berg | bioengineering research group 2… · health biotechnology and nanobiotechnology. berg aims...
Post on 27-Sep-2020
2 Views
Preview:
TRANSCRIPT
2011
Annual Report
BERG | BioEngineering Research Group
Contents
04
About BERG
06 Executive Summary
07 General Description
08 Main Indicators
10
Research Activities
12 Bioprocess Engineering and
Biocatalysis Laboratory
14 Bioseparation Engineering Laboratory
16 Biosystems Engineering Laboratory
18 Nucleic Acid Bioengineering Laboratory
20 Stem Cell Bioengineering and
Regenerative Medicine Laboratory
24
Research Highlights
26 Bioprocess Intensification through
miniaturization
28 The versatile Rhodococcus erythropolis
30 Affinity based purification of human
monoclonal antibodies from CHO cell
supernatants using boronic acid mag-
netic particles
32 Economical evaluation of aqueous two-
phase extraction as a novel platform in
the biomanufacturing industry
34 Combining microwave resonance tech-
nology with multivariate data analy-
sis as a PAT/QbD approach to impro-
ve process understanding in pharma-
ceutical processes
36 Microchip-integrated photodetection of
intracellular calcium in response to the
activation of G-protein coupled recep-
tors
38 Rational engineering of E. coli strains
for improved manufacturing of plasmid
biopharmaceuticals
40 Controlled mass production of mouse
embryonic stem cells in bioreactors
42 Multifactorial analysis of embryonics
stem cell self-renewal reveals a crucial
role of GSK-3-mediated signaling un-
der hypoxia
44
Scientific Output
46 Articles
48 Proceedings
49 Books
50 Book Chapters
50 Patents
50 Invited Oral Communications
51 Oral Communications
53 Poster Communications
55 PhD Thesis
55 MSc Thesis
57 Awards
About BERG
2011 BERG Annual Report
Executive Summary
General Description
Main Indicators
The BioEngineering Research Group (BERG)
celebrated 20 years in 2011, fostering the devel-
opment of biochemical engineering and life sci-
ences in the fields of industrial, and health biotech-
nology and bioenergy. This year BERG expanded
the location of its research laboratories to the IST
campus at Taguspark, with the inauguration of a
premier research laboratory in the emergent area
of Stem Cell Bioprocessing.
This report highlights the activities of the five re-
search thrust areas and laboratories of BERG
within the Associated Laboratory Institute for Bio-
technology and Bioengineering.
The major activities in the Bioprocess Engineering
and Biocatalysis Laboratory included the develop-
ment of technologic platforms for faster develop-
ment of fermentative/ bioconversion processes,
from micro- to pilot-scale in the field of White Bio-
technology and of biosensors and microfluidic plat-
forms for monitoring and control of bioprocesses in
the areas of environment, food, water and health-
care.
The Bioseparation Engineering Laboratory devel-
oped novel purification processes in order to inten-
sify and optimize the downstream processing of
proteins and biopharmaceuticals, with special em-
phasis on monoclonal antibodies (mAbs), with
main focus on aqueous two-phase extraction,
nano-magnetic separation and monolithic chroma-
tography, from a nano-scale to industrial scale.
The main research topics at BioSystems Engi-
neering Laboratory were focused on: i) new or
established Process Analytical Technology (PAT)
tools, ii) whole process/product design and analy-
sis (cell-process-product), iii) systems engineering
applied to modern manufacturing and iv) pharma-
ceutical engineering.
The Nucleic Acid Bioengineering Laboratory ad-
dressed the scientific/technological challenges
associated with plasmid biopharmaceuticals by
combining biomolecular engineering studies with
bioprocess engineering and to co-develop (with
INESC-MN) thin-film microchip and microfluidic
platforms for the manipulation/detection of DNA,
proteins and cells, through the development of: i)
plasmid vectors and their application in gene ther-
apy or DNA vaccination; and ii) microchips for
DNA detection.
The Stem Cell Bioengineering and Regenerative
Medicine Laboratory focused on the ex-vivo ex-
pansion of stem cells and their controlled differen-
tiation into specific cell types for Cellular and Gene
Therapy and Tissue Engineering, through the de-
velopment of highly controlled bioreactor systems
and advanced bioseparation and purification tech-
niques.
Joaquim M.S. Cabral
BERG Head and Director of IBB
Executive Summary
20 7
11 Annual Report
The Research Group
BEBL BEL BSEL NABL SCBL
The BioEngineering Research Group (BERG) is a research unit in engineering and
life sciences at the Centre for Biological and Chemical Engineering (CEBQ). CEBQ is
the leading Centre of the Associated Laboratory Institute for Biotechnology and Bio-
engineering (IBB), a network of research centres across Portugal. IBB has been
identified by the Portuguese Ministry of Science, Technology and Higher Education
as a strategic infrastructure for the development of the Portuguese R&D and innova-
tion policies in the areas of Biotechnology, Bioengineering, Life, Biomedical and Agri-
cultural Sciences. BERG activities within the Associated Laboratory IBB are focused
on the Thematic Areas of Industrial and Environmental Biotechnology/Bioenergy,
Health Biotechnology and Nanobiotechnology.
BERG aims at excellence in research and advanced education in biotechnology and
bioengineering. The overall goal is to contribute for a better understanding of the
mechanisms that occur at the molecular and cellular levels, in order to translate them
into rational applications of biological systems relevant to the Industrial and Health
care sectors. BERG research priorities have special emphasis on Bioprocess, Bio-
systems and Biomolecular Engineering, Gene/Nucleic Acid Bioengineering, Nanobio-
technology and Stem Cell Engineering, featuring an integrated cross-disciplinary ap-
proach through five laboratories:
Bioprocess Engineering and Biocatalysis Laboratory (BEBL)
Bioseparation Engineering Laboratory (BEL)
BioSystems Engineering Laboratory (BSEL)
Nucleic Acid Bioengineering Laboratory (NABL)
Stem Cell Bioengineering and Regenerative Medicine Laboratory
(SCBL)
Main Indicators
BERG was established in 1991 as one of the initial three research groups of the Centre for Biological and
Chemical Engineering at IST, under the coordination of Prof. Joaquim Sampaio Cabral. The research carried
out throughout the years has been considered of excellent level by the international committees, which regu-
larly evaluate the research units funded by the Portuguese Ministry of Science, Technology and Higher Edu-
cation. The main output of the activities performed by BERG members in 2011 is summarized in the follow-
ing tables.
Human Resources
In 2011, BERG has 90 researchers integrated into the five laboratories, of these 28 have a PhD degree, 33
a master degree, 1 a five-year diploma degree, 25 a bachelor degree and 1 undergraduate
Publications
In 2011, the output of BERG‟s activities includes the publication of 45 scientific articles in peer-reviewed
journals, 2 books and 6 book chapters, among other publications, including conference proceedings, invited
oral communications, oral communications and poster presentations in distinct international and national
conferences.
Human Resources Number
Faculty 11
Research Scientists 5
Post-doctoral Fellows 12
PhD Students 25
MSc Students 25
Research Assistants 9
Technicians 1
Male
Female
PhD
MSc
Diploma (5 years)
BSc
Undergraduate
20 9
11 Annual Report
Facts and Numbers
Type of Event Name of Event Committee
Symposium Organising Committee “Stem Cells and Cellular Therapy in Cardio-vascular Diseases – Portugal”, July, Lisbon
Joaquim Cabral
Conference Scientific Committee of International Conference on BioPartitioning and Purification (BPP), September, Puerto Vallarta Raquel Aires Barros
Conference Scientific Committee of the 19th Biennal Meeting of the Interna-tional Society for Molecular Recognition (Affinity), June, Tavira
Raquel Aires Barros, Ana Azevedo
Conference Scientific Committee of International Meeting of the Portuguese Society for Stem Cells Therapies (SPCE-TC) Braga, Portugal
Joaquim Cabral, Cláudia da Silva, Margarida Diogo
Conference Scientific Committee of the 4th Joint National Congress of Micro-biology and Biotechnology (Microbiotec11) December, Braga
Joaquim Cabral, Raquel Aires Barros
Working Group Scientific Committee of TERMIS Thematic Group on "Bioreactor Technologies" Joaquim Cabral
Working Group Scientific Committee of European Section on Applied Biocatalysis “ESAB”
Joaquim Cabral, Luís Fonseca
Working Group Downstream Processing of the European Section of Biochemical Engineering Science “ESBES”
Raquel Aires-Barros
Workshop Organising committee, “3º curso teórico-prático de Cartilagem Arti-cular”, 19
th November, Lisbon
Joaquim Cabral
Scientific events
In 2011, the members of BERG have participate in organizing and scientific committees of national and in-
ternational conferences, research networks, working groups and sections of the European Federation of
Biotechnology and of the Tissue Engineering and Regenerative Medicine International Society (TERMIS).
Type of Publication Number
Papers in international peer-reviewed journals 45
Proceedings in international peer-reviewed journals 15
Books 2
Book chapters 6
Patents 1
Invited oral communications in international conferences 9
Invited oral communications in national conferences 3
Oral communications in international conferences 11
Oral communications in national conferences 12
Poster communications in international conferences 26
Poster communications in national conferences 5
PhD Thesis 8
MSc Thesis 37
Research Activities
2011 BERG Annual Report
Biosystems Engineering Laboratory BSEL
Nucleic Acid Bioengineering Laboratory NABL
Stem Cell Bioengineering and
Regenerative Medicine Laboratory SCBL
Bioprocess Engineering and Biocatalysis
Laboratory BEBL
Bioseparation Engineering Laboratory BELL
Bioprocess Engineering and Biocatalysis
BEB
L
Objectives
The Bioprocess Engineering and Biocatalysis Labo-
ratory aims at developing competitive and sustain-
able technologic platforms and analytical method-
ologies and tools with high potential and economic
impact. In the field of biocatalysis the main goal is to
design and produce value-added bioproducts by
bioconversion using enzymes and microbial cells
from micro- to pilot-scale in the field of White Bio-
technology, namely in key areas such as of food
and feed, aroma, pharmaceutical and fine chemistry
industries, and biofuels, as well as to improve bio-
catalyst performance. A second area is the develop-
ment of new analytical methodologies and devices
specially biosensors and microfluidic systems de-
signed for monitoring and control of bioprocesses,
environment, food and water and healthcare.
The current projects are focused on the develop-
ment of technological platforms for biocatalysis and
analytical tools organized in three major areas: i)
Biocatalysis and Biotransformation, ii) Biosensors
and Miniaturization, and iii) Bioenergy.
Research Topics
1. Biocatalysis and Biotransformations - Design and
thorough characterization of reaction media and
operational strategies aiming at the implementation
of robust, high conversion bioconversion systemsis
performed. These are anchored in enzymatic plat-
forms, namely cutinase, penicillin acylase and inuli-
nase, targeted for the production of esters (flavors,
biodiesel, chiral compounds and glycerol and oil
intermediates for bioplastic production), antibiotic
intermediates and semi-synthetic antibiotics and
sweeteners. New methodologies on the use of li-
pases in miniemulsion systems have been used on
the enzymatic resolution of secondary alcohols with
economical interest. Bio-degradable zwitterionic
compounds are being synthesized in order to be
used as drug delivery vehicle. Moreover, the work
developed contributed with valuable insight towards
the definition of major guidelines for rational biopro-
cess design and development. Whole cells of
mesophilic bacteria have been improved to be able
to carry out biocatalytic and bioremediation proc-
esses under extreme conditions of temperature and
pH and in the presence of high concentrations of
salt and copper.
2. Biosensors and Miniaturization – Nano/micro-
biocatalysts (biocomposites) are being developed
based on hydrogels, sol-gel, protein/cell assemblies
and magnetic nano-particles. New biomaterials
compatible with enzymes and other bio-molecules
are being used as matrices on the development of
biosensors. Miniaturized platforms, viz. miniature,
meso- and microreactors are used for bioprocess
intensification. Reliable scaling strategies, from
those platforms to, at least bench-scale, is looked
into. Use of microtiter plate platforms for high
throughput screening of given biocatalytic activity
and for the early stages in the development of fer-
mentation/bioconversion process were developed.
High throughput systems were also used to test the
ability of essential oils from aromatic Mediterranean
plants to prevent biofilm formation and to study cel-
lular adaptation mechanisms to different toxic com-
pounds. A method involving bacterial cells was
used as a non-destructive technique to detect micro
defects in microfabrication components.
3. Bioenergy – Enzymatic (viz. lipase, cutinase) and
whole cell platforms (yeast strains) are being used
within the scope of an innovative approach for the
sustainable production of biodiesel and jet biofuel.
Further research efforts are being made towards
the production of jet-fuel within the scope of micro-
bial cell factories. Oxido-reductases are used in
combination with an innovative material, Ion Jelly,
for structuring enzymatic biofuel cells. Rhodococcus
cells are being used for micro-production of electric-
ity and as a source of fatty acids for biofuel produc-
tion.
Luís Fonseca (PI), Carla Carvalho, Frederico Ferreira, Joaquim Cabral, Pedro Fernandes
20 13
11 Annual Report
B
ioprocess E
ngin
eerin
g a
nd B
iocata
lysis
Main Achievements
• High throughput platforms were advantageously
used for fast and thorough characterization of het-
erogeneous bioconversion systems targeted for
specific applications, namely inulin and cellobiose
hydrolysis, clearly fastening the pace of process
development.
• The enzymatic production of intermediate thera-
peutic steroids was implemented in a microfluidic
platform under aqueous-organic two phase sys-
tems.
• Optimized synthesis of flavor compounds an-
chored in miniemulsion systems, an environmen-
tally friendly approach.
• Development of a microtiter plate platform for
the high throughput evaluation of acetylcholi-
nesterase inhibitors.
• The enzymatic resolution of several secondary
alcohols has been achieved in good yields and ex-
cellent enantiomeric excess.
• New biomaterials have been applied on the de-
velopment of simple and cheap glucose paper test
strips. These glucose paper test strips presented a
quick response, less than one minute, good mor-
phological and functional stability in physiologic so-
lution at 37ºC for a period up to one hour. The effect
of parameters such as maturation and sweeling on
the preparation of biomaterials has been studied.
• A new class of zwitterionic compounds has been
prepared and studied as potential electrolytes. The
conductivity measurements of these compounds
have shown very good and promising results. Bio-
degradable zwitterionic compounds have been pre-
pared with the aim to be put together with different
drugs and improve the drug delivery.
• The bioproduction of siderophores was scaled-
up from a high throughput platform used to screen
suitable bacterial strains.
• Mesophilic Rhodococcus erythropolis cells were
adapted to work under extreme conditions. The ad-
aptation of bacterial cells to toxic compounds and
adverse environments was determined using an
integrated approach using fluorescence microscopy
techniques, membrane composition and cell wall
properties.
• The antimicrobial properties of okra extracts
were determined and the essential oils of Portu-
guese aromatic plants were used as inhibitors of
cell adhesion and biofilm formation.
Selected Publications
Badenes, S.M., Lemos, F., Cabral, J.M.S., Biotech-
nol. Bioeng., 108, 1279-1289
de Carvalho, C.C.C.R., Biotechnol. Adv., 29, 75-83,
2011
Coutinho, C.P., de Carvalho, C.C.C.R., Madeira, A.,
Pinto-de-Oliveira, A., Sá-Correia, I., Infection and
Immunity, 79, 2950-2960
de Barros, D.P.C., Fernandes, P., Cabral, J.M.S.,
Fonseca, L.P., Catal. Today, 173, 95-102
de Barros, D.P.C., Azevedo, A.M., Cabral, J.M.S.,
Fonseca, L.P., J. Food Biochem., 58, 545-556
Lourenço, N.M.T., Oesterreicher, J., Vidinha, P.,
Barreiros, S., Afonso, C.A.M., Cabral, J.M.S.,
Fonseca, L.P., React. Funct. Polym., 71, 489-495
Marques, M.P.C., Fernandes, P., Molecules, 16,
8368-8401
Santa, G.L.M., Bernardino, S.M.S.A., Magalhães,
S., Mendes, V., Marques, M.P.C., Fonseca, L.P.,
Fernandes, P., Appl. Biochem. Biotechnol., 165, 1-
12
Bioseparation Engineering BEL
Objectives
The Bioseparation Engineering Laboratory aims at
the design and development of novel purification
processes in order to intensify and optimize the
downstream processing of proteins and biopharma-
ceuticals, with special emphasis on monoclonal
antibodies (mAbs). Several alternatives to the cur-
rently established downstream processing platforms
of recombinant proteins are being explored, with
main focus on aqueous two-phase extraction, nano-
magnetic separation and monolithic chromatogra-
phy, from a nano-scale to industrial scale. Addition-
ally, tailor-made synthetic ligands are being used
aiming to improve protein purification, stability and
function.
Research Topics
1. Aqueous two-phase systems (ATPS) for biophar-
maceuticals purification – The feasibility of using
aqueous two-phase extraction as a general platform
for the purification of biopharmaceuticals, especially
of mAbs is being studied. The performance of a
pilot scale packed differential contactor for the con-
tinuous countercurrent aqueous two-phase extrac-
tion (ATPE) of human immunoglobulin G (IgG) from
a Chinese hamster ovary (CHO) cells supernatant
is being evaluated and compared to the batch IgG
extraction. The economical and environmental sus-
tainability of an ATPE based capture process is
been evaluated and compared to the currently es-
tablished platform. Efficient models are being devel-
oped in order to predict protein partition in ATPS,
contributing for a better understanding of the
mechanisms responsible for partitioning of bio-
molecules and the parameters governing partition.
A novel cell separation process based on immu-
noaffinity aqueous two phase systems to isolate
and purify human hematopoietic stem/progenitor
cell directly from the whole umbilical cord blood
(UCB) is being developed in collaboration with
SCBL.
2. Bio-inspired affinity polymer systems for antibody
recognition – Novel biomimetic affinity nanoparti-
cles, based on the conjugation of a Protein L-mimic
affinity ligand with thermosensitive amino-
functionalised PNIPAM microgels, were designed
and synthesised. Adsorption screening with three
different model-proteins (bovine serum albumin, a
commercial monoclonal antibody (mouse IgG1 iso-
type) and human IgG) demonstrated that such bio-
inspired nanoparticles are able to selectively recog-
nize and capture antibody molecules in both pure
and impure/complex media.
3. Nano-magnetic separation of biopharmaceuticals
– The main focus is on the development of mag-
netic nanoparticles suitable for the purification of
mAbs. The feasibility of using boronic acid function-
alized magnetic particles in the adsorption of mAbs
under conditions typically observed in mammalian
cell culture is being evaluated and compared with
analogous supports coated with Protein A.
4. Monolithic chromatography for integration of cell
separation and antibody purification – Novel affinity
and mixed-mode ligands for the purification of
mAbs, with particular focus on phenyl boronate de-
rivatives, have been designed and developed. The
immobilization of the ligands on to the surface of
supramacroporous monoliths (cryogels) will allow
the integration of both clarification and capture in
just one step, and thus the capture of mAbs directly
from cell culture media without any cell removal
step upstream.
5. High throughput bioseparation platforms – A lab-
on-a-chip device is being designed and tested for
mAbs extraction using ATPS in a microfluidic plat-
form, as an effective tool to accelerate bioprocess
design and optimization. The partition of IgG tagged
with fluorescein isothiocyanate in ATPS is being
investigated in a PDMS microfluidic device fabri-
cated using soft lithographic techniques in collabo-
ration with INESC-MN and BEBL. Process simula-
tion will predict IgG diffusion and partitioning behav-
M. Raquel Aires-Barros (PI), Ana Azevedo, M. Ângela Taipa
20 15
11 Annual Report
B
ioseparation E
ngin
eerin
g
iour fitting the experimental results.
Main achievements
• The suitability of a packed differential contactor
for the ATPE of human antibodies from a CHO cells
supernatant has been shown. Higher IgG recovery
yields and purities were obtained when compared to
the batch IgG extraction.
