Download Ch 3. The drug manufacturing process

Bioprocess
A whole process that uses complete living cells or their components
(e.g., bacteria, yeast, mammalian cells, enzymes, plant cells) to obtain
desired products with required quality
• Manufacturing process for a final product
- Starting from a raw material to a final product
- Industrial scale process: Scale-up
- Process optimization
- Purification and formulation: Quality control
- Packaging
• Economic feasibility:
- Cost analysis for an entire bioprocess:
Bioprocess for production of therapeutic agents
• Manufacture of therapeutic agents : one of the most highly
regulated and rigorously controlled bioprocesses
• To gain a manufacturing license, the producer proves that not
only the product itself is safe and effective, but all aspects of the
proposed bioprocess comply with the highest safety and quality
standards
Elements contributing to the safe production
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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
Example: Heparin
• A highly-sulfated glycosaminoglycan with highest negative charge density of any
known biological molecule. It is most abundant in connective tissues, and
produced by basophils and mast cells
• Widely used as an injectable anticoagulant and an inner anticoagulant surface
on various experimental and medical devices such as renal dialysis machines.
• Binds to the enzyme inhibitor anti-thrombin III (AT), activating it.
The activated AT then inactivates thrombin and other proteases involved in
blood clotting, most notably factor Xa.
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Derived from mucosal tissues of slaughtered meat animals such as porcine (pig)
intestine or bovine (cow) lung.
• Native heparin: a polymer with a molecular weight ranging from 3 kDa to 40
kDa. The average molecular weight of most commercial heparin preparations:
12 kDa ~ 15 kDa.
• A variably-sulfated repeating disaccharide unit.
- The most common disaccharide unit is composed of a 2-O-sulfated
iduronic acid and 6-O-sulfated, N-sulfated glucosamine, IdoA(2S)-GlcNS(6S).
Tainted Drugs Put Focus on the FDA
• Contaminated medicine from China was linked to as many as 19 deaths in
the United States  Members of Congress clamored for changes while
regulators defended their actions.
• Nineteen deaths have been linked to contaminated heparin, a crucial
blood thinner manufactured in China.
• The FDA admitted that it violated its own policies by failing to inspect
the China plant, altering border agents to detain suspect heparin
shipments.
• Eighty percent of the active pharmaceutical ingredients consumed in the
United States are manufactured abroad;
- 40 % are made in China and India. The FDA has cut back on its foreign drug
inspections, mainly because of lack of budget.
• There are 566 plants in China that export drugs to the United States,
but the agency inspected just 13 of them last year.
Plants in China and India are rarely inspected by Western governments,
which can reduce costs dramatically.
• Even the Chinese did not inspect the plant making contaminated heparin
because everything made at the plant was shipped overseas.
Overall manufacturing process
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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
• Upstream process: The first step in which microbes/cells are grown for production
of target products.
- Involves all the steps related with inoculum development, media development,
improvement of inoculum by genetic engineering process, optimization of growth
kinetics of cells.
• Downstream process: The next step where the cell mass from the upstream are
processed to produce the target products with required purity and quality.
- Downstream processing: includes three main sections:
cell disruption, a purification section, and a polishing section(formulation, packaging etc.)
Cell culture process for therapeutic proteins
CIP: Cleaning-in-process
Overall bioprocess
Protein purification process
• Increase in the number of purification steps
- Increase in purity
- Decrease in yield
ex) Yield in each step: ~ 90%
Total yield in 7 steps?
