Biotecnología y bioingenieria en México

Biotecnología y bioingenieria en México

Biotecnología y Bioingeniería en Mexico:
Experiencias y Oportunidades
Marco Rito-Palomares
Centro de Biotecnología-FEMSA
Octubre, 2013
¿Qué es la Biotecnología?
Es la conjunción de disciplinas
científicas y tecnológicas
orientadas a la utilización de
células vivas y/o sus
componentes para la
producción de bienes y
servicios.
Biotecnología en el tiempo
• 1750 A.C. – Somerios utilizan levadura
para obtener cerveza.
• 1863 – Mendel descubre la transmisión
de información de genes.
• 1906 – El término genética es introducido.
• 1919 – El término biotecnología es
utilizado por primera vez.
• 1928 – La Penicilina es descubierta.
• 1953 – Watson y Crick descubren la
estructura de doble hélice del DNA.
El progreso rápido en
Biotecnología inicia
•
•
•
•
•
1960 –
1965 –
1966 –
1973 –
1981 –
Primer antibiótico sintético.
Fusión de celulas de ratón y humanas.
Entendimiento del codigo genético.
Desarrollo de la técnica de manipulación de genes.
Primer animal transgénico.
Biotecnología en los últimos 30 años
 1983 – Producción comercial de insulina
 1985 – Plantas modificadas genéticamente son probadas en campo.
 1986 – Uso de microorganismos para la limpieza de derrames de
aceite.
 1988 – Primer patente de un animal alterado genéticamente – Ratón
transgénico.
 1997 – Clonación de la oveja Dolly.
 2002 – Proyecto del genoma humano terminado.
 2010 - Aplicaciones con células madres
La nueva economía del siglo XXI
Biotecnología es uno de los sectores de
mayor crecimiento a nivel mundial
Promueve la llamada
nueva “economía basada
en el conocimiento”.
Áreas de aplicación de la
Biotecnología
– Salud:
Medicamentos y vacunas.
– Alimentos: Nutracéuticos y alimentos funcionales, probióticos.
– Agricultura: Plantas y animales modificadas genéticamente más
resistentes y rendidores bioreguladores y pesticidas.
– Empaques: Envases plásticos biodegradables de almidón y otras fuentes.
– Textil:
Vestimentas faded de mezclilla y prendas de poliester.
– Química:
Pinturas, detergentes, cosméticos, elaboración de papel.
Áreas de aplicación de la
Biotecnología
–
–
–
–
Fármacos:
Bebidas:
Combustibles:
Medicina clínica:
– Servicios:
Vitaminas, antibióticos, enzimas.
Fructosa y otros productos edulcorantes.
Etanol y otros combustibles sofisticados.
Diagnósticos rápidos y en línea para la identificación
de enfermedades y agentes causantes.
Sanidad, toxicología, inocuidad alimentaría,
propagación masiva de plantas.
Productos biotecnológicos
Proteínas
Hormonas
Vacunas
Antibióticos
Aminoácidos
Vitaminas
Esteroides
Enzimas
Colorantes
Aromas
Productos
biotecnologicos
Tamaño de mercado y costo
El costo de desarrollo de un producto biotecnológico es superior a los
$100 millones solo en I&D, cuando se requieren pruebas clínicas.
Resumen de ventas de Productos
biotecnologicos
La contribución total al mercado de este tipo de productos
es del orden de billones de dólares
Bioingeniería:
Procesos Biotecnológicos
Extra cellular
concentration
Primary
recovery
Production
Purification
Cell disruption
Intracellular
Purification cost is up to 85 % of total processing costs
(Bio) Process technology
Purification cost is up to 85 % of total processing costs
Procesos biotecnológicos
Insulina humana
Paso Proceso
1
2
3
4
5-6
7
8
9
10
11-12
14
15
16
17
18
19
20
Flujo (Kg/dia)
Fermentación
Centrifugación
Homogenización
Centrifugación
Solubilización/precipitación
Centrifugación
Degradación química
Ultrafiltración
Evaporación
Solubilización/oxidación
Intercambio aniónico
Degradación enzimática
Cromatografía HPLC
Ultrafiltración
Cristalización
Centrifugación
Evaporación
Rendimiento total
11.16
11.10
10.99
10.93
8.20
8.16
7.75
7.71
7.48
5.61
5.33
5.06
3.54
3.52
3.17
3.15
3.13
Eficiencia (%)
100
99.5
99.0
99.5
75.0
99.5
95.0
99.5
97.0
75.0
95.0
95.0
70.0
99.5
90.0
99.5
99.5
28%
Procesos biotecnológicos
Hormona de crecimiento bovino
Paso
1
2
3
4-5
6
7
8-9
10
11
12
13
14
15
16
Proceso
Separación celular
Ruptura celular
