Technology

PROTEOMES & PROTEOMICS

Therapeutic Protein Production An Over view

a report by

D r N i l a n j a n R o y and S h r u t i A g a r w a l
Head and Researcher, Proteomics Laboratory, National Institute of Pharmaceutical Education and Research (NIPER)

So far, pharmaceuticals used for the treatment of diseases have been based largely on the natural or synthetic organic molecules produced by microbes or synthesised by organic chemists. However, the postgenomic-era scenario projects a remarkable change in the therapeutics market, with the realisation that proteins have many potential therapeutic advantages for preventing and curing diseases and disorders. Proteins are the ultimate players of cellular function. Information stored in DNA directs the protein-synthesising machinery of the cell to produce specific proteins required for its structure, function and regulation. There are many proteins that are essential for good health that some people cannot produce because of genetic defects. The advent of sophisticated genomics and proteomicsbased functional identification has unravelled large numbers of candidate proteins with a potential for therapeutic intervention. Several market research groups have predicted that the growth of the biopharmaceutical market might reach as high as 15% annually and result in a critical shortage in protein-manufacturing capacity in the near future. Short peptide chains consisting of fewer than 30 amino acids can be synthesised chemically. Proteins larger than that are best produced by living cells. These natural bioreactors support the sustained yield of target protein. The protein-encoding DNA is inserted into cells. As the cells grow, they synthesise the protein, which is subsequently harvested and purified. Production of proteins by micro-organisms is well entrenched in the industry. More recent knowledge of the molecular and cellular mechanism of diverse hosts has led to envisioning the art of expression of foreign proteins in mammalian cell culture and transgenic animals and plants. However, several key issues, namely complexity of the protein, quantity requirement and reliability of the expression system,

need to be assessed before choosing a host system for a particular therapeutic protein. Microbial and plant hosts can be utilised for a moderate and large quantity of low-complexity proteins. Complex proteins can be produced in relatively small and large quantities in animal cell culture and transgenic animals, respectively.
Dr Nilanjan Roy

Since 1982, more than 100 therapeutic proteins and peptides have been licensed for production using bacterial, fungal and mammalian cells, and many therapeutic proteins are currently being developed and tested in a variety of host cells.1 An overview of the technologies available and technical hurdles that are encountered in these processes is presented here.
Production of Low-complexity Proteins
Microbial Cell Factories
Shruti Agarwal Dr Nilanjan Roy is Head of the Proteomics Laboratory of the National Institute of Pharmaceutical Education and Research (NIPER). In addition to research, he is actively engaged in teaching in the areas of advanced techniques in biotechnology, genomics, proteomics, bio and pharma informatics. As well as his research articles, he is the author of several reports and popular articles. Dr Roy holds a PhD from Bose Institute, Kolkata, India, and has seven years of research experience from one of the premier hospitals of the US, The Cleveland Clinic Foundation, Cleveland, OH. Shruti Agarwal is an active researcher at NIPERs Proteomics Laboratory. She has been involved in deciphering the role of oxidative stress regulators in the ageing process.

Humans have exploited microbial transformations for centuries initially only in the food industry, but, lately, they have provided immense benefits to the pharmaceutical industry as bioreactors for antibiotic production. The birth of genetic engineering in the late 1970s, and subsequent technological expansion, has provided molecular tools for producing heterologous proteins in a wide range of micro-organisms including bacteria, yeast and filamentous fungi, thus largely extending their manufacturing capabilities.2 Following transformation of a best proteinexpressing host (for example Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris), best protein-expressing clones are identified and further optimised for protein expression. Because of scalability and well-characterised genetics, microbial host systems remain the best option for lowcomplexity, moderate-volume protein production at relatively low cost.3 A major hurdle in microbial