• The economical and environmental sustainability
of an ATPE based capture process has been suc-
cessfully evaluated and compared to the currently
established platform (Protein A). The ATPE process
has shown to be considerably advantageous in
terms of process economy and operation, especially
when processing high titer cell culture supernatants.
This alternative process is able to purify continu-
ously the same amount of mAbs reducing the an-
nual variable operating costs by at least 39% when
cell culture supernatants with mAb titers higher than
2.5 g/L are processed.
• PEG/dextran/NaCl aqueous two-phase system
(ATPS) was successfully used for the specific parti-
tioning and recovery of CD34+ stem/progenitor cells
from UCB. Purification factors up to 245 were
achieved with a single step partitioning experiment,
demonstrating the feasibility of using ATPS as an
alternative step to the traditional techniques for
UCB processing.
• The feasibility of using phenyl boronate as an
alternative ligand to protein A for the direct capture
of mAbs from clarified cell culture supernatants has
been demonstrated. Boronic acid magnetic particles
provided higher binding capacity and identical affin-
ity towards IgG when compared with magnetic parti-
cles coated with Protein A. Complete recovery of
bound IgG was achieved after optimization of the
elution conditions. Considering the substantially
lower cost and higher stability at alkaline conditions
of the boronic acid, this synthetic ligand could be an
alternative to Protein A.
Selected Publications
Rosa, P.A.J. Azevedo, A.M., Sommerfeld, S.,
Baecker, W., Aires-Barros, M.R., Biotechnol.
Adv., 29, 559-567
Borlido, L., Azevedo, A.M., Roque, A.C.A., Aires-
Barros, M.R., J. Chromatogr. A, 1218, 7821-7827
Nascimento, K.S., Yelo, S., Cavada, B.S., Azevedo,
A.M., Aires-Barros, M.R., J. Chem. Eng. Data, 56,
190-194
Sousa, A.G., Andrade, P.Z., Pirzgalska, R.M.,
Galhoz, T.M., Azevedo, A.M., da Silva, C.L., Aires-
Barros, M.R., Cabral, J.M.S., Biotechnol. Lett., 33,
2373-2377
BioSystems Engineering
BSEL
Objectives
To link process and product throughout design, de-
velopment and biomanufacturing, systems engi-
neering approaches must be used or developed
anew. Process analytical technology (PAT) repre-
sents the combined use of different tools applicable
through many of those stages. Though PAT is still
mostly process centred, it can be used within the
QbD (quality by design) context to link process to
product. The main research topics at BioSystems
Engineering Laboratory (BSEL) are focused on: i)
work with new or established PAT tools, ii) whole
process/product design and analysis (cell-process-
product), iii) systems engineering applied to modern
manufacturing, and iv) pharmaceutical engineering.
Research Topics
1. Process Analytical Technology Tools – PAT in-
volves the application of process analytical chemis-
try (i.e., in-process monitoring techniques and
chemometrics), multivariate data analysis (MVDA;
e.g., data-based modelling techniques), and proc-
ess control techniques (namely, use of process data
with multivariate supervision and diagnosis strate-
gies). All these activities are done with the aim of
characterizing the state of a system at any given
time real-time and to be used in process optimiza-
tion and control. The perspective taken in PAT is
that of the process (not the sample or that of a sin-
gle parameter over time). In this research topic the
use of spectroscopy techniques specially suited for
industrial applications is explored in diverse con-
texts (pharma/biopharma) and within the overall
PAT context and aims.
2. Whole Process/Product Design and Analysis –
Systems biology and „omic‟ approaches have pro-
vided a general physiological and metabolic engi-
neering understanding of several important microor-
ganisms, while bioprocess systems engineering has
benefited from the integration of monitoring, model-
ling, control and optimization. In this topic the links
within and between USP (up-stream processing:
biotransformation) and DSP (down-stream process-
ing: bioseparation) are explored at the three levels
of cell-process-product. Concepts such as process
and product design spaces and all aspects related
to quality-by-design are examined for pharma and
biopharma processing.
3. Systems Engineering Applied to Manufacturing –
While product innovation has been the key issue in
pharma and biopharma in the past, manufacturing
has remained relatively static (e.g., locked-validated
processes). Continuous improvement and opera-
tional excellence practices are entering pharma/
biopharma manufacturing and transforming these
industries as happened elsewhere decades ago. In
this research track, science and technology driven
manufacturing paradigms are the main topics exam-
ined, as the way forward to achieve operational ex-
cellence and sustainability throughout process/
product life-cycle. Adapting tools and metrics from
the disciplines of operation excellence in other in-
dustries to biomanufacturing, is the main focus on
this topic.
4. Pharmaceutical Engineering – New paradigms in
design and manufacturing of small and large thera-
peutically active (API) molecules and drug products
(formulated APIs), include continuous microreaction
technologies (MRT). It is relatively straightforward
to scale-down and operate in continuous mode
some API chemical synthesis reactions in micro-
reactors. It is still very complex or yet unfeasible to
operate in continuous mode most unit operations
involving suspended solids (e.g., crystallization),
biotransformations and some bioseparations. In
this research topic work with off-the-shelf MRTs is
being initiated to examine both feasibility and oper-
ability issues of plant miniaturization and process
intensification of small API molecules manufactur-
ing. As experience and knowledge are established
more complex types of products and unit operations
will be examined.
José Menezes (PI)
20 17
11 Annual Report
B
iosyste
ms E
ngin
eerin
g
Main Achievements
• Developing and demonstrating the industrial fea-
sibility for GMP monitoring of near-infrared spectro-
scopic for at-line multiparametric monitoring large
biomolecules‟ manufacturing processes with scale,
clone and media independent calibrations.
• Whole process analysis with PAT techniques in
pharma and biopharma manufacturing.
• Proposing a general framework for developing
and applying QbD through process analytical tech-
nology tools across diverse problems and platforms.
• Developing new algorithms for data fusion and
improved information extraction of PAT monitoring
tools.
• Implementing microwave resonance to pilot and
industrial scale pharmaceutical granulators for in-
situ PAT monitoring and QbD studies.
Selected Publications
Hakemeyer, C., Strauss, U., Jose, G.E., Folque, F.,
Menezes, J .C., Talanta, doi :10.1016/
j.talanta.2011.12.042
Schewitz, J., Herdling, T., Lochmann, D., Reich, G.,
Menezes, J.C., “A Pharmaceutical Industry Per-
spective”, 8th Eur. Congr. Chemi. Eng., Sept. 25-
27th, Berlin, Germany
Menezes, J.C., “Bioprocess Development and
Manufacturing, pp 501-509, Vol.3, Industrial Bio-
technology, Ed. A Moreira, in Comprehensive Bio-
technology (2nd Edition), Ed. M Moo-Young, El-
sevier
“PAT Applied in Biopharmaceutical Process Devel-
opment and Manufacturing An Enabling Tool for
Quality-by-Design”. Eds Cenk Undey, Duncan Low,
Jose C. Menezes, Mel Koch, CRC Press
Jose, G.E., Folque, F., Menezes, J.C., Werz, S.,
Strauss, U., Hakemeyer, U., Biotechnol. Prog., 27,
1339–1346
Puchert, T., Lochmann, D., Menezes, J.C., Reich,
G., Eur. J. Pharm. Biopharm., 78,117–124
Puchert, T., Holzhauer, C.V., Menezes, J.C., Loch-
mann, D., Reich, G., Eur. J. Pharm. Biopharm., 78,
173–182
Lourenço, V., Herdling, T., Reich, G., Menezes,
J.C., Lochmann, D., Eur. J. Pharm. Biopharm., 78,
513-521
NAB
L
Nucleic Acid Bioengineering
Objectives
Nucleic Acid Bioengineering Laboratory (NABL) is
focused on: i) plasmid vectors and their application
in gene therapy or DNA vaccination; and ii) micro-
chips for DNA detection. The specific objectives are
to address the scientific/technological challenges
associated with plasmid biopharmaceuticals by
combining biomolecular engineering studies with
bioprocess engineering and to co-develop (with
INESC-MN) thin-film microchip and microfluidic plat-
forms for the manipulation/detection of DNA, pro-
teins and cells.
Research Topics
In the case of plasmids, the following research top-
ics are pursued:
1. Design, stability and delivery of plasmids – Pa-
rental plasmids are designed to improve the manu-
facturing of minicircles. Marine organisms are
screened for drugs that inhibit nucleases, stabilise
plasmids and hence lead to higher transfection ac-
tivity. Delivery systems (electroporation, liposomes,
carbon tubes, polymeric microparticles) are devel-
oped to increase DNA uptake and transcription lev-
els.
2. Manufacturing of plasmid vectors - Processes for
the production of plasmids are conceptually de-
signed, developed, optimised and compared. E. coli
strains are engineered to produce high amounts of
plasmid DNA by mutating key genes on the glyco-
lytic pathway. The impact of the downstream proc-
essing on the overall quality and biological activity
of plasmids is studied. Downstream processes are
combined with strain engineering and parental plas-
mid design to facilitate the purification of minicircles.
Membranes are designed to improve plasmid chro-
matography. Covalent immobilization of plasmids
and assembling of molecular probes on AFM canti-
levers is pursued to characterize binding interac-
tions with membrane adsorbers. Synthetic protein-
mimic affinity ligands are screened and used to pu-
rify plasmid DNA. Analytical procedures (HPLC) to
monitor manufacturing and product quality are also
developed.
3. DNA vaccines and gene therapy – DNA vaccine
candidates are constructed by cloning antigenic
proteins associated with sleeping sickness/avian flu
and tested in mice models for their ability to gener-
ate cellular and humoral responses, and to provide
immunisation. The possibility of using plasmids to
deliver the cytotoxic bacterial protein azurin to can-
cer cell models is under evaluation (collaboration
with BSRG).
In the case of microchips for DNA detection the fol-
lowing topics are addressed:
1. Immobilization and handling of DNA proteins and
cells – Thin film technologies, chemical modifica-
tion, microfluidics and electronic addressing are
used to develop microchips for the molecular recog-
nition of specific analytes via hybridization. The core
of the chips is a flat surface with immobilized probe
molecules or cells. Other features include the pres-
ence of micro-electrodes to generate electric fields
that accelerate the kinetics of binding/recognition.
2. Photodetectors – Amorphous silicon photodetec-
tors are developed for the optoelectronic detection
of coloured, chemiluminescent and fluorescent
molecules in thin film chips. The presence of these
molecules ultimately reports specific biorecognition
events such as DNA hybridization or metabolic cell
activity.
Main Achievements
• The role played by charge transfer interactions in
the clearance of cell-derived impurities (RNA, DNA,
proteins, lipopolysaccharides) from plasmid-
containing E. coli lysates by phenyl boronate chro-
matography at acidic pH was described.
Duarte Miguel Prazeres (PI), Gabriel Monteiro, José Santos, M. Ângela Taipa, Marília Mateus
20 19
11 Annual Report
Nucle
ic A
cid
Bio
engin
eerin
g
• E. coli strains that produce high amounts ofplas-
mid DNA were created by systematically mutating
key genes on the glycolytic pathway.
• A parental plasmid for minicircle production was
constructed by inserting two identical MRS sites
and the gene for ParA resolvase in a commercial
eukaryotic expression vector pVax.
• The critical influence of the downstream proc-
essing on the ability of plasmids to form lipoplexes
and transfect mammalian cells was demonstrated.
• Liposome-immobilized membranes were devel-
oped and their feasibility as HIC adsorbers in a
plasmid DNA downstream purification protocol was
assessed.
• The surface chemistry of several membrane ad-
sorbers was determined by X-ray photoelectron
spectroscopy (collaboration with CQFM-IST).
• Microfluidic systems were developed to carry out
microspot-based ELISA in with chemiluminescence
and colorimetry detection using integrated thin-film
amorphous silicon photodiodes (in collaboration
with INESC-MN).
• Label-free electrical detection of surface DNA
immobilization and hybridization via streaming cur-
rent measurements in a microchannel was demon-
strated.
Selected Publications
Gomes, G.A., Azevedo, A.M., Aires-Barros, M.R.,
Prazeres, D.M.F., J. Chromatogr. A, 1218, 8629-
8637
Oliveira, P.H., Prather, K.L.J., Prazeres, D.M.F.,
Monteiro, G.A., Biotechnol. J., 6, 378-391
Prazeres, D.M.F., “Plasmid Biopharmaceuticals:
Basics, Applications and Manufacturing”, John
Wiley&Sons
Novo, P., Prazeres, D.M.F., Chu, V., Conde, J.P.,
Lab-on-a-chip, 11, 4063-4071
Martins, D.C., Prazeres, D.M.F., Chu, V., Conde,
J.P., App. Physics Lett., 99, 183702
SCB
L
Stem Cell Bioengineering and
Regenerative Medicine Laboratory
Objectives
The Stem Cell Bioengineering and Regenerative
Medicine Laboratory aims at developing highly con-
trolled bioreactor systems for the ex-vivo expansion
of stem cells and their controlled differentiation into
specific cell types, as well as their integration with
advanced bioseparation and purification techniques.
As stem cells (SC) are rare, their isolation and ex-
pansion/differentiation in vitro significantly increases
the cell population available for Cellular and Gene
Therapy, Tissue Engineering, high-throughput drug
screening and stem cell research. Human hemato-
poietic stem cells (HSC), human mesenchymal
stem cells (MSC), as well as human and mouse
pluripotent stem cells (both embryonic (ESC) and
induced pluripotent stem cells (iPSC)) and neural
stem cells (NSC) are used as model systems.
Research Topics
1. Ex-vivo expansion of HSC in co-culture with MSC
under serum-free conditions - Current research is
focused on: (i) the definition of optimal culture con-
ditions namely concerning cytokine combinations,
enrichment procedures and initial cell concentra-
tions used to provide an amplification of HSC, espe-
cially those obtained from the umbilical cord blood
(UCB); and (ii) the understanding of the mecha-
nisms underlying the hematopoietic supportive ca-
pacity of MSC. These will have implications in terms
of bioreactor design towards the maximization of
human HSC expansion in vitro. Current research
also focuses on platelet production from the ex-vivo
expanded HSC. Isolation and purification methods
of human hematopoietic stem/progenitor cells are
being developed in collaboration with BEL, to obtain
highly enriched cell populations at large-scale.
2. Clinical-scale production of MSC for Cellular
Therapies - Culture protocols are being optimized
for the isolation and expansion of MSC under serum
-/xeno(geneic)-free conditions, while maintaining
their multilineage differentiation and immunosup-
pressive capacities, as well as their genetic stability.
MSC are isolated from adult bone marrow (BM),
adipose tissue (AT), umbilical cord matrix (UCM)
and synovial membrane. Culture of MSC in fully
controlled bioreactors using microcarriers, under
defined, xeno-free conditions, is currently being
exploited to maximize MSC yield. In addition, a pro-
teomic analysis platform is being established in col-
laboration with BSRG, IBB/CEBQ, to understand
how the ex-vivo culture process affects MSC fea-
tures at the proteome level.
3. Bioprocessing of pluripotent and neural stem
cells - The ex-vivo expansion of pluripotent stem
cells (PSC) and PSC-derived NSC is studied to-
wards the definition of highly controlled bioreactor
systems to establish an efficient, reproducible and
cost-effective large-scale bioprocess to obtain the
starting material to generate mature cells (i.e. neu-
rons) for potential use in the treatment of neurologi-
cal disorders, as well as for drug screening. Bio-
separation and purification methods of human PSC-
derived cells are addressed to ensure the quality
control for cellular therapies.
4. Micro-Scale culture of pluripotent stem cells -
High-throughput microarray systems, as well as
microfluidic devices are being developed to eluci-
date important microenvironmental factors (i.e.
chemical, physical) affecting mouse and human
pluripotent stem cell self-renewal and differentiation,
while providing the basis for rapid identification of
signals and conditions that can be used to direct
cellular responses.
Joaquim Cabral (PI), Cláudia Lobato da Silva, Frederico Ferreira, Margarida Diogo, T. Catarina Madeira
20 21
11 Annual Report
Ste
m C
ell B
ioengin
eerin
g
5. Gene delivery strategies to stem cells - Safe and
effective non-viral strategies to genetically-engineer
stem cells are being developed to enhance the
therapeutic efficacy in different clinical settings.
DNA vectors encoding for reporter and/or specific
proteins involved in ex-vivo expansion/
differentiation of stem cells are being delivered to
these cells by microporation or associated to cati-
onic lipids. Novel gene carriers such as minicircles
and miniplasmids are currently being exploited, in
collaboration with NABL, to extend gene expression
and augment cell survival and proliferation, foresee-
ing the maximization of stem cells for applications in
Cellular and Gene Therapy, as well as Tissue Engi-
neering.
6. Tailoring biomaterials to support stem cell cultiva-
tion - Synthetic polymeric supports are developed to
assist scalable culture systems for maximization of
ex-vivo stem cell expansion or differentiation. Elec-
trospinning is currently being used to produce nano-
fiber scaffolds to mimic aspects of the extracellular
matrix, promoting cell-cell and cell-material interac-
tions and cellular adhesion. Moreover, some bio-
medical applications require cell recovery from the
polymeric support at the end of the cell cultivation
stage. Polymers sensitive to harmless stimuli (e.g.
glucose, temperature) are being tailored to release
cells, without affecting cell viability and function, at
physiologic conditions.
Main Achievements
• By studying the effect of the initial degree of
CD34+ cell enrichment on the expansion of hemato-
poietic stem/progenitor cells from UCB in co-culture
with human BM MSC, it was demonstrated the exis-
tence of highly dynamic culture events regarding
CD34 modulation, prior to cell division, affecting cell
cycle and proliferation status in culture and ulti-
mately the final hematopoietic cell yield. These
events point to the need to establish a balance be-
tween the cell recovery upon purification and the
stem/progenitor cell proliferative potential of cul-
tured cells.
• A novel cell separation process based on a im-
munoaffinity aqueous two phase system (ATPS)
composed of polyethylene glycol (PEG) and dextran
was successfully established to isolate and purify
CD34+ stem/progenitor cells directly from whole
UCB. This system is expected to pave a new way to
purify hematopoietic stem/progenitor, at a process
scale, for use in a variety of clinical settings.
• The microcarrier-based stirred culture system
previously developed for human MSC was success-
fully adapted to xeno-free conditions. Furthermore,
this xeno-free stirred culture system was able to
support the expansion of both BM MSC and adi-
pose-derived stem cells (ASC), while maintaining
the characteristic immunophenotype and multipo-
tency differentiation potential. These results repre-
sent a major step towards the GMP compliant large-
scale production of a safe and effective MSC for
Cellular Therapy.
• A two-dimensional gel electrophoresis (2-DE)
based quantitative proteomic study was performed
in collaboration with BSRG, IBB/CEBQ for unveiling
the molecular mechanisms underlying the com-
monly observed decrease on proliferative and
clonogenic potential of human BM MSC upon con-
secutive passages. Proteins of the functional cate-
gories “Structural components and cellular cy-
toskeleton” and “Folding and stress response pro-
teins” were found to be less abundant in later pas-
sages, while the levels of proteins involved in
“Energy metabolism”, “Cell cycle regulation and
aging” and “Apoptosis” were increased. This plat-
form paves the way to establish a proteomic analy-
sis platform as a quality control for MSC products
towards the development of safer and more effec-
tive cellular therapies.
• Hypoxic conditions (2% O2 versus atmospheric
air) were found to induce an immediate and con-
certed downregulation of genes involved in DNA
repair and damage response pathways in human
BM MSC and ASC, concomitantly with the occur-
rence of genomic instability in microsatellite mark-
ers, while maintaining telomere length. These re-
sults provide evidence on how hypoxia selectively
impacts the cellular response of BM MSC and ASC,
thus pointing towards the need to optimize oxygen
tension ex-vivo according to the cell source.
