BIOPROCESOS

INGENIERÍA BIOQUÍMICA
SÉPTIMO NIVEL

Patricio Orozco Freire

MSc Plant Genetic Manipulation

Marzo – Septiembre 2017

Introducción a los bioprocesos
 Un bioproceso es cualquier reacción química o física de un sustrato que
usa organismos o microorganismos vivos, subcomponentes celulares o
metabolitos secundarios (por ejemplo bacterias, levaduras, enzimas,
cloroplastos, etc.) como intermediarios de la reacción para obtener los
cambios físicos o químicos deseados en un proceso generando varios
subproductos y biomasa.
Otras definiciones
 La bioingeniería o Ingeniería de Bioprocesos Industriales es una
rama interdisciplinaria que integra los conocimientos químicos,
biológicos y principios tradicionales de la ingeniería, con el fin de
solucionar diversos problemas a nivel de producción, salud y
energía

 Trata del manejo de los equipos y los procesos que mediante la

propagación de pequeños seres vivos, se pueden generar
productos interesantes para el ser humano (antibióticos, alimentos,
bebidas, enzimas, productos industriales obtenidos por
fermentación, cultivos celulares, tisulares y parenquimáticos)

 Incluye diferentes disciplinas, como la ingeniería bioquímica, la

ingeniería biomédica, la ingeniería de procesos biológicos, la
ingeniería de biosistemas, entre otras
BIOTRANSFORMACIÓN
• Son los procesos en los que los sustratos naturales o sintéticos
son modificados por medio de una ACTIVIDAD ENZIMÁTICA
Elaboración de Cerveza
¿Cómo se denomina a este
bioproceso?
¿Cómo se denomina a este
bioproceso?
Fermentación alcohólica
 La fermentación alcohólica, también conocida como,
fermentación etílica, o del etanol, es un proceso de tipo
biológico, en el cual se lleva a cabo una fermentación sin
presencia de oxígeno. Este tipo de fermentación se debe a las
actividades de ciertos microorganismos, los cuales se
encargan de procesar azúcares, como la glucosa, la fructosa,
etc. (hidratos de carbono), dando como resultado un alcohol a
modo de etanol, CO2 (gas) y ATP (adenosín trifosfato),
moléculas que son utilizadas por los propios microorganismos
en sus metabolismos energéticos.

 Numerosos hongos, bacterias, algas y algunos protozoos,

fermentan azúcares, transformándolos en etanol y CO2.
Finalidad de la fermentación
alcohólica
• La fermentación alcohólica, al igual que otro tipo de
fermentaciones, como es el caso de la fermentación láctica,
es de gran utilidad para el hombre, pues por ejemplo, la
fermentación alcohólica llevada a cabo por las levaduras, sirve
para la fabricación de bebidas alcohólicas (como el vino o la
cerveza), y el CO2 procedente de la fermentación, es utilizado
para hacer crecer el pan y otros alimentos.
¿Cuál es la levadura utilizada
para elaborar cerveza?

Saccharomyces cerevisiae
Desde la Antigüedad
• “ La fermentación alcohólica es utilizada desde antiguo para
realizar productos como la cerveza o el vino. Los griegos
otorgaban el descubrimiento de este proceso al dios Dionisio.
Y algunos procesos similares, como la destilación de alcohol,
se llevaban a cabo ya en el año 1150. Sin duda, dichos
procesos fueron esenciales para el desarrollo de la alquimia
en la Edad Media ”
EN LA ACTUALIDAD
• Hoy en día existen bioprocesos con usos diversos en la
industria, como por ejemplo:

• La producción de cosméticos,
• Productos de limpieza,
VIDEO DE CARNE
• Biocombustibles, DE LABORATORIO
• Pesticidas biológicos,
• Vacunas recombinantes,
• Enzimas para la industria,
• Fármacos: antibióticos, proteínas terapéuticas
• Biorremediación,
• Biomasa, etc.
BIOPRODUCTOS MICROALGAS
1. Las microalgas son consideras una fuente
prometedora de nuevos Bioproductos: son una
fuente más sustentable de ácidos grasos
(omega 3) que el aceite de pescado y una
fuente tan poderosa de antioxidantes como
los carotenoides. Así mismo son una fuente
natural rica en antioxidantes, los cuales las
protegen de radicales libres generados durante
la fotosíntesis

http://nopr.niscair.res.in/bitstream/123456789
/42258/1/IJMS%2046%287%29%201239-
1244.pdf
2. Las microalgas son la única
fuente de biocombustible no
emisora de carbono (huella
ecológica cero) que tiene el
http://www.sciencedirect.com/science/art
potencial de sustituir una parte
icle/pii/S0196890410002207
significativa de nuestra demanda
de combustible sin influenciar
negativamente a la agricultura
para la producción de alimento