- Increase in cost
Industrial scale protein purification column
Affinity purification
Loading
A
Affinity ligand
Column
Binding
Washing
Elution
Affinity chromatography
Therapeutic antibody production and purification
IgG binding domain of Protein A
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Protein A: Surface protein of bacterium Staphylococcus aureus : 42 kDa
- Composed of five IgG binding domains that fold into a three-helix bundle
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Binds to the heavy chain within the Fc of most immunoglobulins : KD= ~30 nM
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Used for affinity purification of IgG
- Immobilization of protein A on resin
- Major market share by GE Healthcare
Antibody purification using Protein A column
A
Ethanol fermentation process
Microorganism fermentation system
Ethanol fermentation tanks
Host cells for the production of valuable molecules
• Use of appropriate expression system for specific products :
Each expression system displays its own unique set of
advantages and disadvantages
• Many of therapeutic agents currently on the market : produced
by recombinant DNA technology using various expression
systems such as bacteria, yeast, fungi, and mammalian cells
• Many therapeutic proteins are produced by recombinant DNA
technology
Escherichia coil
• Most common microbial species used to produce heterologous proteins
- Heterologous protein : protein that does not occur in host cells
• Some important molecules: Amino acids, organic acids, vitamins
ex) recombinant human insulin (Humulin) in 1982
tPA (tissue plasminogen activator in 1996
L-Threonine, L-Tryptophan, L-lactic acid
• 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 fermentation
technology, large scale cultivation
Drawbacks of E. coli
• 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
- 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
Corynebacterium
• Gram-positive, aerobic, rod-shaped bacteria
• Nonpathogenic species of Corynebacterium: very important industrial
applications, such as the production of amino acids, nucleotides, bioconversion
of steroids, cheese aging, and production of enzymes
- One of the most studied species is C. glutamicum
- Glutamic acid: food additive (1.5 million tons/ year)
Lysine : animal feed (~ 700,000 tons /year)
Threonine
• Cell factory
- Robust and reliable process
- High-cell density
- Genome sequence
Yeast
• Saccharomyces cerevisiae, Pichia pastoris :
• Major use : Glycoproteins, Alcohols
• Major advantages
- Well-characterized molecular biology easy 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
• Application : Production of Artemisinin and Opiate precursors
• 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
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 glycosylation pattern compared to that in human
Animal cells
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Major advantage : Post-translational modifications, especially glycosylation
Chinese Hamster Ovary (CHO) and Baby Hamster Kidney (BHK) cells
Many glycoproteins are produced in animal cells
Genome sequence
• Drawbacks
- Complex nutritional requirements : Many kinds of growth factors
 Expensive
 Complicate the purification procedure
- Slow growth rate
- Far more susceptible to physical damage or contamination
- Increased production cost
CHO cells
Transgenic animals
• Transgenic animals : live bioreactor
• Generation of transgenic animals : Direct microinjection of exogenous DNA into
an egg cell
 Stable integration of the DNA into the genetic complement of the cell
 After fertilization, the ova are implanted into a surrogate mother
 Transgenic animal harboring 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 and easy way is to produce a target protein in a mammary gland
 Simple recovery of a target protein from milk
• Mammary-specific expression : Fusing the gene of interest with the promotercontaining 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), β-casein, α- and β-lacto-globulin 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
• Goats and sheep : 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 animals replace 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 (separation and purification) processing due to wellcharacterized properties of major native milk proteins
• Issues to be addressed for practical use
- Variability in 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
- 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
• 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
• 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
Nuclear transfer technology
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A form of cloning
General steps:
- Removal of the DNA from an oocyte (unfertilised egg)
- Injection of the nucleus which contains the DNA to be cloned.
- Implantation into a surrogate mother
Cloning of a sheep
Transgenic plants
• Transgenic plants : genes inserted into them that are derived from another
species
• The inserted genes can come from species within the same kingdom (plant
to plant) or between kingdoms (for example, bacteria to plant).
- The inserted DNA has to be modified slightly in order to correctly and
efficiently express in the host organism.
• 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 of Agarobacterium Ti plasmid is trans-located
to the plant cell and is integrated into the plant cell genome
- Expression of therapeutic proteins in plant tissue
- Regeneration of plant : plant expressing heterologous proteins
• Non-food crops : production of pharmaceutical agents, biofuels, and other
industrially useful goods, as well as for bioremediation
• Potentially attractive recombinant protein producer
- Low cost of plant cultivation
- Harvest equipment/methodologies are inexpensive and well established
- Ease of scale-up: Proteins expressed in seeds are generally stably expressed
- 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
• Most likely focus of future transgenic plant :
- Production of oral vaccines in edible plants or fruit, such as tomatoes and
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
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Several hurdles
- Immunogenicity of orally administered vaccines vary widely
- Stability of antigens in the digestive tract varies widely
- Genetics of many potential systems remain poorly characterized
 Inefficient transformation systems and low expression levels
• Expression of toxins from B. thuringiensis, herbicide resistant genes
• Transgenic carrots: production of the drug Taliglucerase alfa, which is used to
treat Gaucher's disease.