• A robust and quality-controlled large-scale cul-
ture system, under serum-free conditions, was de-
veloped for the mass production of mouse ESC
(mESC) in a fully-controlled stirred tank bioreactor.
Importantly, cells expanded under these conditions
retained the expression of pluripotency markers and
their differentiation potential into cells of the three
embryonic germ layers. This controlled bioprocess
is potentially adaptable to other cell types including
human ESC and iPSC, thus representing a promis-
ing tool for the controlled production of specific cell
types for applications in tissue regeneration and
drug screening.
• A multifactorial design approach was success-
fully used to elucidate the sole and interactive influ-
ence of different signaling pathways in the regula-
tion of the effect of oxygen tension towards mESC
expansion under chemically defined conditions.
MEK/ERK pathway inhibition, activation of Wnt/β-
Catenin by GSK-3 inhibition and activation of
STAT3 were evaluated. These results add new in-
sights into the mechanisms by which oxygen ten-
sion influences mESC fate with GSK-3 inhibition
showing a crucial role towards maintenance of
mESC pluripotency under a low oxygen tension.
• A microcarrier-based culture platform was devel-
oped for scaling-up the expansion of both mouse
and human PSC-derived NSC, under adherent se-
rum-free conditions, in spinner flasks and using
xeno-free microcarriers. This culture system was
able to support PSC-derived NSC expansion while
maintaining their neural stem/progenitor phenotype
and neuronal differentiation potential.
• A feeder-free and serum-free culture platform
was successfully established for the expansion of
human iPSC under static conditions allowing the
maintenance of the pluripotency phenotype after a
successive sub-culturing procedure. This platform
encompasses completely xeno-free culture condi-
tions and a single-cell passaging methodology to-
wards a more accurate control and characterization
of human iPSC cell expansion.
• Novel DNA vectors devoid of bacterial se-
quences – Minicircles - were used to genetically
engineer human MSC and mouse NSC. The ob-
tained results have shown that stem cells trans-
fected with these vectors exhibit higher survival and
transgene expression, for a longer period of time,
using lower amounts of DNA when compared to the
respective plasmid. These findings provide evi-
dence for the advantages of using minicircles for
over-expressing therapeutic proteins, mainly envis-
aging clinical applications.
20 23
11 Annual Report
• An electrospinning system was assembled, key
parameters adjusted and different collectors built
and tested for the production of matrices with differ-
ent nanofiber alignments. A range of materials, in-
cluding cellulose acetate, dextran, polycaprolactone
and polyhydroxybutyrate were used to produce nan-
ofibers with diameters between 75 and 1750 nm.
These matrices were successfully tested as cellular
supports for cultivation of human stem cells.
Selected Publications
Andrade, P.Z., da Silva, C.L., dos Santos, F., Almei-
da-Porada, G., Cabral, J.M.S., J. Cell. Biochem.,
112, 1822-1831
Fernandes-Platzgummer, A., Diogo, M.M., Baptista,
R.P., da Silva, C.L., Cabral, J.M.S., Biotechnol.
Progr., 27, 1421-1432
Madeira, C., Ribeiro, S.C., Pinheiro, I.S.M., Martins,
S.A.M., Andrade, P.Z., da Silva, C.L., Cabral,
J.M.S., J. Biotech., 151, 130-136
Rodrigues, C.A.V., Fernandes, T.G., Diogo, M.M.,
da Silva, C.L., Cabral, J.M.S., Biotechnol. Adv., 29,
815-829
Santos, F.D., Andrade, P.Z., Abecasis, M.M., Gim-
ble, J.M., Chase, L.G., Campbell, A.M., Boucher,
S., Vemuri, M.C., Silva, C.L., Cabral, J.M.S., Tissue
Eng. Part. C Methods, 17, 1201-1210
Sousa, A.G., Andrade, P.Z., Pirzgalska, R.M.,
Galhoz, T.M., Azevedo, A.M., da Silva, C.L., Aires-
Barros, M.R., Cabral, J.M.S., Biotechnol. Lett., 33,
Ste
m C
ell B
ioengin
eerin
g
Research Highlights
2011 BERG Annual Report
Bioprocess Intensification through miniaturization
The versatile Rhodococcus erythropolis
Affinity based purification of human monoclonal antibodies from CHO
cell supernatants using boronic acid magnetic particles
Economical evaluation of aqueous two-phase extraction as a novel
platform in the biomanufacturing industry
Combining microwave resonance technology with multivariate data analysis as a PAT/QbD approach to improve process understanding
in pharmaceutical processes
Microchip-integrated photodetection of intracellular calcium in re-
sponse to the activation of G-protein coupled receptors
Rational engineering of E. coli strains for improved manufacturing of
plasmid biopharmaceuticals
Controlled mass production of mouse embryonic stem cells in biore-
actors
Multifactorial analysis of embryonics stem cell self-renewal reveals a
crucial role of GSK-3-mediated signaling under hypoxia
P. Fernandes, M. Marques and J.M.S. Cabral
Globalization brought along increased competi-tiveness, which has further stressed the need for fast development of more cost-effective and sus-tainable (bio)processes. Process intensification, where large and expensive equipments/processes are replaced with cheaper, smaller and more effi-cient ones is an acknowledged approach to com-ply with such demand. The use of miniaturized devices clearly fits within the scope of process intensification, since they require minimal amounts of chemicals and biological; allow for high level of parallelization; and, in given configu-rations enable scale-out rather than scale-up [1]. Although miniaturization can be implemented from upstream to downstream of a (bio)process, its application in fermentation/bioconversion steps clearly stands out, where it relies in an assorted type of reactor configurations [1,2]. With volumes under 100 mL to a few μL, these reactors may or may not display a microstructured nature. The former configuration abridges microchannel plate and monolith type reactors, whereas the latter encompasses miniature stirred tanks and micro-titer plates (MTP). Miniaturized reactors can be used in different stages of bioconversion/fermentation processing, more specifically during process development or at production scale. Once rationally used, evidence on the advantages of the use of miniaturized reactors in the former stage have been increasing; on the other hand, and de-spite some examples of successful applications in production scale when purely chemical processes are involved, the use of microreactors at produc-tion scale when biologicals are used, the potential for application is still under evaluation [1,2]. The work developed has contributed to further consoli-date the relevance of miniaturized reactors for the early stages of bioconversion process develop-ment..
Microfluidic reactors for bioconversion of ster-
oids
The production of intermediate steroids from sterol substrates is a multi-enzymatic reaction. The first step is the conversion of the 3β-hydroxy function into a 3-keto derivative, which is per-formed by cholesterol oxidase (CO). Given the lipophilic nature of sterol and steroid molecules, the use on non-conventional media, such as or-ganic-aqueous two phase systems, is a common approach to overcome the low volumetric produc-tivities of aqueous bioconversion systems [3]. Moreover, the use of microfluidic reactors when enzymatic catalysis requiring transport across phase boundaries is clearly favored, due to the enhanced mass transfer typical of said microreac-tors. Since the selected bioconversion yields as by-product H2O2, which may deactivate CO, a second enzymatic reaction was added, involving catalase (CAT), resulting on H2O2 decomposition (Fig.1).
The assembled set-up comprised a Y-shaped microfluidic reactor for cholesterol oxidation cou-pled to a packed bed mesoreactor, where polyvi-nyl alcohol (PVA) immobilized CAT decomposed H2O2. The microfluic reactor operated in a hep-tane-phosphate buffer environment, where the organic phase was a pool for substrate and prod-uct. Hydrogen peroxide, dissolved in the aqueous phase, was pumped through the packed bed reac-tor (Fig.2).
The whole allowed for continuous operation over 300 h, where despite the decay of catalytic activity of CO, an overall production of 36 M of choleste-none is expected (Table 1).
Fast characterization of immobilized enzyme
systems
BER
G
Bioprocess Intensification through miniaturi-
zation
Cholesterol
HO
H H
H
H
Cholestenone
O
H H
H
H
+ O2
CO+ H2O2
H2O
+
½ O2
CAT
Cholesterol
HO
H H
H
H
Cholestenone
O
H H
H
H
+ O2
CO+ H2O2
H2O
+
½ O2
CAT
Figure 1. Two-step enzyme reaction. The oxidation of cholesterol is catalyzed by cholesterol oxidase (CO). The by-product hydrogen peroxide is decomposed by catalase (CAT).
20 27
11 Annual Report
Experimental set-ups, anchored in either batteries of temperature controlled, miniature stirred reac-tors (under 2 ml volume) or in microtiter plates (MTP), coupled to high throughput analytical methods, preferably anchored in spectophotomet-
ric methods, were established. These can be eas-ily adapted for the fast characterization of enzyme or whole cell bioconversion systems. Such set-ups were used for the characterization of a sol-gel immobilized inulinase [5] and for the rational screening of strategies for the immobilization of β-glucosidase [6]. In the former case, the biocatalyst formulation never reported at the date, was used for the hydrolysis of inulin to fructose. The porous xerogel particles of about 10 µm size, displayed an immobilization efficiency of 80%. As a result of immobilization, activity was displayed over a broader range of temperature and pH. Further-more, immobilization did not tamper with the na-tive enzyme structure, although it brought along some mass transfer limitations [5]. Still, the sol-gel biocatalyst displayed high operational stability, since it was re-used over more than 20 consecu-tive batch runs, while retaining high conversion yields (Fig. 3).
Using the retention of the catalytic activity follow-ing immobilization.as starting criterion for the se-
lection of promising supports for β-glucosidase, sol-gel and PVA-base supports were selected [6].
In either case, immobilization did not change the pH/activity profile, but the use of the sol-gel sup-port improved the temperature/activity profile. Im-mobilization led to enhanced thermal and pH sta-bility. Nevertheless, immobilization brought along mass transfer limitations. Both enzyme formula-tions displayed operational stability (Fig. 4).
References
[1] Marques, M.P.C., Fernandes, P., Molecules, 16, 8368-8401 (2011)
[2] Fernandes, P., Carvalho, F., Marques, M.P.C., Recent Pat. Biotechnol., 5, 160-173 (2011)
[3] Fernandes, P., Cabral, J.M.S., in: Encyclopedia of Industrial Biotechnology, Vol. 7, M. Flickinger (ed.). John Wiley & Sons, New York, 4610-4628 (2010).
[4] Marques, M.P.C., Fernandes, P., Cabral, J.M.S., et al, New Biotechnol. (2011).
[5] Santa, G.L.M., et al., Appl. Biochem. Biotechnol.,
165, 1-12 (2011)
[6] Figueira, J.A., Dias, F.F.G., Sato, H.H., Fernan-des, P., Enzyme Res., 2011, 1-8, Article ID 642460 (2011).
R
esearch H
ighlights
Table 1 - Production profile of cholestenone in continu-ous operation of the microchannel reactor [4].
Figure 2. Experimental set-up for the aqueous-organic two phase bioconversion system. Cholesterol oxidation is catalyzed by CO in the microfluidic reactor, whereas by-product H2O2 is decomposed in a packed-bed mesoreac-tor filled with PVA immobilized CAT.
CholesterolCholestenone
Organic (heptane) phase
Aqueous (pH 7 buffered) phase
Microfluidic reactor
Packed-bed reactor
Cholestenone
H2O2
CO
CholesterolCholestenone
Organic (heptane) phase
Aqueous (pH 7 buffered) phase
Microfluidic reactor
Packed-bed reactor
Cholestenone
H2O2
CO
Time of operation
(h) Total cholestenone production
(M) 0 0
100 14
200 26
300 36
Figure 3. Repeated use of inulinase immobilized in sol–gel particles, in consecutive 24-hour batch runs. In each run, a 5% (w/v) inulin solution in pH 5.0 acetate buffer was used as substrate. Runs were performed at 50ºC. Fructose concentration in the final of the first cycle was 47 g l−1[5].
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Batch runs
Re
lative
pro
duct
yie
ld (
%)
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Batch runs
Re
lative
pro
duct
yie
ld (
%)
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10 11 12
Batch run
Re
lative
act
ivity
(%
)
Sol-gel
PVA
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10 11 12
Batch run
Re
lative
act
ivity
(%
)
Sol-gel
PVA
Figure 4. Repeated use of immobilized β-glucosidase in sol-gel (¾) and Lentikats (¾) on relative activity. Batch runs were carried out at 50ºC and pH 5.0, using as sub-strate 5 mM p- nitrophenyl-β-D-glucopyranoside.
C.C.C.R. de Carvalho
Rhodococcus erythropolis are able to carry out a wide array of bioconversions and degradations due to a large set of enzymes (e.g. oxidases, ep-oxidases, dehydrogenases, dehalogenases and hydrolases), allowing the production of commer-cially interesting compounds and the metabolism of recalcitrant organic compounds [1]. These cells are very hydrophobic as a result of a mycolic acid-containing cell wall, allowing the adhesion of cells to oil/water interfaces and the direct uptake of hydrophobic compounds such as hydrocarbons [2]. R. eryhtropolis can even adapt the fatty acid composition of the cellular membrane, the mycolic acid content and the cell wall permeability as a response to the carbon source [2,3].
Natural tolerance
R. erythropolis cells are able to degrade aliphatic (Fig. 1) and aromatic hydrocarbons, including benzene, toluene, xylene and ethylbenzene, as well as polyaromatic hydrocarbons such as an-thracene [1,4]. The cells present a particular abil-ity to carry out biotransformations and bioconver-sions in organic-phase systems [5].
When non-adapted R. erythropolis DCL14 cells
were placed in contact with toluene, 10.5% of the cells were still viable after 1h exposure. However, adapted cells were able to degrade 52.4% (v/v) toluene in n-dodecane, toluene being consumed at 10.7 mg/(h
mg protein) [6]. Rhodococcus sp. cells can even be active under starvation conditions and the deg-radation of toxic compounds may not be nega-tively affected by the presence of more easily de-gradable carbon sources such as n-dodecane [7]. The cells are also usually more tolerant to antim-icrobials. For example, a fresh extract of Abelmo-schus esculentus at a concentration of 97.7 mg/mL was sufficient to kill all Staphylococcus aureus cells, which is a worldwide source of nosocomial infection, as well as Mycobacterium, but was inef-fective against R. erythropolis [8].
Adaptation to improve cellular performance
Extremophiles can grow at extreme values of tem-perature, pH, ionic strength and metal concentra-tions, but it may be difficult to find and isolate those possessing the required metabolic activities. On the other hand, R. erythropolis cells possess a large number of catabolic activities and may be
Figure 1. Growth rates observed using n-alkanes as sole carbon and energy sources at 15 and 28ºC.
The versatile Rhodococcus erythropolis
BER
G
0
0.02
0.04
0.06
0.08
0.1
0.12
Penta
ne
Hexane
Cyclo
hexane
Hepta
ne
Tolu
ene
Octa
ne
Iso-o
cta
ne
Nonane
Undecane
Dodecane
Tetr
adecane
Hexadecane
Carbon source
Gro
wth
rate
(h
-1)
15ºC, 0.125%
15ºC, 0.25%
28ºC, 0.125%
28ºC, 0.25%
20 29
11 Annual Report
easily adapted to extreme conditions. The physio-logical adaptations undertaken by these cells, when exposed to conditions sequentially further away from the optimum growth conditions, al-lowed the activity of the cells to be maintained at conditions previously sufficient to kill non-adapted cells [9] (Fig. 2). The cells were able to grow and degrade C6-C16 n-alkanes and alcohols at 4-37ºC, pH 3-11 and in the presence of up to 7.5% sodium chloride and 1% copper sulphate. The large majority of adapted cells were able to main-tain polarization of the membrane under the most difficult conditions tested, to adjust the net surface charge and changed the composition of the fatty acids of the cellular membrane according to the growth condition. Changes in the relative propor-tion of straight, methyl and cyclopropyl saturated, unsaturated and hydroxyl substituted fatty acids were observed, as well as production of polyun-saturated fatty acids unusual in bacteria.
References
[1] de Carvalho, C.C.C.R., da Fonseca, M.M.R. Appl. Microbiol. Biotechnol., 67, 715-726 (2005) [2] de Carvalho C.C.C.R., Wick L.Y., Heipieper H.J., Appl. Microbiol. Biotechnol., 82, 311-320 (2009)
[3] de Carvalho, C.C.C.R., In: Biology of Rhodococcus (Hector M. Alvarez Ed.). Microbiol-ogy Monographs, Vol. 16 (Alexander Steinbüchel, Series Ed.), p. 109-131. Springer Verlag (2010) [4] Tyagi, M., da Fonseca, M.M.R., de Carvalho C.C.C.R., Biodegradation, 22, 231-241 (2011) [5] Fernandes, P., Marques, M.P.C., Carvalho, F., de Carvalho, C.C.C.R., In: Ryan E. Carter (Editor), Organic solvents: properties, toxicity and industrial effects. Nova Science Publishers, Inc., pp. 173 (2011) [6] de Carvalho, C.C.C.R., Fatal, V., Alves, S.S., da Fonseca, M.M.R. Appl. Microbiol. Biotechnol., 76, 1423-1430 (2007) [7] de Carvalho, C.C.C.R., Biotechnol. Adv., 29, 75-83 (2011) [8] de Carvalho, C.C.C.R., Cruz, P.A., Xavier-Filho, L., da Fonseca, M.M.R., Biotechnol. Biopro-
cess Eng., 16, 971-977 (2011)
[9] de Carvalho, C.C.C.R., Res. Microbiol., doi: 10.1016/j.resmic.2011.11.003 (2012)
R
esearch H
ighlights
Figure 2. Optimum, moderate and extreme conditions for R. erythropolis DCL14 growth in terms of tempera-ture, pH, sodium chloride and copper sulphate concentrations for non-adapted and adapted cells (grey areas). Extreme conditions did not allow cell growth.
Extreme
Extreme
Optimum
Optimum
Extreme
Extreme
0 10 20 30 40 50 ºC
Extreme
Extreme
Optimum
Optimum Extreme
0 2 4 6 8 10 12 14
Optimum
Optimum
Extreme
Extreme
0 2 4 6 8 10
Extreme
Extreme
0 2 4 6 8 10
Extreme
Extreme
Extreme
Optimum
Optimum
Extreme
Extreme
0 10 20 30 40 50 ºC
Extreme
Extreme
Optimum
Optimum Extreme
0 2 4 6 8 10 12 14
Optimum
Optimum
Extreme
Extreme
0 2 4 6 8 10
Extreme
Extreme
0 2 4 6 8 10
Extreme
Temperature
pH
NaCl
CuSO4
after adaptation
after adaptation
after adaptation
after adaptation
L. Borlido, A.M. Azevedo, A.C.A. Roque a and M.R. Aires-Barros
a Requimte, FCT, UNL, Caparica, Portugal
Antibodies for therapeutic applications are a fast growing market with increasingly pressing de-mands. The combination of a large potential mar-ket (>500,000 patients) with therapies requiring high doses and/or chronic administration (>1 g per patient per year) served as the driving force to-wards process optimization [1]. Given the inability of Protein A chromatography to directly purify samples with high monoclonal antibody titers (titers greater than 10 g/l are now possible), alter-native and more cost effective purification proc-esses are needed. In this regard, magnetic sepa-rations offer fast, gentle and highly selective (non-magnetic impurities) separation conditions with the potential for high binding capacities (small particles, typically < 2 µm). A great effort has been given in the development of fully synthetic ligands to substitute Protein A. Ideally, this ligand would provide selectivity, increased capacity and chemi-cal stability while decreasing the costs. The bo-ronic acid ligand is capable of selectively captur-ing cis-diol containing molecules, such as carbo-hydrates and glycoproteins, through the formation of a reversible covalent ester bond. Antibodies are glycoproteins as they bear oligosaccharides in both the Fc and Fv regions. In the former, despite some heterogeneity, the 1,2-cis diol saccharides fucose, manose and galactose can be typically found.