Centrifugación
Lavado/centrífuga
Extracción
Centrifugación
Lavado/centrifugación
Desnaturación/oxidación
Centrifugación
Ultrafiltración
Filtración en gel(alim)
Filtración en gel (elución)
Diálisis (alim)
Diálisis (dializado)
Centrifugación
Ultrafiltración
Rendimiento total
Volumen (L)
20
3X20
20
15
12
12
12
70
70
70
4
18
97
2640
97
97
Eficiencia (%)
98
98
98
98
100
98
98
100
92
95
47
72
72
100
19%
Áreas de oportunidad para desarrollo de
Procesos Biotecnológicos
Reducción del número
de etapas
Optimización de
procesos
(técnicas novedosas)
Integración de procesos
Experiencias y Oportunidades
Experiencias y Oportunidades
Bioproducts from Algae
Development of a
prototype ATPS process for
c-phycocyanin recovery
from Spirulina maxima
Spirulina maxima
(Arthrospira)
• Cyanobacteria
• Photosynthetic system
• Optimal temperature and
pH for cultivation 35°C
and 8-11, respectively
• Growth easily in high
content of sodium
carbonate
Uses for c-phycocyanin (CPC)
Colorant for food
products.
Colorant for
cosmetics.
Lab reagent.
Cells coloring
Commercial value of c-phycocyanin
C-phycocyanin food grade
$ 130 USD / g
C-phycocyanin reagent grade
$ 1000-5000 USD / g
C-phycocyanin highly purified
$ 15,000 USD / g
Existing protocols for the recovery of cphycocyanin produced by Spirulina maxima
Fermentation
(Spirulina
maxima)
Gel filtration
Harvesting
Dialysis
Drying
Precipitation
Cell
disruption
Adsorption
CaCl2
Extraction
Centrifugation
Existing protocols for the recovery of cphycocyanin produced by Spirulina maxima
The maximum purity of the product is not
reached.
Excessive number of unit operations.
Low product recovery.
Difficult to scale up.
An alternative approach is needed
Fermentation
S. maxima concentration (0.26 g/L
dry weight)
Fermentation conditions:
Temperature 30 - 35 ° C
CO 2 y air supplied
Culture media composition
H2O
NaCl
CaCl2
Na2SO4
FeSO4
KNO3
MgSO4
NaHCO3
K2HPO4
4.00 L
4.00 g
0.16 g
8.32 g
0.04 g
8.00 g
0.83 g
36.0 g
2.00 g
Process development for the recovery of cphycocyanin produced by Spirulina maxima
Model systems
Effect of TLL
Effect of volume ratio
Effect of MW of PEG
Prototype process for the recovery of cphycocyanin produced by Spirulina maxima
Fermentation
(Spirulina
maxima)
Cell
disruption
1st ATPS
Extraction
PEG + Salt
2nd ATPS
Extraction
Ultrafiltration
PEG and
contaminants
PEG + Salt
Concentrate
Contaminants
Contaminants
Highly purified CPC
Product yield 28%
Commercial value: $15,000 USD/g
Precipitation
C-phycocyanin
Prototype process for the recovery of cphycocyanin produced by Spirulina maxima
A prototype process for B-phycoerythrin
purification from Porphyridium cruentum
B-phycoerythrin (BPE)
• B-phycoerythrin, a pink-coloured protein.
• Produced by red algae as accessory pigment.
• The commercial value of highly purified Bphycoerythrin (> 4, defined as the
relationship of 545 – 280 nm absorbances)
for pharmaceutical or fluorescent uses can
be more than US$ 50/mg.
A prototype process for B-phycoerythrin
purification from Porphyridium cruentum
BPE applications
• Pigment for food industry
• Pigment for cosmetic industry
• Pharmaceutics
• Fluorescent marker
Improved recovery of B-phycoerythrin produced
by the red microalga Porphyridium cruentum
Develop a process to further increase the purity
of BPE above 4.0 (A545nm/A280nm)
BPE recovery (%)
Purity of BPE (A545nm/A280nm)
Recovery of natural colorants from
microbial origin: B-phycerythrin BPE
System pH
A patent granted
Recovery of natural colorants
from microbial origin: Bphycerythrin BPE
A pilot plant for the validation of this prototype process has been established.
Scaling up of the prototype process for the
purification of BPE
Scale up from 10mL to 8.5Lts
(850X extraction system)
Scaling up of the prototype process for the
purification of BPE
Preliminary economic analysis
$ 1.17 US/mg highly purified BPE
Considering raw material, reagents and energy requirements
Carotenoids bioproducts from
Algae
Lutein produced by Chlorella
protothecoides
 Carotenoid (xanthophyll)





