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.

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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 percent are made in China and India. The F.D.A. 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.

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.

• Heparin 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. • 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).

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

• • • • • 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

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

Cell culture process for therapeutic proteins

Overall process

Protein purification process

Affinity purification

Ethanol fermentation process

Ethanol fermentation tanks

Microorganism fermentation system

Host cells for the production of therapeutic agents

• 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 • The use of appropriate expression system for specific products : Each expression system displays its own unique set of advantages and disadvantages

E. coil

• Most common microbial species used to produce heterologous proteins - Heterologous protein : protein that does not occur in host cells ex) 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 fermentation technology, large scale cultivation

Drawbacks of

E. coli

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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

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Prevention of inclusion body formation

growth at lower temperature (30 o C) - 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

Yeast

• Saccharomyces cerevisiae, Pichia pastoris : •

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

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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

Fungal production system

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Aspergillus niger

• Mainly used for production of industrial enzymes : a-amylase, glucoamylase, cellulase, lipase, protease etc..

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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

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

CHO cells

Transgenic animals

• Transgenic animal : 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  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

• 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), β-casein, α- and β-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

• 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 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 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

• 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

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 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

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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)

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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 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

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 virus Autographa californica nuclear polyhedrovirus(AcNPV) in conjunction with cultured armyworm cells

• 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

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 ω

Guides to Good Manufacturing Practice

• Producer must comply with the most rigorous standards to ensure consistent production of a safe and effective drugs • • 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

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An adequate number of sufficiently qualified, experiences personnel

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Key personnel, such as the heads of production and quality control, must be independent of each other

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Personnel should have well-defined job descriptions, and should receive adequate training

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Issues of personal hygiene should be emphasized to prevent product contamination

Premises and equipment

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All premises and equipments should be designed, operated, and serviced to carry out their intended functions

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Facility and equipment should be designed and used to avoid cross contamination or mix-up between different products

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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..)

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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

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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

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Environmentally controlled areas for injectable/sterile drugs: specifically designed to protect the product from contamination

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Designed in a way that allows tight control of entry of all substances ( e.g., equipment, personnel, in-process product, air etc..)

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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

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The flow motion promotes continued flushing from room of any particulates generated during processing

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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 Max permitted No. of particles per m 3 Max. permitted No. of microorganisms

A 3,500 <1 B 3,500 5 C 350,000 100 D 3,500,000 500

Water for therapeutic agents

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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

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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

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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

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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

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increased resistivity with purity

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1- 10 MΩ

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Filters : 0.22 µm, 0,45 µm Reverse osmosis (RO) membrane : semi-permeable membrane (permeable to the solvent, water, but impermeable to solute, i.e., contaminant

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General process Depth filtration

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Organic Trap

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Activated charcoal

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Anion exchange Cation exchange

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Filtration

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Distillation or reverse osmosis

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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