• Transgenic plants have been modified to increase photosynthesis (currently
about 2% at most plants versus the theoretic potential of 9–10%).
Genetically modified crops
• Crops used in agriculture, the DNA of which has been modified using
genetic engineering techniques.
- In most cases, the aim is to introduce a new trait to the crop which does
not occur naturally in the species
- Resistance to certain pests, diseases, or environmental conditions,
reduction of spoilage, or resistance to chemical treatments
(e.g. resistance to a herbicide), or improving the nutrient profile of the crop
• Use of GM crops expanded rapidly in developing countries, with about
18 million farmers growing 54% of worldwide GM crops by 2013
• There is general scientific agreement that food on the market derived from
GM crops poses no greater risk to human health than conventional food,
but should be tested on a case-by-case basis
- Environmental concerns, safety of foods produced from GM crops,
necessity of GM crops to address the world's food needs, and concerns raised
by intellectual property law
Bt corn
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Variant of maize that has been genetically altered to express one or more proteins from the
bacterium Bacillus thuringiensis.
- The protein is poisonous to certain insect pests and is widely used in organic gardening.
- The European corn borer causes about a billion dollars in damage to corn crops each year.
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In recent years, traits have been added to ward off Corn ear worms and root worms,
the latter of which annually causes about a billion dollars in damages.
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The Bt protein is expressed throughout the plant.
- When a vulnerable insect eats the Bt-containing plant, the protein is activated in its gut,
which is alkaline. In the alkaline environment the protein partially unfolds and is cut by
proteases , yielding a toxin that paralyzes the insect's digestive system and forms holes in the
gut wall. The insect stops eating within a few hours and eventually starves.
In 1996, the first GM maize producing a Bt Cry protein was approved, which killed the European
corn borer and related species; subsequent Bt genes were introduced that killed corn rootworm
larvae.
Transgenic crops
Sugar from genetically modified sugar beets
• Currently half of all sugar in the US comes from sugar beets and the other half
from sugar cane
• Recently, such sugars are treated as two different commodities with two
different prices
• The GMO beets by Monsanto since 2005: tolerate the weed killer, known as
Glyphosate or Roundup, which is used to get rid of weeds.
 GM-sugar beets allow farmers to kill weeds with fewer chemicals than
wild type, but concerns about the over use of wood killer.
 Impact on environments
sugar beets
• Since 2014, sugar industries such Hershey, which use sugar as an
ingredient in cereals, candies and chocolate, tend to label their products
as non-GMO.
 Move from beet sugar to cane sugar.
- Sugar cane is not genetically modified.
 Shortage of cane sugar
 Cane sugar is 10 to 15 % more expensive than beet sugar
Engineered mushroom using CRISPR–Cas9
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Engineering of the fungus, the common white button mushroom (Agaricus bisporus),
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to resist browning.
Editing the family of genes that encodes polyphenol oxidase (PPO), an enzyme
that causes browning.
- By deleting just a handful of base pairs in the mushroom’s genome, one of six
PPO genes was knocked out, reducing the enzyme’s activity by 30%.
• Fruits and vegetables that resist browning : valuable because they keep their color longer
when sliced, which lengthens their shelf life.
- commercialization of genetically engineered non-browning apples and potatoes
• Several of the plants that bypassed the USDA: made using gene-editing techniques
such as the zinc-finger nuclease (ZFN) and transcription activator-like effector
nuclease (TALEN) systems.
• Major advantage of using gene-editing tools:
- they contain no foreign DNA from ‘plant pests’
such as viruses or bacteria.