The phenylboronic acid ligand which was used in this work is able to operate as a multi-modal ligand as it is able to promote affinity, electro-static, hydrophobic, aromatic π-π, charge transfer and hydrogen bonding interactions. Depending on the pH value, the boronic acid moiety might be in a trigonal or tetrahedral form. At pH values lower than the pKa of phenylboronic acid, the trigonal form is predominant and thus charge transfer in-teractions between the boron sp2 empty orbital and any Lewis base (e.g. unprotonated amines) can occur [2]. Conversely, at alkaline pH values, this type of interaction may be disregarded as the boronic acid is converted in to the hydroxyboro-nate form, which no longer interacts with Lewis bases but is able to promote electrostatic interac-tions. Contrary to the initial general belief that the optimal pH for the complexation of cis-diol con-taining molecules with boronic acids is above the pKa of the latter, recent reports demonstrated that this is highly dependent on the molecule-ligand pair used [3,4].
Initial batch adsorption studies with commercially available non-porous silica based boronic acid magnetic particles (SiMAG-Boronic acid) showed the binding pH to be an important factor in the adsorption isotherms of human antibodies (Fig. 1a). The maximum binding capacity was found to
Affinity based purification of human mono-
clonal antibodies from CHO cell supernatants
using boronic acid magnetic particles
BER
G
Figure 1. Adsorption behavior of human IgG on SiMAG-Boronic acid and SiMAG-ProteinA magnetic particles. A) Human IgG adsorption isotherms of SiMAG-Boronic acid (pH 7.4 ●, pH 8.5 ● and 9.5 ●) and SiMAG-ProteinA (pH 7.4 ■) particles. The lines represent the fitted Freundlich isotherms. B) Adsorption kinetics, q (full symbols) and percentual variation of the binding capacity with time, dq/dt (empty symbols) of human IgG in SiMAG-Boronic acid (circles) and SiMAG-ProteinA (squares) particles at pH 7.4.
20 31
11 Annual Report
be higher at neutral pH than at pH 9.5. Under the same conditions, non-porous silica based Protein A magnetic particles (SiMAG-ProteinA) showed approximately only half of the binding capacity while exhibiting identical affinities to those of Si-MAG-Boronic particles at pH 7.4 and 8.5. Further-more, the adsorption kinetics (Fig. 1b) were found to be very fast with 70% of the maximum binding capacity obtained at 30 min of incubation, being observed in less than 30 s. Such is only possible due to the high affinity of the particles towards the target molecule and to the small size (1 µm) and non-porous nature of the support.
To test the feasibility of using boronic acid mag-netic particles as an alternative to Protein A a fully human monoclonal antibody was directly purified from a clarified CHO cell culture supernatant.
The most important factor influencing the overall process yield and product purity in boronic acid particles was found to be the binding pH. Basic pH values promoted higher purities while resulting in decreased yields due to the competing effects of molecules such as glucose and lactate present in the cell culture supernatant. After optimization, the particles were successfully used in a multi-cycle purification process of the mAb from the CHO feedstock. Boronic acid particles were able to achieve an average overall yield of 86% with 88% removal of CHO host cell proteins (HCP)
when the binding was performed at pH 7.4, while at pH 8.5 these values were 58% and 97%, re-spectively. In both cases, genomic DNA removal was in excess of 97%. Comparatively, Protein A particles recorded an average overall yield of 80% and an HCP removal greater than 99%. Boronic acid based purification processes can offer a cost-effective alternative to Protein A as the direct cap-turing step from the mammalian cell culture.
References
[1] Farid, S.S. , J. Chromatogr., B 848, 8 (2007)
[2] Azevedo, A.M., Gomes, A.G., Borlido, L., San-tos, I.F.S., Prazeres, D.M.F., Aires-Barros, M.R., J. Mol. Recognit., 23, 569 (2010)
[3] Springsteen, G., Wang, B., Tetrahedron, 58, 5291 (2002)
[4] Yan, J., Springsteen, G., Deeter, S., Wang, B., Tetrahedron, 60, 11205 (2004)
[5] Borlido, L., Azevedo, A.M., Roque, A.C.A., Aires-Barros, M.R., J. Chromatogr. A, 1218, 7821(2011)
R
esearch H
ighlights
Figure 2. Reducing SDS-PAGE analysis of the purification process with SiMAG-Boronic acid particles at pH 7.4 and 8.5 and SiMAG-Protein A particles at pH 7.4. Last 3 lanes represent the corresponding elution fractions.
The biotech industry is currently facing unparal-
leled challenges due to the increasing demand
for biotechnology-based human therapeutic prod-
ucts, such as monoclonal antibodies (mAbs).
This has led companies to improve considerably
their upstream processes, with production yields
increasing to mAbs titers never seen before. The
downstream processes have, however, been
overlooked, causing a production bottleneck at
the downstream level. Although chromatography
remains the workhorse of most purification proc-
esses, several limitations, such as low capacity,
scale-related packing problems, low chemical
and proteolytic stability and resins' high cost,
have arisen. Aqueous two-phase systems
(ATPS) have shown to be a valuable option for
the downstream processing of biopharmaceuti-
cals, combining a high biocompatibility and se-
lectivity with an easy and reliable scale up and
capability of continuous operation. In this work,
the economical sustainability of the aqueous two-
phase extraction process is evaluated and com-
pared to the currently established protein A affin-
ity chromatography (ProA) [1].
The proposed downstream ATPE process is
based on a pilot scale validation previously re-
ported by the authors [2], which is depicted in
Fig. 1. This ATPE-based capture process con-
sists of three main steps: i) extraction (E), ii) back
extraction (BE) and iii) washing (W). In the ex-
traction step, most of the higher molecular weight
contaminants are removed, while the washing
step allows not only the removal of lower molecu-
lar weight contaminants and polymer-rich phase
component (PEG), but may also enables the re-
cycling of the polymer for future uses.
The annual operating costs (AOC) required to
process 840 m3/year of cell culture supernatant
containing 2.5 g/L mAb were estimated to be
US$8.7 and 14.4 millions for ATPE and ProA-
P.A.J. Rosa, A.M. Azevedo, S. Sommerfeld a, W. Bäcker
a, M.R. Aires-Barros
a Bayer Technology Services, Bayer Leverkusen, Germany
Figure 1. Process flow diagram for the continuous ATPE-based capture of human antibodies from a cell culture supernatant (BP: bottom phosphate-rich phase, TP: top PEG-rich phase, MS: mixer–settler) [2]
Economical evaluation of aqueous two-phase
extraction as a novel platform in the bio-
manufacturing industry
BER
G
20 33
11 Annual Report
based capture processes, respectively (Fig. 2).
The AOC of the protein A-based process are
1.65-fold higher, which is mainly due to the very
high costs of the protein A resin (US$16,250 per
liter) that accounts for 79% of the total AOC. On
the contrary , in the APTE process, the raw ma-
terials are the major contributors corresponding
to 58% of the total AOC (Fig. 2A). A 4.5-fold
higher raw material consumption per kg mAb is,
indeed, observed for the ATPE process, conse-
quently, leading to about 10- and 25-fold higher
raw materials and waste treatment and disposal
costs, respectively.
The annual operating costs (AOC) required to
process 840 m3/year of cell culture supernatant
containing 2.5 g/L mAb were estimated to be
US$8.7 and 14.4 millions for ATPE and ProA-
based capture processes, respectively (Fig. 2).
The AOC of the protein A-based process are
1.65-fold higher, which is mainly due to the very
high costs of the protein A resin (US$16,250 per
liter) that accounts for 79% of the total AOC. On
the contrary , in the APTE process, the raw ma-
terials are the major contributors corresponding
to 58% of the total AOC (Fig. 2A). A 4.5-fold
higher raw material consumption per kg mAb is,
indeed, observed for the ATPE process, conse-
quently, leading to about 10- and 25-fold higher
raw materials and waste treatment and disposal
costs, respectively.
WFI (water for injection) is the most consumed
raw material for both processes representing the
major contributor to the raw materials costs in
case of ProA (84%) and the second major in
case of ATPE (28%). In the ATPE-based capture
process, PEG 3350 is the main contributor to the
plant raw materials costs (53%) due to the large
amount required per year.
According to this study, the ATPE process was
shown to be considerably advantageous in terms
of process economics, especially when process-
ing high titer cell culture supernatants. In fact,
this alternative process is able to purify continu-
ously the same amount of mAbs reducing the
annual operating costs from 14.4 to 8.5 million
(US$/kg), which represents a reduction of 39%,
when cell culture supernatants with mAb titers
higher than 2.5 g/L are processed [1].
References
[1] Rosa, P.A.J., Azevedo, A.M., Sommerfeld, S.,
Baecker, W., Aires-Barros, M.R., Biotechnol.
Adv., 29, 559-567 (2011)
[2] Baecker, W., Sommerfeld, S., Mutter, M.,
Rosa, P.A.J., Aires-Barros, M.R., Azevedo, A.M.,
World Patent WO/2009/112149 (2009)
R
esearch H
ighlights
Figure 2. Annual operating costs required to process 840 m3/year of cell culture supernatant containing 2.5 g/L mAb using (A) ATPE and (B) ProA-based capture processes. The total annual operating costs were US$8.7 and 14.4 million for the ATPE and ProA (DBC of 30 g/L) capture processes, respectively [1].
Fixed18%
Plant overhead
5%
Raw materials
58%
Consumables0%
Labour dependent
10%
Laboratory quality control
1%
Waste treatment
and disposal8%
Fixed12%
Plant overhead
3% Raw materials
4%
Consumables79%
Labour dependent
1%
Laboratory quality control
1%
Waste treatment
and disposal0%
BER
G Combining microwave resonance technology with multi-
variate data analysis as a PAT/QbD approach to improve
process understanding in pharmaceutical processes
V. Lourenço, T. Herdlinga, D. Lochmann
a, J. Schewitz
a, J.C. Menezes
a Quality Operations, PAT – Laboratory, Merck-Serono KGaA, Darmstadt, Germany
The pharmaceutical industry is encouraged within
Quality by Design (QbD) to apply science-based
manufacturing principles to assure quality not
even of new but also of existing processes. We
have developed a general strategy based on QbD
principles to be applied to existing industrial phar-
maceuticals fluid bed granulation processes. The
three steps involved are: 1) implementation of
Process Analytical Technology (PAT) monitoring
tools at the industrial scale process, combined
with multivariate data analysis (MVDA) of process
and PAT data to increase the process knowledge;
2) execution of scaled-down designed experi-
ments at a pilot-scale, with adequate PAT moni-
toring tools, to investigate the process response to
intended changes in Critical Process Parameters
(CPPs); and finally 3) the definition of a process
Design Space linking CPPs to Critical to Quality
Attributes (CQAs), within which product quality is
ensured by design, and after scale-up enabling its
use at the industrial process scale.
The proposed strategy was tested in an existing
industrial process. Through enhanced process
knowledge established a significant reduction of
product CQAs variability already within quality
specifications ranges was achieved by a better
choice of CPPs values. The results of such step-
wise development and implementation are de-
scribed.
The novel PAT monitoring tools included a micro-
wave resonance probe to measure in-situ real-
time the granules density, moisture and tempera-
ture and a spatial filter velocimetry (SFV) probe to
measure real-time the particle size distribution of
the granules population (Fig. 1).
Acquiring data over a year of manufacturing
batches using the on-line system of Fig. 1, and
applying PCA (principal component analysis) to
the multivariate signal obtain from such instru-
ment, it was found that process performance var-
ied significantly and showed seasonality effects
(Fig. 2).
A scale-down campaign of several designed ex-
periments (Fig. 3) examining the factors with the
greatest impact on process performance
(obtained via a preliminary risk-assessment to the
industrial process), over the period of a year, en-
hanced process knowledge and lead to the pro-
posal of an improved set of process conditions
(Fig. 4) [2-4].
Under the new set of process conditions granules
properties were optimized and the process is now
consistently operated at a much higher and stable
Figure 1. The in-line probes used: microwave resonance probe (left) and spatial filter velocimetry (right).
20 35
11 Annual Report
R
esearch H
ighlights
throughput (Fig. 4). The full account has been
described in the already concluded PhD disserta-
tion [5].
References:
[1] Lourenço, V., Herdling, T., Reich, G., Mene-
zes, J.C., Lochmann, D., Eur. J. Pharm. Bio-
pharm., 78, 513-521 (2011)
[2] Schewitz, J., Herdling, T., Lochmann, D.,
Reich, G., Menezes, J.C., “A Pharmaceutical In-
dustry Perspective”, IFPAC – 25th Int. Process
Analytical Technology Forum, January 17-21, Bal-
timore, Maryland (USA), 2011
[3] Schewitz, J., Herdling, T., Lochmann, D.,
Reich, G., Menezes, J.C., “A Pharmaceutical In-
dustry Perspective”, 8th Eur. Congr. Chemi. Eng.,
September 25-27th, Berlin (Germany), 2011
[4] Lourenço, V., Herdling, T., Reich, G., Mene-
zes, J.C., Schewitz, J., AIChE Annual Meeting,
October 16-21, Minneapolis (USA), 2011
[5] V.M.C.L. Lourenço, PhD Thesis in Chemical
Engineering (2011). Supervised by TUL / IBB
Prof. J.C. Menezes and Merck-Serono / Germany
Dr D.Lochmann
Figure 2. Multivariate process trajectories ob-tained from PCA of granules density, tempera-ture and moisture time profiles. Profiles in blue relate to batches produced in colder winter/spring months, while profiles in red to warmer summer/autumn months [2-4].
Figure 3. Outline of the screening DOE campaign carried out at the industrial pilot scale (1:10).
Figure 4. Designed granulations have similar yields compared to nominal industrial ones and the tablets manu-factured with the designed granules are in specification. Granules properties such as flowability (FTO, flow through an orifice) show much higher consistency.
BER
G Microchip-integrated photodetection of intra-
cellular calcium in response to the activation
of G-protein coupled receptors
S.A.M. Martins, J.R.C. Trabuco, G.A. Monteiro and D.M.F. Prazeres
G-protein coupled receptors (GPCRs) are a large class of ubiquitous receptors expressed in eu-karyotic cells. Signaling molecules like hormones, neurotransmitters and small peptides can bind to GPCRs thus regulating a variety of cell functions ranging from gene expression levels to cell shape and function. Consequently, these receptors play a major role on the pharmaceutical industry. In-deed, 30% of the current market drugs are GPCRs targets. Still, new approaches for the identification of novel modulators are being devel-oped not only to access new functions but also to increase throughput and thus accelerate drug dis-covery. These issues are being addressed at BERG in collaboration with INESC-MN (J.P. Conde, V. Chu) by integrating microfluidic technol-ogy and silicon photodiodes. The major goal is to develop miniaturized devices capable of monitor-ing GPCR activation in living cells.
GPCR screening assays rely on the recording of the average signal from thousands of cells upon addition of a candidate drug target. Typically, changes in the intracellular levels of key elements in the signaling cascade are monitored using fluo-rescence read-out systems such as microscopy or CCD cameras [1]. Major challenges of the inher-ent miniaturization process, is the scaling-down of
the optical apparatus. Photodiodes are character-ized by presenting high photosensitivity, low dark current and high frequency response. In particu-lar, thin film photodiodes based on hydrogenated amorphous silicon (a-Si:H) are readily compatible with microfabrication techniques and conse-quently easily integrated “On-Chip” for acquisition of the optical signal.
For proof-of concept studies, HEK 293T cell lines, endogenously expressing the Muscarinic M1GPCR was the chosen biological model. Acti-vation of the M1 receptor can be easily monitored by following the rise in intracellular calcium (iCa
2+) upon addition agonist. Typically, cells are stained with calcium sensitive fluorophores that exhibit enhanced fluorescence upon calcium binding. A positive signal is characterized by a steep rise in cell‟s fluorescence, followed by a slow decay as desensitization of the GPCR occurs and calcium levels are restored to basal values (Fig 1A).
The intensity of the maximum signal can be corre-lated with different agonist concentrations in order to generate a dose response curve (Fig. 1B), thus enabling the calculation of the EC50 i.e. the con-centration of agonist that elicits 50% of the maxi-mum signal, which, in turn, represents a measure
Figure 1. HEK 293 cells were allowed to adhere overnight in fibronectin-coated wells (100 µl). Cell staining with the calcium sensitive Fluo4 was performed at 37ºC for 1 h. A- Addition of 1 mM carbachol triggered the activation of the endogenous receptor M1, which promotes the release of Ca2+ into the cytosol. Consequently, the cells fluorescence increased; B - Dose response curve for the agonist carbachol. The fluorescence signals were obtained by fluorescence microscopy and quantified with the imaging software ImageJ
A B
20 37
11 Annual Report
R
esearch H
ighlights
of agonist potency.
The a-Si:H p-i-n photodiodes consist of a mesa junction obtained by sequentially depositing layers of 200 Å n+-a-Si:H, 5000 Å intrinsic a-Si:H and 200 Å p+-a-Si:H]. However, the use of photodi-odes for fluorescence measurements rely on the availability of adequate filters that cut the excita-tion light while allowing the transmission of the emission light. This was accomplished by deposit-ing a 2 µm layer of amorphous silicon carbon-alloy (a-SiC:H)[2]. Figure 2A represents a schematic cross section of the device. Figure 2B represents the ratio of transmitted light of the a-SiC:H filter, showing a two order magnitude increase in trans-mittance of the filter on the emission wavelength when compared to the excitation wavelength of the fluorophore Fluo4.
For the monitoring of GPCR activation, HEK 293 Cells (50 µl) were cultured overnight on PDMS wells, previously coated with fibronectin, followed by an incubation with Fluo4. After performing all
the electronic connections, the cell-containing wells were placed on top of the photodiode and stimulated with 1 mM carbachol. Figure 3 shows the current density, J, obtained for a different set of experiments. An increase in J is observed when carbachol is added to the cell-containing wells. Conclusions The monitoring of GPCR M1 receptor activation was confirmed using a-Si:H photodiodes with inte-grated optical filters. On-going work is focusing on the miniaturization of both the cell and optical ap-paratus. Acknowledgments BERG/NABL acknowledges Prof. João P. Conde, Virginia Chu and INESC-MN group for the fabrication of the photodiodes.
[1] Kenakin, T.P., Nature Reviews Drug Discov-ery, 8, 617-626 (2009)
[2] Pimentel, A.C., et al., Appl. Phys. Lett., 94 (2009)
Figure 3. Curent density measured
using the a-Si:H photodiode with an
integrated fluoresecent filter. An in-
crease in current density is observed
when the agonist is added to the sys-
tem thus indicating an increase in cell
fluorescence due to Ca2+ release into
the cytosol.
Figure 2. A.Schematic cross section of the device. The bottom contact is a 1000 Å layer of indium tin oxide (ITO). The top contact (TiW + Al) was defined with a physical mask to have an area of 2 mm2 which repre-sents the actual sensing area. The a-Si:H filter supresses part of the excitation light whereas the emisson light is transmitted; B- Filter transmitance as function of wavelength. The light transmitted at 516 nm ( lem Fluo4) is two orders of magnitude higher twhen compared with the transmitted light at 490 nm ( lex Fluo4).