3. El tratamiento de aguas residuales

http://www.sciencedirect.com/sci
ence/article/pii/S09596526150070
39
usando microalgas es más eficiente
energéticamente que las tecnologías de
tratamiento de aguas residuales que se
utilizan en la actualidad. En lugar de
remover nutrientes valiosos esta técnica
permite que los nutrientes puedan ser
reciclados a biomasa que puede ser usada
como fuente de alimento para animales,
energía o material de construcción para la
industria química.
CHALLENGES
 El avance de los BIOPROCESOS se ha venido dando gracias al
continuo desarrollo de otras ciencias que apoyan y en conjunto al
ser interdisciplinarias intervienen para el desarrollo de nuevas
tecnologías.

 Microbiología, bioquímica, fisiología, genética, biología molecular.

 Herramientas de la biotecnología moderna: DNA recombinante,

fusión celular (híbridos somáticos, sexuales), gene probes, cultivo de
tejidos animales y vegetales.

 Han permitido la creación de medicinas sofisticadas, el cultivo de

tejidos humanos y órganos, biochips para una nueva era en la
computación, pesticidas compatibles con el ambiente y poderosos
microorganismos que degradan contaminantes.
 REGENERACIÓN DE TEJIDOS Y ÓRGANOS HUMANOS

 Sin embargo estos nuevos productos y procesos pueden ser

desarrollados parcialmente en el laboratorio y algunos de
estos llevan muchos años para finalizarlos, como por ejemplo
los fármacos.

 Se requiere de habilidades en ingeniería y como se dice en

inglés el “know how”.

 Los sistemas biológicos pueden ser complejos y muy difíciles

de controlar.

 Hay que apoyarse de la ingeniería: diseño y operación de

bioreactores, equipos de recuperación de los productos,
desarrollo de sistemas para la automatización y control, etc.
ETAPAS EN EL DESARROLLO DE LOS
BIOPROCESOS
 En general el desarrollo de un bioproceso está comprendido de
las siguientes etapas:
 UPSTREAM PROCESS: elección de los microorganismos, repique de
cepas, medio de cultivo (estéril), preparación del inóculo, etc.
 PROCESS: BIOCATALIZADOR: enzimas, virus, bacterias, levaduras,
hongos filamentosos, algas, células de tejidos vegetales y animales.
BIORREACTOR: elección del tipo y operación del biorreactor,
automatización y control, nutrición y metabolismo, estequiometria,
cinética, etc.
 DOWNSTREAM PROCESS: purificación y recuperación del producto.
Separación sólido-líquido, ruptura celular, solubilización, extracción
en dos fases, precipitación selectiva, separación con membranas,
cromatografía. ACONDICIONAMIENTO FINAL: secado, cristalización,
estabilización en medio líquido
PLANT SEEDS AS GREEN FACTORIES FOR
RECOMBINANT THERAPEUTIC PROTEINS

 “Molecular farming has been considered as one of the

promising approaches for the production of recombinant
proteins (RP) including not only therapeutic compounds to
treat human or animal pathologies but also proteins used in
the agriculture and industry areas” (Xu et al., 2012).”
Benefits to use plants
 Plants are well known because of their capacity to synthetize high
protein levels and also great amounts of biomass just using the sun
energy which is exploited through photosynthesis (Paul and Ma, 2011).

 Post-translational machinery required for performing all of the

necessary modifications to synthetize many eukaryotic proteins, giving
remarkable flexibility in bio-production platforms (Pogue et al., 2010; Xu
et al., 2012).

 Plants cannot suffer human or animal diseases or carry foreign

pathogens in their system; therefore, synthesized proteins have low risk
of contamination, providing more safety to patients. On the other side,
this not occurs in mammalian and bacterial systems (Shih and Doran,
2009).

 Biosafety is imperative when RP are commercialized because it helps to

reduce the purification costs, minimizes the decontamination processes
and avoid possible patient demands (Sharma and Sharma, 2009; Xu et
al., 2012).
Seed platform system advantages

 “Seed platform provides several advantages, for example,

higher protein yields and stable storage compared with other
systems. It is estimated that in the near future, there will be a
tremendous demand for recombinant therapeutic proteins
(Mett et al., 2008).”

