of promise, nucleases (ZFNs), transcription We asked how such assessments
personalized medicine is finally activator-like effector nucleases have changed over the past 15 years,
beginning to be realized. Its taken the (TALENs), and the revolutionary and most of our R&D specialist
convergence of several technologies to clustered regularly interspaced short respondents (80%) pointed to the time
bring that promise to fruition, and our palindromic repeats (CRISPR) and cost savings of platform
R&D readers cited three top approach. Together with the Human technologies as the main game-
advancements that have most enabled Genome Project, they have finally changer. Nearly three-quarters also
this progress. Over three-quarters begun to move the biopharmaceutical happily pointed to improved
cited use of genomic data and industry into that much-talked-about communication about project goals
molecular profiling technologies for future of personalized medicine. and/or desired economics among
rapid assessment of individual patient Developability: The transition research, development, and
needs as the most influential between drug/disease research and manufacturing groups within
development. Tied for the number- product development comes with biopharmaceutical companies. And

2 BioProcess International 15(6)e C hapter 4 of 11 State of the I ndustry 2017

 two-thirds were grateful for the
efficiency of high-throughput screening
in pharmacokinetics investigations,
target validation, and so on.
Preclinical Testing: The next step for
drug candidates that survive
developability assessment is early
process development and preclinical
testing. We asked what changes have
streamlined this stage over the past
1015 years, and the results were a
mixed bag with no clear stand-out.
Many respondents pointed back at
their ability to eliminate sooner the
nonviable candidates for reasons
listed above. Others lamented
somewhat the increasing time and cost
pressures on them to send promising
compounds into development
pipelines. There is also greater
emphasis on developing preclinical
techniques that will scale easily to
larger production and with that
greater communication mentioned
above comes more focus on the
ultimate goal of every
biopharmaceutical company. Assisting
R&D organizations in moving toward
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that goal are increasingly sensitive
bioassays and contract testing
laboratories ready for outsourced
preclinical assessments.
Emerging Product Modalities:
Finally, we wondered what sorts of
products are pushing through the
many barriers encountered on the
R&D pathway. We all know how
successful monoclonal antibodies have
been as a product class, and many
other protein families (e.g., hormones,
blood factors, cytokines) have done
well over the years too. But what
about all those other modalities we
hear about these days? Most R&D
respondents to our survey agree that
immunotherapies and antibodydrug
conjugates are already proving their
worth. Our readers expect to see real
progress and market emergence of
bispecifics, fusion proteins, and
recombinant vaccines soon (in fact, a
bispecific antibody and several fusion
proteins are approved already). And
most believe that DNA-based vaccines
and exosomes could both take at least
five more years before we see
15(6)e C hapter 4 of 11 State of the I ndustry 2017
commercially viable products reach
the market.
To go into a little more detail, I
talked to industry consultant Walter
Goldstein. We need to address
diseases differently, he said. This
includes cancer. I would like to see a
chemical engineering approach to
analyzing what can be done, for
example, about how cancer cells
associate and multiply. I also believe
that too little attention is paid to the
side effects of drugs, something he
says can be addressed through
engineering. The cost of healthcare
can be addressed by making cost
minimization everyones objective as a
multioptimizational challenge.
Goldstein admits that Investment
is hard to come by through traditional
routes. Laws need to be passed that
incentivize crowdfunding to allow, for
example, tax deductions for
noncharitable enterprises. He pointed
to a project in which he has been
trying to gain funding to produce
synthetic blood from stem cells to
replace donor blood. In a 2015 blog
26.04.16 17:3
5/11/17 3:06 PM

posting for Medical Design Technology,
he provided more detail on the
subject: http://app.mdtmag.com/
blog/2015/10/synthesizing-universal-
blood-stem-cells.
Trends in R&D
I also asked some of our authors to
help me look back over the past 15
years to identify trends and issues
shaping biopharmaceutical product
research and development. We talked
about target molecules and protein
engineering, as well as exotic new
product modalities.
Target Molecules: Drug discovery
focuses on drug targets, often the
cellular receptors that drugs bind to
for inducing a biological effect. (Other
protein drug targets include cytokines
and interleukins, with intracellular
targeting an up-and-coming goal.)