350 400 450 500 550 600 650 7001E-5
1E-4
1E-3
0.01
0.1
1
10
100
1000
0.334
% T
Wavelength (nm)
0.012
-200
0
200
400
600
800
1000
1200
1400
1600Ex: 490 nm
( Flu
ore
sce
nce
)
Em: 516 nm
B A
a-SiC:H filter ITO p-i-n TiW + Al
Sensing area: 2 mm2
glass
λex
cell sample
λem
5,0E-11
6,0E-11
7,0E-11
8,0E-11
9,0E-11
1,0E-10
1,1E-10
1,2E-10
1,3E-10
1,4E-10
1,5E-10
light 490 nm PDMS cells cells+assaybuffer Cells+ drug
J(A
/cm
-2)
BER
G Rational engineering of E. coli strains for im-
proved manufacturing of plasmid biopharma-
ceuticals
G.A.L. Goncalves, K.L.J. Prathera, G.A. Monteiro, D.M.F. Prazeres aDepartment of Chemical Engineering, MIT, Cambridge, MA 02139 USA
Plasmid DNA (pDNA) biopharmaceuticals are being developed for veterinary and human appli-cations in gene therapy and vaccination. Although significant advances have been made in plasmid design and downstream processing, the need to improve fermentation processes and pDNA pro-duction strains remains largely unmet. One of the key challenges is the achievement of a high-yield and cost effective manufacturing process. The focus of this project, which is being developed in collaboration with the Prather Research Group at MIT, in the context of the MIT-Portugal Program, is to create improved pDNA production strains.
The gram-negative bacteria Escherichia coli is a well-studied and largely explored microorganism in the industry, and is the most common host for the propagation of pDNA. Nevertheless, most of the strains of E. coli used for pDNA production were created through a series of mutations to fa-cilitate cloning of heterologous genes and produc-tion of recombinant proteins. Recently, new pDNA production strains were developed in order to ob-tain high pDNA yields. However, the new muta-tions were usually done in strains with highly
mutagenized genetic background, such as DH5α, and it is not known if the strain genetic back-ground would impact the effect of a new gene knockout or overexpression (Fig. 1) [1].
Genes in the central carbon metabolism are obvi-ous targets for the engineering of high-yield pDNA strains, since the manipulation of such genes could enhance nucleotide synthesis, increase pro-duction of energy and reducing power, and mini-mize acetate formation. On the other hand, genes related to improving pDNA quality have also been common targets, as have genes that are involved in various other cellular processes relevant to pDNA production such as the stringent response and DNA replication [1].
To enhance the production of nucleotides and reduce organic acids synthesis, key genes on the glycolytic pathway were knocked out. The impact of host strain genetic background was investi-gated as well as the carbon source effect on pDNA production. Genes in the glycolytic path-way, which had been already proved to increase pDNA yields, such as pykF and pykA genes [2-3],
Figure 1. E. coli K12 and derivatives -creation of new strains and relationship between different strains. (A) Lineage of MG1655 and W3110, close relatives of wild-type E. coli K12 [66]. (B) Generation of strains con-taining multiple mutations from MC1061, DH1 and JM101 [67-68] . Dark boxes represent commonly-used E. coli strains for plasmid DNA production and recent developments in E. coli strains designed for high yield pDNA processes. Full line arrows represent the relationship between the strains and dashed line arrows rep-resent mutations carried from one strain to the other.
20 39
11 Annual Report
R
esearch H
ighlights
were first deleted in two different strains, MG1655ΔendAΔrecA (wild-type genetic back-ground) and DH5α (highly mutagenized genetic background).
The deletion of endA and recA were also done in MG1655 in order to minimize recombination and non-specific digestion of DNA [4-5]. We observed that host strain genetic background impacts the effect of a specific gene knockout, since the dou-ble mutation pykF and pykA was efficient only in MG1655ΔendAΔrecA and not in DH5α. Plasmid DNA yields were higher in glycerol than glucose for the wild-type strain MG1655ΔendAΔrecA. However, all the strains containing mutation in the glycolytic pathway were more efficient in glucose [6].
Finally, we created a new pDNA production strain, starting from the wild-type MG1655ΔendAΔrecA, with the introduction of the knockout of pgi gene, to completely redirect the carbon flow into the pentose phosphate pathway (PPP). This strategy enhances NADPH formation and nucleotide syn-thesis, which were demonstrated to favor pDNA production (fig. 2). For the first time, a pgi mutant strain, GALG20 (MG1655ΔendAΔrecAΔpgi), was identified as a potential high-yield pDNA produc-tion strain. GALG20 produced 25-fold more pDNA (mg/g DCW) than MG1655ΔendAΔrecA in high concentration of glucose. The top-three strains in terms of high-yield pDNA production are identified in table 1 [6]. The main achievement of this study was thus the creation of high-yield pDNA produc-tion strains from the wild-type strain MG1655.
References
[1] Goncalves, G.A.L., Bower, D.M., Prazeres, D.M.F., Monteiro, G.A., Prather, K.L.J., Biotechnol J., 7, 251-261 (2012)
[2] Cunningham, D.S., Liu, Z., Domagalski, N., Koespsel, R.R., et al., J. Bacteriol., 191, 3041- 3049 (2009)
[3] Pablos, T.E., Soto, R., Mora, E.M., J. Biotech-nol., 2011, doi:10.1016/j.jbiotec.2011.04.015
[4] Summers, D., Mol. Microbiol., 29, 1137-1141 (1998)
[5] Phue, J.-N., Lee, S.J., Trinh, L., Shiloach, J., Biotechnol. Bioeng., 101, 831-836 (2008) [6] Goncalves, G.A.L., Prazeres, D.M.F., Mon-teiro, G.A., Prather, K.L.J., “Design of Escherichia coli host strains specifically for plasmid DNA pro-
Figure 2. Gene knockout strategy to improve plasmid DNA production in E. coli, pgi knockout redirect glu-cose pathway, increasing fluxes in the pentose phos-phate pathway and enhancing nucleotide synthesis and NADPH.
Strain Carbon
source Volumetric yield
(mg/L) Specific yield
(mg/g DCW)
GALG20(MG1655ΔendAΔrecAΔpgi)
Glucose 140.80 ± 0.76 19.08 ± 1.52
GALG11(MG1655ΔendAΔrecAΔpykA)
Glucose 94.10 ± 2.74 13.05 ± 0.20
MG1655ΔendAΔrecA Glycerol 79.31 ± 1.39 11.15 ± 0.48
DH5α Glycerol 34.68 ± 063 4.40 ± 0.29
Table1. Top three high-yield pDNA production strains identified versus a common strain for pDNA production, DH5α
Strains were grown in shake flasks at 37ºC. Standard error of mean (SEM) is shown in parentheses.
BER
G
Controlled mass production of mouse embry-
onic stem cells in bioreactors
A. Fernandes-Platzgummer, M.M. Diogo, C. Lobato da Silva and J.M.S. Cabral
Embryonic stem cells (ESC) are undifferentiated
cells that have the ability to either self-renew, giv-
ing rise to two identical pluripotent „„daughter‟‟
cells, or to differentiate, producing specialized
cells. These properties make them a very attrac-
tive cell source for stem cell-based therapies, for
developmental biology studies and also for drug/
toxicity-screening. Nonetheless, the successful
implementation of stem cell-based technologies
will require the ability to generate high numbers of
cells with well-defined characteristics. For those
reasons, the goal at the BERG-SCBL is to de-
velop efficient scale-up strategies from the com-
monly used static culture systems (e.g. tissue cul-
ture plates) to dynamic culture systems such as
stirred tank reactors (STR) operating under a con-
tinuous perfusion mode with cell retention. One
important parameter in perfused cultures is the
flow rate at which medium is renewed. The con-
centration of growth factors and nutrients is usu-
ally a growth-rate-limiting factor, as well as unfa-
vorable pH, accumulation of inhibitory metabolites
or a combination of some of these factors. An ex-
cessive medium exchange and/or an unnecessar-
ily high perfusion rate would result in wasting
these valuable components and overdilution of
autocrine factors promoters of cell growth. The
aim of this work was to study the influence of the
residence time on the expansion of mouse ESC
(mESC) using serum-free (SF) medium in a STR
operating under a continuous perfusion mode with
cell retention.
Influence of the residence time on mESC pro-
liferation
A microcarrier-based STR system (Figure 1a),
under SF conditions, was previously established
for mESC expansion using a feeding strategy of
50% medium exchange every day (Fernandes-
Platzgummer, 2011). Herein the residence times
under continuous operation studied were 12, 24,
Figure 1. Expansion of mESC in a stirred tank reactor. (a) New Brunswick Bioreactor (1.3L) and controller, (b) Effect of the residence time of culture medium on 46C mESC growth on Cultispher S microcarriers in a perfused bioreactor culture system using SF medium. Residence times of 96h (p, n=1), 48h (, n=3), 32h (, n=2), 24h (r, n=2) and 12h (, n=2) were studied. Growth curve of the cells fed once per day with 50% medium change every 24 hours (, n=3) is also depicted in figure 1. Cells were inoculated at 5×104 cells/mL on 1 mg of microcarriers per mL of culture medium and agitation rate was set to 60 rpm. Values are represented as mean ± SEM.
( a (b)
20 41
11 Annual Report
R
esearch H
ighlights
32, 48 and 96 hours and the respective growth
curves of mESC are represented in Figure 1b, in
comparison to the previously established discon-
tinuous feeding strategy. The operational pa-
rameters were set to: temperature 37ºC, pH 7.2,
agitation rate 60 rpm, airflow rate 100-200 ccm
and dissolved oxygen concentration 20%.
As it can be seen in Figure 1b, with the exception
of 96h, all the residence times supported mESC
expansion. For the residence time of 96h, in which
only 25% of the medium was exchanged per day,
the cells stopped growing after day 7 probably
due to nutrients depletion and accumulation of
inhibitory metabolites. For the other four residence
times studied, growth curves followed the ex-
pected pattern leading to the maximum cell num-
bers and specific growth rates presented in Table
1. Comparing the growth curve of the cells ex-
panded with a culture medium residence time of
48h (i.e. 50% medium renewal/day) and the
growth curve of the cells fed once per day (i.e.
50% medium renewal/change), it can be observed
that shifting the feeding scheme from discontinu-
ous to continuous mode increased the cell density
by 2-fold. An explanation could be that in the dis-
continuous medium exchange protocol, a large
portion of medium is replaced at a time (50%
every 24h) which might affect cell growth in two
ways: if medium exchange is performed too early
in culture or a large portion of medium is replaced
at a time, a dilution of important autocrine factors
can occur; if medium exchange is performed too
late, an accumulation of toxic metabolic by-
products can inhibit cell growth and ultimately lead
to cell death. On the other hand, in the continuous
perfusion mode, the addition and removal of me-
tabolites and other inhibitors can be made in a
controlled way without the dilution of the autocrine
factors necessary to mESC expansion. Perma-
nent medium exchange via perfusion is thus an
important step forward in the automation and
standardization of the culture conditions.
To evaluate which residence time would contrib-
ute to a higher cell yield, the maximum total cell
number achieved was divided by the total volume
of medium used. As it can be seen in Table 1, the
maximum cell yields, 1.4x106 and 1.2x106 cells
per mL of medium used, were attained with the
residence times of 48 hours and 32 hours, respec-
tively.
Importantly, mESC expanded in the fully con-
trolled STR, using SF medium, retained the ex-
pression of pluripotency markers and their differ-
entiation potential into cells of the three embryonic
germ layers (ectoderm, mesoderm and endo-
derm).
The controlled bioprocess developed herein is
potentially adaptable to other cell types, including
human ESC and induced pluripotent stem cells,
thus representing a promising starting point for the
development of novel technologies for the con-
trolled production of differentiated derivatives from
human pluripotent stem cells.
References:
[1] Fernandes-Platzgummer, A., Diogo, M.M.,
Baptista, R., Lobato da Silva, C., Cabral, J.M.S.,
Biotechnol. Progress, 27, 1421-32 (2011)
Residence times
12 hours 24 hours 32 hours 48 hours
Maximum cell number (cells) 4.0x109 5.5x109 6.4x109 5.5x109
Fold increase 114±5 156±10 184±8 156±19
Specific growth rate (day-1) 1.3±0.1 1.6±0.2 1.3±0.2 1.4±0.1
Doubling time (day) 0.6±0.2 0.4±0.1 0.5±0.1 0.5±0.2
Cell Yield (Cells/mL of medium used) 0.4x106 0.8x106 1.2x106 1.4x106
Table 1. Growth kinetic characterization and cell yields for the different residence times.
BER
G Multifactorial analysis of embryonic stem cell
self-renewal reveals a crucial role of GSK-3-
mediated signaling under hypoxia
H.S.C. Barbosa, T.G. Fernandes, T.P. Dias, M.M. Diogo and J.M.S. Cabral
Work previously performed in our group showed that culturing mouse embryonic stem (mES) cells under different oxygen tensions gave rise to differ-ent cell proliferation patterns and commitment stages depending on which signaling pathways are activated or inhibited to support mES cell self-renewal [1]. These findings indicate that mES cell self-renewal and pluripotency, which are depend-ent on multifactorial signaling networks, can be influenced by different oxygen levels. However, the molecular mechanisms that regulate stem cell fate and function under these conditions are not well understood.
Multifactorial Analysis of Signaling
Networks at Different Oxygen Tensions
To elucidate and dissect how each signaling path-way is functioning at physiological and non-physiological oxygen tensions, we have used a multifactorial approach and response surface methodology. The effects of three independent variables LIF, CHIR99021 (CHIR) and PD0325901 (PD) on the specific growth rate (SGR) and the efficiency in colony formation of mES cells were determined using a face-centered composite design (FC-CD) approach. Therefore, the sole and interactive influence of MEK/ERK pathway inhibition, activation of Wnt/β-Catenin by GSK-3 inhibition, and activation of LIF/STAT3 signaling, was statistically evaluated during ex-pansion of mES cells at different oxygen tensions (Fig. 1). The obtained models were then validated to confirm the effects of each signaling molecules in mES cell expansion and pluripotency at differ-ent oxygen tensions.
Effect of Hypoxia on Mouse ES Cell Expansion
According to the models described above, it is possible to observe that generally mES cells sig-nificantly reduce its propagation in serum-free medium at physiological oxygen levels as com-pared to 20% oxygen conditions (Fig. 2). Taking the highest specific growth rate conditions in both oxygen levels this represents a 6.7 reduction in the cumulative fold increase in total cell number at the end of the tenth day as compared to normoxic (20%) oxygen levels. This higher mES cell prolif-eration rate in normoxia was obtained when the culture medium was supplemented with only one
of the three factors: LIF. None of the two small molecule inhibitors (PD and CHIR) had a signifi-cant impact on mES cell expansion at this O2 level, indicating that LIF/STAT3 signaling was dominant over MEK/ERK and Wnt/β-Catenin sig-naling pathways (Fig. 2a).
On the contrary, under hypoxia, the activation of the Wnt/β-Catenin signaling via inhibition of GSK-3β had a significant influence over the mES cell expansion. In hypoxia, the culture conditions that maximize mES cell specific growth rate are ob-tained when LIF is supplemented into culture me-dium at a concentration of 720 U/mL and CHIR at approximately 3 μM (Fig. 2b). These observations indicate that Wnt signaling mediated by the ca-nonical pathway is not absolutely sufficient and requires an synergistic action of LIF to maintain self-renewal of mouse ES cells under low oxygen levels.
Effect of Hypoxia on
Mouse ES Cell Pluripotency
We also evaluated the capacity of different culture conditions to support mES cell pluripotency by growing these cells at clonal densities. High effi-ciencies of colony formation indicate that mES
Figure 1. Experimental design for the multifactorial
analysis of signaling networks at different oxygen
tensions. A face-centered composite design was
used (I&II) and the obtained models (III) were vali-
dated (IV) to confirm the effects of each signaling
molecules in mES cell expansion and pluripotency at
different oxygen tensions.
20 43
11 Annual Report
R
esearch H
ighlights
cells have a high percentage of survival and can generate a high number of colonies, suggesting that the culture conditions employed can better support mES cell pluripotency. Culturing mES cells at low density in 20% O2 tensions resulted
that all three independent variables under study had a significant influence on the cloning forming efficiency. On the other hand, the presence of both CHIR and LIF are fundamental to obtain high colony formation efficiency at low oxygen tensions under low cell densities.
This was further validated by quantitative PCR and immunofluorescence staining of specific pluri-potency markers (Fig. 3). The expression of pluri-potency genes was up-regulated when CHIR or the two inhibitors (2i) were added with LIF to the culture medium at 20% O2. On the other hand, at 2% O2, absence of CHIR resulted in down-regulation of pluripotency markers, while presence of this molecule in combination with LIF causes maintenance or increasing expression of core pluripotency genes, confirming the results pre-dicted by our models (Fig. 3a). Furthermore, at 2% O2 the absence of CHIR results in colonies with differentiated morphology and cytoplasmic localization of Nanog protein, an indication of early commitment of these cells (Fig. 3b).
Collectively, this approach provided new insights into the mechanisms by which oxygen influences mES cell self-renewal and pluripotency while dis-tinct pathways are activated or inhibited. This modeling approach revealed that at lower O2 ten-sions LIF/STAT3 signaling and Wnt/β-Catenin, in particular, show a significant role towards mainte-nance of mES cell self-renewal and pluripotency. Our results add new insights into the mechanisms by which oxygen tension influences mES cell fate, and GSK-3 inhibition in particular showed an im-portant role towards maintenance of ES cell pluri-potency.
References
[1] Fernandes, T.G., et al., Stem Cell Res., 5, 76-89 (2010)
Figure 2. Number of doubling generations of MSC in
culture along 7 days without being submitted to mi-
croporation (A), and microporated with pDNA (B).
Data were acquired by flow cytometry using cells
previously stained with PKH67 membrane dye. Each
bar represents the percentage of cells in each dou-
bling generation (Generations1–8), that MSC have
undergone at each point in culture [4].
Figure 3. Pluripotency markers were evaluated by quantitative PCR (A) or immunofluorescence staining (B), in cells cultured at the indicated conditions. (A) Heat-maps show relative expression levels of key pluripotency genes. (B) Colo-nies stained for Nanog and nuclear marker DAPI. Scale bar: 50 µm.
Scientific Output
2011 BERG Annual Report
Articles
Proceedings
Book Chapters
Invited Oral Communications
Oral Communications
Poster Communications
Dissertations
Prizes
Books
Patents
Articles in Peer-reviewed Journals
Andrade, P.Z., da Silva, C.L., dos Santos, F.,
Almeida-Porada, G., Cabral, J.M.S., “Initial CD34+
cell-enrichment of cord blood determines hemato-
poietic stem/progenitor cell yield upon ex vivo ex-
pansion”, J. Cell. Biochem., 112(7), 1822-1831
Badenes, S.M., Lemos, F., Cabral, J.M.S., “Stability
of cutinase, wild type and mutants, in AOT reversed
micellar system - effect of mixture components of
alkyl esters production, J. Chem. Technol. Biotech-
nol., 86(1), 34-41
Badenes, S.M., Lemos, F., Cabral, J.M.S.,
“Performance of a cutinase membrane reactor for
the production of biodiesel in organic media”, Bio-
technol. Bioeng., 108(6), 1279-1289
de Barros, D.P.C., Fernandes, P., Cabral, J.M.S.,
Fonseca, L.P., “Synthetic application and activity of
cutinase in an aqueous, miniemulsion model sys-
tem: Hexyl octanoate synthesis”, Catal. Today, 173
(1), 95-102
de Barros, D.P.C., Azevedo, A.M., Cabral, J.M.S.,
Fonseca, L.P., “Optimisation of flavour esters syn-
thesis by Fusarium solani pisi cutinase”, J. Food
Biochem., 58(2), 545-556
Bernardino, S., Estrela, N., Ochoa-Mendes, V., Fer-
nandes, P., Fonseca, L.P., “Optimization in the im-
mobilization of penicillin G acylase by entrapment in
xerogel particles with magnetic properties”, J. Sol-
Gel Sci. Technol., 58(2), 545-556
Borlido, L., Azevedo, A.M., Roque, A.C.A., Aires-
Barros, M.R., “Potential of boronic acid functional-
ized magnetic particles in the adsorption of human
antibodies under mammalian cell culture condi-
tions”, J. Chromatogr. A, 1218(43), 7821-7827
de Carvalho, C.C.C.R., “Enzymatic and whole cell
catalysis: finding new strategies for old processes”,
Biotechnol. Adv., 29(1), 75-83, 2011
de Carvalho, C.C.C.R., Cruz, P.A., da Fonseca,
M.M.R., Xavier-Filho, L., “Antimicrobial properties of
the extract of Abelmoschus esculentus”, Biotechnol.
