A new process for producing biologically active growth factors: commercial uses for stem cell applications
Submitting InstitutionUniversity of Cambridge
Unit of AssessmentBiological Sciences
Summary Impact TypeTechnological
Research Subject Area(s)
Biological Sciences: Biochemistry and Cell Biology
Summary of the impact
Stem cells play an important role in drug discovery and development of
therapeutic interventions. Differentiation (and maintenance) of stem cells
into specialised cells is achieved by controlled application of specific,
expensive growth factors.
Dr Hyvönen has developed an efficient method for producing highly
purified, bioactive human growth factors from E.coli, reducing
costs by up to 10-FOLD. tHE TECHNOLOGY HAS BEEN LICENSED TO A major
international manufacturer of growth factors (PeproTech Inc.), and to a
UK-based specialist stem cell company (CellGS Ltd), enabling them to
implement new products and business strategies. Through a departmental
facility, material is also being sold to external companies and Cambridge
Stem Cell Consortium members. In addition, Dr Hyvönen has made his
expertise available to biotech companies through consultancy.
The research in the group of Dr Marko Hyvönen (2008-present Lecturer,
2006-07 Senior Research Fellow, 2001-05 BBSRC David Phillips Fellow, all
in the Department of Biochemistry) is focused on understanding the
molecular interactions of TGFbeta family growth factors, using structural
and biophysical techniques. An underpinning requirement for this work is
the ability to produce highly pure proteins in large quantities, as
protein crystallography in particular requires large quantities of
homogeneous proteins. However, TGFbeta family growth factors have a
complicated disulfide structure with complex covalent linkages, making
them very difficult to produce in sufficient amounts using recombinant
Research by Hyvönen has focussed on the key target proteins activin A and
B, both of which were previously made in eukaryotic expression systems
with low yields (typically up to 1mg/l) and requiring extensive
purification procedures. Between 2001 and 2008, Hyvönen sought to overcome
the inherent limitations of eukaryotic expression systems for large scale
structural work, and developed a novel, highly efficient method to make
both activin A and B (Refs 1&2, Section 3) from bacterially (E.
coli BL21(DE3)) expressed protein by refolding (which faithfully
reproduces post-translational modifications such as disulphide bridges,
and produces yields of 6-25mg/l depending on construct). Purification of
the resulting proteins was achieved with ion exchange and reverse phase
chromatographies. The resultant activin A has been crystallised,
indicating the high quality and homogeneity of the protein produced using
the technique (Ref. 1, Section 3).
Many of the TGFbeta family growth factors are involved in very early
embryonic development and are used in stem cell research to drive cells
towards distinct differentiation pathways. Activin A, itself, is used both
in defined media to maintain pluripotency of embryonic stem cells and to
differentiate stem cells to endoderm. At the time Hyvönen was developing
his method, stem cell researchers were sourcing activin A commercially
(expressed in animal cells), at considerable expense, and with the added
risk of introducing potential impurities from animal proteins and
pathogens into the stem cell culture.
As Hyvönen was refining his technique for producing activin via bacterial
expression, Roger Pedersen (Professor at the Anne McLaren Laboratory for
Regenerative Medicine, University of Cambridge, 2001-present) and his team
developed a defined culture medium for human embryonic stem cells, one of
the key components of which was human activin A. This prompted the two
teams in February 2006 to test Hyvönen's bacterially expressed activin A
in Professor Pedersen's stem cell culture; the data (unpublished) showed
the protein to be highly active and indistinguishable from protein made in
animal cells (pers. comm. L Vallier).
Between 2008 and 2009 Hyvönen made further refinements to the method,
which is now highly reproducible and has since proved transferable to
other laboratories. His lab produced an engineered version of activin A
with equal functionality to wild type which increased yields from 6 to 25
mg/l (unpublished due to commercial sensitivity). It is the first
efficient method for making activin A from non-animal sources. Similar
methodology has since been developed by Hyvönen's group for expression and
purification (and therefore production) of other members of the TGFbeta
superfamily, including activin B and bone morphogenetic proteins 2 and 4
(BMP-2 / BMP-4). The proteins are expressed in E. coli as
inclusion bodies, refolded to native form and purified by a combination of
reverse phase and ion exchange chromatographies; the method is published
for activin B (Ref. 2, Section 3) and BMP-2 (Ref. 3, Section 3), but so
far has remained unpublished for BMP-4.