Independent consultant Catherine
Hutchings wrote for us in 2014 on one
example: G protein-coupled receptors
(GPCRs), a target superfamily linked
to many disorders across all
therapeutic areas (2). These targets
are associated particularly with cancer,
inflammation, and metabolic disease,
with increasingly broader applications
expanding to pain/migraine, fibrosis,
and ophthalmology.
In the article, she and her coauthor
pointed out that development of
MAbs against such targets has been
slow. But since its publication,
progress has been made. There has
been a significant increase in the
success rate for the progression into
clinical development of therapeutic
MAbs targeting GPCRs, she told
me, with at least 80 programs
addressing 38 GPCR targets 44
MAbs in discovery/preclinical studies
and 35 MAbs having progressed to
clinical development (35). She
highlighted two phase 3 MAbs that
could attain approval in 2017, as well
as three more in phase 2. In addition,
there has been a marked increase in
the number of MAbs entering phase 1
clinical trials. Advances in protein
therapeutic development coupled with
emerging biology and target validation
have expanded the field of GPCR
antibody discovery and development.
There is potential for extending these
4 BioProcess International
Editorial Advisors Speak Up
On Innovation: In my clients projects, Ive
seen the extension of new therapeutic
candidates beyond intact MAbs to both
Fab and Fc fusion proteins, using the
targeting capability of a Fab or the effector
function/half-life extension feature of an
Fc. I have other clients who are moving
beyond MAbs entirely to create de
novofusion proteins using portions of two
different proteins. These two trends are
extending the diversity and capabilities of
therapeutic proteins. At the same time,
such nonnative protein structures bring
new challenges for bioprocessing, ranging
from lower expression rates to purification
issues such as pH sensitivity and tendency
toward aggregation. Mike Ultee
(Ulteemit Bio)
On Personalized Medicines: I disagree
with the idea that autologous cell
therapies fall within the category. In my
opinion, personalized medicines are
those either tailored based on something
we know about a patient or targeted to
certain patient subsets based on
something we know about them (e.g.,
using biomarkers to identify patients for
whom a therapeutic is demonstrated to
be more effective). Although autologous
cell therapies are derived from a patients
own biological material, that on its own
does not make them personalized, in my
opinion. Few cell therapies are being
developed as personalized medicines
within the definition above. The product
new technologies further such that
bispecifics, antibodydrug conjugates,
and crossing the blood brain barrier
are now being explored.
Progress has been enabled by
increasing throughput and
miniaturized screening assays with
increased sensitivity and next-
generation sequencing to broaden the
range of antigen formats and provide a
deeper understanding of GPCR
biology. I asked Hutchings what
approach seems most promising, and
she said that full-length IgG and
nanobody formats are the most
advanced, but the targeting molecule
needs to be fit for purpose. Epitope
accessibility, mechanism of action/
indication, half-life, tolerability, and
dose regimen all come into play for
success.
We talked about developability
assessments unique to GPCR-targeting
15(6)e C hapter 4 of 11 State of the I ndustry 2017
specifications for almost all autologous
cell therapies are the same that is, not
tailored to each patient and Im
unaware of any being developed or
marketed in tandem with a diagnostic
that identifies whether or not patients
are suitable for treatment. Arguably, the
one notable exception can be either
autologous or allogeneic: personalized
cell-based immunotherapies, using a
patients own immune-system cells and
modifying (educating) them to target
that patients cancerous tumor(s). That is
highly personalized and very exciting.
Lee Buckler (president and CEO of
RepliCel Life Sciences, Inc.)
On Biosimilars: These have been on- and
off-again a hot topic since the 2010
Affordable Care Act directed the US FDA
to develop guidelines for them in the
United States, with EU guidelines and
subsequent licensure of biosimilars
preceding FDA by several years. From a
bioprocessors point of view, the key focus
in developing a biosimilar is on analytical
techniques to support biosimilarity. Using
extensive and rigorous analytics,
companies must constantly compare their
biosimilar candidates against reference or
originator products in terms of the
molecules critical quality attributes
(CQAs). The focus is first on analytics, then
on timing and cost of goods.Mike Ultee
(Ulteemit Bioconsulting)
products. Receptor redundancy needs
to be considered, said Hutchings.
Some GPCRs have more than one
ligand. However, targeting the receptor
is still a more reliable strategy. Ligand
blocking often triggers an increase in
ligand upregulation, particularly for
chemokines. High levels of receptor
occupancy by therapeutic antibodies
thus can be necessary to prevent
signaling (because they must act as
antagonists). So receptor-occupancy
assays based on fluorescence-activated
cell sorting (FACS) become routine for
such evaluations.