 Several antibodies, vaccine antigens and other therapeutic

proteins can be accumulated in seeds showing high yields,
stability and functionality during long periods of time, under
room temperature (Lau and Sun, 2009).
Disadvantages

 “Despite the advantages that transgenic plant systems provide

in the production of RP, some safety and regulatory directions
have been applied, allowing the use of this technology just
inside contained greenhouses. This is an important concern
because the cost of the downstream phase is directly related
with the upstream process. Also, the scale-up costs are
affected being more expensive (Wilken and Nikolov, 2012).”
Current status of plan molecular
farming
 A diverse number of RP have been effectively developed in
plants, including: vaccine antigens, antibodies, enzymes and
diagnostic proteins (Table 1) (Ma et al.,2003).
Lau and Sun (2009) confirmed the commercialization of eleven non-pharmaceutical
proteins in the market; however, they also affirmed that some plant pharmaceutical
products were in the final stages of development (Table 2).
Recombinant therapeutic proteins
 Various studies have reported the expression of different
mammalian proteins in plants such as vaccines antigens, blood
proteins, growth hormones, growth factors, diagnostic
proteins including therapeutic enzymes and monoclonal
antibodies (mAbs), auxiliary proteins such as Factor VIII for
hemophiliacs or insulin for the treatment of diabetic people,
immune system stimulators or suppressants such as cytokines,
interleukins and interferons (Sharma and Sharma, 2009; Xu et
al., 2012).

 The yield of the RP obtained can vary significantly from 0.01%

of total soluble protein and 0.1 μg/L, up to 25% of TSP and
247 mg/L. There are several strategies to improve the yield
(Xu et al., 2012).
Translational modifications
 There are certain conditions that these proteins require to
have an effective bio-activity in humans including: protein
folding, disulfide bond development, glycosylation, and
subunit assembly; however plants have demonstrated the
capacity to perform these changes through post-translational
modifications (PTMs) ability.

 The second generation of RP in plant systems has been

focused in research and exploits all of the benefits provided by
PTMs, looking for the creation of new biosimilars and high
efficiency proteins with new motifs of fusion, facilitating the
stability, longevity, solubility, assembly, delivery and trafficking
Glycosylation
 One of the most important concerns in the production of RP is
their glycosylation; although plants can glycosylate proteins
effectively, they do not employ similar upstream processing
to complex glycans at the Golgi level; in other words, the
mechanism is different than in mammalian cells.

 To solve this issue, xylose and α-1, 3-fucose sugars have to be

included in the process
Plants cannot perform mammalian sugars

 Furthermore, plants cannot synthesize mammalian sugars, for

example β-1, 4-galactose residues.

 However, recent studies in Arabidopsis thaliana and Nicotiana

benthamiana have tried to decrease the expression of certain
genes, including those that encode for fucosyl- and xylosyl-
transferases, and in the same time overexpressing a human or
chimeric β-1, 4-galactosyl transferase.

 The result allowed the production of antibodies adapted for

the human organism with N-glycans
Plantibodies
 Among the group of therapeutic proteins, antibodies
produced in plants adopted the denomination of Plantibodies
which have a cost of production relatively lower than in
mammalian cells.

 Currently, all of the efforts in this area are focused in the

“humanization” of Plantibodies.
Plant expression platforms

 Based on molecular, tissue culture and genetic studies, various

plant-based production platforms have been developed,
including whole plants specially tobacco, aquatic plants such
as Lemna minor, leafy crops including alfalfa and lettuce,
cellular suspension systems like rice and carrot, and culture of
specific tissues for example hairy roots
Table 3 Comparison between the expression platforms producing RP (Xu et al., 2012)

Commercially viable Time for Regulatory

Expression system Scalability
species production compliance
Whole plants
Stable transgenic plants Corn, soy, safflower, rice 3–6 months Unlimited field culture Difficult
Transient plants Nicotiana sp., lettuce 2–7 days Greenhouse limited Moderate

In vitro cultured plant cells and species

Hairy roots Nicotiana sp. 14–30 days 20,000 L Easy
Cell suspension culture Tobacco BY-2, carrot, rice 7–20 days 100,000 L Easy
Moss Physcomitrella patens 14–30 days 200 L Easy

Aquatic plants
Duckweed (closed system) Lemna sp.,spirodela sp. 20–40 days 10,000 L Moderate
Microalgae Chlamydomonas reinhardtii
Open system 20–40 days Limited by water surface area Difficult
Photobioreactor 14–30 days 10,000 L Moderate
Conventional bioreactor 7–20 days 200 L Easy
Select the appropiate platform
• To choose the appropriate platform, it is necessary to
understand the biochemical characteristics of the target
protein and the regulatory network behind the plant-based
system (Paul and Ma, 2011).