Low molecular weight hydrophobic compound
Proven benefits for human health
Found in vegetables, flowers, algae, etc
Chlorella protothecoides – Sweet water algae
Commonly used for heterotrophic production of
lutein
 Interesting study case for ATPS
Recuperacion de Luteina
Efficient Extraction and Harvesting
of Cyanobacterial Products by TwoPhase Separation
Lutein & Carotene
Recovery of lutein produced by Chlorella
protothecoides
Experience on process development for
particulate purification
Extractive fermentation
Proteins from plants
New devices
Experience on plant based bioprocess
development
Plantas como sistemas productores
The most common used plants for biopharmaceuticals
production are: tobacco, maize, soybean, and alfalfa.
Challenge:
-- High concentration of
contaminant proteins
-- Amount of target protein
Limitations:
-- Low yields.
-- Inconsistent product quality.
-- COMPLEXITY OF HOST PROTEOME
A recent model protein: rhG-CSF
• Human Granulocyte-Colony Stimulating Factor (hG-CSF)
A glycoprotein which stimulates granulocyte colony formation acting
on hematopoietic cells
• Application:
–
–
–
–
–
Treatment of neutropenia in cancer therapy: leukemia
Bone marrow trasplant, BMT
HIV-associated neutrophil defects
USD$800 /mg
aprox. USD $250 per single dose
rhG-CSF
• It has been expressed in: bacteria (E. coli), conventional yeast,
mammalian cells, and plants (tomato and tobacco).
Production
(bioreactor)
Recovery and
primary
purification
Purification
General Bioprocess Diagram
A successful plant-based production
system will be a frequently used
technology when an easy and cheap
purification process is applied.
Available product
from E. coli
Recuperación del producto
1
Protein
Extraction
Buffer
Tris-Borate-EDTA,
pH 8
Model
protein
rhG-CSF
2
3
4
1.
2.
3.
4.
Molecular marker
Alfalfa/rhG-CSF
Upper phase
rhG-CSF
ATPS
Target product
Gel SDSPAGE &
MS
Vr = 1,
pH 7,
Phosphate salts
1
2
3
4
1.
2.
3.
4.
Molecular marker
Alfalfa/rhG-CSF
Bottom phase
rhG-CSF
Modificación de Farmacos
PEGylation of therapeutic proteins
Improved clinical properties:

Better physical and thermal stability

Protection against susceptibility to
enzymatic degradation

Increased solubility

Longer in vivo circulation
Approved for human use by the U.S. Food and Drug
Administration
Greenwald et al, 2003
PEGylation reaction
PEGylation is a reaction in which at least one chain of
polyethylene glycol (PEG) is attached to a molecule or
protein without changes in its properties.
Protein
mPEG
Model Proteins
a - Lactalbumin
Ribonuclease A

Obtained from bovine pancreas.

Present in mammal milk whey.

MW: 13,686 Da.

MW: 14,176 Da.

124 amino acid residues.

126 amino acid residues.

Antitumoral properties.

Antitumoral properties.
Products from the PEGylation reaction
 Three resulting species are reported for the PEGylation
reactions of both model proteins:
Native
Protein
mono-PEGylated
Protein
di-PEGylated
Protein
 The mono-PEGylated protein in both models presents the
best biological activity.
Separation of PEGylated proteins
• Two basic challenges:
– the separation of PEG-proteins from other
reaction products.
– the sub-fractionation of PEG-proteins.
Separation of PEGylated
proteins
Absorbance 210 nm (mAU)
1200
mono
PEGR
Nase
A
1000
800
600
diPEG
RNase
A
400
200
0
0
50
100
150
Volume (mL)
200
Native
RNase
A
250
300
Experiencias en el desarrollo de
procesos para recuperar bioparticulas
Rotavirus-like particles primary
recovery from insect cells
 Virus like-particles (VLP) are composed
of the main structural proteins of a virus,
but lack its genetic material.
 They are produced by the recombinant
expression of the structural proteins
 Applications: vaccination, biosensors,
nanomaterials.
VP6 VP2
Double layered rotavirus-like
particles (dlRLP)
 Consist of two concentric
protein layers.
VP6 VP2
 The inner layer is formed by
protein VP2, while the outer
layer is formed by protein
VP6.
 dlRLP induce immune
response to treat acute
gastroenteritis*.
*Annually, more than 500,000 children die as victims of acute
gastroenteritis caused by rotavirus (Kirkburk and Buttery, 2003).
Rotavirus-like particles primary
recovery from insect cells
Insect cell culture
Centrifugation
Extra cellular dlRLP
Sucrose cushion
CsCl gradient
Ultrafiltration
2% dlRLP recovery
90% dlRLP purity
45 purification factor
Insect cell culture
Biomass
Biomass
Centrifugation
Cell disruption
Extra cellular dlRLP
ATPS extraction
Intracellular dlRLP
Ultrafiltration
85% dlRLP recovery
90% dlRLP purity
30 - 55 purification factor
Strategies for the potential recovery and
purification of stem cells
Background
Background
STEM
CELLS
replace
diseased
damagedcells
dead
Stop /
Reverse
Disease
s
Objective
Develop a scalable and novel bioengineering strategy for the
potential recovery and purification of stem cells.