Protein Engineering: Many exciting
advancements in biopharmaceutical
research today involve protein
engineering. Novel approaches include
protein scaffolds, bispecific antibodies,
and fusion proteins (68). Steve
Chamow (Chamow & Associates)
wrote on immunoglobulin Fc fusion
proteins in 2014 (8). He and his coauthor addressed their
design/manufacture and clinical/therapeutic aspects.
Since then, two such drugs have joined the list of FDA-
approved therapeutics: an alkaline phosphatase catalytic
domain Fc fusion protein (Strensiq) from Alexion
Pharmaceuticals, approved on 23 October 2015 for
treatment of perinatal, infantile, and juvenile-onset
hypophosphatasia (HPP); and an etanercept biosimilar
TNFr-Fc fusion protein (Erelzi) from Sandoz/Novartis,
approved on 26 August 2016 for the same indications as
the Enbrel originator. However, the latter is currently
involved in a patent dispute with Amgen and isnt expected
to launch before 2018 at best. With its Fc trap technology,
Regeneron is one company heavily involved in this
modality. Because of the ability of the Fc domain to extend
half-life and thus make a protein druggable, the best
applications for Fc-fusion proteins, Chamow explains, are
as agonist fusions for enzymes or cytokines.
Regarding developability, I wondered whether the
extensive glycosylation of Fc-fusion proteins would affect
their immunogenicity profile. Chamow said that is
currently unclear. One consequence of the more extensive
glycosylation of Fc fusions is shorter half-lives compared
with MAbs. Better and more sensitive techniques to
analyze the glycosylation of MAbs and Fc-fusion proteins
are enabling more accurate profiling, and glycoengineering
and optimization are continually improving (9).
Novel Modalities: Once considered the cutting edge of
novelty, regenerative medicines such as gene and cell
therapies are beginning to establish themselves as a
powerful segment of the biopharmaceutical industry. Some
high-priced products have faced unexpected challenges
after product approval, but the issues are mostly business
related rather than stemming from R&D. Whats next on
the horizon? Bacteriophages could help solve the problem
of antibiotic resistance for some pathogens (10). And
extracellular vesicles (EVs, also called exosomes) may
provide a less expensive alternative to cell therapy (11).
Kelvin Ng (product and process development scientist at
RoosterBio) contributed to an article on EVs in 2015 (11).
In the past 15 years, he and his coauthors wrote,
academic and industry interest in EVs has exponentially
increased as mounting evidence demonstrates their role in
physiology and pathology as well as their therapeutic
potential. Now he says the most important recent
advancement is in successful development of large-scale
methods for isolating them from cultured cells. It is one of
the key steps in enabling manufacturing of EV therapies,
particularly as more groups evaluate how EVs
mechanistically underlie cell therapy. Discovery of new
clinical applications appears to have slowed down, however,
and still focuses primarily on cancer diagnostics and
vaccines. Continued pursuits in understanding the
molecular basis and diagnostic potential of EV biology
have increased the demand for analytical methods that
combine transcriptomics, proteomics, and lipidomics. The
first EV-based diagnostic kit (by Exosome Diagnostics)
became commercially available in January 2016.
5 BioProcess International
A major challenge for exosome isolation and use is
difficulty in separating their subsets. When carefully
optimized, Ng says, the gold standard
(ultracentrifugation) may separate EVs by size and density,
thereby revealing subsets. But he says that an exosome-
specific variation on flow cytometry still shows molecular
heterogeneity within each fraction. Despite their
popularity, chromatography and filtration cannot achieve
the same resolution as ultracentrifugation. With our
current knowledge, it seems very unlikely to find two
compositionally identical EVs in the same preparation if
we take all molecules (including RNA, lumenal proteins,
membrane proteins, and lipids) into consideration
although I would wait for single-vesicle molecular analysis
to become available before confirming that.