Bioprocess Eng., 16(5), 971-977
de Carvalho C.C.C.R., Marques, M.P.C., Fernan-
des, P., “Recent achievements on siderophore pro-
duction and application”, Recent Pat. Biotechnol., 5
(3), 183-198
Carvalho, J.A., Azzoni, A.R., Prazeres, D.M.F.,
Monteiro, G.A., “Comparative analysis of antigen-
targeting sequences used in DNA vaccines”, Mol.
Biotechnol., 47(1), 94-94
Correia, V.G., Bonifácio, V.D., Raje, V.P., Casimiro,
T., Moutinho, G., da Silva, C.L., Pinho, M.G., Aguiar
-Ricardo, A., “Oxazoline-based antimicrobial oli-
gomers: synthesis by CROP using supercritical
CO2”, Macromol. Biosci., 11(8), 1128-1137
Costa, E., de Carvalho, J., Casimiro, T., da Silva,
C.L., Cidade, M., Aguiar-Ricardo, A., “Tailoring ther-
moresponsive microbeads in supercritical carbon
dioxide for biomedical applications, J. Supercrit.
Fluid, 56(3), 292-298
Coutinho, C.P., de Carvalho, C.C.C.R., Madeira, A.,
Pinto-de-Oliveira, A., Sá-Correia, I., “Burkholderia
cenocepacia clonal phenotypic variation during
three and a half years of residence in the lungs of a
cystic fibrosis patient”, Infection and Immunity, 79
(7), 2950-2960
Fernandes, P., Carvalho, F., Marques, M.P.C.,
“Miniaturization in Biotechnology: speeding up the
development of bioprocesses”, Recent Pat. Biotech-
nol., 5(3), 160-173
Fernandes-Platzgummer, A., Diogo, M.M., Baptista,
R.P., Silva, C.L., Cabral, J.M.S., “Scale-up of
mouse embryonic stem cell expansion in stirred
bioreactors”, Biotechnol. Progr., 27(5), 1421-1432
Gomes, A.G., Azevedo, A.M., Aires-Barros, M.R.,
Prazeres, D.M.F., “Studies on the adsorption of cell
impurities from plasmid-containing lysates to phenyl
boronic acid chromatographic beads”, J. Chroma-
togr. A., 1218, 8629-8637
Guerrero-German, P., Montesinos-Cisneros, R.M.,
Prazeres, D.M.F., Tejeda-Mansir, A., “Purification of
plasmid DNA from Escherichia coli ferments using
anion-exchange membrane and hydrophobic chro-
matography”, Biotechnol. Appl. Biochem., 58(1), 68-
74
Publications
20 47
11 Annual Report
R
esearch H
ighlights
José, G.E., Folque, F., Menezes, J.C., Werz, S.,
Strauss, U., Hakemeyer, C., “Predicting mab prod-
uct yields from cultivation media components, using
NIR and 2D-fluorescence spectroscopies”, Biotech-
nol. Prog., 27(2), 1339-1346
Joskowiak, A., Santos, M.S., Prazeres, D.M.F.,
Chu, V., Conde, J.P., “Integration of thin film amor-
phous silicon photodetector with lab-on-chip for
monitoring protein fluorescence in solution and in
live microbial cells, Sens. Actuators B, 156(2), 662-
667
Lança, A.S.C., de Sousa, K.F., Atouguia, J., Praz-
eres, D.M.F., Monteiro, G.A., Silva, M.S.,
“Trypanosoma brucei: Immunisation with plasmid
DNA encoding invariant surface glycoprotein gene
is able to induce partial protection in experimental
African trypanosomiasis”, Exp. Parasitol., 127(1), 18
-24
Lima, D.M., Fernandes, P., Nascimento, D.S.,
Ribeiro, R.C.L.F., Assis, S.A., “Fructose syrup: A
biotechnology asset”, Food Technol. Biotechnol., 49
(4), 424–434
Loureiro, J., Andrade, P.Z., Cardoso, S., da Silva,
C.L . , Cabral , J .M.S. , Frei tas, P .P. ,
“Magnetoresistive chip cytometer”, Lab on a chip,
11(13), 2255-2261
Loureiro, J., Andrade, P.Z., Cardoso, S., da Silva,
C.L., Cabral, J.M.S, Freitas, P.P., “Spintronic chip
cytometer”, J. Appl. Phys., 109(7), 07B311
Lourenço, N.M.T., Oesterreicher, J., Vidinha, P.,
Barreiros, S., Afonso, C.A.M., Cabral, J.M.S., Fon-
seca, L.P., “Effect of gelatin-ionic liquid functional
polymers on glucose oxidase and horseradish pero-
xidase kinetics”, React. Funct. Polym., 71(4), 489-
495
Lourenço, V., Herdling, T., Reich, G., Menezes,
J.C., Lochmann, D., “Combining microwave reso-
nance technology to multivariate data analysis as a
novel PAT tool to improve process understanding in
fluid bed granulation”, Eur. J. Pharm. Biopharm. 78
(3), 513-521
Madeira, C., Ribeiro, S.C., Pinheiro, I.S.M., Martins,
S.A.M., Andrade, P.Z., da Silva, C.L., Cabral,
J.M.S., “Gene delivery to human bone marrow mes-
enchymal stem cells by microporation”, J. Biotech.,
151(1), 130-136
Madeira, C., Loura, L.M.S., Aires-Barros, M.R.,
Prieto, M., “Fluorescence methods for lipoplex char-
acterization”, Biochim. Biophys. Acta – Biomembr.,
1808(11), 2694-2705
Marques, M.P.C., Fernandes, P., “Microfluidic de-
vices: Useful tools for bioprocess intensification”,
Molecules, 16(10), 8368-8401
Martins, D.C., Prazeres, D.M.F., Chu, V., Conde,
J.P., “Label-free detection of DNA immobilization
and hybridization by streaming current measure-
ments in microchannels”, App. Physics Lett., 99,
183702
Nascimento, K.S., Yelo, S., Cavada, B.S., Azevedo,
A.M., Aires-Barros, M.R., “Liquid-Liquid equilibrium
data for aqueous two-phase systems composed of
ethylene oxide propylene oxide copolymers”, J.
Chem. Eng. Data, 56(2), 190-194
Novo, P., Prazeres, D.M.F., Chu, V., Conde, J.P.,
“Microspot-based ELISA in Microfluidics: Chemilu-
minescence and colorimetry detection using inte-
grated thin-film hydrogenated amorphous silicon
photodiodes”, Lab-on-a-Chip, 11, 4063-4071
Oliveira, P.H., Prather, K.J., Prazeres, D.M.F., Mon-
teiro, G.A., “Mutation detection in plasmid-based
biopharmaceuticals”, Biotechnol. J., 6(4), 378-391
Pereira, A.T., Novo, P., Prazeres, D.M.F., Chu, V.,
Conde, J.P., “Heterogeneous immunoassays in
microfluidic format using fluorescence detection
with integrated amorphous silicon photodiodes”,
Biomicrofluidics, 5(1), 014102
Puchert, T., Holzhauer, C.V., Menezes, J.C., Loch-
mann, D., Reich, G., “A new PAT/QbD approach for
the determination of blend homogeneity: Combina-
tion of on-line NIRS analysis with PC Scores Dis-
tance Analysis (PC-SDA)”, Eur. J. Pharm. Bio-
pharm. 78 (1), 173–182
Puchert, T., Lochmann, D., Menezes, J.C., Reich,
G., “A multivariate approach for the statistical
evaluation of near-infrared chemical images using
symmetry parameter image analysis (SPIA)”, Eur. J.
Pharm. Biopharm. 78 (1), 117–124
Rodrigues, C.A.V., Diogo, M.M., da Silva, C.L.,
Cabral, J.M.S., “Microcarrier expansion of mouse
embryonic stem cell-derived neural stem cells in
stirred bioreactors”, Biotechnol. Appl. Biochem., 58
(4), 231-242
Rodrigues, C.A.V., Fernandes, T.G., Diogo, M.M.,
da Silva, C.L., Cabral, J.M.S., “Stem cell cultivation
in bioreactors”, Biotechnol. Adv., 29(6), 815-829
Rosa, P.A.J., Azevedo, A.M., Sommerfeld, S.,
Baecker, W., Aires-Barros, M.R., “Aqueous two-
phase extraction as a platform in the biomanufactur-
ing industry: Economical and environmental sus-
tainability”, Biotechnol. Adv., 29(6), 559-567
Sampaio, P.N., Sousa, L., Calado, C.R.C., Pais,
M.S., Fonseca, L.P., “Use of chemometrics in the
selection of a Saccharomyces cerevisiae expres-
sion system for recombinant cyprosin B production”,
Biotechnol. Lett, 33(11), 2111-2119
Santa, G.L.M., Bernardino, S.M.S.A., Magalhães,
S., Mendes, V., Marques, M.P.C., Fonseca, L.P.,
Fernandes, P., “From inulin to fructose syrups using
sol-gel immobilized inulinase”, Appl. Biochem. Bio-
technol., 165(1), 1-12
Santos, F.D., Andrade, P.Z., Abecasis, M.M., Gim-
ble, J.M., Chase, L.G., Campbell, A.M., Boucher,
S., Vemuri, M.C., Silva, C.L., Cabral, J.M.S.,
“Toward a clinical-grade expansion of mesenchymal
stem cells from human sources: a microcarrier-
based culture system under xeno-free conditions”,
Tissue Eng. Part. C Methods, 17(12), 1201-1210
Sousa, A.G., Andrade, P.Z., Pirzgalska, R.M., Gal-
hoz, T.M., Azevedo, A.M., da Silva, C.L., Aires-
Barros, M.R., Cabral, J.M.S., “A novel method for
human hematopoietic stem/progenitor cell isolation
from umbilical cord blood based on immunoaffinty
aqueous two-phase partitioning”, Biotechnol. Lett.,
33, 2373-2377
Székely, Gy., Bandarra, J., Heggie, W., Sellergren,
B., Ferreira, F.C., “Organic solvent nanofiltration: A
platform for removal of genotoxins from active phar-
maceutical ingredients”, J. Membr. Sci., 381(1-2),
21-33
Tyagi, M., da Fonseca, M.M.R., de Carvalho,
C.C.C.R., “Bioaugmentation and biostimulation
strategies to improve the effectiveness of bioreme-
diation processes”, Biodegradation, 22(2), 231-241
Editorials about BERG publications
Jungbauer, A. (2011). Improved products and proc-
esses through biochemical engineering science.
Biotechnol. J., 6, 362-363 (Editorial accompanying
article from Oliveira et al., 2011, 6, 378-391)
Articles in National Journals
Ferreira, F., Llussá, F., Moreira, J.N., Prazeres,
D.M., Rocha I., Rodrigues, L., “Bioteams: do Labo-
ratório para o Mercado”, Boletim de Biotecnologia,
Abril, 23
Prazeres, D.M.F., “Biomímica”, Ingenium, Jan/Fev,
86-88
Prazeres, D.M.F., Monteiro, G.A., “Vacinas de DNA
salvam o condor da Califórnia da extinção”, Biologia
e Sociedade, Nº 12, 21-25
Articles in Conference Proceedings
Azevedo, A.M., Aires-Barros, M.R., “Novel strate-
gies for the purification of monoclonal antibodies”,
Proceedings of the 1st Portuguese Meeting in Bio-
engineering: Bioengineering and Medical Sciences -
The Challenge of the XXI century | ENBENG 2011,
Oeiras, Portugal, March (R. Martins, Editor), pp 72-
75
Azevedo, A.M., Aires-Barros, M.R., “New platforms
for the downstream processing of biopharmaceuti-
cals”, Proceedings of the 1st Portuguese Meeting in
Bioengineering: Bioengineering and Medical Sci-
ences - The Challenge of the XXI century | EN-
BENG 2011, Oeiras, Portugal, March (R. Martins,
Editor), pp 80-83
Fernandes, T.G., Diogo, M.M., Dordick, J.S.,
Cabral, J.M.S., “Exploring embryonic stem cell fate
using cellular microarrays”, Proceedings of the 1st
Portuguese Meeting in Bioengineering: Bioengi-
neering and Medical Sciences - The Challenge of
the XXI century | ENBENG 2011, Oeiras, Portugal,
March (R. Martins, Editor), pp 34-37
Femandes, T.G., Diogo, M.M., Fernandes-
Platzgummer, A., Lobato da Silva, C., Cabral,
J.M.S., “Effect of hypoxia on proliferation and neural
commitment of embryonic stem cells at different
stages of pluripotency”, Proceedings of the 1st Por-
tuguese Meeting in Bioengineering: Bioengineering
and Medical Sciences - The Challenge of the XXI
20 49
11 Annual Report
R
esearch H
ighlights
century | ENBENG 2011, Oeiras, Portugal, March
(R. Martins, Editor), pp 45-48
Fernandes, T.G., Kwon, S.J., Bale, S.S., Lee, M.Y.,
Diogo, M.M., Clark, D.S., Cabral, J.M.S., Dordick,
J.S., “3D cell culture microarray for high-throughput
studies of stem cell fate”, Abstracts of Papers of
the American Chemical Society, 241, 168-BIOT
Fonseca, L.P., Martins, V.C.B., Freitas, P.P.,
“Microreactors and microdevices for analytical and
biosensors applications”, Proceedings of the 1st
Portuguese Meeting in Bioengineering: Bioengi-
neering and Medical Sciences - The Challenge of
the XXI century | ENBENG 2011, Oeiras, Portugal,
March (R. Martins, Editor), pp 146-149
da Silva, C.L., Andrade, P.Z., dos Santos, F.,
Cabral, J.M.S., “Ex-vivo expansion of hematopoietic
stem cells from umbilical cord blood”, Proceedings
of the 1st Portuguese Meeting in Bioengineering:
Bioengineering and Medical Sciences - The Chal-
lenge of the XXI century | ENBENG 2011, Oeiras,
Portugal, March (R. Martins, Editor), pp 49-52
Lourenço, N.M.T., Osterreicher, J., Cabral, J.M.S.,
Fonseca, L.P., Vidinha, P., Barreiros, S.,
“Evaluation of Ion Jelly biopolymer on glucose bio-
sensing”, Proceedings of the 1st Portuguese Meet-
ing in Bioengineering: Bioengineering and Medical
Sciences - The Challenge of the XXI century | EN-
BENG 2011, Oeiras, Portugal, March (R. Martins,
Editor), pp 142-145
Madeira, C., Ribeiro, S.C., Mendes, R., Pinheiro,
I.S.M., da Silva, C.L., Cabral, J.M.S., “Genetic engi-
neering of stem cells by non-viral vectors”, Proceed-
ings of the 1st Portuguese Meeting in Bioengineer-
ing: Bioengineering and Medical Sciences - The
Challenge of the XXI century | ENBENG 2011, Oei-
ras, Portugal, March (R. Martins, Editor), pp 41-44
Marques, M.P.C., Fernandes, P., de Carvalho,
C.C.C.R., “Process intensification platforms for ap-
plication in bioengineering”, Proceedings of the 1st
Portuguese Meeting in Bioengineering: Bioengi-
neering and Medical Sciences - The Challenge of
the XXI century | ENBENG 2011, Oeiras, Portugal,
March (R. Martins, Editor), pp. 64-67
Mateus, M., Raiado-Pereira, L., Prazeres, M.,
“Membrane chromatography for therapeutic DNA
production: Adsorption membranes development”,
Proceedings of the 1st Portuguese Meeting in Bio-
engineering: Bioengineering and Medical Sciences -
The Challenge of the XXI century | ENBENG 2011,
Oeiras, Portugal, March (R. Martins, Editor), pp 68-
71
Oliveira, P.H., Boura, J.S., Abecasis, M.M., da
Silva, C.L., Cabral, J.M.S, “An appraisal of genetic
stability in human mesenchymal stem cells”, Pro-
ceedings of the 1st Portuguese Meeting in Bioengi-
neering: Bioengineering and Medical Sciences -
The Challenge of the XXI century | ENBENG 2011,
Oeiras, Portugal, March (R. Martins, Editor), pp. 57-
59
Prazeres, D.M.F., Martins, S.A.M., Trabuco, J.R.C.,
Monteiro, G.A., Juskowiak, A., Conde, J.P., Chu, V.,
“Towards a high-throughput drug discovery platform
for the screening of GPCR targets in cells”, Pro-
ceedings of the 1st Portuguese Meeting in Bioengi-
neering: Bioengineering and Medical Sciences -
The Challenge of the XXI century | ENBENG 2011,
Oeiras, Portugal, March (R. Martins, Editor), pp 1-4
Rodrigues, C.A.V., Diogo, M.M., Lobato da Silva,
C., Cabral, J.M.S., “Design and operation of biore-
actor systems for the expansion of pluripotent stem
cell-derived neural stem cells”, Proceedings of the
1st Portuguese Meeting in Bioengineering: Bioengi-
neering and Medical Sciences - The Challenge of
the XXI century | ENBENG 2011, Oeiras, Portugal,
March (R. Martins, Editor), pp 38-40
Sousa, A.F.,Loureiro, J., Diogo, M.M., Cabral,
J.M.S., Freitas, P.P., “Magnetic separation of undif-
ferentiated mouse Embryonic Stem (ES) cells from
neural progenitor cultures using a microfluidic de-
vice”, Proceedings of the 1st Portuguese Meeting in
Bioengineering: Bioengineering and Medical Sci-
ences - The Challenge of the XXI century | EN-
BENG 2011, Oeiras, Portugal, March (R. Martins,
Editor), pp 23-26
Books
Prazeres, D.M.F., “Plasmid Biopharmaceuticals:
Basics, Applications and Manufacturing”, 2011,
John Wiley & Sons, Inc., New York (ISBN: 978-0-
470-23292-7)
Undey, C., Low, D., Menezes, J.C., Koch, M., “PAT
Applied in Biopharmaceutical Process Development
and Manufacturing An Enabling Tool for Quality-by-
Design”, 2011, Eds Cenk Undey, Duncan Low, J.C.
Menezes, Mel Koch, CRC Press, USA, 327 pp
(ISBN: 978-1439829455)
Book Chapters
de Carvalho, C.C.C.R., da Fonseca, M.M.R.
“Bioreactions and bioreactor operation | Biotransfor-
mations”, in: Engineering Fundamentals in Biotech-
nology, Vol. 2 (Collin Webb, Ed.), Comprehensive
Biotechnology, 2nd Edition (Editor-in-Chief: Murray
Moo-Young), Elsevier, Oxford, pp. 451-460
dos Santos, F., Andrade, P.Z., Eibes, G., da Silva,
C.L., J.M.S. Cabral, “Ex-vivo expansion of human
mesenchymal stem cells on microcarriers”, in: Mes-
enchymal Stem Cell Assays and Applications,
Methods in Molecular Biology, Vol 698, Part 2
(Vemuri, Mohan; Chase, Lucas G.; Rao, Mahendra
S., editors), Humana Press/Springer, New York, pp.
189-198
Felizardo, P., Menezes, J.C., Neiva-Correia, M.J.,
“PAT use in biofuels manufacturing”, in: PAT Ap-
plied in Biopharmaceutical Process Development
and Manufacturing An Enabling Tool for Quality-by-
Design, Eds Cenk Undey, Duncan Low, Jose C.