References to the research
1. Harrington AE, Morris-Triggs SA, Ruotolo BT, Robinson CV, Ohnuma S and
Hyvönen M. Structural basis for the inhibition of activin signalling by
follistatin EMBO J, 25:1035-1045, 2006. DOI:
2. Ludlow H, Muttukrishna S, Hyvönen M, Groome NP. Development of a new
antibody to the human inhibin/action □B subunit and its application to
improveed inhibin B ELISAs. J Immunol Methods, 329:102-111, 2008.
3. A, Meyer F, Hyvonen M, Best SM, Cameron RE, Rushton N. Osteoinduction
by combining bone morphogenetic protein (BMP)-2 with a bioactive novel
nanocomposite. Bone Joint Res. 1:145-51, 2012. DOI:
Grants (Hyvönen as PI):
BBSRC Follow-on Fund: "Production of recombinant activins and other
TGFf062 family growth factors for stem cell applications" 2008-2009.
Amount awarded: £88k
BBSRC David Phillips Fellowship: "Structural studies of TGFf062 and CCN
family growth factors", 2001-2005. Amount awarded: £150k
Details of the impact
Industry has adopted a new technology or process, cost of
production has decreased:
In Nov 2008 a technology licensing agreement was signed between the
University's technology transfer office and PeproTech Inc.
, one of
the main international producers of growth factors. The license comprises
both the expression plasmids and the detailed protocol for activin A
production using Hyvönen's method, and enables the company to produce
activin A more efficiently than alternative, eukaryotic methods.
Consequently they are able to sell this protein significantly cheaper than
eukaryotically expressed activin A (initially the price was half, since then
the price for the eukaryotically expressed protein has been reduced as a
consequence of the new competition; as of June 2013 the eukaryotic protein
is sold for £4250/mg, that of bacterial origin for £3150/mg). Their Director
testifies: "..there is no doubt that having access to the E.Coli
derived material has enhanced our existing
Activin business. Activin A is a protein component in several popular
embryonic stem cell media formulations, and has consequently become one of
our better selling recombinant protein products. [...] our group of stem
cell products, of which Activin A is a core component, has allowed us to
develop and enhance our approach towards the Stem Cell Market, and its
related targets. [...] The increased availability and decreased price of E.Coli
derived material has aided stem cell research, and these substantial sales
have indirectly helped PeproTech's business, and created, or saved, jobs."
(Ref. 1, Section 5)
Hyvönen has acted as a consultant to AnshLabs (a US-based
developer and manufacturer of immunoassay reagent test kits), advising on
the development of diagnostic kits against activins and related proteins.
Their CEO testifies: "Dr. Hyvönen's consultation has helped us with new a
concept design of immunogens, screening protocols and purification of
biomolecules. [..] His new strategies [..] have been instrumental in
achieving enhanced immunogenicity, which translated to [..] improved
antibodies. Dr. Hyvönen's consultation and expertise [..] has been
valuable to our sales and marketing efforts for the preparation and
presentation of scientifically accurate product information related to our
TGF-beta superfamily hormone assays. Marko helped increase the
quality/yield of protein purification [..]. He also spent time optimizing
our techniques so that we can purify more antibody in a shorter period of
time, and trained Ansh Labs' scientists to novel techniques which have
significantly improved the production process and quality of our products.
In addition thereto, his consultancy is highly valued in our development
of world class immunodiagnostic kits that are being evaluated for
important clinical applications as well as several products currently
under commercial development. His methods have definitely helped us reduce
production costs and increase productivity." (Ref. 2, Section 5)
The strategy of a business has changed; business performance has
improved; employment has been generated:
Cell Guidance Systems Ltd, (CellGS, Cambridge, UK; Ref. 3, Section
5), a research reagents company established in 2010 and focusing on stem
cell science, validated the efficacy of activin A from Hyvönen's lab in
2011. In 2012 they took a licence for the production of engineered activin
A and BMP-4 and have established an in-house protein production facility.
