Bioprocess Eng

8/12/2019 Bioprocess Eng

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Bioprocess for production of therapeutic agents

Manufacture of therapeutic agents : one of the most highlyregulated and rigorously controlled bioprocesses

To gain a manufacturing license, the producer proves that not onlythe product itself is safe and effective, but all aspects of the

proposed bioprocess comply with the highest safety and quality

standards


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Design and layout of the manufacturing facility Raw materials utilized in the process

Bioprocess itself

Training and commitment of personnel involved in all aspects

of the manufacturing operation

Existence of a regulatory framework which assures the

establishment and maintenance of the highest quality

standards regarding all aspects of bioprocess

Elements contributing to the safe production


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Overall manufacturing process

Infrastructure of a typical manufacturing facility

Source of therapeutic proteins

Up-stream and down-stream processing of products

Analysis of the final products : quality control


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Overall process


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Host cells for the production of biomolecules

Many of therapeutic agents currently on the market : produced

by recombinant DNA technology using various expressionsystems such as bacteria, yeast, fungi, and mammalian cells

Many therapeutic proteins are produced by recombinant DNA

technology

The use of appropriate expression system for specific products :

Each expression system displays its own unique set of

advantages and disadvantages


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E. coli

Most common microbial species used to produce heterologous proteins

- Heterologous protein : protein that does not occur in host cellsex) recombinant human insulin (Humulin) in 1982

tPA (tissue plasminogen activator in 1996

Major advantages of E. coli

- Its molecular biology is well characterized

- High level expression of heterologous proteins :

-High expression promoters

- ~ 30 % of total cellular protein

- Rapid growth, simple and inexpensive media, appropriate fermentationtechnology, large scale cultivation


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Intracellular accumulation of proteins in the cytoplasm

complicate downstream processing compared to extracellular production

additional primary processing steps : cellular homogenization,

subsequent removal of cell debris by filtration or centrifugation

extensive purification steps to separate the protein of interest

Inclusion body (insoluble aggregates of partially folded protein)formation via intermolecular hydrophobic interactions

- high level expression of heterologous proteins overload the

normal cellular protein-folding mechanisms

- Nonetheless, inclusion body displays one processing advantage

easy and simple isolation by single step centrifugation

denaturation using 6 M urea

refolding via dialysis or diafiltration

Drawbacks of E. coli


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- Prevention of inclusion body formation- growth at lower temperature (30 oC)

- expression with fusion partner : GST, Thioredoxin, GFP,

- high level co-expression of molecular chaperones

Inability to undertake post-translational modification, especially glycosylation

: limitation to the production of glyco-proteins

Typical examples of glyco-proteins

Presence of lipopolysaccharide on its surface : pyrogenic nature

more complicate purification procedure


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Yeast

Saccharomyces cerevisiae, Pichia pastoris :

Major advantages

- Well-characterized molecular biologyeasy genetic manipulation

- Regarded as GRAS-listed organisms (generally regarded as safe)

Long history of industrial applications ( e.g., brewing and baking)

- Fast growth in relatively inexpensive media, outer cell wall

protects them from physical damage

- Suitable industrial scale fermentation equipment/technology is already available

- Post-translational modifications of proteins, especially glycosylation


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Drawbacks

- Glycosylation pattern usually differs from the pattern observed in the

native glycoprotein : highly mannosylation pattern

-Low expression level of heterologous proteins : < 5 %

Many therapeutic proteins are produced in Yeast


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Fungal production system

Aspergillus niger

Mainly used for production of industrial enzymes : a-amylase, glucoamylase,

cellulase, lipase, protease etc..

Advantages

- High level expression of heterologous proteins

- Secretion of proteins into extracellular media

easy and simple separation procedure

- Post-translational modifications : glycosylation

- different glycostlation pattern compared to that in human


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Animal cells

Major advantage : post-translational modifications, especially glycosylation

Chinese Hamster Ovary (CHO) and Baby Hamster Kidney (BHK) cells

Typical glycoproteins produced in animal cells

Drawbacks

- Complex nutritional requirements : growth factors

expensive

complicate the purification procedure

- Slow growth rate

- Far more susceptible to physical damage or contamination- Increased production costs


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CHO cells


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Transgenic animals

Transgenic animal : live bioreactor

Generation of transgenic animals : direct microinjection of exogenous DNA intoan egg cell

stable integration of the DNA into the genetic complement of the cell

after fertilization, the ova are implanted into a surrogate mother

the transgenic animal harbour a copy of the transferred DNA

In order for the transgenic animal system to be practically useful, the

recombinant protein must be easily separable from the animal

simple way is to produce a target protein in a mammary gland

simple recovery of a target protein from milk


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Mammary-specific expression : fusing the gene of interest with the promoter-

containing regulatory sequence of a gene coding for a milk-specific protein

ex) Regulatory sequences of the whey acid protein (WAP, the most abundant

protein in the milk), -casein, - and -lactoglobulin genes

ex) Production of tPA in the milk of transgenic mice

- Fusion of the tPA gene to the upstream regulatory sequence of

the mouse whey acidic protein

- More practical approach : production of tPA in the milk of

transgenic goats

Production of proteins in the milk of transgenic animals


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Goats and sheep : the most attractive host system