Menezes, Mel Koch CRC Press, Seattle, pp 201-
221
Fernandes, P., Marques, M.P.C., Carvalho, F., de
Carvalho, C.C.C.R., “Organic-solvent tolerant Gram
-positive bacteria: applications and mechanisms of
tolerance”, In: Organic solvents: properties, toxicity
and industrial effects (Ryan E. Carter, Ed.), Nova
Science Publishers, Hauppauge, New York, pp. 89-
103
Lourenço, N.M.T., Nunes, A.V.M., Duarte, C.M.M.,
Vidinha, P., “Ionic liquids gelation with polymeric
materials: the ion jelly approach” in: Applications of
Ionic Liquids in Science and Technology (Scott
Handy editor), Middle Tennessee State University,
USA, pp 155-172
Menezes, J.C., “Process analytical technology and
quality by design in bioprocess development and
manufacturing”, in: Industrial Biotechnology, Vol 3,
Ed. A Moreira, in Comprehensive Biotechnology,
2nd Edition (Editor-in-Chief: Murray Moo-Young),
Elsevier, Oxford, pp. 501-509
Patents
de Carvalho, C.C.C.R., Marques, M.P.C.,
“Dispositivo para diluições sucessivas em micro-
placas”, Provisional patent nr 105887. Priority date:
14 September 2011
Invited Oral Communication
International Conferences
Hakemeyer, C., Werz, S., Folque, F., José, G.,
Menezes, J.C., Strauss, U., “At-Line NIR spectros-
copy as a simple and effective PAT monitoring tech-
nique in mab cultivations during process develop-
ment and manufacturing”, AIChE Annual Meeting,
Minneapolis, USA, October
Ferreira, I.F., de Carvalho, C.C.C.R., Wang, D.I.C.,
Aires-Barros, M.R., “Crude oil microbial desulfuriza-
tion: A viable green technology for sulfur elimination
in refineries”, The 11th International Chemical and
Biological Engineering Conference | CHEMPOR
2011, Caparica, Portugal, September
Lourenço, V., Herdling, T., Reich, G., Menezes,
J.C., Schewitz, J., “Combining microwave reso-
nance technology to multivariate data analysis as a
novel PAT tool to improve process understanding in
fluid bed granulation”, AIChE Annual Meeting, Min-
neapolis, USA, October
Menezes, J.C., “From process-centered to product-
centered QbD”, EUROPACT 2011, Glasgow, Scot-
land, April [Keynote Lecture]
Menezes, J.C., “Quality by design: Tools and Plat-
forms”, 5th International Congress Pharma. Engin-
nering, Graz, Austria, September
Menezes, J.C., “PAT in different industries: Chal-
lenges & Opportunities for NIRS”, NIR2011, Cape
Town, South Africa, May [Keynote Lecture]
Menezes, J.C., “Modern pharmaceutical develop-
ment and manufacturing: A decade into using multi-
variate data analysis”, - 11th annual conference of
the European Network for Business and Industrial
Statistics | ENBIS-11, Coimbra, Portugal, Septem-
ber
Menezes, J.C., “Process Analytical Technology
(PAT) across different industries: Challenges and
20 51
11 Annual Report
opportunities in Process Development”, - The 11th
International Chemical and Biological Engineering
Conference | CHEMPOR 2011, Caparica, Portugal,
September
Schewitz, J., Herdling, T., Lochmann, D., Reich, G.,
Menezes, J.C., “Real-Time release strategy in
MERCK SERONO: A pharmaceutical industry per-
spective”, 25th International Process Analytical
Technology Forum | IFPAC, Baltimore, Maryland
(USA), January
Schewitz, J., Herdling, T., Lochmann, D., Reich, G.,
Menezes, J.C., “PAT strategy in Merck Serono: A
pharmaceutical industry perspective”, 8th European
Congress of Chemical Engineering, Berlin, Ger-
many, September
National Conferences
Fernandes, P. “Miniaturization in bioprocesses: a
resilient approach or just another fad?” 4th Joint
National Congress of Microbiology and Biotechnol-
ogy | Microbiotec11, Braga, Portugal, December
Madeira, C., Cabral, J.M.S., “Células estaminais e
cartilagem”, 3º Curso teórico-prático de Cartilagem
Articular, Lisbon, Portugal, November
Madeira, C., Cabral, J.M.S., “Gene delivery to adult
stem cells: pre-clinical studies and clinical trials”, IX
Encontro de Engenharia Biomédica, IST/FMUL,
Hospital de Santa Maria, Lisbon, Portugal, Novem-
ber
Oliveira, P.H., “Biofármacos: Desafios e Limita-
ções”, Tertúlias FNACiência, FNAC Guimarães,
Portugal, June
Oliveira, P.H., “Da sala de aula ao laboratório –
Uma experiência na primeira pessoa”, Jornadas de
Engenharia Química e Biológica (JEQB), Instituto
Superior Técnico, Lisbon, Portugal, March
Oral Communications
International Conferences
Bessa, A., Madeia, P.P., Ribeiro, L.A., Aires-Barros,
M.R., Reis, C.A., Rodrigues, A.E., Zaslavsky, B.Y.,
“Solvnet features of ATPS formed by different poly-
mers and salt additives”, 16th International Confer-
ence on Biopartitioning and Purification | BPP2011,
Puerto Vallarta, Mexico, September
Borlido, L., Azevedo, A.M., Roque, A.C.A., Aires-
Barros, M.R., “Affinity based purification of human
monoclonal antibodies from CHO cell supernatants
using boronic acid magnetic particles”, 19th Biennial
Meeting of the International Society for Molecular
Recognition | Affinity 2011, Tavira, Portugal, June
de Carvalho, C.C.C.R., “Bacterial adaptation to anti-
neoplastic agents involve biofilm formation”, 15th
International Biodeterioration & Biodegradation
Symposium | IBBS-15, Wien, Austria, September
Ferreira, I.F., de Carvalho, C.C.C.R., Wang, D.I.C.,
Aires-Barros, M.R., “Crude desulfurization in or-
ganic aqueous phase biocatalytic systems”, 15th
International Biodeterioration & Biodegradation
Symposium | IBBS-15, Wien, Austria, September
Gomes, A.G., Azevedo, A.M., Aires-Barros, M.R.,
Prazeres, D.M.F., “On the adsorption of cell impuri-
ties from plasmid-containing lysates to phenyl boro-
nate beads”, 19th Biennial Meeting of the Interna-
tional Society for Molecular Recognition | Affinity
2011, Tavira, Portugal, June
Raiado-Pereira, L., Carapeto, A., Mateus, M., Bo-
telho-do-Rego, A.M., “Grafting hydrophobic and
affinity interaction ligands on membrane adsorbers:
a close-up view by X-ray Photoelectron Spectros-
copy”, 11th International Chemical and Biological
Engineering Conference | CHEMPOR 2011, Lisbon,
Portugal, September
Rosa, P.A.J., Azevedo, A.M., Aires-Barros, M.R.,
“New platforms for the downstream processing of
antibodies”, 16th International Conference on
Biopartitioning and Purification | BPP2011, Puerto
Vallarta, Mexico, September
Nascimento, K.S., Santos, J.A., Cavada, B.S.,
Azevedo, A.M., Aires-Barros, M.R., “Polishing
strategies for the purification of Canavalia brasilien-
sis lectin (ConBr) two-phase extracts: Ultrafiltration
vs. multistage extraction”, 16th International Confer-
ence on Biopartitioning and Purification | BPP2011,
Puerto Vallarta, Mexico, September
Nunes, M.A., Fernandes, P.C., Ribeiro, M.H.,
“Microtiter plates as a representative system for
enzymatic hydrolysis with PVA-lens shaped parti-
cles”, 19th Biennial Meeting of the International So-
ciety for Molecular Recognition | Affinity 2011, Ta-
vira, Portugal, June
Rocha, A., Lourenço, N.M.T, “Novel choline based
zwitterions”, International Conference on Materials
and Technologies for Green Chemistry, Tallinn,
Estonia, September
Trabuco, J.R., Martins, S.A.M., Monteiro, G.A.,
Conde, J.P., Chu, V., Prazeres, D.M.F., “Testing G
protein coupled receptor targets in cells at different
scales using fluorescence microscopy: A tool for the
development of microfluidic platforms”, 19th Biennial
Meeting of the International Society for Molecular
Recognition | Affinity 2011, Tavira, Portugal, June
National Conferences
Azevedo, A.M., Aires-Barros, M.R., “Novel strate-
gies for the purification of monoclonal antibodies”,
1st Portuguese Meeting in Bioengineering: Bioengi-
neering and Medical Sciences - The Challenge of
the XXI century | ENBENG 2011, Oeiras, Portugal,
March
Azevedo, A.M., Aires-Barros, M.R., “New platforms
for the downstream processing of biopharmaceuti-
cals”, 1st Portuguese Meeting in Bioengineering:
Bioengineering and Medical Sciences - The Chal-
lenge of the XXI century | ENBENG 2011, Oeiras,
Portugal, March
Barbosa, H.S.C., Fernandes, T.G., Diogo, M.M.,
Cabral, J.M.S., “Application of a central composite
design for modeling mouse embryonic stem cell self
-renewal at different O2 levels”, 6th International
Meeting of the Portuguese Society for Stem Cells
and Cell Therapy | SPCE-TC, Cantanhede, Portu-
gal, April
Borlido, L., Azevedo, A.M., Roque, A.C.A., Aires-
Barros, M.R., “Potential of boronic acid magnetic
particles in the direct purification of human mono-
clonal antibodies from CHO cell supernatants”, 4th
Joint National Congress of Microbiology and Bio-
technology | Microbiotec11, Braga, Portugal, De-
cember
dos Santos, F., Lobato da Silva, C., Andrade, P.Z.,
Abecasis, M.M., Gimble, J.M., Campbell, A.M.,
Boucher, S., Roos, E., Kuligowski, S., Chase, L.G.,
Vemuri, M.C., Cabral, J.M.S., “Clinical grade expan-
sion of human mesenchymal stem cells using a
microcarrier-based system under serum-free and
xeno-free conditions”, 6th International Meeting of
the Portuguese Society for Stem Cells and Cellular
Therapy | SPCE-TC, Cantanhede, Portugal, April
Fernandes, T.G., Rodrigues, C.A.V., Miranda, C.C.,
Diogo, M.M., Cabral, J.M.S., “Towards fully defined
culture systems for human induced pluripotent stem
cell expansion” 4th Joint National Congress of Mi-
crobiology and Biotechnology | Microbiotec11,
Braga, Portugal, December
Madeira, C., Reis, M.S.C., Ferreira, F.F.C.G., Rodri-
gues, C.A.V., Diogo, M.M., Cabral, J.M.S., “Non-
viral gene delivery strategies using minicircles into
Neural Stem Cells” 4th Joint National Congress of
Microbiology and Biotechnology | Microbiotec11,
Braga, Portugal, December
Marques, M.P.C., Fernandes, P., de Carvalho,
C.C.C.R., “Process intensification processes for
application in bioengineering”, 1st Portuguese Meet-
ing in Bioengineering: Bioengineering and Medical
Sciences - The Challenge of the XXI century | EN-
BENG 2011, Oeiras, Portugal, March
Martins, A.I.F., Brogueira, P., Mateus, M., “On the
development of a plasmid DNA probe for interaction
force measurements and characterization of mem-
brane adsorbers by AFM”, 4th Joint National Con-
gress of Microbiology and Biotechnology | Microbio-
tec11, Braga, Portugal, December
Martins, S.M.A, Trabuco, J., Monteiro, G.A., Conde,
J.P., Chu, V., Prazeres, D.M.F., “Microfluidic cell
chips: monitoring GPCR activation”, 4th Joint Na-
tional Congress of Microbiology and Biotechnology |
Microbiotec11, Braga, Portugal, December
Monteiro, G.A., “Rational engineering of E. coli
strains and plasmids for improved manufacturing of
plasmid biopharmaceuticals”, 4th Joint National
Congress of Microbiology and Biotechnology | Mi-
crobiotec11, Braga, Portugal, December [Keynote
Lecture]
Oliveira, P.H., Boura, J.S., Abecasis, M.M., da
Silva, C.L., Cabral, J.M.S., “An appraisal of genetic
stability in human mesenchymal stem cells”, 1st Por-
tuguese Meeting in Bioengineering: Bioengineering
and Medical Sciences - The Challenge of the XXI
century | ENBENG 2011, Oeiras, Portugal, March
Oliveira, P.H., Boura, J.S., Abecasis, M.M., Gimble,
J., da Silva, C.L., Cabral, J.M.S., “Genetic stability
20 53
11 Annual Report
during the ex-vivo expansion of human mesenchy-
mal stem cells for clinical applications”, 6th Interna-
tional Meeting of the Portuguese Society for Stem
Cells and Cellular Therapy | SPCE-TC, Cantan-
hede, Portugal, April
Poster Communications
International Conferences
Azevedo, A.M., Gomes, A.G., Borlido, L., Prazeres,
D.M.F., Aires-Barros, M.R., “Novel capture step for
the purification of human monoclonal antibodies:
Phenyl boronate chromatography”, 16th Interna-
tional Conference on Biopartitioning and Purification
| BPP2011, Puerto Vallarta, Mexico, September
Azevedo, A.M., Lopes, N.S., Gomes, A.G., Borlido,
L., Prazeres, D.M.F., Aires-Barros, M.R., “Phenyl
boronate chromatography as a new platform in the
downstream processing of monoclonal antibodies”,
19th Biennial meeting of the International Society for
Molecular Recognition | Affinity 2011, Tavira, Portu-
gal, June
Barbosa, H.S.C., Fernandes, T.G., Diogo, M.M.,
Cabral, J.M.S, “Modeling mouse embryonic stem
cell self-renewal at different O2 levels using a facto-
rial design approach”, 9th ISSCR Annual Meeting,
Toronto, Canada, June
Bessa, A., Madeia, P.P., Ribeiro, L.A., de Barros,
D.P.C., Azevedo, A., Aires-Barros, M.R., Reis, C.A.,
Rodrigues, A.E., Zaslavsky, B.Y., “Solute descrip-
tors for free amino acids obtained by partitioning in
ATPS formed by different polymers”, 16th Interna-
tional Conference on Biopartitioning and Purification
| BPP2011, Puerto Vallarta, Mexico, September
Borlido, L., Azevedo, A.M., Roque, A.C.A., Aires-
Barros, M.R., “Feasibility og human antibody purifi-
cation by boronic acid magnetic particles”, 16th In-
ternational Conference on Biopartitioning and Purifi-
cation | BPP2011, Puerto Vallarta, Mexico, Septem-
ber
Borlido, L., Azevedo, A.M., Roque, A.C.A., Aires-
Barros, M.R., “Ion-exchange purification of human
antibodies using negatively charged magnetic parti-
cles”, 11th International Chemical and Biological
Engineering Conference | CHEMPOR 2011, Lisbon,
Portugal, September
de Barros, D.P.C., Madeia, P.P., Azevedo, A., Reis,
C.A., Rodrigues, A.E., Baptista, A.M., Aires-Barros,
M.R., “Amino acids partitioning in aqueous two-
phase polymer/polymer systems”, 19th Biennial
meeting of the International Society for Molecular
Recognition | Affinity 2011, Tavira, Portugal, June
Carvalho, R.Jr., Azevedo, A.M., Cramer, S.M., Aires
-Barros, M.R., “Purification of monoclonal antibod-
ies (mAbs) using continuous bed chromatography
with cryogel monoliths support”, 19th Biennial meet-
ing of the International Society for Molecular Recog-
nition | Affinity 2011, Tavira, Portugal, June
de Carvalho, C.C.C.R., “Improving the bioremedia-
tion abilities of Rhodococcus erythropolis”, 15th In-
ternational Biodeterioration & Biodegradation Sym-
posium | IBBS-15, Wien, Austria, September
dos Santos, F., Lobato da Silva, C., Andrade, P.Z.,
Abecasis, M.M., Gimble, J.M., Campbell, A.M.,
Boucher, S., Roos, E., Kuligowski, S., Chase, L.G.,
Vemuri, M.C., Cabral, J.M.S., “Clinical grade expan-
sion of human mesenchymal stem cells using a
microcarrier-based system under serum-free and
xeno-free conditions” 17th International Society for
Cellular Therapy Annual Meeting | ISCT, Rotter-
dam, Netherlands, May
Fernandes-Platzgummer, A., Diogo, M.M., Lobato
da Silva, C., Cabral, J.M.S., ”Culture of embryonic
stem cells in stirred biorreactors”, 2nd ESACT Meet-
ing, Vienna, Austria, May
Figueira, J.A., Sato, H.H., Fonseca, L.P., Fernan-
des, P., “Screening of methods for β-glucosidase
immobilization”, 9th Congress Carbohydrate Bioen-
geneering Meeting | CBM9, Lisbon, Portugal, May
Gomes, A.G., Azevedo, A.M., Santos, J.A.L., Aires-
Barros, M.R., Prazeres, D.M.F., “A novel integrated
plasmid purification process based on phenyl-
boronate adsorption", 16th International Conference
on Biopartitioning and Purification | BPP2011,
Puerto Vallarta, Mexico, September
Gomes, A.M.P., Prazeres, D.M.F., Santos, J.A.L.,
“Plasmid DNA recovery and purification by tangen-
tial flow filtration”, International Congress on Mem-
branes and Membranes Processes | ICOM2011,
Amsterdam, Netherlands, July
Lourenço N.M.T., Monteiro C.M., Afonso C.A.M.,
“One-pot enzymatic resolution of sec-alcohols”, 10th
International Symposium on Biocatalysis | Biotrans
2011, Sicily, Italy, October
Lourenço N.M.T., Rocha, A., “Synthesis of new
ionic liquids based on choline moiety”, International
Conference on Materials and Technologies for
Green Chemistry, Tallinn, Estonia, September
Marques, S., Alves, L., Matos, C.T., Roseiro, J.C.,
Santos, J.A.L., “Development of a membrane-
recycle bioreactor for lactic acid production from
recycled paper sludge”, International Congress on
Membranes and Membranes Processes|
ICOM2011, Amsterdam, Netherlands, July
Martins, J.D., Tavares, E., Gomes, A.M.P., Praz-
eres, D.M.F., Santos, J.A.L., “Mechanical cell lysis
technique for plasmid DNA release”, 8th European
Congress of Chemical Engineering/ 1st European
Congress of Applied Biotechnology | ECCE &
ECAB, Berlin, Germany, September
Martins, S.A.M., Trabuco, J.R.C., Antunes, P., Con-
de, J.P., Chu, V., Prazeres. D.M.F., “A microfluidic
cell-based assay to monitor endogenous GPCR
activation”, European Lab Automation Congress |
ELA 2011, Hamburg, Germany, June-July
Monteiro, C.M., Lourenço, N.M.T., Afonso, C.A.M.,
“Simple and more sustainable enzymatic resolution-
separation of sec-alcohols based on nanofiltration”,
10th International Symposium on Biocatalysis | Bio-
trans 2011, Sicily, Italy, October
Nascimento, K.S., Cavada, B.S., Azevedo, A.M.,
Aires-Barros, M.R., “Purification of the lectin Cana-
valia brasiliensis (ConBr) using aqueous two-phase
extraction: Back-extraction studies”, 19th Biennial
meeting of the International Society for Molecular
Recognition | Affinity 2011, Tavira, Portugal, June
Novo, P., Moulas, G., Chu, V., Prazeres, D.M.F.,
Conde, J.P., “Lab-on-a-Chip ochratoxin a detection
using competitive ELISA in microfluidics with inte-
grated photodiode signal acquisition”, Eurosensors
XXV, Athens, Greece, September
Oliveira, P.H., da Silva, C.L., Cabral, J.M.S.,
“Unusual DNA structures and instability motifs cor-
relate with human mitochondrial deletion break-
points involved in genetic disorders and cancer”,
11th International Symposium on Mutations in the
Genome, Santorini, Greece, June
Oliveira, P.H., Boura, J.S., Abecasis, M.M., Gimble,
J., da Silva, C.L., Cabral, J.M.S., “An appraisal of
genetic stability during the ex-vivo expansion of
human mesenchymal stem cells”, 11th International
Symposium on Mutations in the Genome, Santorini,
Greece, June
Rodrigues, C.A.V., Diogo, M.M., Lobato da Silva,
C., Cabral, J.M.S., “Scaling-up the expansion of
mouse embryonic stem cell-derived neural stem
cells in stirred bioreactors”, 9th Annual Meeting |
ISSCR, Toronto, Canada, June
Sousa, A.F., Pirzgalska, R.M., Galhoz, T.M.,
Andrade, P.Z., Azevedo, A.M., da Silva, C.L., Aires-
Barros, M.R., Cabral, J.M.S., “Novel selection
method for human hematopoietic stem cell isola-
tion”, 19th Biennial meeting of the International Soci-
ety for Molecular Recognition | Affinity 2011, Tavira,
Portugal, June
Székely, G., Bandarra, J., Heggie, W., Sellergren,
B., Ferreira, F., “Organic solvent nanofiltration for
removal of genotoxins from active pharmaceutical
ingredient”, International conference of membranes
and membrane processes | ICOM, Amsterdam,
Netherlands, July
Székely, G., Bandarra, J., Heggie, W., Ferreira, F.,
Sellergren, B., “Molecularly imprinted polymers and
organic solvent nanofiltration – A hybrid process for
removal of 1,3-diisopropylurea impurity from Active
Pharmaceutical Ingredients”, 19th Biennial meeting
of the International Society for Molecular Recogni-
tion | Affinity 2011, Tavira, Portugal, June
National Conferences
Barbosa, H.S.C., Fernandes, T.G., Dias, T.P.,
Diogo, M.M., Cabral, J.M.S., “A factorial design ap-
proach for modeling mouse embryonic stem cell self
-renewal at different O2 levels”, 4th Joint National
Congress of Microbiology and Biotechnology | Mi-
crobiotec11, Braga, Portugal, December
Boura, J.S., dos Santos, F., Gimble, J.M., Cardoso,
C., Madeira, C., Cabral, J.M.S., da Silva, L. C.,
“Non-viral gene delivery to mesenchymal stem cells
of human sources using cationic liposomes”, 6th
International Meeting of the Portuguese Society for
Stem Cells and Cellular Therapy | SPCE-TC, Can-
tanhede, Portugal, April
Coutinho, C.P., de Carvalho, C.C.C.R., Madeira, A.,
Pinto-de-Oliveira, A., Sá-Correia, I., “Burkholderia
20 55
11 Annual Report
cenocepacia clonal phenotypic variation during long
-term colonization of a cystic fibrosis patient lungs”,
4th Joint National Congress of Microbiology and
Biotechnology | Microbiotec11, Braga, Portugal,
December
Fernandes, T.G., Rodrigues, C.A.V., Miranda, C.C.,
Diogo, M.M., Cabral, J.M.S., “Towards fully defined
culture systems for human induced pluripotent stem
cell expansion”, 6th International Meeting of the Por-
tuguese Society for Stem Cells and Cell Therapy |
SPCE-TC, Cantanhede, Portugal, April
Monteiro, M.E., Raiado-Pereira, L., Mateus, M.,
Prazeres, D.M.F., “Design of a liposome-based
chromatographic membrane and its use for final
plasmid DNA purification from Escherichia coli lys-
ate contaminants”, 4th Joint National Congress of
Microbiology and Biotechnology | Microbiotec11,
Braga, Portugal, December
Dissertations
Ph.D. Thesis
Alexandra R. Gonçalves, PhD in Chemistry,
“Developing analytical chemistry knowledge, based
on process impurities detection in pharmaceutical
formulations”, IST/UTL (Supervisors: J.C. Menezes,
J.M. Martins).