Table 4 Plant expression systems developed for the production of therapeutic proteins (Paul and Ma, 2011)
Open-field Glasshouse N. benthamiana
Rhizosecretion Cell culture
tobacco tobacco transient expression
Small proteins Veterinary vaccines,
Typical model PMP or Antibodies, Antibodies,
(CV-N), Antibodies, antigens glucocerebrosidase
product antigens antigens
antibodies (UPLYSO)
++ (15–50 ++ (15–50
+ (0.25–3 µg/mL/ +++ (0.5–4 g/kg leaf fresh
Yield (range) mg/kg leaf fresh mg/kg leaf +++
24 H) weight)
weight) fresh weight)
Scalability +++ + ++ ++ +

Consistency + ++ ++ ++ +++
Downstream purification
+ + +++ + ++
burden
Regulatory development + ++ ++ + +++
Seeds as bioreactor model
 There are many advantages that are mentioned when seeds are
used as bioreactors for protein production, for example, the protein
stability and storage which are variables easier to control compared
with leaf systems

 The proteolytic degradation is commonly avoided and also, with the

adequate cold storage conditions, it is possible to conserve the
seeds for a long period of time, in some cases more than three
years, leading to the minimal reduction of the protein activity

 The reported quantity of protein achieved in seeds is approximately

7% to 10%, transforming them in one of the promising platforms for
the manufacture of several targeted proteins

 Different grains have been used as expression models to produce

therapeutic proteins, such as canola, corn, soybean, rice, wheat,
barley and maize, among others
Example
 One clear example of the benefits provided by seed systems is
the assembly of HIV neutralizing antibody 2 G12 in maize
seeds

 As a potential topical microbicide, not only demonstrating

lower production costs, but also a purification process less
complicated. The downstream processes including the
purification are considered the most expensive procedures
involved in the production of therapeutic proteins
 The use of oil seeds instead of common crops has been
implemented due to the facility to separate the protein from
the complex, via oil body isolation.

 One of the pioneer companies in this area is SemBioSys

Genetics located in Canada, which has created the oleosin-
fusion platform widely used for the production of human
insulin biosimilar at low costs. “The RP is mixed with oleosin
and subsequently targeted to oil bodies, subcellular organelles
which store oils. The protein recovery is performed by a
simple purification of the oil bodies followed by the separation
of the protein from the oleosin by endoprotease digestion”.
 Other examples include Ventria Biosciences and Meristem
Therapeutics that developed rice systems for the manufacture
of lactoferrin and lysozyme, both human proteins

 And ORF Genetics Ltd. located in Iceland, which developed

barley grains as targeted seeds for the expression of growth
factors and cytokines
Recovery and purification methods
 The scientific effort has been focused in the development of
effective recovery and purification processes of RP; however,
the major progress was achieved in the pretreatment and
extraction areas

 The production of specific proteins were evaluated

individually, therefore the extraction protocols have been
standardized case-by-case, creating dependent platforms.

 On the other hand, the advance in the pretreatment was

possible with the knowledge that plant extracts and
homogenates required different conditions after the
purification
Purification
 In the case of purification, scientists developed new protocols
based on prior purification models created for other protein
production hosts; the promising methods as cheap
alternatives are non-chromatographic and include aqueous
two phase partitioning and membrane filtration
Selection of downstream method
 The downstream process is very important because in some
cases, it represents the major percentage of the total costs.

 The selection of the downstream method depends of the

following characteristics: required protein concentration and
purity and the complexity of the plant extract.

 The downstream process can be classified into two stages:

primary recovery and purification. Figure 3 summarizes the
downstream process of different platforms
Primary recovery
 The function of this phase is to increase the product yield in
the extract, decrease the volume of the media and clarify it
for the next step, the purification.

 In the case of primary recovery from seed platforms, the

following steps are required: aqueous extraction or
homogenization to release the product from the biomass,
solid-liquid separation and clarification
Fractionation
 This process is especially used in seed-based systems and
provides a mechanism to increase the RP yield and generate
sub products, reducing the volume and solid composition.