1. R Andersson and R Mynahan, The Protein Production Challenge, http://www.windhover.com 2. Old bugs for new tasks; the microbial offer in the proteomics era, http://www.microbialcellfactories.com/content/1/1/4 3. R Andersson and R Manahan , The Protein Production Challenge, In Vivo, Windhover Information Inc., May 2001, pp. i5.
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PROTEOMES & PROTEOMICS

production is in the post-production recovery of proteins. Examples of strategies for improved bioprocesses are as follows. Use of a secretory signal peptide fused to the N-terminus of the target protein can ensure efficient secretion of the protein. This eliminates the need for cell disruption and reduces interaction with host proteins. However, dilution of protein can be a shortcoming. This can be overcome by tagging a hydrophobic tail to the target protein, which can alter the partitioning of the protein in two-phase systems for efficient purification and concentration. Otherwise, tagging of an affinity tail (for example His-tag) can be an option for low-cost purification. The addition of charged amino acid residues or mutation of amino acids of a protein to alter the isoelectric point can improve the purification by ion exchange chromatography followed by production. However, proteins with complicated folding requirements and post-translational modification steps cannot be produced with this system.
Transgenic Plants

accumulation, the size of transgenic chloroplasts increases and this facilitates purification. The issues surrounding objections and concerns to transgenic crops are as follows. There may be damage to human health due to allerginicity of contaminating plant proteins. Crop-to-crop and crop-to-weed transfer of foreign protein genes and negative effects on herbivorous animals are a risk. Thus, it is of utmost importance to ensure that the gene pool is not contaminated. Adequate quarantine measures can therefore increase production cost.6 There are also some possibilities of disturbances in traditional farming practices and economies, especially in developing countries, where molecular farming can be a lucrative alternative to traditional processes.5 Thus, cost-efficient production of transgenic plants can alter the economics of recombinant protein synthesis. To make best use of this promising opportunity, it is important that government regulatory agencies take active interest in developing the agronomic and manufacturing regulations needed to ensure safety, consistency and potency of plant-made pharmaceuticals.
Production of Complex Proteins
Animal Cell Cultures

The use of genetically engineered plants to produce valuable proteins is increasing slowly. Vaccines, mammalian blood constituents, enzymes, antibodies and low-calorie sweeteners are being produced in tobacco leaves. In order for a plant to be used to produce specific molecules, a novel gene is inserted into its chromosomes. A regulatory code inserted with that gene dictates to the plant where to produce the desired protein in its leaves, roots or seeds. Since the plant proteins are not modified posttranslationally, transgenic plants are likely to be a viable alternative for less complex proteins.4 With well-established transgenic technology and easier expansion by means of clonal propagation, transgenic plants are specially suited to high-volume production of proteins that otherwise cannot be produced costeffectively with conventional microbial systems.5 Protein extraction and purification are required much the same way, as are the requirements in the case of cell culture methods. However, recent chloroplast transformation techniques have been perfected and have achieved high-level protein production in chloroplasts. Because of protein

Animal cell lines have been utilised increasingly as hosts for the production of recombinant proteins and currently dominate some product families such as antibodies. Considerable research effort has been directed towards the development and optimisation of the cell culture process, with the objective of delivering the quantity and quality of protein required by the healthcare industries. Many potential products are already in the clinical trial pipeline. However, many of the mammalian cell lines that are used for industrial-scale recombinant protein production undergo apoptotic death due to deprivation of key nutrients (such as amino acids, glucose and serum), oxygen, the use of virus-based protein expression systems and cytostatic agents in the bioreactor environment.7 This limits culture

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4. R Fischer, et al., Towards molecular farming in the future: transient protein expression in plants, Biotechnol. Appl. Biochem., 30 (1999), pp. 113116. 5. B R Thomas, A V Deynze and K J Bradford, Production of Therapeutic Proteins in Plants, Agricultural Biotechnology in California Series, Pub-8078. 6. R Fischer and N Emans, Molecular farming of Pharmaceutical proteins, Transg. Res., 9 (2000), pp. 279229. 7. B D Kelley, Biochemical engineering: Bioprocessing of therapeutic proteins, Curr. Opin. Biotechnol., 12 (2001), pp. 173174.