Ana Gabriela Gonçalves Neves Gomes, PhD in
Biotechnology, “Intermediate recovery of plasmid
DNA based on aqueous two-phase systems and
phenyl -boronate adsorpt i t ion”, UTL/IST
(Supervisors: D.M.F. Prazeres, M.R. Aires-Barros)
Ana Margarida Pires Fernandes Platzgummer,
PhD in Bioengineering, “Bioreactor culture systems
for the expansion of mouse embryonic stem cells”,
UTL/IST (Supervisors: J.M.S. Cabral, C. Lobato da
Silva, M.M.R. Diogo)
Carlos André Vitorino Rodrigues, PhD in Bioengi-
neering, “Design and operation of bioreactor sys-
tems for the expansion and controlled neural differ-
entiation of stem cells”, UTL/IST (Supervisors:
J.M.S. Cabral, C. Lobato da Silva, M.M.R. Diogo)
Francisco Ferreira dos Santos, PhD in Biotech-
nology, “Isolation and ex-vivo expansion of mesen-
chymal stem cells for supplementation during he-
matopoietic stem cell transplantation”, UTL/IST
(Supervisors: J.M.S. Cabral, C. Lobato da Silva)
Isabel Filipa Prates Acciaioli Hilário Ferreira,
PhD in Bioengineering, “Biodesulfurization of crude
oil by whole cells of Rhodoccocus Erythropolis”,
UTL/IST (Supervisors: M.R. Aires Barros, C.C.C.R
de Carvalho, D.I.C. Wang)
Pedro Miguel Zacarias Andrade, PhD in Bioengi-
neering, “Novel approaches for the isolation and ex-
vivo expansion of hematopoietic stem cells from
human umbilical cord blood for cell therapy”, UTL/
IST (Supervisors: J.M.S. Cabral, C. Lobato da
Silva)
Vera Mónica de Campos Loures Lourenço, PhD
in Chemical Engineering, “A quality by design study
of an industrial fluid bed granulation process”, IST/
UTL (Supervisors: J.C. Menezes, D. Lochmann)
M.Sc. Thesis
Ana Rita de Matos Parente Vasconcelos, MSc in
Biological Engineering, “Concepção e desenvolvi-
mento de um bloqueador de cimento ósseo”, UTL/
IST (Supervisors: M.R. Aires Barros, L. Pinto)
Cátia M.M. Sousa, MSc in Pharmaceutical Engi-
neering, “The Application of Quality by Design to
Evaluate the Robustness of an Analytical Method”,
UTL (Supervisors: J.C. Menezes, S. Queirós)
Cláudia Daniela Canelas Miranda, MSc in Bio-
technology, “Towards fully defined culture systems
for human induced pluripotent Stem Cell expan-
sion”, UTL/IST (Supervisors: M.M. Diogo, T. Fer-
nandes)
David Soares da Conceição, MSc in Bioengineer-
ing and Nanosystems, “Stem Cells in microfluidics -
Controlling the celular environment of microspotted
Stem Cells”, UTL/IST (Supervisors: M.M. Diogo,
J.P. Conde)
Daniel Filipe Camarneiro Silva, MSc in Biotech-
nology, “Antibody separation using aqueous two-
phase systems in a microfluidic”, UTL/IST
(Supervisors: M.R. Aires Barros, J.P. Conde)
Elisabete Marques Ribeiro, “Towards production
scale with microreactors. Early steps to crack the
paradox”, UTL/IST (Supervisor: P. Fernandes)
Filipa Esteves Leal Rodrigues de Carvalho, MSc
in Biological Engineering, “Design of validation mas-
ter plan for pharmaceutical industry and process
validation of lyophilized drug”, UTL/IST
(Supervisors: J.M.C. Menezes, S. Pereira)
Filipa Fiel do Carmo Glórias Ferreira, MSc in Bio-
technology, “Novel plasmid-based vectors for gene
delivery to Neural Stem Cells”, UTL/IST
(Supervisor: C. Madeira)
Francisco Tavares Marinho Mendes, MSc in Bio-
logical Engineering, “Estudo da eficiência de um
processo de produção de bolachas sustentado na
gestão da qualidade”, UTL/IST (Supervisors: M.
Mateus, R. Machado)
Isabela Dodd Gueiros, MSc in Biotechnology,
“Screening enzymatic systems for selective methyl
ester production”, UTL/IST (Supervisors: F.C.
Ferreira, P. Fernandes, C. Fonseca)
Irina Neves Simões, MSc in Biotechnology,
“Isolation, characterization and ex-vivo expansion of
mesenchymal stem cells from umbilical cord ma-
trix” (Supervisors: J.M.S. Cabral, C. Lobato da
Silva)
Joana Baltazar Domingues, MSc in Biological
Engineering, “Stability assessment of biopharma-
ceutical formulations”, UTL/IST (Supervisors: A.M.
Azevedo, J.A. Santos)
Joana da Costa Branco, MSc in Biological Engi-
neering, “Development of a yeast based platform for
the screening of compounds that modulate TTR
toxicity”, UTL/IST (Supervisors: F.C. Ferreira, P.
Calado)
Joana Lopes Pereira, MSc in Biological Engineer-
ing, “Development of meat alternatives - Under-
standing fiber formation of vegetable proteins”, UTL/
IST (Supervisors: M. Mateus, F. van de Velde)
Joana Rita Pires Bentes Gil, MSc in Biotechnol-
ogy, “Development of DNA vacines prototypes
against avian influenza viruses”, UTL/IST,
(Supervisors: G.A. Monteiro, M. Fevereiro)
José Frederico Silva Oliveira, MSc in Biological
Engineering, “An integrated process for the purifica-
tion of antibodies based on magnetic particles and
aqueous two-phase systems”, UTL/IST
(Supervisors: M.R. Aires Barros, A.M. Azevedo)
João Miguel da Costa Medeiros, MSc in Biologi-
cal Engineering, “Elucidation of endogenous
haematopoietic cytokines production in a three-
dimensional biomimicry of human bone marrow”,
(Supervisors: C. Lobato da Silva, A. Mantalaris)
João Pedro dos Santos Borges, MSc in Mechani-
cal Engineering, “Desenvolvimento de técnicas
baseadas em filmes de células bacterianas para
aplicação em ensaios não destrutivos (END) de
materiais de Engenharia”, UNL/FCT (Supervisors:
T. Santos, C.C.C.R. Carvalho)
João Porfírio da Silva Burgal, MSc in Biological
Engineering, “Production of recombinant human
cytochrome P450 (1A1) in E. coli JM109: Fed-batch
fermentation in 20 L scale with a novel phage resis-
tant strain”, (Supervisors: D.M.F. Prazeres, M. Kit-
telmann)
João Rodrigo Cardoso Trabuco, MSc in Biotech-
nology, “Towards the miniaturization of cell assays
for GPCR monitoring”, UTL/IST (Supervisors:
D.M.F. Prazeres J.P. Conde)
Márcia Andreia Faria da Mata, MSc in Biological
Engineering, “Tools for transient manipulation of
HSC using non-integrating retroviral vectors”,
(Supervisors: C. Lobato da Silva, S. Howe)
Marina Eduarda Santos Valada Monteiro, MSc in
Biotechnology, “Design of a liposome-based chro-
matographic membrane and its use for final plasmid
DNA purification from Escherichia coli lysate con-
taminants”, UTL/IST (Supervisor: M. Mateus)
Marta Taveira Santos Castro Silva, MSc in Celu-
lar Biology, “Avaliação da capacidade de extractos
voláteis de plantas aromáticas para inibir a forma-
ção de biofilmes bacterianos”, FCUL (Supervisors:
C.C.C.R. Carvalho, A.C. Figueiredo)
Nancy Hachicho, Master of Science, “Adaptation
of Rhodococcus opacus to different chlorophenols
and carbon sources”, Universität Leipzig
(Supervisors: H.J. Heipieper, C.C.C.R. Carvalho)
Nicolau F. Dehanov, MSc in Pharmaceutical Engi-neering, “Técnicas de calibração espectroscópica baseadas na estimativa do ruído espectral e do sinal de resposta aplicadas a espectros de infraver-melhos médios (MIR) de amostras de culturas de células estaminais”, FF/UL (Supervisor: J.C. Mene-zes)
20 57
11 Annual Report
Núria Catarina Mendes da Silva Lopes, MSc in
Biotechnology, “Purification of monoclonal antibod-
ies by phenyl boronate chromatography”, UTL/IST
(Supervisors: A.M. Azevedo, M.R. Aires Barros)
Pedro Almeida Nolasco, MSc in Bioengineering
and Nanosystems, “Structural and mechanical char-
acterization of Sialoliths, UTL/IST (Supervisor: P.
Almeida de Carvalho, M.M. Diogo)
Raphaël Faustino Canadas, MSc in Bioengineer-
ing and Nanosystems, “Electrospun nanofibers for
human stem cell cultivation”, UTL/IST (Supervisors:
F.C. Ferreira, C. Lobato da Silva)
Roksana Maria Pirzgalska, MSc in Biotechnology,
“Optimization of aqueous two-phase systems for
human hematopoietic stem cells separation”, UTL/
IST (Supervisors: M.R. Aires Barros, A.M. Azevedo)
Sandra Cristina Sarmento Donato dos Santos e
Silva, MSc in Biotechnology, “Dielectrophoresis - A
Biological Approach - Positive and Negative Dielec-
trophoresis of E. coli in a Microfluidic Environment”,
UTL/IST(Supervisors: J.P. Conde, D.M.F. Prazeres)
Sofia Machado Pinheiro, MSc in Biochemistry,
“Desenvolvimento de métodos para estudo de inibi-
dores da acetillcolinesterase (Tratamento sintomáti-
co da Doença de Alzheimer)”, UL/FC (Supervisors:
M.L.M.O.M Serralheiro, P. Fernandes)
Sofia de Oliveira Dias Duarte, MSc in Applied Mi-
crobiology, “The role of calcium in Saccharomyces
sp. In response to etanol stress”, FCUL,
(Supervisors: G.A. Monteiro, A. Tenreiro)
Tatiana Vieira Arriaga, MSc in Biological Engineer-
ing, “Controlled and tailored denaturation and ag-
gregation of whey proteins”, UTL/IST (Supervisors:
M. Mateus, T. Huppertz)
Teresa Margarida da Silveira e Silva Galhoz,
MSc in Biotechnology, “Production of monoclonal
antibodies by hybridoma cell culture”, UTL/IST
(Supervisors: A.M. Azevedo, C. Lobato da Silva)
Tiago Miguel Peixoto Dias, MSc in Biotechnology,
“Molecular Mechanisms Underlying Modulation of
Mouse Embryonic Stem (ES) Cell Self-Renewal
under Different Oxygen Tensions, UTL/IST
(Supervisors: M.M.R. Diogo, T.P.G. Fernandes)
Tomás Miguel de Freitas Dias, MSc in Bioengi-
neering and Nanosystems, “Magnetoresistive Chip-
based platform for the evaluation of cfDNA integrity
as a potential biomarker in cancer diagnosis”, UTL/
IST (Supervisor: G.A. Monteiro)
Vera Sequeira Ribeiro Guerra, MSc in Biotechnol-
ogy, “High throughput in biocatalysis: steroid bio-
conversions”, UTL/IST (Supervisors: P. Fernandes,
M.P.C. Marques)
Awards
UTL/Santander Totta Scientific Award
Pedro Fernandes was distinguished with the UTL/
Santander Totta Scientific Award, in the area of
Biological Engineering.
Carla C.C.R. de Carvalho was distinguished with an
Honorable Mention by UTL/Santander Totta, in the
area of Biological Engineering.
UTL/Deloitte Young Researchers Award
Nuno M.T. Lourenço has been distinguished with
the Young Researchers UTL/Deloitte Award in the
scientific areas of Chemistry and Biochemistry.
Filipa Ferreira has been distinguished with the
Young Researchers UTL/Deloitte Award in the sci-
entific areas of Biological Engineering and Biotech-
nology.
Roche Young Investigator Award
Luis Borlido was distinguished with the Roche
Younger Investigator Award 2011 for best oral com-
munication, at the Affinity 2011, the 19th biennial
meeting of the International Society for Molecular
Recognition, Tavira, Portugal.
João Trabuco was distinguished with the Roche
Younger Investigator Award 2011 for best oral com-
munication, at the Affinity 2011, the 19th biennial
meeting of the International Society for Molecular
Recognition, Tavira, Portugal.
Oral Presentation Award
I. Filipa Ferreira was distinguished with the Best
Oral Presentation Award for the presentation:
I.Filipa Ferreira, Carla C.C.R. de Carvalho, Daniel
I.C. Wang, M. Raquel Aires-Barros, Crude oil micro-
bial desulfurization: a viable green technology for
sulfur elimination in refineries. ChemPor2011, Lis-
bon, 5-7 September 2011.
Poster Presentation Award
Marina Monteiro was distinguished with the Best
Poster Presentation Prize in Bioprocess Engineer-
ing for the presentation: Monteiro, M.E., Raiado-
Pereira, L., Mateus, M., Prazeres, D.M.F., “Design
of a liposome-based chromatographic membrane
and its use for final plasmid DNA purification from
Escherichia coli lysate contaminants”, 4th Joint Na-
tional Congress of Microbiology and Biotechnology |
Microbiotec11, Braga, Portugal, December
Tiago Dias was distinguished with the Best Poster
Presentation Prize in Cell and Tissue Engineering
and Biomaterials for the presentation: Barbosa,
H.S.C., Fernandes, T.G., Dias, T.P., Diogo, M.M.,
Cabral, J.M.S., “A factorial design approach for
modeling mouse embryonic stem cell self-renewal
at different O2 levels”, 4th Joint National Congress
of Microbiology and Biotechnology | Microbiotec11,
Braga, Portugal, December
20 59
11 Annual Report
Staff
Faculty
Joaquim M.S. Cabral
Maria Raquel Aires-Barros
Duarte Miguel Prazeres
Luís Fonseca
José Menezes
Cláudia Lobato da Silva
Gabriel Monteiro
José Santos
Maria Ângela Taipa
Marília Mateus
Frederico Ferreira
Research Scientists
Ana Azevedo
Carla C.C.R. de Carvalho
M. Margarida Diogo
Pedro Fernandes
Teresa Catarina Madeira
Post-doctoral Fellows
Ana Fernandes-Platzgummer
Carlos Rodrigues
Dragana de Barros
Francisco dos Santos
Hélder Barbosa
Marco Marques
Nuno Lourenço
Pedro Oliveira
Pedro Andrade
Sara Badenes
Sofia Martins
Tiago Fernandes
PhD Students
Aruna Santhagunam
Cláudia Miranda
Cláudia O. Silva
David Malta
Filipe Carvalho
Geisa Gonçalves
Irina Simões
Irina Pinheiro
Javad Hatami
Joana Boura
João Guerreiro
Jonathan de la Vega
Luís Borlido
Luís Raiado Pereira
Michaela Simcikova
Miriam Sousa
Mónica Coelho
Nuno Faria
Patrícia Soares
Ricardo Figueiredo
Rimenys Jr. Carvalho
Salomé Magalhães
Tiago Dias
Tomás Dias
Vera Lourenço
Master Students
Ana Rosa
Ana Vencá
Andreia Dias
Andreia Fernandes
Andreia Matos
Antónia Pinto
António Soure
Beatriz Monteiro
Bruno Alves
Elisabete Freitas
Francisco Moreira
Inês Ferreira
Joana Serra
Joana Batista
João Anes
Maria Ana Cortes
Marta Costa
Marta de Castro Silva
Marta Silva
Nadiya Kubasova
Raquel Correia
Rita Costa
Rita Martins
Sara Matias
Sara Rosa
Research Assistants
Ana I. Martins
Ana Maria Gomes
Daniel Silva
João Trabuco
Marina Monteiro
Mário Fonseca
Rui Carvalho
Sara Gomes Pereira
Sofia Duarte
Technician
Ricardo Pereira
BERG
BioEngineering Research Group
Institute for Biotechnology and Bioengineering
Instituto Superior Técnico
Av. Rovisco Pais
1049-001 Lisbon
Portugal
www.ibb.pt/berg
top related