 The common methods include: dry milling, dry fractionation,

and wet milling. In the case of oilseeds, the oil separation is
necessary to avoid the emulsification
Tissue disruption and product isolation
 This stage is crucial because its efficiency is correlated with the total
volume extracted, purity and concentration of the RP and the
magnitude of impurities which have to be removed from the extract
in the purification phase.

 The whole process depends on the effective plant tissue

homogenization and the rupture of the plant cell walls.

 To homogenize seeds with a low content of oil, it is recommended

to apply a method called dry grinding/pulverization and then the
grounded seed are processed with low-shear mixers, applying
approximately four or five liters of buffer per kilogram of dry seed.

 In the case of oil bodies, they require more water for their
extraction
Issues

 The proteolysis and phenol oxidation of the RP is considered

as one of the issues during this process, therefore, it is
necessary to use adequate buffers with protein stabilizing
agents, for example protease inhibitors (EDTA, PMSF), metal
chelators, antioxidants and in some cases detergents (Triton
X-100)
Solid-liquid separation

 The most common approach used to remove the solid content

of an extract is the centrifugation. Using seed systems benefit
this stage due to the difference in the density between the
solids and liquids is large, therefore the required G-force in the
centrifugation is lower.
Conditioning and pretreatment
 In order to improve the extract quality before the purification,
some clarification and pretreatment processes can be
performed, such as the regulation of the pH, buffer
composition, volume reduction and ionic strength.

 Despite of the availability of these methods, new ones were

developed, for example adsorption, precipitation, two-phase
partitioning and membrane filtration.

 In the case of seed extracts the process is less complicated; pH

adjustment and a subsequent clarification through a simple
centrifugation or depth filtration are commonly required,
helping to remove phytic acid precipitates
Purification
 The pre-purification procedure begins with a capture process which
helps to increase the concentration of the RP and also, it removes
the impurity particles that at the end of the purification can affect
the protein quality and yield.

 When a major purity is required, for example in the development of

therapeutic proteins, a polishing procedure is necessary to remove
the remaining impurities.

 Immediately after the capture and polishing, the purification process

is applied, which is selected depending on the protein nature and
the amount of residual impurities. Commonly, the same purification
methods developed for biopharmaceutical products are used, and
the most widely applied is adsorption chromatography due to its
higher resolution compared to other methods
ETAPAS QUE INTERVIENEN EN EL PROCESO DE
RECUPERACIÓN Y PURIFICACIÓN DEL PRODUCTO

 1. Remoción Celular – Centrifugación – Centrifuga

 2. Disrupción celular – Ruptura química; MECÁNICOS
homogeneizador: de alta presión, microfluidizadores;
sonicadores-ultrasonido (cavitación); bead mills; morteros.
Métodos no mecánicos
 Químicos: 1) con solventes orgánicos (cloroformo, tolueno) que
permeabilizan la membrana, disuelven compuestos hidrofóbicos como
los fosfolípidos de la membrana interna en bacterias gram negativas;

 2) Alcalis (hidróxido de sodio e hipoclorito), se da la saponificación de

los lípidos de membrana

 3) Agentes caotrópicos (úrea, clorhidrato de guanidina), interfieren con

los enlaces no covalentes como los puentes de hidrógeno y las fuerzas
de van der Walls

 4) Agentes quelantes (EDTA): quela los iones Ca2+ y Mg2+ que unen los
lipopolisacáridos que contienen proteínas

 5) Antibióticos contra bacterias gram negativas

 6) Detergentes (SDS, tritón) forma micelas con lípidos de membrana

• Físicos: 1) Shock osmótico, utilizando medios hiper e
hipoosmóticos

• 2) Congelamiento y descongelamiento

• 3) Descompresión causada por el uso de un gas presurizado

• 4) Termólisis: altas temperaturas

• Enzimáticos: 1) Disrupción enzimática

• 2) Auto-lisis

• Métodos combinados
ETAPAS QUE INTERVIENEN EN EL PROCESO DE
RECUPERACIÓN Y PURIFICACIÓN DEL PRODUCTO

 3. Concentración: ultrafiltración, evaporación.

 4. Extracción del producto: cromatografía de intercambio
iónico, afinidad, etc.
 5. Purificación de alta resolución: afinidad
 6. Formulación, esterilización:

 Estabilidad: parámetros de calidad

 Actividad biológica: libre de pirógenos
 pH
 Conductividad
 Concentración
Microorganismos utilizados en la
industria