Nature (2016)
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
- Silkworm virus Bombyx mori nuclear polyhedrovirus(BmNPV)
in conjunction with cultured silkworm cells:
- Virus Autographa californica nuclear polyhedrovirus(AcNPV)
in conjunction with cultured armyworm cells
• Advantages
- High level intracellular protein expression
- 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
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
- Use of silkworm for the production of feline interferon ω
- Direct injection of a vector containing a gene coding for a target protein
- protein expression level: ~ 0.4 mg protein /each
Current Good Manufacturing Practice (cGMP)
Guides to Good Manufacturing Practice
• Producer must comply with the most rigorous standards to ensure
consistent production of a safe and effective therapeutic agents
• Principles underlining such standards are summarized in publications
which detail Good Manufacturing Practice (GMP)
- EU guide to Good Manufacturing Practice for Medicinal Products
• Producers must be familiar with the principles, and they are legally
obliged to ensure adoption of these principles to their specific bioprocess
• Regulatory authority assesses compliance of the producer with the
principles by undertaking regular inspections of the facility
Principles outlined in GMP
• An adequate number of sufficiently qualified, experiences
personnel
• Key personnel, such as the heads of production and quality
control, must be independent of each other
• Personnel should have well-defined job descriptions, and
should receive adequate training
• Issues of personal hygiene should be emphasized to prevent
product contamination
Premises and equipment
• All premises and equipments should be designed, operated, and serviced
to carry out their intended functions
• Facility and equipment should be designed and used to avoid crosscontamination or mix-up between different products
• Sufficient storage area must be provided, and clear demarcation must exist
between storage zones for materials at different levels of processing (raw
materials, partially processes products, finished products etc..)
• Quality control labs must be separated from production, and must be
designed equipped to a standard allowing them to fulfill their intended
function
• Some of the principles outlined in the guide are sufficiently general to
render them applicable to most manufacturing industries.
• Most of principles outlined in guides to GMP are equally as applicable to
the manufacture of traditional pharmaceuticals as to new ones
• Many of the guidelines are specific : guidelines relating to the
requirement for dedicated facilities when manufacturing specific
products
Manufacturing facility
• Appropriate design and layout of the facility required for
production of pharmaceuticals : crucial to the production of
safe and effective medicines
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Injectable products
- Clean room technology
- Generation of ultra pure water
- Maintenance of non-critical areas : storage, labeling, and
packing areas
Clean rooms
• Environmentally controlled areas for injectable/sterile drugs: specifically
designed to protect the product from contamination
• Designed in a way that allows tight control of entry of all substances ( e.g.,
equipment, personnel, in-process product, air etc..)
• Installation of high efficiency particulate air(HEPA) in the ceilings :
- Layers of high-density glass fiber : depth-filter
- Flow pattern of HEPA-filtered air
- Air is pumped into the room via the filters, generating a constant
downward sweeping motion
• Flow motion promotes continued flushing from room of any particulates
generated during processing
• Clean rooms with various levels of cleanliness
- Classified based on the number of airborne particles and viable microorganisms in the
room
- A, B, C, D grades in order of decreasing cleanliness in Europe
- Class 100 (grade A/B), Class 10,000(grade C), Class 100,000 (grade D) in the US
A
B
C
D
Max permitted No. of particles / m3
3,500
3,500
350,000
3,500,000
Max. permitted No. of microorganisms
<1
5
100
500
Water for bioprocess and therapeutic agents
• Water : One of the most important raw materials : used as a basic ingredient
- Cell culture media, buffers, solvent in extraction and purification, solvent in
preparation of liquid form and freeze-dried products
- Used for ancillary processes : cleaning
- ~ 30,000 liters of water : production of 1 kg of a recombinant products by
microbial system
- Generation of water of suitable purity : central to successful operation of
facility
• Purified water :
- Used as the solvent in the manufacture of aqueous-based oral products (e.g.,
cough mixtures, )
- Used for primary cleaning of some process equipment/clean room floors
generation of steam in the facilities, autoclaves
- Used for cell culture media
• Water for injection (WFI)
- Extensive application in biopharmaceutical manufacturing
- Extraction/homogenization/chromatography buffers rinsing process
equipment coming into direct contact with the products
Generation of purified water and WFI
• Generated from potable water
• Removal of impurities found in potable water
• Multi-step purification procedure for purified water and WFI: Monitoring of
each step : continuous measurement of the resistivity of the water
 Increased resistivity with purity
 1- 10 MΩ
• Filters for filtration : 0.22 µm, 0,45 µm of pore size
• Reverse osmosis (RO) membrane : semi-permeable membrane (permeable
to the solvent, water, but impermeable to solute, i.e., contaminant
• General process
Depth filtration Organic Trap Activated charcoal Anion exchange
 Cation exchange  Filtration Distillation or reverse osmosis
 Water for injection
Documentation
• Adequate documentation : essential part of GMP
• Essential in order to
- Help prevent errors/misunderstandings associated with verbal
communication
- Facilitate the tracing of the manufacturing history of any batch of product
- Ensure reproducibility in all aspects of bioprocess