Post on 28-Mar-2020
Biotecnología y Bioingeniería en Mexico: Experiencias y Oportunidades
Marco Rito-Palomares
Centro de Biotecnología-FEMSA
Octubre, 2013
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.
¿Qué es la Biotecnología?
• 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.
Biotecnología en el tiempo
• 1960 – Primer antibiótico sintético.
• 1965 – Fusión de celulas de ratón y humanas.
• 1966 – Entendimiento del codigo genético.
• 1973 – Desarrollo de la técnica de manipulación de genes.
• 1981 – Primer animal transgénico.
El progreso rápido en
Biotecnología inicia
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
Promueve la llamada
nueva “economía basada
en el conocimiento”.
Biotecnología es uno de los sectores de
mayor crecimiento a nivel mundial
La nueva economía del siglo XXI
– 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: Vitaminas, antibióticos, enzimas. – Bebidas: Fructosa y otros productos edulcorantes. – Combustibles: Etanol y otros combustibles sofisticados. – Medicina clínica: Diagnósticos rápidos y en línea para la identificación
de enfermedades y agentes causantes. – Servicios: Sanidad, toxicología, inocuidad alimentaría,
propagación masiva de plantas.
Áreas de aplicación de la
Biotecnología
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
Production
concentration
Cell disruption
Primary
recovery Purification
Extra cellular
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 Flujo (Kg/dia) Eficiencia (%) 1 Fermentación 11.16 100
2 Centrifugación 11.10 99.5
3 Homogenización 10.99 99.0
4 Centrifugación 10.93 99.5
5-6 Solubilización/precipitación 8.20 75.0
7 Centrifugación 8.16 99.5
8 Degradación química 7.75 95.0
9 Ultrafiltración 7.71 99.5
10 Evaporación 7.48 97.0
11-12 Solubilización/oxidación 5.61 75.0
14 Intercambio aniónico 5.33 95.0
15 Degradación enzimática 5.06 95.0
16 Cromatografía HPLC 3.54 70.0
17 Ultrafiltración 3.52 99.5
18 Cristalización 3.17 90.0
19 Centrifugación 3.15 99.5
20 Evaporación 3.13 99.5
Rendimiento total 28%
Procesos biotecnológicos Hormona de crecimiento bovino
Paso Proceso Volumen (L) Eficiencia (%)
1 Separación celular 20 98
2 Ruptura celular 3X20 98
3 Centrifugación 20 98
4-5 Lavado/centrífuga 15 98
6 Extracción 12 100
7 Centrifugación 12 98
8-9 Lavado/centrifugación 12 98
10 Desnaturación/oxidación 70 100
11 Centrifugación 70 92
12 Ultrafiltración 70 95
13 Filtración en gel(alim) 4 -
Filtración en gel (elución) 18 47
14 Diálisis (alim) 97 -
Diálisis (dializado) 2640 72
15 Centrifugación 97 72
16 Ultrafiltración 97 100
Rendimiento total 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
Development of a prototype ATPS process for
c-phycocyanin recovery from Spirulina maxima
Bioproducts from Algae
Experiencias y Oportunidades
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
C-phycocyanin food grade
$ 130 USD / g
C-phycocyanin reagent grade
$ 1000-5000 USD / g
C-phycocyanin highly purified
$ 15,000 USD / g
Commercial value of c-phycocyanin
Existing protocols for the recovery of c-
phycocyanin produced by Spirulina maxima
Fermentation
(Spirulina
maxima)
Harvesting Drying Cell
disruption
CaCl2
Extraction
Centrifugation Adsorption Precipitation Dialysis Gel filtration
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
Existing protocols for the recovery of c-
phycocyanin produced by Spirulina maxima
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 4.00 L
NaCl 4.00 g
CaCl2 0.16 g
Na2SO4 8.32 g
FeSO4 0.04 g
KNO3 8.00 g
MgSO4 0.83 g
NaHCO3 36.0 g
K2HPO4 2.00 g
Process development for the recovery of c-
phycocyanin produced by Spirulina maxima
Effect of TLL
Model systems
Effect of MW of PEG
Effect of volume ratio
Fermentation
(Spirulina
maxima)
Cell
disruption
1st ATPS
Extraction
PEG + Salt
2nd ATPS
Extraction
PEG + Salt
Ultra-
filtration PEG and
contaminants
Contaminants
Contaminants
Concentrate
Precipitation
C-phycocyanin
Highly purified CPC
Product yield 28%
Commercial value: $15,000 USD/g
Prototype process for the recovery of c-
phycocyanin produced by Spirulina maxima
Prototype process for the recovery of c-
phycocyanin produced by Spirulina maxima
A prototype process for B-phycoerythrin
purification from Porphyridium cruentum
B-phycoerythrin (BPE)
• B-phycoerythrin, a pink-coloured protein.
• Produced by red algae as accessory pigment.
• The commercial value of highly purified B-
phycoerythrin (> 4, defined as the
relationship of 545 – 280 nm absorbances)
for pharmaceutical or fluorescent uses can
be more than US$ 50/mg.
