biotecnología y bioingeniería en mexico: experiencias y … · 2019-04-01 · •1750 a.c. –...

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


5-6 Solubilización/precipitación 8.20 75.0


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


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


8-9 Lavado/centrifugación 12 98

10 Desnaturación/oxidación 70 100


12 Ultrafiltración 70 95

13 Filtración en gel(alim) 4 -

Filtración en gel (elución) 18 47

14 Diálisis (alim) 97 -

Diálisis (dializado) 2640 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-


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-


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-


Prototype process for the recovery of c-


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


Monterrey

Dr. José González Valdez


Monterrey

Dr. Karla Mayolo Deloisa

Post-doc Post-doc


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

biotecnología y bioingeniería en mexico: experiencias y … · 2019-04-01 · •1750 a.c. –...

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