- high milk production capacities

- ease of handling and breeding

- ease of harvesting of crude product : simply requires

the animal to be milked

- pre-availability of commercial milking systems with

maximum process hygiene

- low capital investment : relatively low-cost animalsreplace high-cost traditional equipment and low running costs

- High expression levels of proteins are potentially attained :

> 1 g protein/liter milk

- on-going supply of product is guaranteed by breeding

- ease downstream processing due to well-characterized properties of major

native milk proteins


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Issues to be addressed for practical use

- variability of expression levels

- different post-translational modifications, especially glycosylation,

from that in human- significant time lag between the generation of a transgenic

embryo and commencement of routine product manufacture :

- gestation period ranging from 1 month to 9 months

- requires successful breeding before beginning to lactate

- the overall time lag : 3 years in the case of cows, 7 months in the case of

rabbits

- inefficient and time-consuming in the use of the micro-injection technique to

introduce the desired gene into the egg


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Another approaches

- Use of replication-defective retroviral vectors : consistent

delivery of a gene into cells and chromosomal integration

- Use of nuclear transfer technology

manipulation of donor cell nucleus so as to harbor a gene coding for a target

protein

substitution of genetic information in un unfertilized egg with donor genetic

information

transgenic sheep, Polly and Molly, producing human blood factor IX, in 1990s


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No therapeutic proteins produced in the milk of transgenic animals had

been approved for general medical use

Alternative approach : production of therapeutic proteins in the blood of

transgenic pigs and rabbits

- Drawbacks

- relatively low volumes of blood

- complicate downstream processing

- low stability of proteins in serum


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Transgenic plants

Expression of heterologous proteins in plant : introduction of foreign genes

into the plant species :Agrobacterium-based vector-mediated gene transfer

-Agarobacterium tumefaciens, A. rhizogenes ; soil-based plant

pathogens

- when infected, a proportion ofAgarobacteriumTi plasmid is

trans-located to the plant cell and is integrated into the plantcell genome

- Expression of therapeutic proteins in plant tissue


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Potentially attractive recombinant protein producer

- low cost of plant cultivation

- harvest equipment/methodologies are inexpensive and well established

- ease of scale-upProteins expressed in seeds are generally stably

- Plant-based systems are free of human pathogens(e.g., HIV)

Disadvantages- variable/low expression levels of proteins

- potential occurrence of post-translational gene silencing

( a sequence specific mRNA degradation mechanism)

- different glycosylation pattern from that in human

- seasonal/geographical nature of plant growth


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Most likely focus of future transgenic plant :

production of oral vaccines in edible plants or fruit, such as tomatoes an bananas

- ingestion of transgenic plant tissue expressing recombinant sub-unit vaccines

induces the production of antigen-specific antibody responses

direct consumption of plant material provides an inexpensive, efficient and

technically straightforward mode of large-scale vaccine delivery

Several hurdles

- the immunogenicity of orally administered vaccines vary widely

- the stability of antigens in the digestive tract varies widely

- genetics of many potential systems remain poorly characterized

inefficient transformation systems and low expression levels

I ll b d


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Insect cell-based system

Laboratory scale production of proteins

Infection of cultured insect cells with an engineered baculovirus

( a viral family that naturally infects insects) carrying the gene coding for a target

protein

Most commonly used systems- the silkworm virus Bombyx mori nuclear polyhedrovirus(BmNPV)

in conjunction with cultured silkworm cells

- the virusAutographa californica nuclear polyhedrovirus(AcNPV)

in conjunction with cultured armyworm cells


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Advantages

- High level intracellular protein expression

- The use of strong promoter derived from the viral polyhedrin :

~30-50 % of total intracellular protein- Cultivation at high growth rate and less expensive media than

animal cell lines

- no infection of human pathogens, e.g., HIV

Drawbacks

- low expression level of targeted extracellular production of protein

- glycosylation patterns : incomplete and different

No therapeutic protein approved for human use


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Alternative insect cell-based system

Use of live insects

- live caterpillars or silkworms

infection with the engineered baculovirus vector

Ex) Veterinary pharmaceutical company, Vibragen Owega

- The use of silkworm for the production of feline interferon

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Bioprocess Eng