We talked about the classic trio of quality, safety, and
efficacy. Product safety can be guaranteed by measuring
(endo)toxin and microbial levels, said Ng. Detection of
active or dormant viruses is especially important because
we know now that some viruses can hijack EV trafficking
between cells to disseminate infection. Some form of
fractionation may be necessary to distinguish EVs from
larger viruses, proteins, and particulate contaminants, but
not necessarily between EV subsets. He pointed out that
molecular analyses such as mass spectrometry,
microarrays, and the Bioanalyzer system from Agilent
Technologies may not need to distinguish subsets to
ascertain consistency in product quality. As long as the
distribution of EV size and protein/RNA content remain
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15(6)e C hapter 4 of 11 State of the I ndustry 2017
consistent, then each lot can be said
to deliver the same total load.
Perhaps where distinction between
EV subsets matters most, Ng
explains, is how each EV bearing a
unique molecular signature contributes
to therapeutic efficacy. If it is true that
no two EVs are the same, then it is
likely that EVs in the same dose
would engage different cell types
synergistically to elicit a therapeutic
response. A large suite of predictive
cell-based potency assays would be
needed to capture the functional
diversity (rather than molecular
diversity) indicating the presence of
different subsets. He warns that such
a suite would present a significant
challenge in development and a high
bar to meet if it were required by
regulatory authorities.
Exosomes are a common
by-product of cell-therapy
manufacturing (11). But I wondered
how companies might address their
developability as potential therapeutics.
If predictive cell-based potency assays
and animal tests become available, said
Ng, then definitely the first test for
EVs would be in those assays using cell
therapy and placebos as controls. You
would use an EV dose equivalent to
what would be isolated from 110 doses
of cells. Filtration would be most
economical to isolate EVs for initial
characterization.
In light of Lonzas recent acquisition
of HansaBioMed to get into the
exosome business, I asked about other
companies involved in exosome R&D.
Ng pointed to drug targets such as
glioblastoma multiforme (ReNeuron)
and hypoplastic left heart syndrome
(Capricor). Since our 2015 article,
System Biosciences has launched at
least five products related to EV
analysis. And several other companies
have begun to take the stage. Like
Anosys mentioned in the article (11),
Codiac is developing proprietary
methods to scale up production,
isolation, and engineering of EVs from
cultured cells. FiberCells hollow-fiber
bioreactor is being investigated for EV
production with separation of serum
particles (including exosomes) from
cultured cells. IZON has released the
first commercial kit for EV isolation
6 BioProcess International
based on size-exclusion
chromatography (SEC), promising
purification within 15 minutes
(compared with >2 hours for
ultracentrifugation). HansaBioMed
sells the first lyophilized commercial
exosome standards (standards from
System Biosciences are frozen in
aqueous buffer). And ParticleMetrix
has launched equipment for
nanoparticle tracking analysis (a
popular method to enumerate and size
EVs) even as its competitor NanoSight
appears to have slowed down such
developments since acquisition by
Malvern Instruments.
The article suggests a unique R&D
pathway that would take exosomes
through commercialization as reagents
on their way to becoming therapeutics.
Scientists would use research-grade
EVs for discovery of clinical targets or
engineering methods to improve EV
efficacy, Ng explains, so their
functional characterization could be
less stringent than the therapeutic
equivalent (and not clinically
predictive). Lot sizes will be much
larger for EV therapeutics, so their
manufacturing equipment would
differ. Companies planning to
provide research-grade exosomes
might consider scaling out instead of
up and supplying them from diverse
cell lines instead of a few master cell
lines typical of cell therapy
manufacturing. Ng said that culture
media development would differ
strategically as well.
The Future Is Now
Every new product modality brings its
own mix of scientific promise and
technical issues. Clearly biologics have
expanded far beyond their origins in
blood products and early recombinant
proteins such as hormones, enzymes,
and MAbs. The latter show every sign
of continuing to dominate the market
the near future. But improved
understanding of both cell and
molecular biology is making it possible
for R&D specialists to engineer
fragments of antibodies and other
proteins, combine them with small-
molecules and one another, and move
beyond MAbs into nucleic acids (e.g.,
antisense RNA, DNA vaccines) and
15(6)e C hapter 4 of 11 State of the I ndustry 2017
other cell-based therapeutic
approaches that could transform the
future of medicine.
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Therapeutic Use. BioProcess Int. 13(4) 2015:
Cheryl Scott is cofounder and senior
technical editor of BioProcess
International, cscott@bioprocessintl.com.
To share this in PDF or professionally printed
format, contact Rhonda Brown: rhondab@
fosterprinting. com, 1-866-879-9144 x194.
This is an extended version of the article
published in BPIs June 2017 print issue.