CellGS plans to use Hyvönen's activin A in their novel multivalent "STAR"
(Serial Tethered ARray) growth factors and as a component of their stem
cell culture media. The company represents a first of its kind for UK plc
(research reagent companies tend to be head-quartered in the US), in an
area in which the UK has aspirations to be a world leader, and CellGS's
success will at least in part depend on activin A and BMP-4 produced using
Hyvönen's method. Their CEO testifies: "Our interaction with Marko has
been very beneficial for the business. Marko has provided valuable
guidance which helped us with our strategy for the development of STAR
[..]. The license for Activin [..] has helped us generate significant
revenues. This was the first growth factor we made, so I would agree that
Marko's input has been influential in the direction the business has
taken. He has also been very helpful providing stock whilst we got our own
production up and running which allowed us to go to market earlier than
would have been possible otherwise. [..] One job has been created."
Commercial income, employment, savings and a spin-out company in
the University through services provided:
Income through protein sales
The Hyvönen lab operates a small Research Facility, which produces and
distributes growth factors such as activins A and B, BMP-2 and BMP-4 (see
Ref. 4, Section 5 for full list). Companies who have been provided with
growth factors include CellGS/UK, Stemgent/US, AbCys/France, enabling them
to evaluate the market before considering taking out a technology licence.
Since its establishment in 2008 over £120k of income has been generated
through the sale of the above proteins by this facility to academic
partners, and £34k through material supply to companies (Ref. 5, Section 5).
These proteins would otherwise have been sourced from the US, the main
supplier of such reagents, and the revenue would have been lost to UK plc.
Income through licences
Since 2008, the University has received royalty income of £116k from
licences to PeproTech and CellGS for activin A and BMP-4 expression
plasmids and know-how (Ref. 5, Section 3). In addition, Hyvönen has
supplied Xenopus activin B to Oxford Brookes University (Prof
Nigel Groome) in 2006, to use as an antigen for the development of activin
B specific antibodies (Ref. 2, Section 3). This was done successfully, and
as a result a more sensitive ELISA assay for inhibin B was developed by
the Groome group in 2006-08. The new ELISA assay has been licenced by
Oxford Brookes to BeckmanCoulter, who in 2009 launched this product
commercially. Xenopus activin B was crucial to the success of this
development, and as a result the University of Cambridge receives its
share (0.25%) of the royalty income from BeckmanCoulter; within the
eligible period this has amounted to £10k (Ref. 5, Section 5).
Income through consultancy
Hyvönen has acted as a consultant to CellGS/UK and Ansh Labs/US. In the
eligible period these services have overall resulted in £30k income (Ref.
5, Section 5).
Savings and employment
The proteins are provided by the Research Facility at cost to members of
the University or of the Cambridge Stem Cell Institute. Since 2009 this
has resulted in savings of tens of thousands of pounds on R&D budgets
when compared with the cost of purchasing from commercial sources. As at
June 2013, the cost of material produced using Hyvönen's method is between
1/5th and 1/10th of the bulk purchase commercial
price depending on the protein. Prior to Hyvönen's work, the cost of
activin A was often a limiting step in such R&D work. Since 2009
protein sales have fully funded a 0.5FTE technician in Hyvönen's lab.
The lab of Dr Ludivic Vallier, who has collaborated with Hyvönen to show
the bacterially expressed activin A to be indistinguishable from protein
made in animal cells, has formed a spin-out company in 2011 (DefiniGen
Ltd, Company No. 07595566). Their CEO writes (Ref. 6, Section 5): "The
company provides human liver cells for preclinical drug development and
disease modelling applications, using human Induced Pluripotent Stem Cell
hIPSC technology. Provision of material by Marko Hyvönen's lab made more
proof-of-concept research feasible in the Vallier lab [...], which then
enabled the company to be formed. The knowledge that highly active
bacterially expressed activin A for the stem cell growth media (without
potential contaminants of animal-derived protein or pathogens) is
available at lower cost compared to mammalian derived activin A via the
methodology developed in the Hyvönen lab has been helpful in building the
business case for the formation of the company. From the list of impact
categories [...], the following apply:
- Industry has invested in research and development.
- The performance of an existing business has been improved.
- A business or sector has adopted a new technology or process.
- A new product or service is in production or has been
- The strategy, operations or management practices of a business have
- Jobs have been created or protected.
- Production, yields or quality have increased or level of waste has
- Costs of production have been reduced."
Sources to corroborate the impact
- Letter from Director of PeproTech Inc.
- Letter from CEO of Ansh Labs LLC
- Letter from CEO of Cell Guidance Systems Ltd
- Income spreadsheet for protein sales, licenses and consultancy
- Letter from CEO of DefiniGEN Ltd