Unique, fast, economic and scalable bioprocess.

Exploiting non-conventional technologies as
Aqueous Two-Phase Systems (ATPS).

Allowing manipulation of high quantities of
sample, reducing losses and processing times.
Production
Primary recovery
Purification
Methodology: model system
Experimental Matrix
Measurement system
Human umbilical cord
blood
Flow Cytometry
employing specific
stem cell marker
(CD133 antibody) and
7AAD for viability.
Lymphoprep
Sample rich
in CD133+
stem cells
used in
ATPS
PUBLISHED ARTICLE:
González-González, M., and M. Rito-Palomares, 2013, Aqueous two-phase systems strategies to establish novel
bioprocesses for stem cells recovery: Critical Reviews in Biotechnology, p. 1-10.
Strategy II. Immunoaffinity ATPS
• PEGylated antibody
1) DEX 70,000-Ficoll 400,000
2) DEX 10,000-PEG 10,000
Phases
formation
PEGylated CD133 antibody
cells addition into ATPS
and
Release of
stem cells
CD133+
Purified
CD133+
stem cells
Comentarios y mensaje final
Biotecnología en México
Áreas
Enfoque
Generación de
Transferencia de
conocimiento
Universidades
VS
tecnología
Industria
Comentarios y mensaje final
La Biotecnología establece una plataforma que nos
permite soñar. Es importante que esos sueños se
conviertan en metas, para impactar la calidad de vida.
 Imaginar
i
4
=
 Investigar
 Innovar
 Incubar
Research Group Members
Prof. Marco A.
Rito-Palomares
Chair Director

SNI III

AMC Member

Line: Bioprocesses and Purification

Post-doc at Cambridge University

Ph.D. At Birmingham University
Dr. Jorge
Benavides Lozano
Dr. Alejandro
Aguilar Jiménez
Researcher - Professor
Researcher - Professor

Line: Bioprocesses and
Purification

Ph.D. At Tecnológico de Monterrey
•
•
Line: Recovery and Purification
Ph.D. At Tecnológico de Monterrey
Dr. Daniel Jacobo
Velázquez
Dr. José Manuel
Aguilar Yáñez
Researcher - Professor
Researcher - Professor

Line: Purification of Nutraceuticals

Line: Molecular Biology

Ph.D. At Texas A&M

Ph.D. At Tecnológico de
Monterrey
Dr. Karla Mayolo
Deloisa
Dr. José González
Valdez
Post-doc

Ph.D. At Tecnológico de
Monterrey
Ph.D. Students
•
•
•
•
•
•
•
•
•
•
•

José Arquímedes Echanove Juan

Andrés Enrique Ramos

Tomás Juan Aguirre González

Edgar Acuña González
Post-doc
Post-doc

Ph.D. At Tecnológico de
Monterrey

Ph.D. At Tecnológico de
Monterrey
Master Students
Jesús Simental Martínez
Celeste Ibarra Herrera
Federico Ruiz Ruiz
Patricia Vázquez Villegas
Juan Carlos Sánchez Rangel
Marco Mata Gómez
Alma Gómez Loredo
Edith Espitia Saloma
Mario Antonio Torres Acosta
Luis Alberto Mejía Manzano
Agustín Hernández Martínez
Support Professionals
Dra. Mirna
González
•
•
•
•
•
Ana Mariel Torres Contreras
Alejandro Becerra
Luis Rodolfo Chavez Castillo
Cesar Ivan Ortiz Alcaraz
Daniel Villarreal Garcia
Collaborations
•
•
•
•
•
•
•
University College London (U.K.)
Carnegie Mellon University (U.S.A.)
Instituto Superior Tecnico (Portugal)
University of Chile (Chile)
University of Houston (U.S.A.)
University of British Columbia (Canada)
Jacobs University-Bremen (Germany)
Thanks for your attention
Questions