BPE applications
• Pigment for food industry
• Pigment for cosmetic industry
• Pharmaceutics
• Fluorescent marker
A prototype process for B-phycoerythrin
purification from Porphyridium cruentum
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)
System pH
BP
E r
ecover
y (
%)
Pu
rity
of
BP
E (
A54
5nm
/A28
0nm)
Recovery of natural colorants from
microbial origin: B-phycerythrin BPE
A pilot plant for the validation of this prototype process has been established.
A patent granted
Recovery of natural colorants
from microbial origin: B-
phycerythrin BPE
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 Two-
Phase 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.
Limitations:
-- Low yields.
-- Inconsistent product quality.
-- COMPLEXITY OF HOST PROTEOME
Challenge:
-- High concentration of
contaminant proteins
-- Amount of target protein
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).
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
Production
(bioreactor)
Purification
Recovery and
primary
purification
General Bioprocess Diagram
Recuperación del producto
ATPS Buffer
Tris-Borate-EDTA, pH 8
Protein
Extraction
Vr = 1, pH 7,
Phosphate salts
rhG-CSF
Model
protein
Gel SDS-PAGE &
MS
1. Molecular marker
2. Alfalfa/rhG-CSF
3. Upper phase
4. rhG-CSF
1 2 3 4
1. Molecular marker
2. Alfalfa/rhG-CSF
3. Bottom phase
4. rhG-CSF
1 2 3 4
Target product
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
Modificación de Farmacos
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
Ribonuclease A a - Lactalbumin
Present in mammal milk whey.
MW: 14,176 Da.
126 amino acid residues.
Antitumoral properties.
Obtained from bovine pancreas.
MW: 13,686 Da.
124 amino acid residues.
Antitumoral properties.
Products from the PEGylation reaction
Three resulting species are reported for the PEGylation reactions of both model proteins:
The mono-PEGylated protein in both models presents the best biological activity.
Native Protein
mono-PEGylated Protein
di-PEGylated Protein
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
0
200
400
600
800
1000
1200
0 50 100 150 200 250 300
Ab
sorb
ance
21
0 n
m (
mA
U)
Volume (mL)
Native RNase
A
monoPEGRNase
A
diPEG RNase
A
Experiencias en el desarrollo de procesos para recuperar bioparticulas
Rotavirus-like particles primary recovery from insect cells
VP6 VP2
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.
Double layered rotavirus-like
particles (dlRLP)
Consist of two concentric
protein layers.
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).
VP6 VP2
Rotavirus-like particles primary
recovery from insect cells
Extra cellular dlRLP
Insect cell culture
Centrifugation
Cell disruption
ATPS extraction
Ultrafiltration
Intracellular dlRLP
Biomass
85% dlRLP recovery
90% dlRLP purity
30 - 55 purification factor
Insect cell culture
Centrifugation
Sucrose cushion
CsCl gradient
Ultrafiltration
2% dlRLP recovery
90% dlRLP purity
45 purification factor
Biomass
Extra cellular dlRLP
Strategies for the potential recovery and purification of stem cells
diseased
damaged
dead
Diseases
replace cells
Stop /
Reverse
Background Background
STEM CELLS
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
Human umbilical cord blood
Lymphoprep
Sample rich
in CD133+
stem cells
used in
ATPS
Measurement system
Flow Cytometry employing specific
stem cell marker (CD133 antibody) and
7AAD for viability.
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.
Phases
formation
1) DEX 70,000-Ficoll 400,000
2) DEX 10,000-PEG 10,000
PEGylated CD133 antibody and
cells addition into ATPS
Release of
stem cells
CD133+
Purified
CD133+
stem cells
Strategy II. Immunoaffinity ATPS
• PEGylated antibody
Comentarios y mensaje final
Biotecnología en México
Áreas
Generación de
conocimiento
Universidades
Enfoque
Transferencia de
tecnología
Industria
VS
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
Investigar
Innovar
Incubar
i 4 =
Chair Director
• Line: Recovery and Purification
• Ph.D. At Tecnológico de Monterrey
Prof. Marco A. Rito-Palomares
Dr. Alejandro Aguilar Jiménez
SNI III
AMC Member
Line: Bioprocesses and Purification
Post-doc at Cambridge University
Ph.D. At Birmingham University
Line: Bioprocesses and
Purification
Ph.D. At Tecnológico de Monterrey
Dr. Jorge Benavides Lozano
Line: Molecular Biology
Ph.D. At Tecnológico de
Monterrey
Dr. José Manuel Aguilar Yáñez
Line: Purification of Nutraceuticals
Ph.D. At Texas A&M
Dr. Daniel Jacobo Velázquez
Researcher - Professor Researcher - Professor
Researcher - Professor Researcher - Professor
Research Group Members
• 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
• Ana Mariel Torres Contreras • Alejandro Becerra • Luis Rodolfo Chavez Castillo • Cesar Ivan Ortiz Alcaraz • Daniel Villarreal Garcia
Ph.D. Students Master Students
Ph.D. At Tecnológico de
Monterrey
Dr. José González Valdez
Ph.D. At Tecnológico de
Monterrey
Dr. Karla Mayolo Deloisa
Post-doc Post-doc
Ph.D. At Tecnológico de
Monterrey
Dra. Mirna
González
Post-doc
José Arquímedes Echanove Juan
Andrés Enrique Ramos
Tomás Juan Aguirre González
Edgar Acuña González
Support Professionals
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