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

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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

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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

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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-

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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

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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)

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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

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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

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• 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

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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.

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• 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,

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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%

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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)

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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

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after adaptation

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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.

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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)

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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

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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)

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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).

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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

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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

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0.334

% T

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0.012

-200

0

200

400

600

800

1000

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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

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1,0E-10

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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.

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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)

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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.

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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

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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