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Technology

PROTEOMES & PROTEOMICS

duration and productivity. Thus, though the technique is fairly well characterised, the capacity constraints call for accurate manufacturing strategy, therefore limiting use to low-volume requirements of high-complexity proteins. Again, the set-up costs are prohibitively expensive. Building or expanding a manufacturing facility to include 10,000-litre bioreactors the minimum size for truly large-scale commercial production takes three to five years and costs US$250500 million.8 Process control equipment, assay development and quality control systems are major expenses. Moreover, the time required to construct largescale facilities has imposed significant business and financial risks on pharmaceutical companies. For the facility to be able to work by the time US Food and Drug Administration (FDA) approval is sanctioned, the company needs to start investing when the drug is somewhere in the Phase III stage of clinical trials.9 Given the uncertainty related to the outcome of clinical trials, pharmaceutical companies face the risk of loss due to underbuilding or over-building capacities. Thus, an appropriate decision needs to be taken before manufacturing proteins with limited markets. Before the successful commercialisation of this technology, significant research inputs are required in the areas of cell line selection, bioreactor configuration, online bioreactor controls and production modes: suspension culture in serum or protein-free media, fed batch or perfusion technology and protein purification techniques. Also, the production of biologics must be managed carefully to avoid unintentional transmission of viral diseases that could infect humans.
Transgenic Animals

with good prospects of large-scale commercialisation in the near future. The process involves microinjection of the DNA solution into the pronucleus of the embryo using a very fine glass needle. The injected zygote is then transferred into a hormonally prepared recipient and brought to term. Finally, positive transgenic animals are matured and the level of expression of the transgene is determined. However, transgenic animals are costly to produce. The cost of making one transgenic animal ranges from US$20,000 to US$300,000 and only a small portion of the attempts succeeds in producing a transgenic animal. The rate of transgenesis is 5% to 25% of live births,10 but the unit cost per protein should be significantly less when animals are used as bioreactors to produce human proteins. Different transgenic species of cow, sheep, goat, pig and rabbit are in use. Although gene expression and heterologous protein production is possible in many different tissues and fluids of the animal, transgenic milk production is currently the method that is most feasible and furthest along in development and the regulatory process, making ruminants the best choice in this regard. Secretion in milk is made possible by coupling a mammary gland-targeting signal sequence with the gene for making protein.11 The transgenic protein can be harvested and purified from milk. The advantages of this method are: a high expression level and volume output; the ability to express complex proteins; low initial capital investment is required; low operational costs; and the production facility is reproducible inbreeding could pass an animals ability to produce transgenic protein to its offspring.

The term transgenic animals describes animals with chromosomes that contain stably integrated copies of genes or gene constructs derived from other species or not normally found in the host animal. The use of transgenics as a technology for producing recombinant proteins has made remarkable strides in the past few years. The production of the first transgenic farm animals was reported in 1985, and biopharmaceutical production by these animals followed shortly thereafter. Pharming is the production of human pharmaceuticals in farm animals, a process still in the developmental stages,

However, as with any other developing technology, a transgenic animal also has its own constraints and shortcomings that need to be resolved. The major issue is regulatory approval. For animal-based transgenic companies, the FDA is expected to be the primary regulatory body, along with regulations that govern moral animal treatment, protein purification and extensive testing.12 Stringent rules are still in the process of development and implementation. Currently, there is a high degree of uncertainty related to long-term policy associated with FDA approval of a transgenically produced protein. s

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8. A Ingley, J Pavlik and T Smith, Transgenic Protein Production as an alternative Manufacturing Technology for Pharmaceutical Companies. 9. Biopharmaceutical outsourcing: will a supply crunch hit its growing market?, http://www.biotech.about.com 10. W G Gavin, The future of Transgenics, Regulatory Affairs Focus, May 2001 11. M Herper, Milking Genetically Modified Cows, http://www.forbes.com 12. FDA Directive 75/318/EEC, Use of Transgenic Animals in the Manufacture of Biological Medicinals.

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